EP1218411A2 - Secreted proteins and uses thereof - Google Patents

Secreted proteins and uses thereof

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Publication number
EP1218411A2
EP1218411A2 EP00965288A EP00965288A EP1218411A2 EP 1218411 A2 EP1218411 A2 EP 1218411A2 EP 00965288 A EP00965288 A EP 00965288A EP 00965288 A EP00965288 A EP 00965288A EP 1218411 A2 EP1218411 A2 EP 1218411A2
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European Patent Office
Prior art keywords
seq
accession number
nucleic acid
number pta
amino acid
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EP00965288A
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German (de)
French (fr)
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EP1218411A4 (en
Inventor
Susan J. Kirst
John D. Sharp
Christopher C. Fraser
Thomas Barnes
Gillian Kingsbury
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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Publication of EP1218411A2 publication Critical patent/EP1218411A2/en
Publication of EP1218411A4 publication Critical patent/EP1218411A4/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 proteins, fragments, derivatives, and variants thereof of the present invention are collectively referred to herein 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 invention features nucleic acid molecules which are at least 30%, 35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of human SEQ ID NO:66, SEQ ID NO:67, the nucleotide sequence of the cDNA insert of an human EpT509 clone deposited August 5, 1999 with the ATCC® as Accession Number PTA-438, or a complement thereof
  • the invention features nucleic acid molecules of at least 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or 2020 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:l, the nucleotide sequence of an INT307 cDNA of ATCC® Accession Number PTA-455, or a complement thereof.
  • the invention features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 645 contiguous nucleotides of nucleic acids 1 to 649 of SEQ ID NO:l, or a complement thereof.
  • the invention features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250 or 300 contiguous nucleotides of nucleic acids 1120 to 1430 of SEQ ID NO:l, or a complement thereof.
  • the invention features nucleic acid molecules of at least 460, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300 or 3340 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:34, the nucleotide sequence of an EpT351 cDNA of ATCC® Accession Number PTA-424, or a complement thereof.
  • the invention features nucleic acid molecules of at least 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:42, the nucleotide sequence of an EpT361 cDNA of ATCC® Accession Number PTA-438, 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 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:3, the amino acid sequence encoded by an INT307 cDNA of ATCC® Accession Number PTA-455, or a complement thereof.
  • nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:68 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 289 contiguous amino acids of SEQ ID NO:68 or the amino acid sequence encoded by a human EpT509 cDNA of ATCC® Accession Number PTA-438.
  • the invention also features isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 25%, preferably 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to a nucleic acid sequence encoding SEQ ID NO:3, 25, 36, 44, 56, 63, 68, or 78, isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76 or 77, or a complement thereof, or the non-coding strand of INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509
  • the nucleic acid molecules are at least 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or 2020 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, an LNT307 cDNA of ATCC® Accession Number PTA-455, or a complement thereof.
  • the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450 or 1490 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 3564 to 5058 of SEQ ID NO:42, or a complement thereof.
  • the invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:76 or 77, or a mouse EpT509 cDNA of ATCC® Accession Number PTA-438, or a complement thereof.
  • biological activities include, e.g., (1) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of immune cells (e.g., B-lymphocytes and monocytes); (2) the ability to modulate, protein-protein interactions (e.g., homophilic and/or heterophilic), and protein-ligand interactions, e.g., in receptor-ligand recognition;
  • immune cells e.g., B-lymphocytes and monocytes
  • protein-protein interactions e.g., homophilic and/or heterophilic
  • protein-ligand interactions e.g., in receptor-ligand recognition
  • biological activities include, e.g., (1) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of mammary cells, e.g., mammary epithelial cells; (2) the ability to modulate the development and progression of cell proliferative disorders such as cancer (e.g. breast or breast-associated cancer); (3) the ability to modulate, protein-protein interactions (e.g.
  • an INTERCEPT 307 protein includes at least one or more of the following domains: a signal sequence, an extracellular domain, a transmembrane domain, a gas vehicle protein GVPc domain, and an intracellular or cytoplasmic domain.
  • the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (e.g., 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 is a fragment of a polypeptide of the invention or a nucleic acid molecule encoding such a polypeptide fragment.
  • 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.
  • an antibody or a fragment thereof i.e., human and non-human antibodies or fragments thereof and/or monoclonal antibodies or fragments thereof of the invention, specifically bind to an extracellular domain having the amino acid sequence of SEQ ID NO:6, 7, 8, 28, 39, 49, 75 or 84.
  • the dashed vertical line separates the signal sequence (amino acids 1 to 23 of SEQ ID NO:3, (SEQ ID NO:4)) on the left from the mature protein (amino acids 24 to 362 of SEQ ID NO:3, (SEQ ID NO:5)) on the right.
  • Figure 6 shows an alignment of the nucleotide sequence of INTERCEPT 307 coding region (SEQ ID NO:2) and the nucleotide sequence of human eosinophil granule major basic protein coding region (SEQ ID NO:87; Accession Number Z26248).
  • the nucleotide sequences of the coding regions are 38.1% identical.
  • the full-length INTERCEPT 307 nucleic acid sequence (SEQ ID NO:l) and human eosinophil granule major basic protein cDNA (SEQ ID NO:88; Accession Number Z26248) have an overall sequence identity of 57.3%>.
  • Figure 26 depicts a hydropathy plot of mouse TANGO 509. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line.
  • the cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace.
  • the dashed vertical line separates the signal sequence (amino acids 1 to 18 of SEQ ID NO:78 (SEQ ID NO:79)) on the left from the mature protein (amino acids 19 to 290 of SEQ ID NO:78) on the right.
  • Figure 27 shows an alignment of the mouse TANGO 509 amino acid sequence (SEQ ID NO:78) with the butyrophilin-like protein amino acid sequence (SEQ ID NO:85; Accession Number AF242780). The alignment shows that there is a 31.9% overall amino acid sequence identity between mouse TANGO 509 and the butyrophilin-like protein. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • the INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 proteins and nucleic acid molecules comprise families of molecules having certain conserved structural and functional features.
  • family or “families” are 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.
  • a MANGO 511 family member includes one or more MANGO 511 Ig-like domains having an amino acid sequence that is at least 55%>, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95%> identical to amino acids 60 to 118 of SEQ ID NO:25 (SEQ ID NO:31), and has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain, has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine, and has at least one MANGO 511 biological activity as described herein.
  • a TANGO 351 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain.
  • a TANGO 351 protein contains an extracellular domain at about amino acid residues 25 to 458 of SEQ ID NO:36 (SEQ ID NO:39), a transmembrane domain at about amino acid residues 459 to 476 of SEQ ID NO:36 (SEQ ID NO:40), and a cytoplasmic domain at about amino acid residues 477 to 482 of SEQ ID NO:36 (SEQ ID NO:41).
  • the mature TANGO 351 protein corresponds to amino acids 25 to 482 of SEQ ID NO:36 (SEQ ID NO:38).
  • a SEA domain typically has the following consensus sequence: h-t-h-Xaa-h-Xaa-Xaa-Xaa-Xaa-Xaa-h-Xaa-a-t-t-t-h-t-t-t-Xaa-o-Xaa-Xaa-a-Xaa-Xaa-Xaa-h-Xaa-t-t-H-Xaa-t- Xaa-h-Xaa-t-t-Xaa-Xaa-Xaa-Xaa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xa-Xaa
  • SEA domains are predominantly found in adhesive proteins present in heavily glycosylated environments.
  • SEA domains are found in a 63 kDa sea urchin sperm protein, agrin, enterokinase, perlecan, the breast cancer marker MUCl (episialin) and the cell surface antigen 114/A10 (Bork and Patthy, (1995) Prot. Sci. 4:1421-1425).
  • a TANGO 361 family member can include a serine protease domain.
  • Serine protease domains are typically found in serine proteases, and can be found in, among other proteins, e.g., blood coagulation factors VII, XI, and X, thrombin, plasminogen, tryptases, such as trypsin, airway trypsin-like proteases, mast cell proteases, and members of the complement system which are known for regulation of energy balance and suppression of infectious agents.
  • serine protease domain refers to a polypeptide sequence that includes about 100-400 amino acid residues, preferably about 150-350 amino acid residues, more preferably about 200-300 amino acid residues, and most preferably about 225-260 amino acid residues.
  • a serine protease typically has two consensus sequences.
  • the Ig-like domain of human TANGO 509 is an Ig-like domain which has the following consensus sequence at the C-terminus of the domain: [FY]-Xaa-C-Xaa-[VAIF]-COO-, wherein [FY] is either a phenylalanine or a tyrosine residue (preferably tyrosine), where "Xaa” is any amino acid, C is a cysteine residue, [VA] is a valine, an alanine, an isoleucine or phenylalanine residue, and COO- is the C-terminus of the domain.
  • a mouse TANGO 509 family member includes one or more such Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81).
  • a mouse TANGO 509 family member includes one or more mouse TANGO 509 Ig-like domains having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81), has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain, and has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine.
  • a mouse TANGO 509 family member includes one or more Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81).
  • a cDNA sequence of human INTERCEPT 307 has a nucleotide at position 54 which is cytosine (C)(SEQ ID NO:l).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is leucine (L)(SEQ ID NO:3).
  • a species variant cDNA sequence of human INTERCEPT 307 has a nucleotide at position 54 which is adenine (A)(SEQ ID NO:l 13).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is isoleucine (I)(SEQ ID NO:l 14), i.e., a conservative substitution.
  • the first has the sequence SLR (at amino acid residues 56 to 58 of SEQ ID NO:3)
  • the second has the sequence TTK (at amino acid residues 102 to 104 of SEQ ID NO:3)
  • the third has the sequence SAK (at amino acid residues 124 to 126 of SEQ ID NO:3)
  • the fourth has the sequence SRR (at amino acid residues 147 to 149 of SEQ ID NO:3)
  • the fifth has the sequence TWK (at amino acid residues 353 to 355 of SEQ ID NO:3).
  • INTERCEPT 307 has three casein kinase II phosphorylation sites.
  • Figure 3 shows an alignment of the human INTERCEPT 307 amino acid sequence (SEQ ID NO:3) with the prostate cancer gene PB39 amino acid sequence (SEQ ID NO:20; Accession Number NM_003627). The alignment shows that there is a 21.0% overall amino acid sequence identity between INTERCEPT 307 and PB39.
  • PB39 is expressed in tissues of the adult colon, small intestine, ovary, prostate, spleen, skeletal muscle and pancreas.
  • PB39 is also expressed in fetal kidney, liver and lung.
  • the expression of PB39 has been shown to be increased early in prostate cancer development and PB39 can play a role in the development of human prostate cancer.
  • INTERCEPT 307, nucleic acids and proteins may be useful, for example, as early markers for the development of prostate cancer (e.g., early markers for the development of prostatic intraepithelial neoplasia (PIN))-
  • PIN prostatic intraepithelial neoplasia
  • a cDNA encoding human MANGO 511 was identified by analyzing the sequences of clones present in a human dendritic cell library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jThxh005c 10, encoding full-length human MANGO 511.
  • the human MANGO 511 cDNA of this clone is 1477 nucleotides long ( Figure 7; SEQ ID NO:23).
  • the signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human MANGO 511 includes a 41 amino acid signal peptide (amino acid 1 to amino acid 41 of SEQ ID NO:25, (SEQ ID NO:26) preceding the mature MANGO 511 protein (corresponding to amino acid 42 to amino acid 299 of SEQ ID NO:25, (SEQ ID NO:27).
  • the molecular weight of MANGO 511 protein without post-translational modifications is 32.8 kDa prior to the cleavage of the signal peptide, and 28.6 kDa after cleavage of the signal peptide.
  • tyrosine phosphorylation of LIR-1 in monocytes has been shown to result in the binding of tyrosine phosphatase SHP- 1, and LIR-1 has been shown to be involved in the inhibition or down-modulation of monocyte activation signals (Fanger et al. (1998) Eur. J. Immunol. 28:3423-3434).
  • MANGO 511 nucleic acids, proteins and modulators thereof are useful in modulating MHC class I binding and monocyte activation.
  • Figure 10 shows an alignment of the coding regions of the nucleotide sequence of LIR-1 (SEQ ID NO:33; Accession Number AF009221) and the nucleotide sequence of human MANGO 511 (SEQ ID NO:23). The alignment shows a 34.0 % overall sequence identity between the two nucleotide sequences.
  • the coding region of the nucleotide sequence of LIR-1 (SEQ ID NO:33; Accession Number AF009221) and the full-length nucleotide sequence of human MANGO 511 cDNA (SEQ ID NO:24) have an overall sequence identity of 44.0%.
  • Such MANGO 511 compositions and modulators thereof can be utilized modulate or treat immune disorders that include, but are not limited to, immune proliferative disorders (e.g., carcinoma, lymphoma, e.g., follicular lymphoma), and disorders associated with fighting pathogenic infections, e.g., bacterial (e.g., chlamydia) infection, parasitic infection, and viral infection (e.g.
  • immune proliferative disorders e.g., carcinoma, lymphoma, e.g., follicular lymphoma
  • pathogenic infections e.g., bacterial (e.g., chlamydia) infection, parasitic infection, and viral infection (e.g.
  • MANGO 511 exhibits homology to LIR-1
  • MANGO 511 nucleic acids, proteins and/or modulators thereof can be used to modulate natural killer cell function, e.g., activation.
  • MANGO 511 nucleic acids, proteins and modulators thereof can be used to treat diseases associated with aberrant natural killer cell activation such as chronic natural killer cell lymphocytosis, aggressive non-T, non-B natural killer cell lymphoma/leukemia (ANKL/L), and Chediak-Higashi syndrome.
  • diseases associated with aberrant natural killer cell activation such as chronic natural killer cell lymphocytosis, aggressive non-T, non-B natural killer cell lymphoma/leukemia (ANKL/L), and Chediak-Higashi syndrome.
  • human TANGO 351 has extracellular domains at amino acid residues contains an extracellular domain at amino acid residues 1 to 458 of SEQ ID NO:36, a transmembrane domain at amino acid residues 459 to 476 of SEQ ID NO:36 (SEQ ID NO:40), and a cytoplasmic domain at amino acid residues 477 to 482 of SEQ ID NO:25 (SEQ ID NO:41).
  • a cDNA sequence of human TANGO 351 has a nucleotide at position 273 which is guanine (G)(SEQ ID NO:34).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position
  • TANGO 351 has a prokaryotic membrane lipoprotein lipid attachment site with the sequence HLGGWWVSSGC (at amino acid residues 260 to 270 of SEQ ID NO:36).
  • TANGO 351 nucleic acids, proteins and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of kidney cells and the kidney.
  • TANGO 351 nucleic acids, proteins and modulators thereof can be used to modulate or 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 diseases (e.g., acute tubular necrosis and acute
  • Figure 14 depicts a hydropathy plot of human TANGO 361. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
  • Human TANGO 361 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 235 to 423 of SEQ ID NO:49, (SEQ ID NO:49), a transmembrane domain at amino acid residues 217 to 234 of SEQ ID NO:44 (SEQ ID NO:47), and a cytoplasmic domain at amino acid residues 36 to 216 of SEQ ID NO:44 (SEQ ID NO:48).
  • a human TANGO 361 protein contains a cytoplasmic domain at amino acid residues 235 to 423 of SEQ ID NO:49, (SEQ ID NO:49), a transmembrane domain at amino acid residues 217 to 234 of SEQ ID NO:44 (SEQ ID NO:47), and an extracellular domain at amino acid residues 36 to 216 of SEQ ID NO:44 (SEQ ID NO:48).
  • a cDNA sequence of human TANGO 361 has a nucleotide at position 117 is thymine (T)(SEQ ID NO:42).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 15 that is phenylalanine (F)(SEQ ID NO:44).
  • a species variant cDNA sequence of human TANGO 361 has a nucleotide at position 117 which is adenine (a)(SEQ ID NO: 141).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 26 that is tyrosine (Y)(SEQ ID NO: 142), i.e., a conservative substitution.
  • a cDNA sequence of human TANGO 361 has a nucleotide at position 122 is thymidine (T)(SEQ ID NO:42).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 28 that is serine (S)(SEQ ID NO:44).
  • a species variant cDNA sequence of human TANGO 361 has a nucleotide at position 122 which is adenine (A)(SEQ ID NO: 143).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 28 that is threonine (T)(SEQ ID NO: 144), i.e., a conservative substitution.
  • TANGO 361 Four N-myristylation sites are present in TANGO 361.
  • the first has the sequence GTRRSK (at amino acid residues 179 to 184 of SEQ ID NO:44, the second has the sequence GSHRCG (at amino acid residues 213 to 218 of SEQ ID NO:44), the third has the sequence GALKND (at amino acid residues 317 to 322 of SEQ ID NO:44), and the fourth has the sequence GSLEGK (at amino acid residues 360 to 365 of SEQ ID NO:44).
  • TANGO 361 has a serine protease, serine active site, consensus sequence with the sequence GDSGG (at amino acid residues 371 to 375 of SEQ ID NO:44.
  • Clone EpT361 which encodes TANGO 361 was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on July 29, 1999 and assigned Accession Number PTA-438. 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.
  • variant 1 is a secreted protein having amino acids 1 to 254 depicted in SEQ ID NO:56.
  • Figure 17 shows an alignment of the human TANGO 499 form 1, variant 1 amino acid sequence SEQ ID NO:56 with the Artemin amino acid sequence SEQ ID NO:59. The alignment shows that there is a 23.5% overall amino acid sequence identity between TANGO 499 form 1, variant 1 and Artemin.
  • the Artemin protein is widely expressed in the nervous system that has been shown to be involved in such processes as peripheral neuron survival and also dopaminergic midbrain neuron survival.
  • variant 3 has an N-glycosylation site with the sequence NISI (at amino acid residues 95 to 98 of SEQ ID NO:63).
  • N-myristylation sites are present in TANGO 499 form 2, variant 3.
  • the first has the sequence GVRQAQ (at amino acid residues 107 to 112 of SEQ ID NO:63)
  • the second has the sequence GCEPSC (at amino acid residues 144 to 149 of SEQ ID NO:63)
  • the third has the sequence GQTFAD (at amino acid residues 153 to 158 of SEQ ID NO:63)
  • the fourth has the sequence GTDLCR (at amino acid residues 159 to 164 of SEQ ID NO:63)
  • the fifth has the sequence GARHCF (at amino acid residues 177 to 182 of SEQ ID NO:63)
  • the sixth has the sequence GSGSGS (at amino acid residues 218 to 223 of SEQ ID NO:63).
  • Figure 21 depicts an alignment of TANGO 499 form 1, variant 1 amino acid sequence and TANGO 499 form 2, variant 3 amino acid sequence.
  • the amino acid sequences are 90.2% identical, the cDNAs are 85.6%> identical, and the ORFs are 90.2%) identical.
  • the alignment clearly demonstrates that the two forms are identical except for the result of a putative alternative exon splicing event in the illustrated region.
  • the invention contemplates additional splice variants of the presently claimed nucleic acids and proteins encoded by such splice variants.
  • the open reading frame of the form 2 TANGO 499 cDNA conserved region comprises nucleotides 1 to 708 of SEQ ID NO:92 (SEQ ID NO:93), and encodes a polypeptide comprising the sequence of SEQ ID NO: 94.
  • TANGO 499 was originally found in a human pituitary library, TANGO 499 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of cells, tissues and/or organs, e.g. , the proliferation of tissues and cells of pituitary origin.
  • TANGO 499 was originally identified in a pituitary library, for example, TANGO 499 nucleic acids, proteins and modulators thereof can be used to regulate processes involved with sexual development and function, including, e.g., normal and abnormal reproductive hormonal function in the fetus, infant, adolescent and adult and also can modulate the effects of pituitary insufficiency and pituitary adenomas on sexual development, reproductive function and sexuality in men and women.
  • TANGO 499 nucleic acids, proteins and modulators thereof can be used to treat pituitary tumors causing Cushing's syndrome, and also hypopituitarism during pregnancy which may be the result of intrasellar adenomas, suprasellar lesions, lymphocytic hypophysitis or antepartum pituitary necrosis, and in the postpartum period may be because of postpartum hemorrhage and pituitary necrosis.
  • TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and function of the eye, such as retinal development and function, ( e.g., photoreceptor disk morphogenesis).
  • TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to treat eye diseases and/or disorders, e.g., autosomal dominant retinitis pigmentosa, autosomal dominant punctata albescens, butterfly-shaped pigment dystrophy, cataracts, macular degeneration, myopia, stigmatism and retinoblastoma.
  • TANGO 499 family members have homology to glial cell line-derived neurotrophic-related factors (e.g., GDNF, artemin, neurturin, and persephin).
  • glial cell line-derived neurotrophic-related factors e.g., GDNF, artemin, neurturin, and persephin.
  • TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to modulate survival, activation, proliferation, motility, and differentiation of peripheral or central neurons.
  • TANGO 499 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of neural organs, e.g., neural tissues and cells, e.g., cells of the central nervous system, e.g., cells of the peripheral nervous system.
  • TANGO 499 nucleic acids, proteins, and modulators thereof can also be used to modulate symptoms associated with abnormal neural signaling and function, e.g., epilepsy, stroke, traumatic injury.
  • TANGO 499 proteins could be useful to treat neural related disorders or neural damage, such as for regenerative neural repair after damage by trauma, degeneration, or inflammation e.g.
  • TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and progression of cancerous and non-cancerous cell proliferative disorders, such as deregulated proliferation (such as hyperdysplasia, hyper- IgM syndrome, or lymphoproliferative disorders), cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hyperplasia), cancers such as neoplasms or tumors (such as carcinomas, sarcomas, adenomas or myeloid lymphoma tumors, e.g., f ⁇ brosarcoma, myxosarcoma,
  • TANGO 499 expression can be utilized as a marker (e.g., an in situ marker) for specific tissues (e.g., the pituitary) and/or cells (e.g., pituitary cells) in which TANGO 499 is expressed.
  • TANGO 499 nucleic acids can also be utilized for chromosomal mapping, or as chromosomal markers, e.g., in radiation hybrid mapping.
  • a cDNA encoding human TANGO 509 was identified by analyzing the sequences of clones present in a mammary epithelium library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jthvb017hl 1, encoding full-length human TANGO 509.
  • the human TANGO 509 cDNA of this clone is 3575 nucleotides long ( Figure 23; SEQ ID NO:66).
  • the signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 509 includes a 18 amino acid signal peptide (amino acid 1 to amino acid 18 of SEQ ID NO:68; SEQ ID NO:69) preceding the mature TANGO 509 protein (corresponding to amino acid 19 to amino acid 290 of SEQ ID NO:68; SEQ ID NO:70).
  • the molecular weight of TANGO 509 protein without post-translational modifications is 33.3 kDa prior to the cleavage of the signal peptide, and 31.0 kDa after cleavage of the signal peptide.
  • a human TANGO 509 protein contains a cytoplasmic domain at amino acid residues 260 to 290 of SEQ ID NO:68, a transmembrane domain at amino acid residues 241 to 259 of SEQ ID NO:68 (SEQ ID NO:73), and an extracelluar domain at amino acid residues 19 to 240 of SEQ ID NO:68.
  • a cDNA sequence of human TANGO 509 has a nucleotide at position 69 which is thymidine (T)(SEQ ID NO:66).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is phenylalanine (F)(SEQ ID NO:68).
  • a species variant cDNA sequence of human TANGO 509 has a nucleotide at position 69 which is adenine (A)(SEQ ID NO: 153).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is tyrosine (Y)(SEQ ID NO: 154), i.e., a conservative substitution.
  • a cDNA sequence of human TANGO 509 has a nucleotide at position 72 which is cytosine (C)(SEQ ID NO:66).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 5 that is alanine (A)(SEQ ID NO:68).
  • a cDNA sequence of human TANGO 509 has a nucleotide at position 132 which is adenine (A)(SEQ ID NO:66).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 25 that is lysine (K)(SEQ ID NO:68).
  • a species variant cDNA sequence of human TANGO 509 has a nucleotide at position 132 which is guanine (G)(SEQ ID NO: 157).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 25 that is arginine (R)(SEQ ID NO: 158), i.e., a conservative substitution.
  • a cDNA sequence of human TANGO 509 has a nucleotide at position 191 which is guanine (G)(SEQ ID NO:66).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 45 that is glutamate (E)(SEQ ID NO:68).
  • a species variant cDNA sequence of human TANGO 509 has a nucleotide at position 191 which is cytosine (C)(SEQ ID NO: 159).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 45 that is glutamine (Q)(SEQ ID NO: 160), i.e., a conservative substitution.
  • Human TANGO 509 has four N-glycosylation sites with the first sequence NMTI (at amino acid residues 35 to 38 of SEQ ID NO:68), the second has the sequence NVTS (at amino acid residues 192 to 195 SEQ ID NO:68), the third has the sequence NTTT (at amino acid residues 200 to 203 SEQ ID NO:68), and the fourth has the sequence NHTA (at amino acid residues 219 to 222 SEQ ID NO:68).
  • Two cAMP and cGMP-dependent protein kinase phosphorylation sites are present in human TANGO 509.
  • the first has the sequence KRIT (at amino acid residues 124 to 127 SEQ ID NO:68), and the second has the sequence KKQS (at amino acid residues SEQ ID NO:68).
  • Human TANGO 509 has five casein kinase II phosphorylation sites. The first has the sequence SEHE (at amino acid residues 149 to 152 of SEQ ID NO:68), the second has the sequence TSSD (at amino acid residues 168 to 171 of SEQ ID NO:68), the third has the sequence SKRE (at amino acid residues 184 to 187 of SEQ ID NO:68), the fourth has the sequence TTNE (at amino acid residues 202 to 205 SEQ ID NO:68), and the fifth has the sequence THLE (at amino acid residues 285 to 288 SEQ ID NO:68).
  • SEHE amino acid residues 149 to 152 of SEQ ID NO:68
  • TSSD at amino acid residues 168 to 171 of SEQ ID NO:68
  • the third has the sequence SKRE (at amino acid residues 184 to 187 of SEQ ID NO:68)
  • the fourth has the sequence TTNE (at amino acid residues 202 to 205 SEQ ID NO:68)
  • Human TANGO 509 has a tyrosine kinase phosphorylation site with the sequence KLQDAGVY (at amino acid residues 105 to 112 of SEQ ID NO:68). Human TANGO 509 has four N-myristoylation sites. The first has the sequence GSNMTI (at amino acid residues 33 to 38 of SEQ ID NO:68), the second has the sequence GVYRCM (at amino acid residues 110 to 115 of SEQ ID NO:68), the third has the sequence GVALTF (at amino acid residues 252 to 257 SEQ ID NO:68), and fourth has the sequence GIQDTN (at amino acid residues 273 to 278 of SEQ ID NO:68).
  • Figure 24 shows an alignment of the human TANGO 509 amino acid sequence SEQ ID NO:68 with the butyrophilin-like amino acid sequence SEQ ID NO:85; Accession Number: AF 142780).
  • the alignment shows that there is a 31.9% overall amino acid sequence identity between TANGO 509 and Butyrophilin-like protein.
  • the Butyrophilin-like protein is expressed in dendritic cells which are involved in such processes as antigen presentation and immune stimulation.
  • nucleic acids and modulators thereof could be useful in immune modulation, for example in antigen presentation and immune stimulation.
  • a cDNA encoding mouse TANGO 509 was identified by analyzing the sequences of clones present in an alveolar macrophage cell line library. This analysis led to the identification of a clone, jtmca053b03, encoding mouse TANGO 509.
  • the mouse TANGO 509 cDNA of this clone is 3637 nucleotides long ( Figure 25; SEQ ID NO:76).
  • Figure 26 depicts a hydropathy plot of mouse TANGO 509. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
  • Mouse TANGO 509 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 261 to 290 of SEQ ID NO:78, (SEQ ID NO: 84), a transmembrane domain at amino acid residues 240 to 260 of SEQ ID NO:78, (SEQ ID NO:82), and a cytoplasmic domain at amino acid residues 19 to 239 of SEQ ID NO:78, (SEQ ID NO:83).
  • mouse TANGO 509 contains an extracellular domain at amino acid residues 261 to 290 of SEQ ID NO:78, (SEQ ID NO: 84), a transmembrane domain at amino acid residues 240 to 260 of SEQ ID NO:78, (SEQ ID NO:82), and a cytoplasmic domain at amino acid residues 1 to 239 of SEQ ID NO:78.
  • a cDNA sequence of mouse TANGO 509 has a nucleotide at position 132 which is cytosine (C)(SEQ ID NO:76).
  • the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position
  • Mouse TANGO 509 has six N-glycosylation sites with the first sequence NVTM (at amino acid residues 35 to 38 of SEQ ID NO:78), the second has the sequence NVTS (at amino acid residues 191 to 194 SEQ ID NO: 78), the third has the sequence NAT A (at amino acid residues 199 to 202 SEQ ID NO:78), the fourth has the sequence NHTA (at amino acid residues 218 to 221 SEQ ID NO:78), the fifth has the sequence NRTH (at amino acid residues 236 to 239 SEQ ID NO:78), and the sixth has the sequence NDTQ (at amino acid residues 283 to 286 SEQ ID NO:78).
  • Mouse TANGO 509 has one cAMP and cGMP-dependent protein kinase phosphorylation site, having the sequence KRIT (at amino acid residues 124 to 127 SEQ ID NO:78).
  • Mouse TANGO 509 has five casein kinase II phosphorylation sites.
  • the first has the sequence SEHE (at amino acid residues 148 to 151 of SEQ ID NO:78), the second has the sequence TNSD (at amino acid residues 167 to 170 of SEQ ID NO:78), the third has the sequence SRTE (at amino acid residues 183 to 186 of SEQ ID NO:78), the fourth has the sequence TAND (at amino acid residues 201 to 204 SEQ ID NO:78), and the fifth has the sequence TQFE (at amino acid residues 285 to 288 SEQ ID NO:78).
  • TANGO 509 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, activation, development, differentiation, and/or function of mammary cells, tissues and/or organs, e.g., tissues and cells of mammary epithelium origin.
  • TANGO 509 nucleic acids, proteins and modulators thereof can be used to treat mammary-related disorders, e.g. , breast cancer.
  • Antibodies may activate or inhibit the cell adhesion, proliferation and activation, and may help in treating infection, autoimmunity, inflammation, and cancer by affecting these cellular processes.
  • TANGO 509 nucleic acids, proteins and modulators thereof can also be utilized to modulate intercellular signaling in the immune system, e.g., modulate intercellular signal transduction in immune stimulation or suppression and modulate immune cell membrane adhesion to ECM components, during development, e.g., late stages of development.
  • TANGO 509 nucleic acids, proteins and modulators thereof can be used for immune cell receptor co-stimulation via CD28 to modulate IL-2 expression in addition to modulating the expression of other lymphokines.
  • TANGO 509 nucleic acids, proteins and modulators thereof can be used to modulate diseases of the immune system, in particular AIDS, asthma or chronic viral diseases such as hepatitis C virus or hepatitis B virus infections, or to modulate the immune system in cancer patients, or patients undergoing organ or tissue transplantation procedures, or inflammatory disorders, e.g. , bacterial or viral infection, psoriasis, septicemia, arthritis, allergic reactions.
  • 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 1, 2, 23, 24, 34, 35,
  • nucleic acid molecules encoding proteins of the invention from other species which have a nucleotide sequence which differs from that of the human or mouse protein described herein are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologs 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 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 contiguous nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO:l, 23, 34, 42, 54, 61, 66, or 76 or a complement thereof.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77, 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).
  • the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.
  • 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).
  • 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.
  • DNA mimics in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.
  • 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.
  • 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.
  • 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.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • 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.
  • 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.
  • the expression characteristics of an endogenous 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 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509) and controls, modulates or activates the endogenous gene.
  • 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 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509) and controls, modulates or activates the endogenous gene.
  • 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 PL
  • Cre/loxP recombinase system of bacteriophage PL
  • Cre/loxP recombinase system of Saccharomyces cerevisiae
  • FLP recombinase system of Saccharomyces cerevisiae
  • 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.
  • 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.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF; Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • 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 prefe ⁇ ed 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 earner is applied orally and swished and expectorated or swallowed.
  • 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.
  • the antibodies and cytotoxic factor(s), chemotherapeutic drug(s) and/or cytokine(s) can be administered by the same or separate routes, for example, by intravenous, intranasal or intramuscular administration.
  • Cytotoxic factors include, but are not limited to, TNF- ⁇ , TNF- ⁇ , IL-1, IFN- ⁇ and IL-2.
  • Chemotherapeutic drugs include, but are not limited to, 5-fluorouracil (5FU), vinblastine, actinomycin D, etoposide, cisplatin, methotrexate and doxorubicin.
  • 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.
  • 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 drug combination, and the degree of expression or activity to be modulated.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologs, 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).
  • polypeptides of the invention can to used to (i) modulate cellular proliferation; (ii) modulate cellular differentiation; and/or (iii) modulate cellular adhesion.
  • 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 drugs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or abe ⁇ ant activity compared to the wild type protein.
  • the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention.
  • 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.
  • 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
  • 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.
  • glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • glutathione sepharose beads Sigma Chemical; St. Louis, MO
  • glutathione derivatized microtitre plates which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e
  • 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).
  • the candidate compound when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression.
  • the candidate compound when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression.
  • the level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
  • 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. 47:31-41 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.
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • 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.
  • Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (refe ⁇ ed to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent).
  • Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of a polypeptide of the invention in clinical trials.
  • agents e.g., drugs or other compounds
  • An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample.
  • a prefe ⁇ ed agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention.
  • the nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NO:l, 23, 34, 42, 54, 61, 66, or 76, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 contiguous nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention.
  • a full-length cDNA such as the nucleic acid of SEQ ID NO:l, 23, 34, 42, 54, 61, 66, or 76, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 contiguous 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 prefe ⁇ ed 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.
  • 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 abe ⁇ ant expression of a polypeptide of the invention as discussed, for example, in sections above relating to uses of the sequences of the invention.
  • kits can be used to determine if a subject is suffering from or is at risk for brain-related disorders such as peripheral nerve survival, cerebral edema, hydrocephalus, brain herniations, iatrogenic 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 hemo ⁇ hage 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, which are associated with abe ⁇ ant TANGO 499 activity and/or expression.
  • iatrogenic disease due to, e.g., infection
  • 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 instructions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with abe ⁇ ant 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) 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 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 abe ⁇ ant 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 abe ⁇ ant expression or activity of a polypeptide of the invention, e.g., an immunologic disorder, or embryonic disorders.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder.
  • bacterial infection e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g. , asthma, psoriasis), which are associated with abe ⁇ ant INTERCEPT 307, MANGO 511 and TANGO 361 activity and/or expression.
  • prognostic assays described herein can be used to identify a subject having or at risk of developing renal disorders e.g., glomerular diseases, tubular diseases and tubulo-interstitial diseases, embryonic disorders and fetal disorders, and which are associated with abe ⁇ ant INTERCEPT 307 activity and/or expression.
  • renal disorders e.g., glomerular diseases, tubular diseases and tubulo-interstitial diseases, embryonic disorders and fetal disorders, and which are associated with abe ⁇ ant INTERCEPT 307 activity and/or expression.
  • prognostic assays described herein can be used to identify a subject having or at risk of developing brain-related disorders such as cerebral edema, hydrocephalus, brain hemiations, iatrogenic 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 hemo ⁇ hage 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, which are associated with abe ⁇ ant TANGO 509 and/or expression.
  • iatrogenic disease due to, e.g., infection
  • prognostic assays described herein can be used to identify a subject having or at risk of developing prostate-related disorders (e.g, prostate cancer, prostatis, benign prostatic hypertrophy, benign prostatic hype ⁇ lasmia and atypical prostatic stromal lesions) which are associated with abe ⁇ ant INTERCEPT 307, and TANGO 361 activity and/or expression.
  • prostate-related disorders e.g, prostate cancer, prostatis, benign prostatic hypertrophy, benign prostatic hype ⁇ lasmia and atypical prostatic stromal lesions
  • prognostic assays described herein can be used to identify a subject having or at risk of developing pituitary-related disorders (e.g., Cushing's disease, hype ⁇ rolactinemia, acromegaly-gigantism, and precocious and delayed puberty disorders), or Parkinson's disease or peripheral neuropathy-related disorders (e.g., amylothrophic lateral sclerosis) which are associated with abe ⁇ ant TANGO 499 activity and/or expression.
  • pituitary-related disorders e.g., Cushing's disease, hype ⁇ rolactinemia, acromegaly-gigantism, and precocious and delayed puberty disorders
  • Parkinson's disease or peripheral neuropathy-related disorders e.g., amylothrophic lateral sclerosis
  • 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 abe ⁇ ant 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
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abe ⁇ ant 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 abe ⁇ ant expression or activity of the polypeptide).
  • 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 rea ⁇ angement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an abe ⁇ ant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene.
  • assay techniques known in the art which can be used for detecting lesions in a gene.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 :1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 :360-364), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • 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 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759).
  • genetic mutations can be identified in two-dimensional a ⁇ ays containing light-generated DNA probes as described in Cronin et al., supra.
  • a first hybridization a ⁇ ay 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 a ⁇ ays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization a ⁇ ay that allows the characterization of specific mutations by using smaller, specialized probe a ⁇ ays complementary to all variants or mutations detected.
  • Each mutation a ⁇ ay 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 co ⁇ esponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Bio/Techniques 19:448), including sequencing by mass spectrometry ( see, e.g., PCT Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a prefe ⁇ ed embodiment, the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a selected sequence is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in genes.
  • single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc. Natl.
  • RNA rather than DNA
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a "GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are refe ⁇ ed to as "altered drug action.” Genetic conditions transmitted as single factors altering the way the body acts on drugs are refe ⁇ ed to as "altered drug metabolism”. These pharmacogenetic conditions can occur either as rare defects or as polymo ⁇ hisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitro furans
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite mo ⁇ hine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply geno typing of polymo ⁇ hic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype.
  • 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.
  • increased administration of the agent may be desirable to increase the expression or activity of the polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abe ⁇ ant expression or activity of a polypeptide of the invention.
  • disorders characterized by abe ⁇ ant expression or activity of the polypeptides of the invention include proliferative disorders such as cancer.
  • an agonist or antagonist agent can be used for treating the subject.
  • an antagonist of an INTERCEPT 307, MANGO 511 or TANGO 509 protein may be used to treat an immunological disorder.
  • the appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule.
  • the agent stimulates one or more of the biological activities of the polypeptide.
  • stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell.
  • the agent inhibits one or more of the biological activities of the polypeptide of the invention.
  • inhibitory agents include antisense nucleic acid molecules and antibodies.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or abe ⁇ ant expression or activity of the polypeptide.
  • Clones containing cDNA molecules encoding MANGO 511 were deposited with the American Type Culture Collection (Manassas, VA) on July 23, 1999 as Accession Number PTA-425, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid 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 lOO ⁇ 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 1.5 kb fragments that co ⁇ espond to MANGO 511 (511).
  • the identity of the strain containing MANGO 511 can be infe ⁇ ed from the liberation of a fragment of the above identified size.
  • 15 aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with lOO ⁇ 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 2 Not I and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
  • INTERCEPT 307 (307): 2.0 kb TANGO 361 (361): 5.1 kb 25 TANGO 509 (509): 3.6 kb
  • strains can be infe ⁇ ed from the fragments liberated.
  • Clones containing cDNA molecules encoding TANGO 499 form 1, variant 1 (clone EpT499 form 1, variant 1), were deposited with the American Type Culture r . Collection (Manassas, VA) on August 5, 1999 as Accession Number PTA-455, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
  • an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB 35 plates) supplemented with lOO ⁇ 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 1.1 kb fragments that co ⁇ espond to TANGO 499 form 1, variant 1 (EpT499 form 1, variant 1).
  • the identity of the strain containing TANGO 499 form 1, variant 1 can be infe ⁇ ed from the liberation of a fragment of the above identified size.
  • an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with lOO ⁇ 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 1.1 kb fragments that co ⁇ espond to TANGO 499 form 2, variant 3 (EpT499 form 2, variant 3).
  • the identity of the strain containing TANGO 499 form 2, variant 3 can be infe ⁇ ed from the liberation of a fragment of the above identified size.

Abstract

The invention provides isolated INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 AND TANGO 509, nucleic acid molecules and polypeptide molecules. INTERCEPT 307 encodes a transmembrane polypeptide related to the prostate cancer upregulated PB36 gene product. MANGO 511 encodes transmembrane polypeptides related to the leukocyte Ig-like receptors (LIRs). TANGO 351 encodes a transmembrane polypeptide that is involved in signaling. TANGO 499 variants encode secreted polypeptides related to glial cell line-derived neurotrophic factor (GDNF)-like polypeptides. Human and mouse TANGO 509 encode transmembrane polypeptides that contain immunoglobulin domains. The invention also provides antisense nucleic acid molecules, expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a nucleic acid molecule of the invention has been introduced or disrupted. The invention still further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

Description

SECRETED PROTEINS AND USES THEREOF
Cross Reference to Related Applications This application is a continuation-in-part of U.S. patent application Serial No.
09/399,723, filed September 20, 1999, the contents of which are incorporated herein by reference in its entirety.
Background of the Invention Many secreted proteins, for example, cytokines and cytokine receptors, play a vital role in the regulation of cell growth, cell differentiation, and a variety of specific cellular responses. A number of medically useful proteins, including erythropoietin, granulocyte- macrophage colony stimulating factor, human growth hormone, and various interleukins, are secreted proteins. Thus, an important goal in the design and development of new therapies is the identification and characterization of secreted and transmembrane proteins and the genes which encode them.
Many secreted proteins are receptors which bind a ligand and transduce an intracellular signal, leading to a variety of cellular responses. The identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction.
Summary of the Invention The present invention is based, at least in part, on the discovery of cDNA molecules which encode the INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 proteins, all of which are either wholly secreted or transmembrane polypeptides.
The INTERCEPT 307 proteins are transmembrane polypeptides related to the prostate cancer upregulated PB39 gene product.
The MANGO 511 proteins are transmembrane polypeptides related to the leukocyte Ig-like receptors (LIRs) which bind MHC class I.
The TANGO 351 proteins are transmembrane polypeptides involved in signaling, e.g., signaling in renal cells. The TANGO 361 proteins are transmembrane polypeptides related to serine proteases which belong to the trypsin-like protease family. The TANGO 499 proteins are secreted polypeptides related to the glial cell line- derived neurotrophic factor (GDNF)-like polypeptides, such as human artemin and GDNF, which are cell surface antigens associated with embryogenesis and development.
The TANGO 509 proteins are transmembrane polypeptides related to butyrophilin-like proteins and containing immunoglobulin domains.
The INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 proteins, fragments, derivatives, and variants thereof of the present invention are collectively referred to herein 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 58%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, the nucleotide sequence of the cDNA insert of an INT307 clone deposited July 29, 1999 with the American Type Culture Collection (ATCC®) as Accession Number PTA-455, or a complement thereof.
The invention features nucleic acid molecules which are at least 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NO:23, SEQ ID NO:24, the nucleotide sequence of the cDNA insert of an EpM511 clone deposited July 23, 1999 with the ATCC® as Accession Number PTA-425, or a complement thereof.
The invention features nucleic acid molecules which are at least 30%, 35%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NO:34, the nucleotide sequence of the cDNA insert of an EpT351 clone deposited July 23, 1999 with the ATCC® as Accession Number PTA-424, or a complement thereof The invention also features nucleic acid molecules which are at least 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NO:35 or a complement thereof.
The invention features nucleic acid molecules which are at least 30%, 35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NO: 42, SEQ ID NO:43, the nucleotide sequence of the cDNA insert of an EpT361 clone deposited July 29, 1999 with the ATCC® as Accession Number PTA-438, or a complement thereof.
The invention features nucleic acid molecules which are at least 80%, 85%, 90%, 95%, or 98% identical to the nucleotide sequence of form 1 SEQ ID NO:54, the nucleotide sequence of the cDNA insert of an EpT499 form 1 clone deposited August 5, 1999 with the ATCC® as Accession Number PTA-455, or a complement thereof. The invention features nucleic acid molecules which are at least 75%, 80%, 85%, 90%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NO:55 or a complement thereof. The invention features nucleic acid molecules which are at least 30%, 35%, 40%, 45%, 50% 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotides 301 to 480 of SEQ ID NO: 55 or a complement thereof.
The invention features nucleic acid molecules which are at least 80%, 85%, 90%, 95%ι, or 98% identical to the nucleotide sequence of form 2 SEQ ID NO:61, the nucleotide sequence of the cDNA insert of an EpT499 form 2, variant 3 clone deposited August 5, 1999 with the ATCC® as Accession Number PTA-454, or a complement thereof. The invention features nucleic acid molecules which are at least 75%, 80%, 85%, 90%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NO:62 or a complement thereof The invention features nucleic acid molecules which are at least 30%, 35%, 40%, 45%, 50% 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotides 240 to 344 of SEQ ID NO: 62 or a complement thereof.
The invention features nucleic acid molecules which are at least 30%, 35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotide sequence of human SEQ ID NO:66, SEQ ID NO:67, the nucleotide sequence of the cDNA insert of an human EpT509 clone deposited August 5, 1999 with the ATCC® as Accession Number PTA-438, or a complement thereof
The invention features nucleic acid molecules which are at least 85%, 95%, or 98% identical to the nucleotide sequence of mouse SEQ ID NO: 76, the nucleotide sequence of the cDNA insert of a mouse EpT509 clone, or a complement thereof. The invention features nucleic acid molecules which are at least 90%, 95%, or 98% identical to the nucleotide sequence of mouse SEQ ID NO:77, or a complement thereof.
The invention features isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 90%, 95%, or 98% identical to the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62 66, 67, 76, or 77, a complement thereof, or the non-coding strand of INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-454, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention.
The invention features nucleic acid molecules of at least 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or 2020 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:l, the nucleotide sequence of an INT307 cDNA of ATCC® Accession Number PTA-455, or a complement thereof. The invention features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 645 contiguous nucleotides of nucleic acids 1 to 649 of SEQ ID NO:l, or a complement thereof. The invention features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250 or 300 contiguous nucleotides of nucleic acids 1120 to 1430 of SEQ ID NO:l, or a complement thereof.
The invention features nucleic acid molecules comprising at least 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or 1085 contiguous nucleotides of nucleic acids 1 to 1086 of SEQ ID NO:2, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 contiguous nucleotides of nucleic acids 1 to 604 of SEQ ID NO:2, or a complement thereof.
The invention features nucleic acid molecules of at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400 or 1450 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:23, the nucleotide sequence of a EpM511 cDNA or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 896 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:24, or a complement thereof.
The invention features nucleic acid molecules of at least 460, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300 or 3340 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:34, the nucleotide sequence of an EpT351 cDNA of ATCC® Accession Number PTA-424, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2050 or 2071 contiguous nucleotides of nucleic acids 1 to 2071 of SEQ ID NO:34, or a complement thereof. The invention features nucleic acid molecules which include a fragment of at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400 or 1440 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:35, or a complement thereof.
The invention features nucleic acid molecules of at least 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:42, the nucleotide sequence of an EpT361 cDNA of ATCC® Accession Number PTA-438, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1150 or 1170 contiguous nucleotides of nucleic acids 1 to 1176 of SEQ ID NO:42, or a complement thereof. The invention features nucleic acid molecules comprising at least 25, 50, 100, 150 or 165 contiguous nucleotides of nucleic acids 1653 to 1821 of SEQ ID NO:42, or a complement thereof. The invention features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400 or 1450 contiguous nucleotides of nucleic acids 2035 to 3506 of SEQ ID NO:42, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1450 or 1490 contiguous nucleotides of nucleic acids 3564 to 5058 of SEQ ID NO:42, or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 135, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or 1250 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:43, or a complement thereof. The invention features nucleic acid molecules which include a fragment of at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100 or 1130 contiguous nucleotides of nucleic acids 1 to 1136 of SEQ ID NO:43, or a complement thereof.
The invention features nucleic acid molecules of at least 500, 525, 550, 600, 650, 700, 750, 800, 850, 1000, or 1100 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:54, the nucleotide sequence of an EpT499 form 1, variant 1 cDNA of ATCC® Accession Number PTA-455, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, or 174 contiguous nucleotides of nucleic acids 385 to 559 of SEQ ID NO: 54, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25 contiguous nucleotides of nucleic acids 1072 to 1106 of SEQ ID NO:54, or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 285, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 760 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:55, or a complement thereof. The invention features nucleic acid molecules which include a fragment of at least 25, 50, 100, 150 or 175 contiguous nucleotides of nucleic acids 301 to 480 of SEQ ID NO:55, or a complement thereof.
The invention features nucleic acid molecules of at least 500, 525, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or 1075 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:61, the nucleotide sequence of an EpT499 form 2, variant 3 cDNA of ATCC® Accession Number PTA-454, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 30, 50, 100, 150 or 175 contiguous nucleotides of nucleic acids 310 to 488 of SEQ ID NO:61, or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 275, 300, 350, 400, 450, 500, 550, 600, 650 or 675 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:62, or a complement thereof.
The invention features nucleic acid molecules of at least 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500 or 3575 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:66, the nucleotide sequence of an human EpT509 cDNA of ATCC® Accession Number PTA-438, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 contiguous nucleotides of nucleic acids 1 to 3023 of SEQ ID NO:66 or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 25, 50, 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 860 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:67, or a complement thereof.
The invention features nucleic acid molecules of at least 265, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3100, 3200, 3300, 3400, 3500, 3600 or 3637 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 76, the nucleotide sequence of a mouse EpT509 cDNA or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50 or 100 contiguous nucleotides of nucleic acids 1 to 106 of SEQ ID NO:76, or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 265, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 860 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:77, or a complement thereof. The invention features nucleic acid molecules which include a fragment of at least 25 or 50 contiguous nucleotides of nucleic acids 1 to 52 of SEQ ID NO:77, or a complement thereof.
The invention features isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 25, 35, 50, 100, 150, 200, 250, 300, 400 or more contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77, a complement thereof or the non-coding strand of INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-454, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention.
The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:3, the amino acid sequence encoded by an INT307 cDNA of ATCC® Accession Number PTA-455, 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 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:25, the amino acid sequence encoded by a EpM511 cDNA of ATCC® Accession Number PTA-425, 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 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:36, the amino acid sequence encoded by an EpT351 cDNA of ATCC® Accession Number PTA-424, 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 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:44, the amino acid sequence encoded by an EpT361 cDNA of ATCC® Accession Number PTA-438, 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 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:56, the amino acid sequence encoded by an EpT499 forml cDNA of ATCC® Accession Number PTA-455, 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 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:63, the amino acid sequence encoded by an EpT499 form 2, variant 3 cDNA of ATCC® Accession Number PTA-454, 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 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:68, the amino acid sequence encoded by a human EpT509 cDNA of ATCC® Accession Number PTA-438, 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 35%, 40%), 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:78, the amino acid sequence encoded by a mouse EpT509 cDNA of ATCC® Accession Number PTA-438, 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 25% preferably 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, or 78, the amino acid sequence encoded by INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-454, respectively, or a complement thereof, wherein the protein encoded by the nucleotide sequence also exhibits at least one structural and/or functional feature of a polypeptide of the invention.
In preferred embodiments, the nucleic acid molecules have the nucleotide sequence of SEQ ID NO: l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77 or the nucleotide sequence of the cDNA clones of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, or Accession Number PTA-454. Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:3 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 or 360 contiguous amino acids of SEQ ID NO:3 or the amino acid sequence encoded by a INT307 cDNA of ATCC® Accession Number PTA-455.
Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:25 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 298 contiguous amino acids of SEQ ID NO:25 or the amino acid sequence encoded by a human EpM511 cDNA of ATCC® Accession Number PTA-425.
Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:36 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 481 contiguous amino acids of SEQ ID NO:36 or the amino acid sequence encoded by a EpT351 cDNA of ATCC® Accession Number PTA-424.
Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:44 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 422 contiguous amino acids of SEQ ID NO:44 or the amino acid sequence encoded by a EpT361 cDNA of ATCC® Accession Number PTA-438.
Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:56 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225 or 253 contiguous amino acids of SEQ ID NO:56 or the amino acid sequence encoded by a EpT499 form 1, variant 1 cDNA of ATCC® Accession Number PTA-455.
Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:63 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200 or 228 contiguous amino acids of SEQ ID NO:63 or the amino acid sequence encoded by an EpT499 form 2, variant 3 cDNA of ATCC® Accession Number PTA-454.
Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:68 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 289 contiguous amino acids of SEQ ID NO:68 or the amino acid sequence encoded by a human EpT509 cDNA of ATCC® Accession Number PTA-438.
Also within the invention are nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:78 or a fragment thereof including at least 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 270 or 278 contiguous amino acids of SEQ ID NO:78 or the amino acid sequence encoded by a mouse EpT509 cDNA.
The invention also features nucleic acid molecules which encode a polypeptide fragment of at least 10, 15, 20, 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 or more contiguous amino acids of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, or 78 or the amino acid sequence encoded by INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, or Accession Number PTA-454, wherein the fragment exhibits at least one structural and/or functional feature of a polypeptide of the invention.
The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, or 78 or the amino acid sequence encoded by a cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-454, or a complement thereof
Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 25%, preferably 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:l or 36 or the amino acid sequence encoded by an LNT307 cDNA of ATCC® Accession Number PTA-455 or encoded by an EpT351 cDNA of ATCC® Accession Number PTA-424.
Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 30%, preferably 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:25 or 44 or the amino acid sequence encoded by an EpM511 cDNA of ATCC® Accession Number PTA-425 or encoded by an EpT361 cDNA of ATCC® Accession Number PTA-438.
Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 35%, preferably 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:56, 63, 68 or 78 or the amino acid sequence encoded by an EpT499 form 1, variant 1 cDNA of ATCC® Accession Number PTA-455, encoded by an EpT499 form 2, variant 3 cDNA of ATCC® Accession Number PTA-438, encoded by a human EpT509 cDNA of ATCC® Accession Number PTA-425 or encoded by a mouse EpT509 cDNA.
The invention also features isolated polypeptides or proteins having an amino acid sequence that is at least about 25%, preferably 30%, 35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, or 78 or the amino acid sequence encoded by INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, or Accession Number PTA-454, wherein the protein or polypeptides also exhibit at least one structural and/or functional feature of a polypeptide of the invention.
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:3, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or 2, a complement thereof, or the non-coding strand of an INT307 cDNA of ATCC® Accession Number PTA-455.
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 58%, preferably 60%, 65%, 75%, 85%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:25, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:23 or 24, a complement thereof, or the non-coding strand of an EpM511 cDNA of ATCC® Accession Number PTA-425.
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 45%, preferably 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:36, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:34 or 35, a complement thereof, or the non-coding strand of an EpT351 cDNA of ATCC® Accession Number PTA-424.
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:44, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:42 or 43, a complement thereof, or the non-coding strand of an EpT361 cDNA of ATCC® Accession Number PTA-438.
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 85%, preferably 90%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:56, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:54 or 55, a complement thereof, or the non-coding strand of an EpT499 form 1, variant 1 cDNA of ATCC® Accession Number PTA-455.
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 80%, preferably 85%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:63, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:61 or 62, a complement thereof, or the non-coding strand of an EpT499 form 2, variant 3 cDNA of ATCC® Accession Number PTA-454.
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:68, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:66 or 67, a complement thereof, or the non-coding strand of a human EpT509 cDNA of ATCC® Accession Number PTA-438.
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:78, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:76 or 77, a complement thereof, or the non-coding strand of a mouse EρT509 cDNA.
The invention also features isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 25%, preferably 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95% or 98% identical to a nucleic acid sequence encoding SEQ ID NO:3, 25, 36, 44, 56, 63, 68, or 78, isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76 or 77, or a complement thereof, or the non-coding strand of INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, or Accession Number PTA- 454, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention.
Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that include the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, or 78 or the amino acid sequence encoded by a cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, or Accession Number PTA-454, respectively, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77, or a complement thereof under stringent conditions.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or 2 or an INT307 cDNA of ATCC® Accession Number PTA-455, or a complement thereof. In one embodiment, the nucleic acid molecules are at least 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or 2020 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, an LNT307 cDNA of ATCC® Accession Number PTA-455, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 645 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 649 of SEQ ID NO:l, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250 or 300 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1120 to 1430 of SEQ ID NO:l, or a complement thereof.
In another embodiment, the nucleic acid molecules are at least 475, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1050 or 1085 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 604 of SEQ ID NO:2, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:23 or 24, or an EpM511 cDNA of ATCC® Accession Number PTA-425, or a complement thereof. In one embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or 1440 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:23, an EpM511 cDNA of ATCC® Accession Number PTA-425, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 896 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:24, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:34 or 35, or an EpT351 cDNA of ATCC® Accession Number PTA-424, or a complement thereof. In one embodiment, the nucleic acid molecules are at least 460, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300 or 3340 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:34, an EpT351 cDNA of ATCC® Accession Number PTA-424, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400 or 2500 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 2501 of SEQ ID NO:34 or a complement thereof.
In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1440 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:35, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:42 or 43 or an EpT361 cDNA of ATCC® Accession Number PTA-438, or a complement thereof In one embodiment, the nucleic acid molecules are at least 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900 or 5000 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:42, an EpT361 cDNA of ATCC® Accession Number PTA-438, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1150 or 1170 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 1176 of SEQ ID NO:42, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150 or 165 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1653 to 1821 of SEQ ID NO:42, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1450 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 2035 to 3506 of SEQ ID NO:42, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450 or 1490 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 3564 to 5058 of SEQ ID NO:42, or a complement thereof.
In another embodiment, the nucleic acid molecules are at least 135, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200 or 1250 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:43, or a complement thereof. In yet another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100 or 1130 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 1136 of SEQ ID NO:43, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 54 or 55, or an EpT499 form 1, variant 1 cDNA of ATCC® Accession Number PTA-455, or a complement thereof. In one embodiment, the nucleic acid molecules are at least 500, 550, 600, 650, 700, 750, 800, 850, 1000 or 1100 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:54, an EpT499 form 1, variant 1 cDNA of ATCC® Accession Number PTA-455, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150 or 175 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 385 to 563 of SEQ ID NO:54, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 20 or 30 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1072 to 1106 of SEQ ID NO:54, or a complement thereof.
In another embodiment, the nucleic acid molecules are at least 285, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 760 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:55, or a complement thereof. In yet another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150 or 175 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 301 to 480 of SEQ ID NO:55, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:61 or 62, or an EpT499 form 2, variant 3 cDNA of ATCC® Accession Number PTA-454, or a complement thereof. In one embodiment, the nucleic acid molecules are at least 500, 550, 600, 650, 700, 750, 800, 850, 1000, 1050 or 1075 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:61, an EpT499 form 2, variant 3 cDNA of ATCC® Accession Number PTA-454, or a complement thereof.
In another embodiment, the nucleic acid molecules are at least 275, 300, 350, 400, 450, 500, 550, 600, 650 or 675 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:62, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150 or 175 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 240 to 344 of SEQ ID NO: 62, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:66 or 67 or a human EpT509 cDNA of ATCC® Accession Number PTA-438, or a complement thereof. In one embodiment, the nucleic acid molecules are at least 925, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, or 3574 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:66, a human EpT509 cDNA of ATCC® Accession Number PTA-438, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 3023 of SEQ ID NO:66, or a complement thereof.
In another embodiment, the nucleic acid molecules are at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 850 or 860 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:67, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:76 or 77, or a mouse EpT509 cDNA of ATCC® Accession Number PTA-438, or a complement thereof. In one embodiment, the nucleic acid molecules are at least 265, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3100, 3200, 3300, 3400, 3500, 3600 or 3637 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:76, a mouse EpT509 cDNA of ATCC® Accession Number PTA-438, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25, 50 or 100 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 106 of SEQ ID NO:76, or a complement thereof.
In another embodiment, the nucleic acid molecules are at least 265, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 860 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:77, or a complement thereof. In another embodiment, the nucleic acid molecules are at least 25 or 50 contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising nucleic acids 1 to 52 of SEQ ID NO:77, or a complement thereof.
The invention also features nucleic acid molecules at least 15, preferably 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600 or more contiguous nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77 of INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, or TANGO 509 cDNAs of ATCC® Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-454, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
In preferred embodiments, the isolated nucleic acid molecules encode a cytoplasmic, transmembrane, or extracellular domain of a polypeptide of the invention.
In one embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.
Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In another embodiment, the invention provides host cells containing such a vector or engineered to contain and/or express 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 encoding a polypeptide of the invention such that the polypeptide of the invention is produced.
Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide. An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo or in vitro, according to standard techniques. Such activities can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein.
For INTERCEPT 307 or modulators thereof, biological activities include, e.g., (1) the ability to modulate (this term, as used herein, includes, but is not limited to, stabilize, increase or promote, suppress, decrease, inhibit, or disrupt) the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of immune cells (e.g., T-lymphocyte function); (2) the ability to modulate the development and progression of cell proliferative disorders such as cancer (e.g., prostate cancer); (3) the ability to modulate, protein-protein interactions (e.g., homophilic and/or heterophilic), and protein-ligand interactions, e.g., in receptor-ligand recognition; (4) the ability to modulate cell-cell interactions and/or cell-extracellular matrix interactions; (5) the ability to modulate hematopoietic processes; (6) the ability to modulate intracellular signaling cascades (e.g., signal transduction cascades); (7) the ability to modulate the proliferation, differentiation, and/or function of prostate cells; and (8) the ability to modulate infections, e.g., infections mediated by eosinophil granule release; and (9) the ability to modulate the function, e.g., activation, of eosinophils.
For MANGO 511 or modulators thereof, biological activities include, e.g., (1) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of immune cells (e.g., B-lymphocytes and monocytes); (2) the ability to modulate, protein-protein interactions (e.g., homophilic and/or heterophilic), and protein-ligand interactions, e.g., in receptor-ligand recognition;
(3) ability to modulate cell-cell interactions and/or cell-extracellular matrix interactions;
(4) the ability to modulate hematopoietic processes; (5) the ability to modulate intracellular signaling cascades (e.g., signal transduction cascades); (6) the ability to modulate MHC class I recognition and binding; (7) the ability to modulate ligand-receptor interactions in proteins with immunoglobulin domains; (8) the ability to modulate immunoglobulin binding to antigens; and (9) the ability to modulate lymphocyte selection (such as modulation of B-cell receptor or T-cell receptor stimulation in developing lymphocytes, e.g., through modulation of antigen interaction with immunoglobulin domains of the receptors).
For TANGO 351 or modulators thereof, biological activities include, e.g., (1) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of kidney cells and the kidney; (2) the ability to modulate, protein-protein interactions (e.g., homophilic and/or heterophilic), and protein- ligand interactions, e.g., in receptor-ligand recognition; (3) ability to modulate cell-cell interactions and/or cell-extracellular matrix interactions; (4) the ability to modulate intracellular signaling cascades (e.g., signal transduction cascades); (5) the ability to modulate the development of organs, tissues and/or cells in an embryo and/or fetus.
For TANGO 361 or modulators thereof, biological activities include, e.g., (1) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of prostate cells (e.g., prostate epithelial cells) or adipocytes; (2) the ability to modulate the development and progression of cell proliferative disorders such as cancer (e.g. prostate or prostate-associated cancer); (3) the ability to modulate, protein-protein interactions (e.g., homophilic and/or heterophilic), and protein-ligand interactions, e.g. , in receptor-ligand recognition; (4) ability to modulate cell-cell interactions and/or cell-extracellular matrix interactions; (5) the ability to act as a protease (e.g., serine protease) and/or modulate protease (e.g., serine protease) activities, such as serine protease activity involved in platelet function, (e.g., activation and aggregation), serine protease activity involved in progression of Alzheimer's disease (e.g., formation of Alzheimer's plaques), or serine protease activity involved in activation of the complement system (e.g., C3b cleavage); (6) the ability to modulate intracellular signaling cascades (e.g., signal transduction cascades); (7) the ability to modulate intercellular signaling (e.g., in the prostate); (8) the ability to modulate suppression of infectious diseases or cancer (e.g., bacteria, viruses, parasites, or neoplastic cells); (9) the ability to modulate autoimmunity (e.g., as associated with multiple sclerosis, psoriasis, arthritis, lupus); (10) the ability to modulate transplant rejections (e.g., graft rejections, or allograft rejections); (11) the ability to modulate carbohydrate binding; and (12) the ability to modulate systemic energy balance.
For TANGO 499 or modulators thereof, biological activities include, e.g., (1) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation, survival and/or activity of neurons, (e.g., peripheral neurons and/or central neurons), glial cells, (e.g., oligodendrocytes or astrocytes) or endocrine cells (e.g., pituitary cells or pineal gland cells); (2) the ability to modulate the development and progression of cell proliferative disorders such as cancer (e.g., glial associated cancers such as glioblastoma) or neural associated cancer; (3) the ability to modulate, protein-protein interactions (e.g., homophilic and/or heterophilic), and protein-ligand interactions, e.g., in receptor-ligand recognition; (4) ability to modulate cell-cell interactions and/or cell-extracellular matrix interactions; (5) the ability to modulate intracellular signaling cascades (e.g., signal transduction cascades); (6) the ability to modulate intercellular signaling (e.g., in the nervous system); (7) the ability to modulate the development of neural organs and tissues; (8) the ability to modulate riboflavin delivery to the embryo; and (9) the ability to modulate the development of an embryo and/or fetal development.
For TANGO 509 or modulators thereof, biological activities include, e.g., (1) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of mammary cells, e.g., mammary epithelial cells; (2) the ability to modulate the development and progression of cell proliferative disorders such as cancer (e.g. breast or breast-associated cancer); (3) the ability to modulate, protein-protein interactions (e.g. , homophilic and/or heterophilic), and protein-ligand interactions, e.g., in receptor-ligand recognition; (4) ability to modulate cell-cell interactions and/or cell-extracellular matrix interactions; (5) the ability to modulate mammary processes (e.g., milk secretion or fat secretion in milk); (6) the ability to modulate intracellular signaling cascades (e.g., signal transduction cascades); (7) the ability to modulate intercellular signaling (e.g., hormonal signals to secrete milk); (8) the ability to modulate the development of embryonic organs, tissues and/or cells; (9) the ability to modulate the development, differentiation, morphology, migration or chemotaxis, proliferation and/or activity of immune cells (e.g., B-lymphocytes, T- lymphocytes and monocytes); (10) the ability to modulate hematopoietic processes (e.g., immune response); (11) the ability to modulate MHC class I recognition and binding; (12) the ability to modulate ligand-receptor interactions in proteins with immunoglobulin domains; (13) the ability to modulate immunoglobulin binding to antigens; (14) the ability to modulate lymphocyte selection such as modulation of B-cell receptor or T-cell receptor stimulation in developing lymphocytes, e.g., through modulation of interaction of antigens with the immunoglobulin domain(s) of the immune cell's antigen receptors; (15) the ability to modulate immunoglobulin production; and (16) the ability to modulate cell killing, such as, the ability to modulate production of cytokines or activation of cytotoxic T-cell killing.
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 or encode a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences which contain or encode 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, an INTERCEPT 307 protein includes at least one or more of the following domains: a signal sequence, an extracellular domain, a transmembrane domain, a gas vehicle protein GVPc domain, and an intracellular or cytoplasmic domain.
In one embodiment, a MANGO 511 protein includes at least one or more of the following domains: a signal sequence, an extracellular domain, a transmembrane domain, an Ig-like domain, and an intracellular or cytoplasmic domain.
In one embodiment, a TANGO 351 protein includes at least one or more of the following domains: a signal sequence, an extracellular domain, a transmembrane domain, and an intracellular or cytoplasmic domain.
In one embodiment, a TANGO 361 protein includes at least one or more of the following domains: a signal sequence, an extracellular domain, a transmembrane domain, a serine protease domain, a SEA-like domain and an intracellular or cytoplasmic domain.
In one embodiment, a TANGO 499 protein includes at least one or more of the following domains: a signal sequence, an extracellular domain, a transmembrane domain and an intracellular or cytoplasmic domain.
In one embodiment, a TANGO 509 protein includes at least one or more of the following domains: a signal sequence, an extracellular domain, a transmembrane domain, an Ig-like domain and an intracellular or 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.
The invention further features antibodies that specifically bind a polypeptide of the invention, such as monoclonal or polyclonal antibodies or fragments thereof.
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, 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 the presence, activity or expression such that the presence activity or expression of a polypeptide of the invention 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 (e.g., 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 is a fragment of a polypeptide of the invention or a nucleic acid molecule encoding such a polypeptide fragment.
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 polypeptide (e.g., an antibody or a fragment of a polypeptide of the invention), a peptidomimetic, or other small molecule (e.g., a 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 another aspect, the invention provides human or non-human antibodies or fragments thereof which specifically bind to a protein of the invention.
In a preferred embodiment, an antibody or a fragment thereof, i.e., human and non-human antibodies or fragments thereof and/or monoclonal antibodies or fragments thereof of the invention, specifically bind to an extracellular domain having the amino acid sequence of SEQ ID NO:6, 7, 8, 28, 39, 49, 75 or 84.
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 and instructions for use. Such kits can also comprise 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
Figure 1 depicts the cDNA sequence of INTERCEPT 307 (SEQ ID NO:l) and the predicted amino acid sequence of human INTERCEPT 307 (SEQ ID NO: 3). The open reading frame of INTERCEPT 307 extends from nucleotides 45 to 1130 of SEQ ID NO:l, (SEQ ID NO:2). Figure 2 depicts a hydropathy plot of human INTERCEPT 307. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 23 of SEQ ID NO:3, (SEQ ID NO:4)) on the left from the mature protein (amino acids 24 to 362 of SEQ ID NO:3, (SEQ ID NO:5)) on the right.
Figure 3 depicts an alignment of the amino acid sequence of PB39 (SEQ ID NO:20; Accession Number NM 003627) and the amino acid sequence of human INTERCEPT 307 (SEQ ID NO:3). The amino acid sequences of human PB39 and human INTERCEPT 307 are 21.0% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 4 depicts an alignment of the nucleotide sequence of the coding region of PB39 (SEQ ID NO:21; Accession Number AF045584) and the nucleotide sequence of the coding region of human INTERCEPT 307 (SEQ ID NO:2). The nucleotide sequences of the coding regions of PB39 and human INTERCEPT 307 are 40.9% identical. The full- length nucleic acid sequences of PB39 (SEQ ID NO:22; Accession Number NM_003627) and human INTERCEPT 307 are 44.0% identical. These alignments were performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 5 depicts an alignment of the human INTERCEPT 307 amino acid sequence (SEQ ID NO:3) with the human eosinophil granule major basic protein amino acid sequence (SEQ ID NO:86; Accession Number Z26248). The amino acid sequences of INTERCEPT 307 and human eosinophil granule major basic protein are 13.8% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 6 shows an alignment of the nucleotide sequence of INTERCEPT 307 coding region (SEQ ID NO:2) and the nucleotide sequence of human eosinophil granule major basic protein coding region (SEQ ID NO:87; Accession Number Z26248). The nucleotide sequences of the coding regions are 38.1% identical. The full-length INTERCEPT 307 nucleic acid sequence (SEQ ID NO:l) and human eosinophil granule major basic protein cDNA (SEQ ID NO:88; Accession Number Z26248) have an overall sequence identity of 57.3%>. These alignments were performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4. Figure 7 depicts the cDNA sequence of human MANGO 511 (SEQ ID NO:23) and the predicted amino acid sequence of human MANGO 511 (SEQ ID NO:25). The open reading frame of human MANGO 511 extends from nucleotide 108 to 1004 of SEQ ID NO:23 (SEQ ID NO:24).
Figure 8 depicts a hydropathy plot of human MANGO 511. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 41 of SEQ ID NO:25 (SEQ ID NO:26)) on the left from the mature protein (amino acids 42 to 299 of SEQ ID NO:25 (SEQ ID NO:27)) on the right.
Figure 9 depicts a local alignment of the amino acid sequence of leukocyte Ig-like receptor-1 (LIR-1; SEQ ID NO:32; Accession Number AAB63522) and the amino acid sequence of human MANGO 511 (SEQ ID NO:3). The amino acid sequences of human LIR-1 and human MANGO 511 are 59.2% identical over the 233 amino acid overlap region that was analyzed. This alignment were performed using the LALIGN version 2.0, July 1996, local alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4. A global alignment of the amino acid sequence of leukocyte Ig-like receptor-1 (LIR-1; SEQ ID NO:32; Accession Number AAB63522) and the amino acid sequence of human Mango 511 (SEQ ID NO:3) reveals that the amino acid sequences of human LIR-1 and human MANGO 511 are 24.2% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 10 depicts an alignment of the nucleotide sequence of the coding region of LIR-1 (SEQ ID NO:33; Accession Number AF009221) and the nucleotide sequence of the coding region of human MANGO 511 (SEQ ID NO:2). The nucleotide sequences of the coding regions of LIR-1 and human MANGO 511 are 34.0% identical. The full-length nucleic acid sequence of MANGO 511 and the coding region of LIR-1 are 44.0% identical. These alignments were performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 11 depicts the cDNA sequence of human TANGO 351 (SEQ ID NO:34) and the predicted amino acid sequence of human TANGO 351 (SEQ ID NO:36). The open reading frame of human TANGO 351 extends from nucleotides 143 to 1588 of SEQ ID NO:34 (SEQ ID NO:35).
Figure 12 depicts a hydropathy plot of human TANGO 351. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 24 of SEQ ID NO:36 (SEQ ID NO:37) on the left from the mature protein (amino acids 25 to 482 of SEQ ID NO:36 (SEQ ID NO:38)) on the right.
Figure 13 depicts the cDNA sequence of TANGO 361 (SEQ ID NO:42) and the predicted amino acid sequence of TANGO 361 (SEQ ID NO:44). The open reading frame of TANGO 361 extends from nucleotides 41 to 1309 of SEQ ID NO:42 (SEQ ID NO:43).
Figure 14 depicts a hydropathy plot of TANGO 361. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 35 of SEQ ID NO:44 (SEQ ID NO:45)) on the left from the mature protein (amino acids 36 to 423 of SEQ ID NO:44 (SEQ ID NO:46)) on the right.
Figure 15 depicts the cDNA sequence of TANGO 499 form 1, variant 1 (SEQ ID NO: 54) and the predicted amino acid sequence of TANGO 499 form 1, variant 1 (SEQ ID NO:56). The open reading frame of TANGO 499 form 1, variant 1 extends from nucleotides 83 to 844 of SEQ ID NO:54 (SEQ ID NO:55).
Figure 16 depicts a hydropathy plot of TANGO 499 form 1, variant 1. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 30 of SEQ ID NO:56 (SEQ ID NO:57)) on the left from the mature protein (amino acids 31 to 254 of SEQ ID NO:58)) on the right.
Figure 17 shows an alignment of the human TANGO 499 form 1, variant 1 amino acid sequence SEQ ID NO:56 with the artemin amino acid sequence (SEQ ID NO:59). The alignment shows that there is a 23.5% overall amino acid sequence identity between TANGO 499 form 1 , variant 1 and Artemin. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 18 shows an alignment of the human TANGO 499 form 1, variant 1 amino acid sequence SEQ ID NO:56 with the riboflavin binding protein amino acid sequence (SEQ ID NO:60). The alignment shows that there is a 19.9% overall amino acid sequence identity between TANGO 499 form 1, variant 1 and riboflavin binding protein. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4. Figure 19 depicts the cDNA sequence of TANGO 499 form 2, variant 3 (SEQ ID NO:61) and the predicted amino acid sequence of TANGO 499 form 2, variant 3 (SEQ ID NO:63). The open reading frame of TANGO 499 form 2, variant 3 extends from nucleotides 144 to 830 of SEQ ID NO:61 (SEQ ID NO:62).
Figure 20 depicts a hydropathy plot of TANGO 499 form 2, variant 3. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 30 of SEQ ID NO:63 (SEQ ID NO:64) on the left from the mature protein (amino acids 31 to 1085 of SEQ ID NO:65) on the right.
Figure 21 shows an alignment of the TANGO 499 form 1, variant 1 amino acid sequence (SEQ ID NO: 56) with the TANGO 499 form 2, variant 3 amino acid sequence (SEQ ID NO:63). The alignment shows an alternative spliced exon which is present in form 1 and absent in form 2 and that there is a 90.2% overall amino acid sequence identity between human TANGO 499 form 1, variant 1 and the TANGO 499 form 2, variant 3. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 22 depicts the cDNA sequence of human TANGO 509 (SEQ ID NO: 66) and the predicted amino acid sequence of human TANGO 509 (SEQ ID NO:68). The open reading frame of human TANGO 509 extends from nucleotides 59 to 928 of SEQ ID NO:66 (SEQ ID NO:67).
Figure 23 depicts a hydropathy plot of human TANGO 509. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 18 of SEQ ID NO:68 (SEQ ID NO:69)) on the left from the mature protein (amino acids 19 to 290 of SEQ ID NO:70)) on the right.
Figure 24 shows an alignment of the human TANGO 509 amino acid sequence (SEQ ID NO:68) with the butyrophilin-like protein amino acid sequence (SEQ ID NO:85; Accession Number AF242780). The alignment shows that there is a 33.0% overall amino acid sequence identity between human TANGO 509 and the butyrophilin-like protein. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 25 depicts the cDNA sequence of mouse TANGO 509 (SEQ ID NO:76) and the predicted amino acid sequence of mouse TANGO 509 (SEQ ID NO:78). The open reading frame of mouse TANGO 509 extends from nucleotide 49 to 918 of SEQ ID NO:76 (SEQ ID NO:77).
Figure 26 depicts a hydropathy plot of mouse TANGO 509. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N-glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 18 of SEQ ID NO:78 (SEQ ID NO:79)) on the left from the mature protein (amino acids 19 to 290 of SEQ ID NO:78) on the right.
Figure 27 shows an alignment of the mouse TANGO 509 amino acid sequence (SEQ ID NO:78) with the butyrophilin-like protein amino acid sequence (SEQ ID NO:85; Accession Number AF242780). The alignment shows that there is a 31.9% overall amino acid sequence identity between mouse TANGO 509 and the butyrophilin-like protein. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Detailed Description of the Invention
The INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 proteins and nucleic acid molecules comprise families of molecules having certain conserved structural and functional features. As used herein, the terms "family" or "families" are 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, the INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 proteins of the invention can have signal sequences. As used herein, a "signal sequence" includes a peptide of at least about 15 or 20 amino acid residues in length which occurs at the N-terminus of secretory and membrane-bound proteins and which contains at least about 70% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. In a preferred embodiment, a signal sequence contains at least about 10 to 40 amino acid residues, preferably about 19-34 amino acid residues, and has at least about 60-80%, more preferably 65-75%, and more preferably at least about 70% hydrophobic residues. A signal sequence serves to direct a protein containing such a sequence to a lipid bilayer. A signal sequence is usually cleaved during processing of the mature protein.
Thus, in one embodiment, an INTERCEPT 307 protein can contain a signal sequence of about amino acids 1 to 23 of SEQ ID NO:3 (SEQ ID NO:4).
In another embodiment, a MANGO 511 protein can contain a signal sequence of about 1 to 41 of SEQ ID NO:41 (SEQ ID NO:42). In another embodiment, a TANGO 351 protein contains a signal sequence of about amino acids 1 to 24 of SEQ ID NO:25 (SEQ ID NO:26).
In another embodiment, a TANGO 361 protein can contain a signal sequence of about amino acids 1 to 35 of SEQ ID NO:44 (SEQ ID NO:45).
In another embodiment, a TANGO 499 form 1 , variant 1 protein can contain a signal sequence of about amino acids 1 to 30 of SEQ ID NO:56 (SEQ ID NO:57).
In another embodiment, a TANGO 499 form 2, variant 3 protein can contain a signal sequence of about amino acids 1 to 30 of SEQ ID NO:63 (SEQ ID NO:64).
In another embodiment, a human TANGO 509 protein can contain a signal sequence of about amino acids 1 to 18 of SEQ ID NO:68 (SEQ ID NO:69).
In another embodiment, a mouse TANGO 509 protein can contain a signal sequence of about amino acids 1 to 18 of SEQ ID NO:78 (SEQ ID NO:79).
An INTERCEPT 307 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. In one embodiment, an INTERCEPT 307 protein contains extracellular domains at about amino acid residues 24 to 153, 211 to 228 and 319 to 330 of SEQ ID NO:3 (SEQ ID NO: 6, 7 and 8, respectively), transmembrane domains at about amino acid residues 154 to 175, 192 to 210, 229 to 252, 296 to 318 and 331 to 348 of SEQ ID NO:3 (SEQ ID NO:9, 10, 11, 12, and 13, respectively) and cytoplasmic domains at about amino acid residues 176 to 191, 253 to 295 and 349 to 362 of SEQ ID NO:3 (SEQ ID NO:14, 15, and 16, respectively). In this embodiment, the mature INTERCEPT 307 protein corresponds to amino acids 24 to 362 of SEQ ID NO:3 (SEQ ID NO:5).
An INTERCEPT 307 family member can include a signal sequence. In certain embodiments, a INTERCEPT 307 family member has the amino acid sequence of SEQ ID NO:3, and the signal sequence is located at amino acids 1 to 21, 1 to 22, 1 to 23, 1 to 24 or 1 to 25. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 23 results in an extracellular domain consisting of amino acids 24 to 153 of SEQ ID NO:3 and the mature INTERCEPT 307 protein corresponding to amino acids 24 to 362 of SEQ ID NO:3 (SEQ ID NO:5). An INTERCEPT 307 family member can include one or more Gas vesicle protein GVPc-like domains. A gas vesicle protein GPVc-like domain as described herein can have the following consensus sequence: F-L-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-A- Xaa-Q-Xaa-Xaa-Xaa-L-Xaa-Xaa-F, wherein F is phenylalanine, L is leucine, "Xaa" is any amino acid, A is alanine, and Q is glutamine.
In one embodiment, an INTERCEPT 307 family member has the amino acid sequence of SEQ ID NO:3 and, preferably, a gas vesicle protein GPVc-like domain is located at about amino acid positions 112 to 141 of SEQ ID NO:3 (SEQ ID NO:17). In another embodiment, an INTERCEPT 307 family member has the amino acid sequence of SEQ ID NO:3 and, preferably, a gas vesicle protein GPVc-like consensus sequence is located at about amino acid positions 122 to 141 of SEQ ID NO:3 (SEQ ID NO:18). In another embodiment, an INTERCEPT 307 family member includes one or more gas vesicle protein GPVc-like consensus sequences having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 112 to 141 of SEQ ID NO:3 (SEQ ID NO: 17). In yet another embodiment, an INTERCEPT 307 family member includes one or more gas vesicle protein GPVc-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 122 to 141 of SEQ ID NO: 3 (SEQ ID NO: 18).
In another embodiment an INTERCEPT 307 family member includes one or more gas vesicle protein GPVc-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 112 to 141 of SEQ ID NO:3 (SEQ ID NO: 17), and has at least one INTERCEPT 307 biological activity as described herein. In yet another embodiment, an INTERCEPT 307 family member includes one or more gas vesicle protein GPVc-like domain consensus sequences having an amino acid sequence that is at least about 55%), preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 122 to 141 of SEQ ID NO:3 (SEQ ID NO: 18), and has at least one INTERCEPT 307 biological activity as described herein.
In another embodiment, the gas vesicle protein GVPc-like domain of INTERCEPT 307 is a gas vesicle protein GVPc domain. A gas vesicle protein GVPc domain typically has the following consensus sequence: F-L-Xaa-Xaa-T-Xaa-Xaa-Xaa-R-Xaa-Xaa-Xaa-A- Xaa-Xaa-Q-Xaa-Xaa-Xaa-L-Xaa-Xaa-F, wherein F is phenylalanine, L is leucine, "Xaa" is any amino acid, T is threonine, R is arginine, A is alanine and Q is glutamine. Gas vesicle protein GVPc domains are found in cyanobacterial and Archaebacteria microorganisms. Gas vesicles are small, hollow, gas filled protein structures that enable the bacteria to position themselves at a favorable depth in a liquid medium for growth. In this embodiment, an INTERCEPT 307 family member includes one or more gas vesicle protein GVPc domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 112 to 141 of SEQ ID NO:3 (SEQ ID NO: 17).
A MANGO 511 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. In one embodiment, a MANGO 511 protein contains an extracellular domain at about amino acid residues 42 to 265 of SEQ ID NO:25 (SEQ ID NO:28), a transmembrane domain at about amino acid residues 266 to 284 of SEQ ID NO:25 (SEQ ID NO:29), and a cytoplasmic domain at about amino acid residues 285 to 299 of SEQ ID NO:25 (SEQ ID NO:30). In this embodiment, the mature MANGO 511 protein corresponds to amino acids 42 to 299 of SEQ ID NO:29 (SEQ ID NO:27).
A MANGO 511 family member can include a signal sequence. In certain embodiments, a MANGO 511 family member has the amino acid sequence of SEQ ID NO:25, and the signal sequence is located at amino acids 1 to 39, 1 to 40, 1 to 41, 1 to 42 or 1 to 43. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 41 results in an extracellular domain consisting of amino acids 42 to 265 of SEQ ID NO:25 and a mature MANGO 511 protein corresponding to amino acids 42 to 299 of SEQ ID NO:25 (SEQ ID NO:27).
A MANGO 511 family member can include one or more Ig-like domains. A MANGO 511 Ig-like domain as described herein has the following consensus sequence, beginning about 1 to 15 amino acid residues, more preferably about 3 to 10 amino acid residues, and most preferably about 5 amino acid residues from the domain C-terminus: [FY]-Xaa-C, wherein [FY] is either a phenylalanine or a tyrosine residue (preferably tyrosine), where "Xaa" is any amino acid, and C is a cysteine residue. In one embodiment, a MANGO 511 family member includes one or more Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%>, yet more preferably at least about 85%o, and most preferably at least about 95% identical to amino acids 60 to 118 of SEQ ID NO:25 (SEQ ID NO:31).
In another embodiment, a MANGO 511 family member includes one or more MANGO 511 Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95%> identical to amino acids 60 to 118 of SEQ ID NO:25 (SEQ ID NO:31), and has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain. In another embodiment, a MANGO 511 family member includes one or more Ig-like domains having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 60 to 118 of SEQ ID NO:25 (SEQ ID NO:31), has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain, and has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine.
In yet another embodiment, a MANGO 511 family member includes one or more MANGO 511 Ig-like domains having an amino acid sequence that is at least 55%>, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95%> identical to amino acids 60 to 118 of SEQ ID NO:25 (SEQ ID NO:31), and has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain, has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine, and has at least one MANGO 511 biological activity as described herein.
In another embodiment, the Ig-like domain of MANGO 511 is an Ig domain. An Ig domain as used in the context of MANGO 511 has the following consensus sequence, beginning at about 1 to 15 amino acid residues, more preferably about 3 to 10 amino acid residues, and most preferably about 5 amino acid residues from the C-terminal end of the domain: [FY]-Xaa-C-Xaa-[VA]-COO-, wherein [FY] is either a phenylalanine or a tyrosine residue (preferably tyrosine), where "Xaa" is any amino acid, C is a cysteine residue, [VA] is either valine or an alanine residue (preferably alanine), and COO- is the C-terminus of the domain. In this embodiment, a MANGO 511 family member includes one or more Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 60 to 118 of SEQ ID NO:25 (SEQ ID NO:31).
A TANGO 351 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. In one embodiment, a TANGO 351 protein contains an extracellular domain at about amino acid residues 25 to 458 of SEQ ID NO:36 (SEQ ID NO:39), a transmembrane domain at about amino acid residues 459 to 476 of SEQ ID NO:36 (SEQ ID NO:40), and a cytoplasmic domain at about amino acid residues 477 to 482 of SEQ ID NO:36 (SEQ ID NO:41). In this embodiment, the mature TANGO 351 protein corresponds to amino acids 25 to 482 of SEQ ID NO:36 (SEQ ID NO:38).
A TANGO 351 family member can include a signal sequence. In certain embodiments, a TANGO 351 family member has the amino acid sequence of SEQ ID NO:36, and the signal sequence is located at amino acids 1 to 22, 1 to 23, 1 to 24, 1 to 25 or 1 to 26. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 24 results in an extracellular domain consisting of amino acids 25 to 458 of SEQ ID NO:36 (SEQ ID NO:39) and the mature TANGO 351 protein corresponding to amino acids 25 to 482 of SEQ ID NO:36 (SEQ ID NO:38).
A TANGO 361 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. In one embodiment, a TANGO 361 protein contains an extracellular domain at about amino acid residues 235 to 423 of SEQ ID NO:44 (SEQ ID NO:49) a transmembrane domain at about amino acid residues 217 to 234 of SEQ ID NO:44 (SEQ ID NO:47), and a cytoplasmic domains at about amino acid residues 36 to 216 of SEQ ID NO:44 (SEQ ID NO:48). In this embodiment, the mature TANGO 361 protein corresponds to amino acids 36 to 423 of SEQ ID NO:44 (SEQ ID NO:46).
A TANGO 361 family member can include a signal sequence. In certain embodiments, a TANGO 361 family member has the amino acid sequence of SEQ ID NO:44, and the signal sequence is located at about amino acids 1 to 33, 1 to 34, 1 to 35, 1 to 36 or 1 to 37. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a TANGO 361 signal sequence consisting of amino acids 1 to 35 results in an extracellular domain consisting of amino acids 235 to 423 of SEQ ID NO:44 (SEQ ID NO:49) and the mature TANGO 361 protein corresponding to amino acids 36 to 423 of SEQ ID NO:44 (SEQ ID NO:46).
A TANGO 361 family member can include one or more SEA domains. As used herein, the term "SEA domain" refers to a protein domain that can be found in TANGO 361 proteins and can regulate protein-protein or protein binding to carbohydrate side chains. A SEA domain typically has about 50-200 amino acid residues, preferably about 75-150 amino acid residues, more preferably about 80-140 amino acid residues, and most preferably about 115-135 amino acid residues.
A SEA domain typically has the following consensus sequence: h-t-h-Xaa-h-Xaa- h-Xaa-Xaa-Xaa-Xaa-h-Xaa-a-t-t-t-h-t-t-t-Xaa-o-Xaa-Xaa-a-Xaa-Xaa-h-Xaa-t-t-H-Xaa-t- Xaa-H-Xaa-t-Xaa-a-t-t-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-h-h-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- Xaa-Xaa-t-t-Xaa-Xaa-h-Xaa-Xaa-Xaa-h-Xaa-h-t-h-h-Xaa-t-Xaa-Xaa-Xaa-Xaa-Xaa-t-t-t-h- t-Xaa-Xaa-Xaa-Xaa-t-Xaa-h-t-Xaa-Xaa-Xaa-Xaa-Xaa-h-Xaa-Xaa-Xaa-t-Xaa-Xaa-Xaa- Xaa-t-Xaa-Xaa-t-h-Xaa-Xaa-Xaa-Xaa-t, wherein t is a glycine, proline or polar amino acid, h is a hydrophobic amino acid, a is an aromatic amino acid and o is a serine or threonine. These domains are predominantly found in adhesive proteins present in heavily glycosylated environments. For example, SEA domains are found in a 63 kDa sea urchin sperm protein, agrin, enterokinase, perlecan, the breast cancer marker MUCl (episialin) and the cell surface antigen 114/A10 (Bork and Patthy, (1995) Prot. Sci. 4:1421-1425).
In one embodiment, a TANGO 361 family member has the amino acid sequence of SEQ ID NO:44 and, preferably, a SEA domain is located at about amino acids 47 to 170 of SEQ ID NO:44 (SEQ ID NO:50). In another embodiment, a TANGO 361 family member includes one or more SEA domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 47 to 170 of SEQ ID NO:44 (SEQ ID NO:50).
In another embodiment, a TANGO 361 family member includes one or more SEA domains having an amino acid sequence that is at least about 55%>, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 44 to 170 of SEQ ID NO:44 (SEQ ID NO:50), and has at least one TANGO 361 biological activity as described herein.
A TANGO 361 family member can include a serine protease domain. Serine protease domains are typically found in serine proteases, and can be found in, among other proteins, e.g., blood coagulation factors VII, XI, and X, thrombin, plasminogen, tryptases, such as trypsin, airway trypsin-like proteases, mast cell proteases, and members of the complement system which are known for regulation of energy balance and suppression of infectious agents. As used herein, the term "serine protease domain" refers to a polypeptide sequence that includes about 100-400 amino acid residues, preferably about 150-350 amino acid residues, more preferably about 200-300 amino acid residues, and most preferably about 225-260 amino acid residues. A serine protease typically has two consensus sequences. The first consensus sequence is a histidine active site and has the following sequence: [LIVM]-[ST]-A-[STAG]-H-C, wherein [LIVM] is a leucine, isoleucine, valine, or methionine, [ST] is a serine or threonine, A is alanine, [STAG] is serine, threonine, alanine or glycine, H is histidine and C is cysteine. The second consensus sequence is a serine active site and has the following consensus sequence: [DNSTAGC]-[GSTAPIMVQH]-x(2)-G-[DE]-S-G-[GS]-[SAPHV]-[LIVMFYWH]- [LIVMFYSTANQH], wherein [DNSTAGC] is an aspartic acid, asparagine, serine, threonine, alanine, glycine, or cysteine, [GSTAPIMVQH] is a glycine, serine, threonine, alanine, proline, isoleucine, methionine, valine, glutamine, or histidine, x(2) is two consecutive amino acids, G is a glycine, [DE] is an aspartic acid or glutamic acid, S is a serine, G is a glycine, [GS] is a glycine or serine, [SAPHV] is a serine, alanine, proline, histidine or valine, [LIVMFYWH] is a leucine, isoleucine, valine, methionine, phenylalanine, tyrosine, tryptophan or histidine, [LIVMFYSTANQH] is a leucine, isoleucine, valine, methionine, phenylalanine, tyrosine, serine, threonine, alanine, asparagine, glutamine or histidine.
In one embodiment, a TANGO 361 family member has the amino acid sequence of SEQ ID NO:44 in which a serine protease domain appears at about amino acids 192 to 417 of SEQ ID NO:44 (SEQ ID NO:51). Preferably, a histidine active site consensus sequence is located at about amino acids 228 to 233 of SEQ ID NO:44 (SEQ ID NO:52) and a serine active site consensus sequence is located at 367 to 378 of SEQ ID NO:44 (SEQ ID NO:53). In another embodiment, a TANGO 361 family member includes one or more serine protease domain consensus sequences having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%), and most preferably at least about 95% identical to amino acids 228 to 233 of SEQ ID NO:44 (SEQ ID NO:52) or 367 to 378 of SEQ ID NO:44 (SEQ ID NO:53).
In another embodiment, a TANGO 361 family member includes one or more serine protease domain consensus sequences having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%>, and most preferably at least about 95% identical to amino acids 228 to 233 of SEQ ID NO:44 (SEQ ID NO:52) or 367 to 378 of SEQ ID NO:44 (SEQ ID NO:53), and has at least one TANGO 361 biological activity as described herein.
A TANGO 499 family member can include one or more of the following domains: 1) a signal sequence; and 2) a secreted protein. In one embodiment, a TANGO 499 protein is a secreted protein containing a signal sequence of 1 to 30 amino acids of SEQ ID NO:56 (SEQ ID NO:57) and is an immature protein of 254 amino acids of SEQ ID NO:56. In this embodiment, the mature TANGO 499 protein corresponds to amino acids 31 to 254 of SEQ ID NO:56 (SEQ ID NO:58). In certain embodiments, a TANGO 499 family member has the amino acid sequence of SEQ ID NO:56, and contains a signal sequence that is preferably located at about amino acids 1 to 28, 1 to 29, 1 to 31 or 1 to 32. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 31 results in a mature protein comprising a secreted protein of amino acids 32 to 254 of SEQ ID NO:56 (SEQ ID NO:58). In certain embodiments, a TANGO 499 family member has the amino acid sequence of SEQ ID NO:63, and contains a signal sequence that is preferably located at about amino acids 1 to 28, 1 to 29, 1 to 31 or 1 to 32. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 31 results in a mature protein comprising a secreted protein of amino acids 32 to 229 of SEQ ID NO:63 (SEQ ID NO:65).
In one embodiment, a TANGO 499 family member is a polypeptide comprising the amino acid sequence of SEQ ID NO:56. In another embodiment, a TANGO 499 family member is a polypeptide comprising the amino acid sequence of SEQ ID NO:63.
In another embodiment, a TANGO 499 family member is a polypeptide comprising the amino acid sequence of SEQ ID NO:91. In another embodiment, a TANGO 499 family member is a polypeptide comprising the amino acid sequence of SEQ ID NO:92.
In another embodiment, a TANGO 499 family member is a polypeptide comprising the amino acid sequence of SEQ ID NO:94. In another embodiment, a TANGO 499 family member is a polypeptide comprising the amino acid sequence of SEQ ID NO:96.
A human TANGO 509 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. In one embodiment, a human TANGO 509 protein contains an extracellular domain at about amino acid residues 19 to 240 of SEQ ID NO:68 (SEQ ID NO:74), a transmembrane domain at about amino acid residues 241 to 259 of SEQ ID NO:68 (SEQ ID NO:73), and a cytoplasmic domain at about amino acid residues 260 to 290 of SEQ ID NO:68 (SEQ ID NO:75). In this embodiment, the mature TANGO 509 protein corresponds to amino acids 19 to 290 of SEQ ID NO:68 (SEQ ID NO:70).
A human TANGO 509 family member can include a signal sequence. In certain embodiments, a human TANGO 509 family member has the amino acid sequence of SEQ ID NO: 68, and the signal sequence is located at about amino acids 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 18 results in a mature human TANGO 509 protein corresponding to amino acids 19 to 290 of SEQ ID NO:68 (SEQ ID NO:70).
A human TANGO 509 family member can include one or more Ig-like domains. A TANGO 509 Ig-like domain as described herein has the following consensus sequence, beginning about 1 to 15 amino acid residues, more preferably about 3 to 10 amino acid residues, and most preferably about 5 amino acid residues from the domain C-terminus: [FY]-Xaa-C, wherein [FY] is either a phenylalanine or a tyrosine residue (preferably tyrosine), where "Xaa" is any amino acid, and C is a cysteine residue. In one embodiment, a human TANGO 509 family member includes one or more Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 or 148 to 211 of SEQ ID NO:68 (SEQ ID NO:71 or SEQ ID NO:72).
In another embodiment, a human TANGO 509 family member includes one or more TANGO 509 Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 or 148 to 211 of SEQ ID NO:68 (SEQ ID NO:71 or SEQ ID NO:72), and has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain. In another embodiment, a human TANGO 509 family member includes one or more TANGO 509 Ig-like domains having an amino acid sequence that is at least 55%), preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 or 148 to 211 of SEQ ID NO:68 (SEQ ID NO:71 or SEQ ID NO:72), has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig- like domain and has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine.
In yet another embodiment, a human TANGO 509 family member includes one or more TANGO 509 Ig-like domains having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 or 148 to 211 of SEQ ID NO:68 (SEQ ID NO:71 or SEQ ID NO:72), and has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain which has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine, and has at least one human TANGO 509 biological activity as described herein.
In another embodiment, the Ig-like domain of human TANGO 509 is an Ig-like domain which has the following consensus sequence at the C-terminus of the domain: [FY]-Xaa-C-Xaa-[VAIF]-COO-, wherein [FY] is either a phenylalanine or a tyrosine residue (preferably tyrosine), where "Xaa" is any amino acid, C is a cysteine residue, [VA] is a valine, an alanine, an isoleucine or phenylalanine residue, and COO- is the C-terminus of the domain. In this embodiment, a human TANGO 509 family member includes one or more of these Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 or 148 to 211 of SEQ ID NO:68 (SEQ ID NO:71 or SEQ ID NO:72).
A mouse TANGO 509 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. In one embodiment, a mouse TANGO 509 protein contains an extracellular domain consisting of amino acids 19 to 239 of SEQ ID NO:78 (SEQ ID NO:83), a transmembrane domain at amino acids 240 to 260 of SEQ ID NO:78 (SEQ ID NO:82), a cytoplasmic domain at amino acids 261 to 290 of SEQ ID NO:78 (SEQ ID NO:84) and a mature mouse TANGO 509 protein at amino acids 19 to 290 (SEQ ID NO:80).
A mouse TANGO 509 family member can include a signal sequence. In certain embodiments, a TANGO 509 family member has the amino acid sequence of SEQ ID NO: 78, and the signal sequence is located at about amino acids 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 18 results in a mature mouse TANGO 509 protein corresponding to amino acids 19 to 290 of SEQ ID NO: 78 (SEQ ID NO:80).
A mouse TANGO 509 family member can include one or more Ig-like domains. A mouse TANGO 509 Ig-like domain as described herein has the following consensus sequence, beginning about 1 to 15 amino acid residues, more preferably about 3 to 10 amino acid residues, and most preferably about 5 amino acid residues from the domain C- terminus: [FY]-Xaa-C, wherein [FY] is either a phenylalanine or a tyrosine residue (preferably tyrosine), where "Xaa" is any amino acid, and C is a cysteine residue. In one embodiment, a mouse TANGO 509 family member includes one or more such Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81).
In another embodiment, a mouse TANGO 509 family member includes one or more mouse TANGO 509 Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81), and has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain. In another embodiment, a mouse TANGO 509 family member includes one or more mouse TANGO 509 Ig-like domains having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81), has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain, and has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine.
In yet another embodiment, a mouse TANGO 509 family member includes one or more Ig-like domains having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81), and has a conserved cysteine residue about 8 residues downstream from the N-terminus of the Ig-like domain, has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine, and has at least one mouse TANGO 509 biological activity as described herein.
In another embodiment, the Ig-like domain of mouse TANGO 509 is an Ig domain which has the following consensus sequence at the C-terminus of the domain: [FY]-Xaa- C-Xaa-[VAIF]-COO-, wherein [FY] is either a phenylalanine or a tyrosine residue (preferably tyrosine), where "Xaa" is any amino acid, C is a cysteine residue, [VA] is a valine, an alanine, an isoleucine or phenylalanine residue, and COO- is the C-terminus of the domain. In this embodiment, a mouse TANGO 509 family member includes one or more Ig-like domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 33 to 116 of SEQ ID NO:78 (SEQ ID NO:81).
Various features of INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509 are summarized below.
HUMAN INTERCEPT 307
A cDNA encoding human INTERCEPT 307 was identified by analyzing the sequences of clones present in a human TH-2 induced T-cell library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jthtg033cl0, encoding full-length human INTERCEPT 307. The human INTERCEPT 307 cDNA of this clone is 2021 nucleotides long (Figure 1; SEQ ID NO:l). The open reading frame of this cDNA, nucleotides 45 to 1130 of SEQ ID NO:l (SEQ ID NO:2), encodes a 362 amino acid transmembrane protein (Figure 1; SEQ ID NO:3)
Figure 2 depicts a hydropathy plot of human INTERCEPT 307. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering JO: 1-6) predicted that human INTERCEPT 307 includes a 23 amino acid signal peptide (amino acid 1 to amino acid 23 of SEQ ID NO:3; SEQ ID NO:4) preceding the mature INTERCEPT 307 protein (corresponding to amino acid 24 to amino acid 362 of SEQ ID NO:3 (SEQ ID NO:5)). In instances wherein the signal peptide is cleaved, the molecular weight of INTERCEPT 307 protein without post-translational modifications is 40.6 kDa prior to the cleavage of the signal peptide, and 38.1 kDa after cleavage of the signal peptide.
Human INTERCEPT 307 protein is a transmembrane protein that contains extracellular domains at amino acid residues 24 to 153, 211 to 228, and 319 to 330 of SEQ ID NO:3, (SEQ ID NO:6, 7 and 8, respectively), transmembrane domains at amino acid residues 154 to 175, 192 to 210, 229 to 252, 296 to 319, and 331 to 348 of SEQ ID NO:3, (SEQ ID NO: 9, 10, 11, 12, and 13, respectively), and cytoplasmic domains at amino acid residues 176 to 191, 253 to 295, and 349 to 362 of SEQ ID NO:3, (SEQ ID NO:14, 15, and 16, respectively).
In instances wherein the signal peptide is not cleaved, a human INTERCEPT 307 protein is a transmembrane protein that contains extracellular domains at amino acid residues 1 to 153, 211 to 228, and 319 to 330 of SEQ ID NO:3, transmembrane domains at amino acid residues 154 to 175, 192 to 210, 229 to 252, 296 to 319, and 331 to 348 of SEQ ID NO:3, and cytoplasmic domains at amino acid residues 176 to 191, 253 to 295, and 349 to 362 of SEQ ID NO:3.
Alternatively, in another embodiment, a human INTERCEPT 307 protein contains cytoplasmic domains at amino acid residues 24 to 153, 211 to 228, and 319 to 330 of SEQ ID NO:3, transmembrane domains at amino acid residues 154 to 175, 192 to 210, 229 to 252, 296 to 319, and 331 to 348 of SEQ ID NO:3, (SEQ ID NO:9, 10, 11, 12, and 13, respectively), and extracellular domains at amino acid residues 176 to 191, 253 to 295, and 349 to 362 of SEQ ID NO:3.
In one embodiment a cDNA sequence of human INTERCEPT 307 has a nucleotide at position 54 which is cytosine (C)(SEQ ID NO:l). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is leucine (L)(SEQ ID NO:3). In an alternative embodiment, a species variant cDNA sequence of human INTERCEPT 307 has a nucleotide at position 54 which is adenine (A)(SEQ ID NO:l 13). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is isoleucine (I)(SEQ ID NO:l 14), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human INTERCEPT 307 has a nucleotide at position 76 which is thymine (T)(SEQ ID NO:l). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 11 that is phenylalanine (F)(SEQ ID NO:3). In an alternative embodiment, a species variant cDNA sequence of human INTERCEPT 307 has a nucleotide at position 76 which is adenine (A)(SEQ ID NO:l 15). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 11 that is tyrosine (Y)(SEQ ID NO: 116), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human INTERCEPT 307 has a nucleotide at position 87 which is adenine (A)(SEQ ID NO:l). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 15 that is asparagine (N)(SEQ ID NO:3). In an alternative embodiment, a species variant cDNA sequence of human INTERCEPT 307 has a nucleotide at position 87 which is guanine (G)(SEQ ID NO:l 17). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 15 that is aspartate (D)(SEQ ID NO: 118), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human INTERCEPT 307 has a nucleotide at position 123 which is thymidine (T)(SEQ ID NO:l). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 27 that is serine (S)(SEQ ID NO:3). In an alternative embodiment, a species variant cDNA sequence of human INTERCEPT 307 has a nucleotide at position 180 which is adenine (A)(SEQ ID NO: 119). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 27 that is threonine (T)(SEQ ID NO: 120), i.e., a conservative substitution.
Human INTERCEPT 307 includes a gas vesicle protein GVPc-like domain (at amino acids 112 to 141 of SEQ ID NO:3, (SEQ ID NO: 17)).
Two N-glycosylation sites are present in INTERCEPT 307. The first has the sequence NYSY (at amino acid residues 91 to 94 of SEQ ID NO:3) and second has the sequence NGTT (at amino acid residues 100 to 103 of SEQ ID NO:3). Five protein kinase C phosphorylation sites are present in INTERCEPT 307. The first has the sequence SLR (at amino acid residues 56 to 58 of SEQ ID NO:3), the second has the sequence TTK (at amino acid residues 102 to 104 of SEQ ID NO:3), the third has the sequence SAK (at amino acid residues 124 to 126 of SEQ ID NO:3), the fourth has the sequence SRR (at amino acid residues 147 to 149 of SEQ ID NO:3), and the fifth has the sequence TWK (at amino acid residues 353 to 355 of SEQ ID NO:3). INTERCEPT 307 has three casein kinase II phosphorylation sites. The first has the sequence TTKE (at amino acid residues 102 to 105 of SEQ ID NO:3), the second has the sequence SAKE (at amino acid residues 124 to 127 of SEQ ID NO:3), and the third has the sequence TWKE (at amino acid residues 353 to 356 of SEQ ID NO:3). Eight N-myristylation sites are present in INTERCEPT 307. The first has the sequence GNLFGQ (at amino acid residues 19 to 24 of SEQ ID NO:3), the second has the sequence GAFDSS (at amino acid residues 35 to 40 of SEQ ID NO: 3), the third has the sequence GLCPGN (at amino acid residues 95 to 100 of SEQ ID NO:3), the fourth has the sequence GTLNSL (at amino acid residues 169 to 174 of SEQ ID NO:3), the fifth has the sequence GGDMAR (at amino acid residues 180 to 185 of SEQ ID NO:3), the sixth has the sequence GSNAAF (at amino acid residues 278 TO 283 of SEQ ID NO:3), the seventh has the sequence GLVMAL (at amino acid residues 298 to 303 of SEQ ID NO:3), and the eighth has the sequence GSLQND (at amino acid residues 320 to 325 of SEQ ID NO:3). INTERCEPT 307 has a leucine zipper pattern with the sequence LFGLVMALSAVVSLLQFPIFTL at amino acid residues 296 to 317 of SEQ ID NO:3, (SEQ ID NO:20).
The INTERCEPT 307 gene maps to human chromosome 11 between markers Dl lS1357 and Dl lS1765.
Figure 3 shows an alignment of the human INTERCEPT 307 amino acid sequence (SEQ ID NO:3) with the prostate cancer gene PB39 amino acid sequence (SEQ ID NO:20; Accession Number NM_003627). The alignment shows that there is a 21.0% overall amino acid sequence identity between INTERCEPT 307 and PB39. PB39 is expressed in tissues of the adult colon, small intestine, ovary, prostate, spleen, skeletal muscle and pancreas. PB39 is also expressed in fetal kidney, liver and lung. The expression of PB39 has been shown to be increased early in prostate cancer development and PB39 can play a role in the development of human prostate cancer. As such, INTERCEPT 307, nucleic acids and proteins may be useful, for example, as early markers for the development of prostate cancer (e.g., early markers for the development of prostatic intraepithelial neoplasia (PIN))-
Figure 4 shows an alignment of the nucleotide sequence of INTERCEPT 307 coding region (SEQ ID NO:2) and the nucleotide sequence of PB39 coding region (SEQ ID NO:21; Accession Number AF045584). The alignment shows a 40.9 % overall sequence identity between the two nucleotide sequences. The full-length INTERCEPT 307 nucleic acid sequence (SEQ ID NO:l) and PB39 cDNA (SEQ ID NO:22; Accession Number NM_003627) have an overall sequence identity of 44.0%.
Figure 5 shows an alignment of the human INTERCEPT 307 amino acid sequence (SEQ ID NO:3) with the human eosinophil granule major basic protein amino acid sequence (SEQ ID NO:86; Accession Number Z26248). The alignment shows that there is a 13.8 % overall amino acid sequence identity between INTERCEPT 307 and human eosinophil granule major basic protein. Human eosinophil granule major basic protein (MBP) is expressed in eosinophils and has toxic effects on many targets, including helminths, protozoa and bacteria and surrounding cells (Gleich, A.J. et al. (1993) Annu. Rev. Med. 44:85-101). MBP mediates damage to the respiratory epithelium (e.g., desquamation and destruction of sputum ciliated cells) in individuals with asthma, and increased MBP concentration has been shown to be a good marker for asthma (Frigas, E. et al. (1986) J. Allergy Clinical Immunol. 77:537-537).
Figure 6 shows an alignment of the nucleotide sequence of INTERCEPT 307 coding region (SEQ ID NO:2) and the nucleotide sequence of human eosinophil granule major basic protein amino acid sequence coding region (SEQ ID NO:87); Accession Number Z26248). The alignment shows a 38.1 % overall sequence identity between the two nucleotide sequences. The full-length INTERCEPT 307 nucleic acid sequence (SEQ ID NO:l) and human eosinophil granule major basic protein cDNA (SEQ ID NO:88; Accession Number Z26248) have an overall sequence identity of 57.3%>.
Clone INT307, which encodes human INTERCEPT 307, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110- 2209) on July 29, 1999 and assigned Accession Number PTA-455. 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.
Uses of INTERCEPT 307 Nucleic acids. Polypeptides. and Modulators Thereof
As INTERCEPT 307 was originally found in a human TH2-induced T-cell library, INTERCEPT 307 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of lymphocytes, e.g., T- lymphocytes. INTERCEPT 307 nucleic acids, proteins and modulators thereof can be utilized to modulate immune-related processes, e.g. , the host immune response by, for example, modulating the formation of and/or binding to immune complexes, detection and defense against surface antigens and bacteria, and immune surveillance for rapid removal or pathogens. Such INTERCEPT 307 compositions and modulators thereof can be utilized, e.g., to ameliorate incidence of any symptoms associated with disorders that involve such immune-related processes, including, but not limited to, viral or bacterial infection, and inflammatory disorders (e.g., bacterial or viral infection, psoriasis, allergies and inflammatory bowel diseases). INTERCEPT 307 nucleic acids, proteins and modulators thereof can be used to modulate or treat immune related disorders, e.g., immunodeficiency disorders (e.g., HIV), viral disorders, cancers, and inflammatory disorders (e.g., bacterial or viral infection, psoriasis, septicemia, arthritis, allergic reactions). INTERCEPT 307 nucleic acids, proteins and modulators thereof can be used to treat atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g., leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.
INTERCEPT 307 exhibits homology to PB39. Therefore, INTERCEPT 307 nucleic acids, proteins and modulators thereof can be utilized to modulate the proliferation, differentiation, and/or function of prostate cells. INTERCEPT 307 nucleic acids, proteins and modulators thereof can be utilized to modulate processes involved in prostate development, differentiation and activity, including, but not limited to development, and differentiation and activation of prostate tissues and cells as well as any function associated with such cells, and amelioration of one or more symptoms associated with abnormal function of such cell types or disorders associated with such cell types. Such disorders can include, but are not limited to, malignant or benign prostate cell growth or inflammatory disorders (e.g. , prostatitis, benign prostatic hypertrophy, benign prostatic hyperplasia (BPH), prostatic paraganglioma, prostate adenocarcinoma, prostatic intraepithelial neoplasia, prostato-rectal fistulas, and/or atypical prostatic stromal lesions).
In further light of the fact that INTERCEPT 307 exhibits homology to PB39 which is expressed by tumor cells, INTERCEPT 307 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and progression of cancerous and non-cancerous cell proliferative disorders, such as deregulated proliferation (such as hyperdysplasia, hyper-IgM syndrome, or lymphoproliferative disorders), cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hyperplasia), or cancers, such as neoplasms or tumors (such as carcinomas, sarcomas, adenomas or myeloid lymphoma tumors, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, colon sarcoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hematoma, bile duct carcinoma, melanoma, choriocarcinoma, semicoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependynoma, pinealoma, hemangioblastoma, retinoblastoma), leukemias, (e.g. acute lymphocytic leukemia), acute myelocytic leukemia (myelolastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), or polycythemia vera, or lymphomas (Hodgkin's disease and non-Hodgkin's diseases), multiple myelomas and Waldenstrδm's macroglobulinemia.
In particular, INTERCEPT 307 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and progression of prostate cancer, e.g., prostatic intraepithelial neoplasia, prostatic paraganglioma, and prostate adenocarcinoma.
As INTERCEPT 307 has a gas vesicle protein-like domain, INTERCEPT 307 nucleic acids and protein fragments that contain the gas vesicle protein-like domain can be used to produce gas vesicles which can be used for protein, drug, and antigen presentation (e.g., vaccines).
INTERCEPT 307 has a leucine zipper pattern, therefore, INTERCEPT 307 nucleic acids, proteins and modulators thereof can be used to modulate protein-protein interactions (e.g., stabilize, promote, inhibit or disrupt protein-protein interactions).
As INTERCEPT 307 has homology to eosinophil granule basic protein, INTERCEPT 307 nucleic acids, proteins and modulators thereof can be used to modulate eosinophil function and activity, e.g., the ability to kill targets such as helminth, protozoa, and bacteria. INTERCEPT 307 nucleic acids, proteins and modulators thereof can be used to treat asthma, allergies (e.g., ocular allergies), nonallergic ophthalmic diseases (e.g., Wegener's granulomatosis, orbital pseudo-tumor and histiocytosis X), and helminth infection. INTERCEPT 307 nucleic acids, proteins and modulators thereof can be used to monitor an individual's asthma condition.
INTERCEPT 307 expression can be utilized as a marker (e.g., an in situ marker) for specific tissues (e.g., the prostate) and/or cells (e.g., prostatic cells) in which INTERCEPT 307 is expressed. INTERCEPT 307 expression can also be utilized as a marker for the development and/or progression of diseases and disorders such as prostate cancer and asthma. Further, INTERCEPT 307 nucleic acids be utilized for chromosomal mapping, or as chromosomal markers, e.g. , in radiation hybrid mapping. HUMAN MANGO 511
A cDNA encoding human MANGO 511 was identified by analyzing the sequences of clones present in a human dendritic cell library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jThxh005c 10, encoding full-length human MANGO 511. The human MANGO 511 cDNA of this clone is 1477 nucleotides long (Figure 7; SEQ ID NO:23). The open reading frame of this cDNA, nucleotides 108 to 1004 of SEQ ID NO:23, (SEQ ID NO:24), encodes a 299 amino acid transmembrane protein (Figure 7; SEQ ID NO:25).
Figure 8 depicts a hydropathy plot of human MANGO 511. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human MANGO 511 includes a 41 amino acid signal peptide (amino acid 1 to amino acid 41 of SEQ ID NO:25, (SEQ ID NO:26) preceding the mature MANGO 511 protein (corresponding to amino acid 42 to amino acid 299 of SEQ ID NO:25, (SEQ ID NO:27). In instances wherein the signal peptide is cleaved, the molecular weight of MANGO 511 protein without post-translational modifications is 32.8 kDa prior to the cleavage of the signal peptide, and 28.6 kDa after cleavage of the signal peptide.
Human MANGO 511 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 42 to 265 of SEQ ID NO:25, (SEQ ID NO:28), a transmembrane domain at amino acid residues 266 to 284 of SEQ ID NO:25, (SEQ ID NO:29), and a cytoplasmic domain at amino acid residues 285 to 299 of SEQ ID NO:25 (SEQ ID NO:30).
In instances wherein the signal peptide is not cleaved, a human MANGO 511 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 1 to 265 of SEQ ID NO:25, a transmembrane domain at amino acid residues 266 to 284 of SEQ ID NO:25, (SEQ ID NO:29), and a cytoplasmic domain at amino acid residues 285 to 299 of SEQ ID NO:25 (SEQ ID NO:30).
Alternatively, in another embodiment, a human MANGO 511 protein is a transmembrane protein that contains a cytoplasmic domain at amino acid residues 42 to 265 of SEQ ID NO:25, a transmembrane domain at amino acid residues 266 to 284 of SEQ ID NO:25, (SEQ ID NO:29), and an extracellular domain at amino acid residues 285 to 299 of SEQ ID NO:25.
In one embodiment a cDNA sequence of human MANGO 511 has a nucleotide at position 138 which is cytosine (C)(SEQ ID NO:23). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 11 that is leucine (L)(SEQ ID NO:25). In an alternative embodiment, a species variant cDNA sequence of human MANGO 511 has a nucleotide at position 138 which is adenine (A)(SEQ ID NO:121). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 11 that is isoleucine (I)(SEQ ID NO: 122), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human MANGO 511 has a nucleotide at position 156 which is guanine (G)(SEQ ID NO:23). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 17 that is aspartate (D)(SEQ ID NO: 25). In an alternative embodiment, a species variant cDNA sequence of human MANGO 511 has a nucleotide at position 156 which is adenine (A)(SEQ ID NO: 123). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 17 that is asparagine (N)(SEQ ID NO:124), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human MANGO 511 has a nucleotide at position 202 which is guanine (G)(SEQ ID NO:23). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 32 that is serine (S)(SEQ ID NO:25). In an alternative embodiment, a species variant cDNA sequence of human MANGO 511 has a nucleotide at position 202 which is cytosine (C)(SEQ ID NO: 125). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 32 that is threonine (T)(SEQ ID NO: 126), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human MANGO 511 has a nucleotide at position 214 which is guanine (G)(SEQ ID NO:23). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 36 that is arginine (R)(SEQ ID NO:25). In an alternative embodiment, a species variant cDNA sequence of human MANGO 511 has a nucleotide at position 214 which is adenine (A)(SEQ ID NO: 127). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 36 that is lysine (K)(SEQ ID NO: 128), i.e., a conservative substitution.
Human MANGO 511 includes an Ig-like domain at amino acids 60 to 118 of SEQ ID NO:25 (SEQ ID NO:31). Human MANGO 511 has three N-glycosylation sites. The first has the sequence NLSK (at amino acid residues 43 to 46 of SEQ ID NO:25), the second has the sequence NVTL (at amino acid residues 157 to 160 of SEQ ID NO:25), and the third has the sequence NKSD (at amino acid residues 248 to 251 of SEQ ID NO:25). Four protein kinase C phosphorylation sites are present in MANGO 511. The first has the sequence TIR (at amino acid residues 64 to 66 of SEQ ID NO:25), the second has the sequence SHR (at amino acid residues 207 to 209 of SEQ ID NO:25), the third has the sequence SRR (at amino acid residues 217 to 219 of SEQ ID NO:41), and the fourth has the sequence SQR (at amino acid residues 289 to 291 of SEQ ID NO:25). MANGO 511 has three casein II kinase phosphorylation sites. The first has the sequence SMTE (at amino acid residues 105 to 108 of SEQ ID NO:25) and the second has the sequence TSGE (at amino acid residues 153 to 156 of SEQ ID NO:25). Five N-myristylation sites are present in MANGO 511. The first has the sequence GSVISR (at amino acid residues 54 to 59 of SEQ ID NO:25), the second has the sequence GNSVTI (at amino acid residues 60 to 65 of SEQ ID NO:25), the third has the sequence GTLEAQ (at amino acid residues 69 to 74 of SEQ ID NO:25), the fourth has the sequence GQFQAL (at amino acid residues 193 to 198 of SEQ ID NO:25), and the fifth has the sequence GAADNL (at amino acid residues 238 to 243 of SEQ ID NO:25)
Figure 9 shows a local alignment of the human MANGO 511 amino acid sequence (SEQ ID NO:25) with the leukocyte Ig-like receptor-1 (LIR-1) amino acid sequence (SEQ ID NO:32; Accession Number AAB63522). The alignment shows that there is a 59.2% local identity over the 233 amino acids that were compared from the sequences of MANGO 511 and LIR-1.
LIR-1 is a expressed by lymphocytes, natural killer cells, monocytes, and dendritic cells and has been shown to be a major histocompatibility complex (MHC) class I binding protein (Cosman et al. (1997) Immunity 7:273-282). LIR-1 can function as a broad HLA class I-specific inhibitory receptor that recognizes different alleles coded for by different HLA loci (Vitale et al. (1999) Int. Immunol. 11 :29-35). The tyrosine phosphorylation of LIR-1 in monocytes has been shown to result in the binding of tyrosine phosphatase SHP- 1, and LIR-1 has been shown to be involved in the inhibition or down-modulation of monocyte activation signals (Fanger et al. (1998) Eur. J. Immunol. 28:3423-3434). As such MANGO 511 nucleic acids, proteins and modulators thereof are useful in modulating MHC class I binding and monocyte activation.
Figure 10 shows an alignment of the coding regions of the nucleotide sequence of LIR-1 (SEQ ID NO:33; Accession Number AF009221) and the nucleotide sequence of human MANGO 511 (SEQ ID NO:23). The alignment shows a 34.0 % overall sequence identity between the two nucleotide sequences. The coding region of the nucleotide sequence of LIR-1 (SEQ ID NO:33; Accession Number AF009221) and the full-length nucleotide sequence of human MANGO 511 cDNA (SEQ ID NO:24) have an overall sequence identity of 44.0%.
Clone EpM511 , which encodes human MANGO 511, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110- 2209) on July 23, 1999 and assigned Accession Number PTA-425. 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.
Uses of MANGO 511 Nucleic acids. Polypeptides. and Modulators Thereof
As MANGO 511 was originally found in a dendritic cell library, and including one or more Ig-like domains, MANGO 511 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of immune cells, e.g. B-cells, dendritic cells, natural killer cells and monocytes, and/or immune function. MANGO 511 nucleic acids, proteins and modulators thereof can be utilized to modulate immunoglobulins and formation of antibodies, and immune-related processes, e.g., the host immune response by, for example, modulating the formation of and/or binding to immune complexes, detection and defense against surface antigens and bacteria, and immune surveillance for rapid removal or pathogens. Such MANGO 511 compositions and modulators thereof can be utilized modulate or treat immune disorders that include, but are not limited to, immune proliferative disorders (e.g., carcinoma, lymphoma, e.g., follicular lymphoma), and disorders associated with fighting pathogenic infections, e.g., bacterial (e.g., chlamydia) infection, parasitic infection, and viral infection (e.g. , HSV or HIV infection), and pathogenic disorders associated with immune disorders (e.g., immunodeficiency disorders, such as HIV), autoimmune disorders, such as arthritis, graft rejection (e.g., allograft rejection), multiple sclerosis, Grave's disease, or Hashimoto's disease, T cell disorders (e.g., AIDS) and inflammatory disorders, such as septicemia, cerebral malaria, inflammatory bowel disease, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma, psoriasis), apoptotic disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus, insulin- dependent diabetes mellitus), cytotoxic disorders, septic shock, and cachexia.
MANGO 511 nucleic acids, proteins, and modulators thereof can also be used to modulate leukocyte trafficking, cancer, Type I immunologic disorders, e.g. , anaphylaxis and/or rhinitis, by, for example, modulating the interaction between antigens and cell receptors, e.g., high affinity IgE receptors. As MANGO 511 exhibits homology to leukocyte Ig-like receptor-1 (LIR-1), MANGO 511 nucleic acids, proteins and modulators thereof can be used modulate MHC class I binding. For example, MANGO 511 nucleic acids, proteins and modulators thereof can be used to modulate or treat disorders associated with aberrant MHC class I binding, such as autoimmune disorders, bacterial infections and viral infections. MANGO 511 nucleic acids, proteins and modulators thereof can be used to modulate monocyte activation signals and may be used to inhibit unwanted bystander responses mediated by antigen-specific T-cells. For example, antagonists of MANGO 511 action, such as peptides, antibodies or small molecules, that decrease or prevent MANGO 511 signaling can be used as modulators of monocyte activation. MANGO 511 nucleic acids, proteins and modulators thereof can be used to modulate or treat disorders associated with aberrant monocyte activation including, but not limited to, Wegener's granulomatosis (WG), hemophagocytic lymphohistiocytosis (HLH), histiocytic medullary reticulosis (HM), sarcoidosis, polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes (POMEMS) syndrome, and systemic sclerosis (Ssc).
As MANGO 511 exhibits homology to LIR-1, MANGO 511 nucleic acids, proteins and/or modulators thereof can be used to modulate natural killer cell function, e.g., activation. MANGO 511 nucleic acids, proteins and modulators thereof can be used to treat diseases associated with aberrant natural killer cell activation such as chronic natural killer cell lymphocytosis, aggressive non-T, non-B natural killer cell lymphoma/leukemia (ANKL/L), and Chediak-Higashi syndrome.
MANGO 511 expression can be utilized as a marker (e.g., an in situ marker) for specific tissues (e.g., the spleen) and/or cells (e.g., immune cells such as dendritic cells and natural killer cells) in which MANGO 511 is expressed. MANGO 511 nucleic acids can also be utilized for chromosomal mapping, or as chromosomal markers, e.g., in radiation hybrid mapping.
HUMAN TANGO 351
A cDNA encoding human TANGO 351 was identified by analyzing the sequences of clones present in a human fetal kidney library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jthKa90dl2, encoding full-length human TANGO 351. The human TANGO 351 cDNA of this clone is 3345 nucleotides long (Figure 11; SEQ ID NO:34). The open reading frame of this cDNA, nucleotides 143 to 1588 of SEQ ID NO:22 (SEQ ID NO:35), encodes a 482 amino acid transmembrane protein (Figure 11 ; (SEQ ID NO:36).
Figure 8 depicts a hydropathy plot of human TANGO 351. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 351 includes a 24 amino acid signal peptide (amino acid 1 to amino acid 24 of SEQ ID NO:36; SEQ ID NO:37) preceding the mature TANGO 351 protein (corresponding to amino acid 25 to amino acid 482 of SEQ ID NO:36; SEQ ID NO:38). In instances wherein the signal peptide is cleaved, the molecular weight of TANGO 351 protein without post-translational modifications is 53.4 kDa prior to the cleavage of the signal peptide, and 51.1 kDa after cleavage of the signal peptide.
Human TANGO 351 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 25 to 458 of SEQ ID NO:36 (SEQ ID NO:39), a transmembrane domain at amino acid residues 459 to 476 of SEQ ID NO:36 (SEQ ID NO:40), and a cytoplasmic domain at amino acid residues 477 to 482 of SEQ ID NO:25 (SEQ ID NO:41).
In instances wherein the signal peptide is not cleaved, human TANGO 351 has extracellular domains at amino acid residues contains an extracellular domain at amino acid residues 1 to 458 of SEQ ID NO:36, a transmembrane domain at amino acid residues 459 to 476 of SEQ ID NO:36 (SEQ ID NO:40), and a cytoplasmic domain at amino acid residues 477 to 482 of SEQ ID NO:25 (SEQ ID NO:41).
Alternatively, in another embodiment, a human TANGO 351 protein contains a cytoplasmic domain at amino acid residues 25 to 458 of SEQ ID NO:36, a transmembrane domain at amino acid residues 459 to 476 of SEQ ID NO:36 (SEQ ID NO:40), and an extracellular domain at amino acid residues 477 to 482 of SEQ ID NO:25.
In one embodiment a cDNA sequence of human TANGO 351 has a nucleotide at position 153 which is cytosine (C)(SEQ LD NO:34). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is alanine (A)(SEQ ID NO:36). In an alternative embodiment, a species variant cDNA sequence of human TANGO 351 has a nucleotide at position 153 which is thymine (T)(SEQ ID NO: 129). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is valine (V)(SEQ ID NO: 130), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 351 has a nucleotide at position 165 which is adenine (A)(SEQ ID NO:34). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 8 that is tyrosine (Y)(SEQ ID NO:36). In an alternative embodiment, a species variant cDNA sequence of human TANGO 351 has a nucleotide at position 165 which is thymine (T)(SEQ ID NO: 131). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 8 that is phenylalanine (F)(SEQ ID NO: 132), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 351 has a nucleotide at position 269 which is guanine (G)(SEQ ID NO:34). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position
43 that is aspartate (D)(SEQ ID NO:36). In an alternative embodiment, a species variant cDNA sequence of human TANGO 351 has a nucleotide at p sition 269 which is adenine (A)(SEQ ID NO: 133). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 43 that is asparagine (N)(SEQ ID NO:134), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 351 has a nucleotide at position 273 which is guanine (G)(SEQ ID NO:34). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position
44 that is arginine (R)(SEQ ID NO:36). In an alternative embodiment, a species variant cDNA sequence of human TANGO 351 has a nucleotide at position 273 which is adenine (A)(SEQ ID NO: 135). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 44 that is lysine (K)(SEQ ID NO:136), i.e., a conservative substitution.
Two N-glycosylation sites are present in TANGO 351. The first has the sequence NQTR (at amino acid residues 131 to 134 of SEQ ID NO:36) and the second has the sequence NFSE (at amino acid residues 353 to 356 of SEQ ID NO:36).
Two cAMP- and cGMP-dependent protein kinase phosphorylation sites are present in TANGO 351. The first has the sequence RRAS (at amino acid residues 94 to 97 of SEQ ID NO: 36) and the second has the sequence KRPT (at amino acid residues 424 to 427 of SEQ ID NO:36).
TANGO 351 has seven protein kinase C phosphorylation sites. The first has the sequence TGR (at amino acid residues 62 to 64 of SEQ ID NO:36), the second has the sequence SRR (at amino acid residues 119 to 121 of SEQ ID NO: 36), the third has the sequence TGR (at amino acid residues 127 to 129 of SEQ ID NO:36), the fourth has the sequence SAR (at amino acid residues 159 to 161 of SEQ ID NO:36), the fifth has the sequence SPR (at amino acid residues 193 to 195 of SEQ ID NO:36), the sixth has the sequence STR (at amino acid residues 323 to 325 of SEQ ID NO:36), and the seventh has the sequence TER (at amino acid resides 370 to 372 of SEQ ID NO:36). Six casein kinase II phosphorylation sites are present in TANGO 351. The first has the sequence TEAD (at amino acid residues 104 to 107 of SEQ ID NO:36), the second has the sequence TMHE amino acid residues 173 to 176 of SEQ ID NO: 36), the third has the sequence SGCE (at amino acid residues 268 to 271 of SEQ ID NO:36), the fourth has the sequence TPMD (at amino acid residues 342 to 345 of SEQ ID NO:36), the fifth has the sequence TERD (at amino acid residues 370 to 373 of SEQ ID NO:36), and the sixth has the sequence SSPD (at amino acid residues 417 to 420 of SEQ ID NO:36).
TANGO 351 has three tyrosine kinase phosphorylation sites. The first has the sequence RGATEADY (at amino acid residues 101 to 108 of SEQ ID NO:36), the second has the sequence RLVEELY (at amino acid residues 195 to 201 of SEQ ID NO:36), and the third has the sequence RSWEGYY (at amino acid residues 290 to 296 of SEQ ID NO:36). Four N-myristylation sites are present in TANGO 351. The first has the sequence GATEAD (at amino acid residues 102 to 107 of SEQ ID NO:36), the second has the sequence GTPSTR (at amino acid residues 320 to 325 of SEQ ID NO:36), the third has the sequence GGAGAW (at amino acid residues 361 to 366 of SEQ ID NO:36), and the fourth has the sequence GQRPTD (at amino acid residues 410 to 415 of SEQ ID NO:36).
TANGO 351 has a prokaryotic membrane lipoprotein lipid attachment site with the sequence HLGGWWVSSGC (at amino acid residues 260 to 270 of SEQ ID NO:36).
Clone EpT351, which encodes human TANGO 351 , was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110- 2209) on July 23, 1999 and assigned Accession Number PTA-424. 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.
Uses of TANGO 351 Nucleic acids. Polypeptides. and Modulators Thereof
As TANGO 351 was originally found in a human fetal kidney library, TANGO 351 nucleic acids, proteins and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of kidney cells and the kidney. TANGO 351 nucleic acids, proteins and modulators thereof can be used to modulate or 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 hemo lytic anemia, atheroembolic renal disease, diffuse cortical necrosis, and renal infarcts), or tumors (e.g., renal cell carcinoma and nephroblastoma).
In view of the fact that TANGO 351 is expressed in fetal cells, TANGO 351 nucleic acids, proteins and modulators thereof can be used for the development of an embryo and/or fetus.
TANGO 351 expression can be utilized as a marker (e.g., an in situ marker) for specific tissues (e.g., the kidney) and or cells (e.g., renal cells) in which TANGO 351 is expressed. TANGO 351 nucleic acids can also be utilized for chromosomal mapping, or as chromosomal markers, e.g., in radiation hybrid mapping.
HUMAN TANGO 361
A cDNA encoding human TANGO 361 was identified by analyzing the sequences of clones present in a prostate epithelium library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jthqb014c05, encoding full-length human TANGO 361. The human TANGO 361 cDNA of this clone is 5058 nucleotides long (Figure 13; SEQ ID NO:42). The open reading frame of this cDNA, nucleotides 41 to 1309 of SEQ ID NO:42, (SEQ ID NO:43), encodes a 423 amino acid transmembrane protein (Figure 13; SEQ ID NO:44).
Figure 14 depicts a hydropathy plot of human TANGO 361. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering K):l-6) predicted that human TANGO 361 includes a 35 amino acid signal peptide (amino acid 1 to amino acid 35 of SEQ ID NO:44, (SEQ ID NO:45) preceding the mature TANGO 361 protein (corresponding to amino acid 36 to amino acid 423 of SEQ ID NO:44, (SEQ ID NO:46). In instances wherein the signal peptide is cleaved, the molecular weight of TANGO 361 protein without post-translational modifications is 47.7 kDa prior to the cleavage of the signal peptide, and 43.6 kDa after cleavage of the signal peptide.
Human TANGO 361 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 235 to 423 of SEQ ID NO:49, (SEQ ID NO:49), a transmembrane domain at amino acid residues 217 to 234 of SEQ ID NO:44 (SEQ ID NO:47), and a cytoplasmic domain at amino acid residues 36 to 216 of SEQ ID NO:44 (SEQ ID NO:48).
In instances wherein the signal peptide is not cleaved, human TANGO 361 contains an extracellular domain at amino acid residues 235 to 423 of SEQ ID NO:49, (SEQ ID NO:49), a transmembrane domain at amino acid residues 217 to 234 of SEQ ID NO:44 (SEQ ID NO:47), and a cytoplasmic domain at amino acid residues 1 to 216 of SEQ ID NO:44.
Alternatively, in another embodiment, a human TANGO 361 protein contains a cytoplasmic domain at amino acid residues 235 to 423 of SEQ ID NO:49, (SEQ ID NO:49), a transmembrane domain at amino acid residues 217 to 234 of SEQ ID NO:44 (SEQ ID NO:47), and an extracellular domain at amino acid residues 36 to 216 of SEQ ID NO:44 (SEQ ID NO:48).
In one embodiment a cDNA sequence of human TANGO 361 has a nucleotide at position 63 which is thymine (T)(SEQ ID NO:42). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 8 that is valine (V)(SEQ ID NO:44). In an alternative embodiment, a species variant cDNA sequence of human TANGO 361 has a nucleotide at position 63 which is cytosine (C)(SEQ ID NO: 137). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 8 that is alanine (A)(SEQ ID NO: 138), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 361 has a nucleotide at position 66 which is guanine (G)(SEQ ID NO:42). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 9 that is arginine (R)(SEQ ID NO:44). In an alternative embodiment, a species variant cDNA sequence of human TANGO 361 has a nucleotide at position 66 which is adenine (A)(SEQ ID NO: 139). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 9 that is lysine (K)(SEQ ID NO: 140), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 361 has a nucleotide at position 117 is thymine (T)(SEQ ID NO:42). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 15 that is phenylalanine (F)(SEQ ID NO:44). In an alternative embodiment, a species variant cDNA sequence of human TANGO 361 has a nucleotide at position 117 which is adenine (a)(SEQ ID NO: 141). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 26 that is tyrosine (Y)(SEQ ID NO: 142), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 361 has a nucleotide at position 122 is thymidine (T)(SEQ ID NO:42). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 28 that is serine (S)(SEQ ID NO:44). In an alternative embodiment, a species variant cDNA sequence of human TANGO 361 has a nucleotide at position 122 which is adenine (A)(SEQ ID NO: 143). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 28 that is threonine (T)(SEQ ID NO: 144), i.e., a conservative substitution.
Human TANGO 361 includes a serine protease domain at amino acids 192 to 417 of SEQ ID NO:44 (SEQ ID NO:51).
TANGO 361 has three N-glycosylation sites with the first sequence NFTE at amino acid residues 75 to 78 (of SEQ ID NO:44), the second sequence NKTE at amino acid residues 166 to 169 (of SEQ ID NO:44), NATW at amino acid residues 223 to 226 (of SEQ ID NO:44).
Ten protein kinase C phosphorylation sites are present in TANGO 361. The first has the sequence TDK (at amino acid residues 61 to 63 of SEQ ID NO:44, the second has the sequence SQR (at amino acid residues 80 to 82 of SEQ ID NO:44, the third has the sequence SVK (at amino acid residues 159 to 161 of SEQ ID NO:44, the fourth has the sequence TRR (at amino acid residues 180 to 182 of SEQ ID NO:44, the fifth has the sequence SLR (at amino acid residues 189 to 191 of SEQ ID NO:44, the sixth has the sequence SHR (at amino acid residues 214 to 216 of SEQ ID NO:44, the seventh has the sequence TYK (at amino acid residues 236 to 238 of SEQ ID NO:44, the eighth has the sequence TIK (at amino acid residues 250 to 252 of SEQ ID NO:44, the ninth has the sequence TPR (at amino acid residues 353 to 355 of SEQ ID NO:44, and the tenth has the sequence TSK (at amino acid residues 418 to 420 of SEQ ID NO:44.
TANGO 361 has seven casein kinase II phosphorylation sites. The first has the sequence STED (at amino acid residues 127 to 130 of SEQ ID NO:44, the second has the sequence TETD (at amino acid residues 168 to 171 of SEQ ID NO:44, the third has the sequence TEVE (at amino acid residues 196 to 199 of SEQ ID NO:44, the fourth has the sequence SLAE (at amino acid residues 279 to 282 of SEQ ID NO:44, the fifth has the sequence TLID (at amino acid residues 335 to 338 of SEQ ID NO:44, the sixth has the sequence TCNE (at amino acid residues 341 to 344 of SEQ ID NO:44, and the seventh has the sequence SWGD (at amino acid residues 393 to 396 of SEQ ID NO:44. Four N-myristylation sites are present in TANGO 361. The first has the sequence GTRRSK (at amino acid residues 179 to 184 of SEQ ID NO:44, the second has the sequence GSHRCG (at amino acid residues 213 to 218 of SEQ ID NO:44), the third has the sequence GALKND (at amino acid residues 317 to 322 of SEQ ID NO:44), and the fourth has the sequence GSLEGK (at amino acid residues 360 to 365 of SEQ ID NO:44).
TANGO 361 has a ATP/GTP binding site motif with the sequence AGSLEGKT (at amino acid residues 359 to 366 of SEQ ID NO:44). TANGO 361 has a serine protease, histidine active site, consensus sequence with the sequence VSAAHC (at amino acid residues 228 to 233 of SEQ ID NO:44.
TANGO 361 has a serine protease, serine active site, consensus sequence with the sequence GDSGG (at amino acid residues 371 to 375 of SEQ ID NO:44.
Clone EpT361, which encodes TANGO 361, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on July 29, 1999 and assigned Accession Number PTA-438. 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.
Uses of TANGO 361 Nucleic acids. Polypeptides, and Modulators Thereof
As TANGO 361 was originally found in a prostate epithelium library, TANGO 361 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, differentiation, and/or function of prostate cells and tissues, and to ameliorate of one or more symptoms associated with abnormal function of such cells or tissues or disorders associated with such cells or tissues. Such disorders can include, but are not limited to, malignant or benign prostate cell growth or inflammatory disorders (e.g., prostatitis, benign prostatic hypertrophy, benign prostatic hyperplasia (BPH), prostatic paraganglioma, prostate adenocarcinoma, prostatic intraepithelial neoplasia, prostato-rectal fistulas, atypical prostatic stromal lesions).
TANGO 361 has structural homology with serine proteases. As such TANGO 361 nucleic acids, proteins and modulators thereof can be utilized to modulate activities, processes or disorders associated with protease activity, e.g. , serine protease activity. For example, TANGO 361 nucleic acids, proteins or modulators thereof can be used to modulate serine protease activities, such as those activities associated with such serine proteases (or, where appropriate, human homologs thereof), e.g., adipsin (complement factor D), acrosin, thrombin, plasminogen, protein C, cathepsin G, chymotrypsin, complement components and signaling, cytotoxic cell proteases, duodenase I, elastases 1, 2, 3A, 3B and medullasin, enterokinase, hepatocyte growth factor activator, hepsin, kallikreins, gamma-renin, prostate specific antigen, mast cell proteases, myeloblastin, Alzheimer's plaque-related proteases, tryptases, ancrod, batroxobin, cerastobin, flavoxobin, apolipoprotein, blood fluke cercarial protease, Drosophila trypsin like protease (e.g., alpha, easter, and snake locus), Drosophila protease stubble, or major mite fecal antigen.
TANGO 361 nucleic acids, proteins and modulators thereof can be used to modulate processes and/or diseases involved with serine protease response activity. For example, such processes and/or diseases can include, but are not limited to cellular activation, cellular proliferation, motility and differentiation, the alternative complement pathway, e.g., disturbances of the complement regulation system, such as complement regulator deficiencies, which include, for example, hereditary angioedema (an allergic disorder) and proxysmal nocturnal hemoglobinuria (the presence of hemoglobin in the urine), modulate body weight or body weight disorders, e.g., obesity or cachexia, systemic energy balance and diabetes.
TANGO 361 nucleic acids, proteins and modulators thereof can also be used to modulate immune related diseases and disorders, such disorders can include, e.g., autoimmune disorders (e.g., arthritis, graft rejection (e.g., allo graft rejection), and T cell autoimmune disorders (e.g., AIDS) and inflammatory disorders (e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma, psoriasis).
TANGO 361 nucleic acids, proteins, and modulators thereof can also be used to rid the body of invading or infecting agents, e.g. , bacteria, viruses, parasites, neoplastic cells, cellular platelet function, e.g., activation. Antagonists of TANGO 361 nucleic acids, proteins and modulators thereof, such as peptides, antibodies or small molecules that decrease or block TANGO 361 action, can be used as platelet antagonists, e.g., activation and aggregation blockers. In another example, agonists that mimic TANGO 361 activity, such as peptides, antibodies or small molecules, can be used to induce platelet function, e.g., activation and aggregation. TANGO 361 nucleic acids, proteins and modulators thereof can be utilized to modulate platelet-related processes and disorders, e.g., Glanzmann's thromboasthemia, which is a bleeding disorder characterized by failure of platelet aggregation in response to cell stimuli, and hereditary hemophilia. TANGO 361 nucleic acids, proteins and modulators thereof can be used to modulate formation of Alzheimer's plaques, treatment of Alzheimer's disease, treatment of Fanconi's anemia (FA), and symptoms associated with FA (e.g., bone marrow failure, aplastic anemia, infection, fatigue and/or spontaneous hemorrhage or bleeding). TANGO 361 expression can be utilized as a marker (e.g., an in situ marker) for specific tissues (e.g., the prostate) and/or cells (e.g., prostatic cells) in which TANGO 361 is expressed. TANGO 361 nucleic acids can also be utilized for chromosomal mapping, or as chromosomal markers, e.g., in radiation hybrid mapping.
HUMAN TANGO 499 form 1. variant 1
A cDNA encoding human TANGO 499 was identified by analyzing the sequences of clones present in a human pituitary library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jthbbl23cl0, encoding human TANGO 499. This form of human TANGO 499 is referred to herein as human TANGO 499 form 1, variant 1. The human TANGO 499 form 1, variant 1 cDNA of this clone is 1106 nucleotides long (Figure 15; SEQ ID NO:54).
In one embodiment, the open reading frame of a TANGO 499 cDNA is nucleotides 83 to 844 of SEQ ID NO:54, (SEQ ID NO:55), and encodes a human TANGO 499, form 1, variant 1 polypeptide comprising a 254 amino acid polypeptide (SEQ ID NO:56; Figure 15).
Figure 16 depicts a hydropathy plot of human TANGO 499 form 1, variant 1 (SEQ ID NO: 56). Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGN ALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 499 form 1, variant 1 includes a 30 amino acid signal peptide (amino acid 1 to amino acid 30 of SEQ ID NO:56; SEQ ID NO: 57) preceding the mature TANGO 499 form 1, variant 1 protein (coπesponding to amino acid 31 to amino acid 254 of SEQ ID NO:56; SEQ ID NO:58). In instances wherein the signal peptide is cleaved, the molecular weight of TANGO 499 form 1, variant 1 protein without post-translational modifications is 27.2 kDa prior to the cleavage of the signal peptide, and 23.8 kDa after cleavage of the signal peptide, thus TANGO 499 form 1, variant 1 polypeptides can be secreted and contain a sequence of amino acids 31 to 254 of SEQ ID NO:56 (SEQ ID NO:58).
In instances wherein the signal peptide is not cleaved, human TANGO 499 form 1, variant 1 is a secreted protein having amino acids 1 to 254 depicted in SEQ ID NO:56.
In one embodiment a cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 134 which is cytosine (C)(SEQ ID NO: 54). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 18 that is leucine (L)(SEQ ID NO:56). In an alternative embodiment, a species variant cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 134 which is guanine (G)(SEQ ID NO: 145). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 18 that is valine (I)(SEQ ID NO: 146), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 137 which is adenine (A)(SEQ ID NO:54). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 19 that is threonine (T)(SEQ ID NO:56). In an alternative embodiment, a species variant cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 137 which is thymine (T)(SEQ ID NO: 147). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 19 that is serine (S)(SEQ ID NO: 148), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 192 which is guanine (G)(SEQ ID NO:54). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 37 that is arginine (R)(SEQ ID NO:56). In an alternative embodiment, a species variant cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 192 which is adenine (A)(SEQ ID NO: 149). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 37 that is lysine (K)(SEQ ID NO:150), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 197 which is cytosine (C)(SEQ ID NO:54). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 39 that is glutamine (Q)(SEQ ID NO:56). In an alternative embodiment, a species variant cDNA sequence of human TANGO 499 form 1, variant 1 has a nucleotide at position 197 which is guanine (G)(SEQ ID NO:151). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 39 that is glutamate (E)(SEQ ID NO: 152), i.e., a conservative substitution.
TANGO 499 form 1, variant 1 (SEQ ID NO:56) has an N-glycosylation site with the sequence NISI (at amino acid residues 95 to 98 of SEQ ID NO:56). Three glycosaminoglycan attachments sites are present in TANGO 499 form 1 , variant 1. The first has the sequence SGPG (at amino acid residues 95 to 98 of SEQ ID NO:56), the second has the sequence SGSG (at amino acid residues 244 to 247 of SEQ ID NO:56), and the third has the sequence SGSG (at amino acid residues 248 to 251 of SEQ ID NO:56).
Three protein kinase C phosphorylation sites are present in TANGO 499 form 1 , variant 1. The first has the sequence SEK (at amino acid residues 165 to 167 of SEQ ID NO:56), and the second has the sequence SRR (at amino acid residues 228 to 230 of SEQ ID NO:56), and the third has the sequence SPR (at amino acid residues 233 to 235 of SEQ ID NO:56). TANGO 499 form 1, variant 1 has four casein kinase II phosphorylation sites. The first has the sequence SEMD (at amino acid residues 87 to 90 of SEQ ID NO:56), the second has the sequence SFLE (at amino acid residues 113 to 116 of SEQ ID NO:56), the third has the sequence TFAD (at amino acid residues 180 to 183 of SEQ ID NO:56), and the fourth has the sequence SILD (at amino acid residues 237 to 240 of SEQ ID NO:56).
Six N-myristylation sites are present in TANGO 499 form 1, variant 1. The first has the sequence GVRQAQ (at amino acid residues 132 to 137 of SEQ ID NO:56), the second has the sequence GCEPSC (at amino acid residues 169 to 174 of SEQ ID NO:56), the third has the sequence GQTFAD (at amino acid residues 178 to 183 of SEQ ID NO:56), the fourth has the sequence GTDLCR (at amino acid residues 184 to 189 of SEQ ID NO: 56), the fifth has the sequence GARHCF (at amino acid residues 202 to 207 of SEQ ID NO: 56), and the sixth has the sequence GSGSGS (at amino acid residues 243 to 248 of SEQ ID NO:56).
TANGO 499 form 1 , variant 1 has an amidation site with the sequence PGRR (at amino acid residues 219 to 222 of SEQ ID NO:56).
Figure 17 shows an alignment of the human TANGO 499 form 1, variant 1 amino acid sequence SEQ ID NO:56 with the Artemin amino acid sequence SEQ ID NO:59. The alignment shows that there is a 23.5% overall amino acid sequence identity between TANGO 499 form 1, variant 1 and Artemin. The Artemin protein is widely expressed in the nervous system that has been shown to be involved in such processes as peripheral neuron survival and also dopaminergic midbrain neuron survival.
Human TANGO 499 form 1, variant 1 contains Glial cell line-derived neurotrophic factor (GDNF) and riboflavin binding protein homology. Figure 18 shows an alignment of the nucleotide sequence of Riboflavin binding protein (SEQ ID NO:60) and the amino acid sequence of TANGO 499 form 1, variant 1 (SEQ ID NO:56). The alignment shows a 44.5 % overall sequence identity between the two nucleotide sequences. The Riboflavin binding protein is expressed in germinal tissues and is involved in the development and maturation of the embryo.
Clone EpT499, which encodes TANGO 499 form 1, variant 1, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on August 5, 1999 and assigned Accession Number PTA-455. 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.
HUMAN TANGO 499 form 2. variant 3
A cDNA encoding human an additional form of TANGO 499 was identified by analyzing the sequences of clones present in a retina library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, AthX435e8, encoding human TANGO 499. This sequence is referred to herein as form 2, variant 3. The human TANGO 499 form 2, variant 3 cDNA of this clone is 1085 nucleotides long (Figure 19; SEQ ID NO:61).
In one embodiment, the open reading frame of this cDNA is from nucleotides 144 to 830 of SEQ ID NO:61 (SEQ ID NO:62) and encodes a 229 amino acid secreted protein (Figure 19; SEQ ID NO:63).
Figure 20 depicts a hydropathy plot of human TANGO 499 form 2, variant 3. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 499 form 2, variant 3 includes a 30 amino acid signal peptide (amino acid 1 to amino acid 30 of SEQ ID NO:63; SEQ ID NO:64) preceding the mature TANGO 499 form 2, variant 3 protein (corresponding to amino acid 31 to amino acid 229 of SEQ ID NO:63; SEQ ID NO:65). In instances wherein the signal peptide is cleaved, the molecular weight of TANGO 499 form 2, variant 3 protein without post-translational modifications is 24.6 kDa prior to the cleavage of the signal peptide, and 21.2 kDa after cleavage of the signal peptide, thus TANGO 499 form 2, variant 3 polypeptides can be secreted and contain a sequence of amino acids 31 to 229 of SEQ ID NO:63 (SEQ ID NO:65).
TANGO 499 form 2, variant 3 has an N-glycosylation site with the sequence NISI (at amino acid residues 95 to 98 of SEQ ID NO:63).
Three glycosaminoglycan attachments sites are present in TANGO 499 form 2, variant 3. The first has the sequence SGPG (at amino acid residues 70 to 73 of SEQ ID NO:63), the second has the sequence SGSG (at amino acid residues 219 to 222 of SEQ ID NO:63), and the third has the sequence SGSG (at amino acid residues 223 to 246 of SEQ ID NO:63).
Three protein kinase C phosphorylation sites are present in TANGO 499 form 2, variant 3. The first has the sequence SEK (at amino acid residues 140 to 152 of SEQ ID NO: 63), and the second has the sequence SRR (at amino acid residues 203 to 205 of SEQ ID NO:63), and the third has the sequence SPR (at amino acid residues 208 to 210 of SEQ ID NO:63). TANGO 499 form 2, variant 3 has four casein kinase II phosphorylation sites. The first has the sequence SEMD (at amino acid residues 62 to 65 of SEQ ID NO:63), the second has the sequence SFLE (at amino acid residues 88 to 91 of SEQ ID NO:63), the third has the sequence TFAD (at amino acid residues 155 to 158 of SEQ ID NO:63), and the fourth has the sequence SILD (at amino acid residues 212 to 215 of SEQ ID NO:63).
Six N-myristylation sites are present in TANGO 499 form 2, variant 3. The first has the sequence GVRQAQ (at amino acid residues 107 to 112 of SEQ ID NO:63), the second has the sequence GCEPSC (at amino acid residues 144 to 149 of SEQ ID NO:63), the third has the sequence GQTFAD (at amino acid residues 153 to 158 of SEQ ID NO:63), the fourth has the sequence GTDLCR (at amino acid residues 159 to 164 of SEQ ID NO:63), the fifth has the sequence GARHCF (at amino acid residues 177 to 182 of SEQ ID NO:63), and the sixth has the sequence GSGSGS (at amino acid residues 218 to 223 of SEQ ID NO:63).
TANGO 499 form 2, variant 3 has an amidation site with the sequence PGRR (at amino acid residues 194 to 197 of SEQ ID NO: 63).
Human TANGO 499 form 2, variant 3 includes Glial cell line-derived neurotrophic factor (GDNF) and riboflavin binding protein homology. The Riboflavin binding protein is expressed in germinal tissues and is involved in the development and maturation of the embryo. Loss of expression of the riboflavin binding protein results in the failure of the embryo to develop.
Figure 21 depicts an alignment of TANGO 499 form 1, variant 1 amino acid sequence and TANGO 499 form 2, variant 3 amino acid sequence. The amino acid sequences are 90.2% identical, the cDNAs are 85.6%> identical, and the ORFs are 90.2%) identical. The alignment clearly demonstrates that the two forms are identical except for the result of a putative alternative exon splicing event in the illustrated region. The invention contemplates additional splice variants of the presently claimed nucleic acids and proteins encoded by such splice variants.
Clone EpT499, which encodes TANGO 499 form 2, variant 3, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on August 5, 1999 and assigned Accession Number PTA-454. 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.
ADDITIONAL HUMAN TANGO 499 VARIANTS
Analysis of multiple individual human TANGO 499 clones revealed a complex set of variant transcripts (e.g., alternatively spliced transcripts). One such alternatively spliced form of a human TANGO 499 gene is referred to as a human TANGO 499 form 1 , variant 1, which is also described in detail above. Another such alternatively spliced form of a human TANGO 499 gene is referred to as a human TANGO 499 form 2, variant 3, which is described in detail above. Additional human TANGO 499 form 1 and form 2 variants have been identified and are described below.
Further analysis of the individual human TANGO 499 forms reveals a conserved region. In one embodiment, the open reading frame of the form 1 TANGO 499 cDNA conserved region comprises nucleotides 1 to 783 of SEQ ID NO:89 (SEQ ID NO:90), and encodes a polypeptide comprising the sequence of SEQ ID NO:91.
In another embodiment, the open reading frame of the form 2 TANGO 499 cDNA conserved region comprises nucleotides 1 to 708 of SEQ ID NO:92 (SEQ ID NO:93), and encodes a polypeptide comprising the sequence of SEQ ID NO: 94.
In another embodiment, the open reading frame of a TANGO 499 cDNA comprises nucleotides 26 to 847 of SEQ ID NO: 101 (SEQ ID NO: 102), and encodes a polypeptide comprising the sequence of SEQ ID NO: 103, referred to herein as form 2, variant 1.
In another embodiment, the open reading frame of a TANGO 499 cDNA comprises nucleotides 11 to 794 of SEQ ID NO: 104 (SEQ ID NO: 105), and encodes a polypeptide comprising the sequence of SEQ ID NO: 106, referred to herein as form 1, variant 2. In another embodiment, the open reading frame of this cDNA comprises nucleotides 11 to 719 of SEQ LO NO: 107 (SEQ ID NO: 108), and encodes a polypeptide comprising the sequence of SEQ ID NO: 109, referred to herein as form 2, variant 2.
In another embodiment, the open reading frame of a TANGO 499 cDNA comprises nucleotides 447 to 1230 of SEQ ID NO:l 10 (SEQ ID NO:l 11), and encodes a polypeptide comprising the sequence of SEQ ID NO:l 12, referred to herein as form 1, variant 3.
In another embodiment, the open reading frame of this cDNA comprises nucleotides 95 to 908 of SEQ ID NO:95 (SEQ ID NO:96), and encodes a polypeptide comprising the sequence of SEQ ID NO:97, referred to herein as form 1, variant 4. In another embodiment, the open reading frame of this TANGO 499 comprises nucleotides 95 to 833 of SEQ ID NO:98 (SEQ ID NO:99), and encodes a polypeptide comprising the sequence of SEQ ID NO: 100, referred to herein as form 2, variant 4.
Uses of TANGO 499 Nucleic acids. Polypeptides. and Modulators Thereof
As TANGO 499 was originally found in a human pituitary library, TANGO 499 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of cells, tissues and/or organs, e.g. , the proliferation of tissues and cells of pituitary origin.
Thus, as TANGO 499 was originally identified in a pituitary library, for example, TANGO 499 nucleic acids, proteins and modulators thereof can be used to regulate processes involved with sexual development and function, including, e.g., normal and abnormal reproductive hormonal function in the fetus, infant, adolescent and adult and also can modulate the effects of pituitary insufficiency and pituitary adenomas on sexual development, reproductive function and sexuality in men and women. Furthermore, TANGO 499 nucleic acids, proteins and modulators thereof can be used to treat pituitary tumors causing Cushing's syndrome, and also hypopituitarism during pregnancy which may be the result of intrasellar adenomas, suprasellar lesions, lymphocytic hypophysitis or antepartum pituitary necrosis, and in the postpartum period may be because of postpartum hemorrhage and pituitary necrosis. TANGO 499 nucleic acids, proteins and modulators thereof can also be used to treat posterior pituitary problems in pregnancy manifested by diabetes insipidus, with a pregnancy-specific variety resulting from excessive degradation of arginine vasopressin by placental vasopressinase. Further, TANGO 499 nucleic acids, proteins and modulators thereof can be used to treat pituitary-related disorders, e.g., hormone secretion disorders, (e.g. , Cushing's disease, hyperprolactinemia, acromegaly-gigantism, and precocious and delayed puberty).
In light of the fact that TANGO 499 was identified in a retina library, TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and function of the eye, such as retinal development and function, ( e.g., photoreceptor disk morphogenesis). TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to treat eye diseases and/or disorders, e.g., autosomal dominant retinitis pigmentosa, autosomal dominant punctata albescens, butterfly-shaped pigment dystrophy, cataracts, macular degeneration, myopia, stigmatism and retinoblastoma.
TANGO 499 family members have homology to glial cell line-derived neurotrophic-related factors (e.g., GDNF, artemin, neurturin, and persephin). Thus, TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to modulate survival, activation, proliferation, motility, and differentiation of peripheral or central neurons.
TANGO 499 nucleic acids, proteins and modulators thereof can be used to modulate kidney development or for gene therapy for modulating defects in kidney organogenesis. As such, TANGO 499 nucleic acids, proteins and modulators thereof can also be used to modulate renal disorders, e.g., glomerular disease, (e.g., acute and chronic glomerulonephritis), tubular diseases, and tubulo-interstitial diseases.
TANGO 499 nucleic acids, proteins and modulators thereof can also be used to modulate intercellular signaling in the nervous system, to modulate disorders associated with aberrant signal transduction in response to neurotrophic factors and cell surface receptors such as, e.g., other GDNF proteins, and to modulate myelin-associated processes. For example, TANGO nucleic acids, proteins and modulators thereof can be used to maintain the myelin sheath e.g. , enhance myelin membrane adhesion to extracellular matrices during development, e.g., at late stages of development.
Furthermore, TANGO 499 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, development, differentiation, and/or function of neural organs, e.g., neural tissues and cells, e.g., cells of the central nervous system, e.g., cells of the peripheral nervous system. TANGO 499 nucleic acids, proteins, and modulators thereof can also be used to modulate symptoms associated with abnormal neural signaling and function, e.g., epilepsy, stroke, traumatic injury. In particular, TANGO 499 proteins could be useful to treat neural related disorders or neural damage, such as for regenerative neural repair after damage by trauma, degeneration, or inflammation e.g. , spinal cord injuries, infarction, infection, malignancy, exposure to toxic agents, nutritional deficiency, paraneoplastic syndromes, and degenerative nerve diseases including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's Chorea, amyotrophic lateral sclerosis, progressive supra-nuclear palsy, Hirchsprung's disease, and other dementias or peripheral neuropathy-related disorders, maintenance of the entire myelin sheath.
As TANGO 499 family members have homology to riboflavin binding proteins, TANGO 499 nucleic acids, proteins and modulators thereof can be used to modulate cofactor or vitamin concentrations, in particular TANGO 499 nucleic acids, proteins and modulators thereof can be used to modulate vitamin concentrations in and around an embryo, to modulate the development of an embryo, and to modulate the length of time of the pregnancy (i.e., terminating).
Moreover, TANGO 499 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and progression of cancerous and non-cancerous cell proliferative disorders, such as deregulated proliferation (such as hyperdysplasia, hyper- IgM syndrome, or lymphoproliferative disorders), cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hyperplasia), cancers such as neoplasms or tumors (such as carcinomas, sarcomas, adenomas or myeloid lymphoma tumors, e.g., fϊbrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, colon sarcoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hematoma, bile duct carcinoma, melanoma, choriocarcinoma, semicoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependynoma, pinealoma, hemangioblastoma, retinoblastoma), leukemias, (e.g. acute lymphocytic leukemia), acute myelocytic leukemia (myelolastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), or polycythemia vera, or lymphomas (Hodgkin's disease and non-Hodgkin's diseases), multiple myelomas and Waldenstrδm's macroglobulinemia.
TANGO 499 expression can be utilized as a marker (e.g., an in situ marker) for specific tissues (e.g., the pituitary) and/or cells (e.g., pituitary cells) in which TANGO 499 is expressed. TANGO 499 nucleic acids can also be utilized for chromosomal mapping, or as chromosomal markers, e.g., in radiation hybrid mapping.
HUMAN TANGO 509
A cDNA encoding human TANGO 509 was identified by analyzing the sequences of clones present in a mammary epithelium library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jthvb017hl 1, encoding full-length human TANGO 509. The human TANGO 509 cDNA of this clone is 3575 nucleotides long (Figure 23; SEQ ID NO:66). The open reading frame of this cDNA, nucleotides 59 to 928 of SEQ ID NO:66, (SEQ ID NO:67), encodes a 290 amino acid transmembrane protein (Figure 23; SEQ ID NO:68). Figure 24 depicts a hydropathy plot of human TANGO 509. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 509 includes a 18 amino acid signal peptide (amino acid 1 to amino acid 18 of SEQ ID NO:68; SEQ ID NO:69) preceding the mature TANGO 509 protein (corresponding to amino acid 19 to amino acid 290 of SEQ ID NO:68; SEQ ID NO:70). In instances wherein the signal peptide is cleaved, the molecular weight of TANGO 509 protein without post-translational modifications is 33.3 kDa prior to the cleavage of the signal peptide, and 31.0 kDa after cleavage of the signal peptide.
Human TANGO 509 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 260 to 290 of SEQ ID NO:68 (SEQ ID NO:75), a transmembrane domain at amino acid residues 241 to 259 of SEQ ID NO:68 (SEQ ID NO:73), and a cytoplasmic domain at amino acid residues 19 to 240 of SEQ ID NO:68 (SEQ ID NO:74).
In instances wherein the signal peptide is not cleaved, human TANGO 509 contains an extracellular domain at amino acid residues 260 to 290 of SEQ ID NO:68 (SEQ ID NO:75), a transmembrane domain at amino acid residues 241 to 259 of SEQ ID NO: 68 (SEQ ID NO: 73), and a cytoplasmic domain at amino acid residues 1 to 240 of SEQ ID NO:68.
Alternatively, in another embodiment, a human TANGO 509 protein contains a cytoplasmic domain at amino acid residues 260 to 290 of SEQ ID NO:68, a transmembrane domain at amino acid residues 241 to 259 of SEQ ID NO:68 (SEQ ID NO:73), and an extracelluar domain at amino acid residues 19 to 240 of SEQ ID NO:68.
In one embodiment a cDNA sequence of human TANGO 509 has a nucleotide at position 69 which is thymidine (T)(SEQ ID NO:66). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is phenylalanine (F)(SEQ ID NO:68). In an alternative embodiment, a species variant cDNA sequence of human TANGO 509 has a nucleotide at position 69 which is adenine (A)(SEQ ID NO: 153). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is tyrosine (Y)(SEQ ID NO: 154), i.e., a conservative substitution. In another embodiment a cDNA sequence of human TANGO 509 has a nucleotide at position 72 which is cytosine (C)(SEQ ID NO:66). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 5 that is alanine (A)(SEQ ID NO:68). In an alternative embodiment, a species variant cDNA sequence of human TANGO 509 has a nucleotide at position 72 which is thymine (T)(SEQ ID NO: 155). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 5 that is valine (V)(SEQ ID NO: 156), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 509 has a nucleotide at position 132 which is adenine (A)(SEQ ID NO:66). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 25 that is lysine (K)(SEQ ID NO:68). In an alternative embodiment, a species variant cDNA sequence of human TANGO 509 has a nucleotide at position 132 which is guanine (G)(SEQ ID NO: 157). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 25 that is arginine (R)(SEQ ID NO: 158), i.e., a conservative substitution.
In another embodiment a cDNA sequence of human TANGO 509 has a nucleotide at position 191 which is guanine (G)(SEQ ID NO:66). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 45 that is glutamate (E)(SEQ ID NO:68). In an alternative embodiment, a species variant cDNA sequence of human TANGO 509 has a nucleotide at position 191 which is cytosine (C)(SEQ ID NO: 159). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 45 that is glutamine (Q)(SEQ ID NO: 160), i.e., a conservative substitution.
Human TANGO 509 has four N-glycosylation sites with the first sequence NMTI (at amino acid residues 35 to 38 of SEQ ID NO:68), the second has the sequence NVTS (at amino acid residues 192 to 195 SEQ ID NO:68), the third has the sequence NTTT (at amino acid residues 200 to 203 SEQ ID NO:68), and the fourth has the sequence NHTA (at amino acid residues 219 to 222 SEQ ID NO:68).
Two cAMP and cGMP-dependent protein kinase phosphorylation sites are present in human TANGO 509. The first has the sequence KRIT (at amino acid residues 124 to 127 SEQ ID NO:68), and the second has the sequence KKQS (at amino acid residues SEQ ID NO:68).
Seven protein kinase C phosphorylation sites are present in human TANGO 509. The first has the sequence SYR (at amino acid residues 80 to 82 of SEQ ID NO:68), the second has the sequence TVK (at amino acid residues 127 to 129 of SEQ ID NO:68), the third has the sequence SGK (at amino acid residues 176 to 178 of SEQ ID NO:68), the fourth has the sequence SKR (at amino acid residues 184 to 186 SEQ ID NO:68), the fifth has the sequence TLR (at amino acid residues 196 to 198 SEQ ID NO:68), the sixth has the sequence TFR (at amino acid residues 210 to 212 SEQ ID NO:68), and the seventh has the sequence SKK (at amino acid residues 279 to 281 SEQ ID NO:68).
Human TANGO 509 has five casein kinase II phosphorylation sites. The first has the sequence SEHE (at amino acid residues 149 to 152 of SEQ ID NO:68), the second has the sequence TSSD (at amino acid residues 168 to 171 of SEQ ID NO:68), the third has the sequence SKRE (at amino acid residues 184 to 187 of SEQ ID NO:68), the fourth has the sequence TTNE (at amino acid residues 202 to 205 SEQ ID NO:68), and the fifth has the sequence THLE (at amino acid residues 285 to 288 SEQ ID NO:68).
Human TANGO 509 has a tyrosine kinase phosphorylation site with the sequence KLQDAGVY (at amino acid residues 105 to 112 of SEQ ID NO:68). Human TANGO 509 has four N-myristoylation sites. The first has the sequence GSNMTI (at amino acid residues 33 to 38 of SEQ ID NO:68), the second has the sequence GVYRCM (at amino acid residues 110 to 115 of SEQ ID NO:68), the third has the sequence GVALTF (at amino acid residues 252 to 257 SEQ ID NO:68), and fourth has the sequence GIQDTN (at amino acid residues 273 to 278 of SEQ ID NO:68).
Figure 24 shows an alignment of the human TANGO 509 amino acid sequence SEQ ID NO:68 with the butyrophilin-like amino acid sequence SEQ ID NO:85; Accession Number: AF 142780). The alignment shows that there is a 31.9% overall amino acid sequence identity between TANGO 509 and Butyrophilin-like protein. The Butyrophilin-like protein is expressed in dendritic cells which are involved in such processes as antigen presentation and immune stimulation. As such TANGO 509 proteins, nucleic acids and modulators thereof could be useful in immune modulation, for example in antigen presentation and immune stimulation.
Clone EpT509, which encodes human TANGO 509, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110- 2209) on August 5, 1999 and assigned Accession Number PTA-438. 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. MOUSE TANGO 509
A cDNA encoding mouse TANGO 509 was identified by analyzing the sequences of clones present in an alveolar macrophage cell line library. This analysis led to the identification of a clone, jtmca053b03, encoding mouse TANGO 509. The mouse TANGO 509 cDNA of this clone is 3637 nucleotides long (Figure 25; SEQ ID NO:76). The open reading frame of this cDNA, nucleotides 49 to 918 of SEQ ID NO: 76, (SEQ ID NO:77), encodes a 290 amino acid transmembrane protein (Figure 25; SEQ ID NO:78).
Figure 26 depicts a hydropathy plot of mouse TANGO 509. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that mouse TANGO 509 includes a 18 amino acid signal peptide (amino acid 1 to amino acid 18 of SEQ ID NO:78; SEQ ID NO:79) preceding the mature TANGO 509 protein (coπesponding to amino acid 19 to amino acid 290 of SEQ ID NO:78; SEQ ID NO:80). In instances wherein the signal peptide is cleaved, the molecular weight of TANGO 509 protein without post-translational modifications is 33.3 kDa prior to the cleavage of the signal peptide, and 31.0 kDa after cleavage of the signal peptide.
Mouse TANGO 509 protein is a transmembrane protein that contains an extracellular domain at amino acid residues 261 to 290 of SEQ ID NO:78, (SEQ ID NO: 84), a transmembrane domain at amino acid residues 240 to 260 of SEQ ID NO:78, (SEQ ID NO:82), and a cytoplasmic domain at amino acid residues 19 to 239 of SEQ ID NO:78, (SEQ ID NO:83).
In instances wherein the signal peptide is not cleaved, mouse TANGO 509 contains an extracellular domain at amino acid residues 261 to 290 of SEQ ID NO:78, (SEQ ID NO: 84), a transmembrane domain at amino acid residues 240 to 260 of SEQ ID NO:78, (SEQ ID NO:82), and a cytoplasmic domain at amino acid residues 1 to 239 of SEQ ID NO:78.
Alternatively, in another embodiment, a mouse TANGO 509 protein contains a cytoplasmic domain at amino acid residues 261 to 290 of SEQ ID NO:78, a transmembrane domain at amino acid residues 240 to 260 of SEQ ID NO:78, (SEQ ID NO:82), and an extracellular domain at amino acid residues 19 to 239 of SEQ ID NO:78. In one embodiment a cDNA sequence of mouse TANGO 509 has a nucleotide at position 65 which is thymidine (T)(SEQ ID NO: 76). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is phenylalanine (F)(SEQ ID NO:78). In an alternative embodiment, a species variant cDNA sequence of mouse TANGO 509 has a nucleotide at position 65 which is adenine (A)(SEQ ID NO: 161). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 4 that is tyrosine (Y)(SEQ ID NO: 162), i.e., a conservative substitution.
In another embodiment a cDNA sequence of mouse TANGO 509 has a nucleotide at position 68 which is cytosine (C)(SEQ ID NO:76). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 5 that is alanine (A)(SEQ ID NO: 78). In an alternative embodiment, a species variant cDNA sequence of mouse TANGO 509 has a nucleotide at position 68 which is thymine (T)(SEQ ID NO: 163). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 5 that is valine (V)(SEQ ID NO: 164), i.e., a conservative substitution.
In another embodiment a cDNA sequence of mouse TANGO 509 has a nucleotide at position 128 which is adenine (A)(SEQ ID NO:76). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position
25 that is lysine (K)(SEQ ID NO:78). In an alternative embodiment, a species variant cDNA sequence of mouse TANGO 509 has a nucleotide at position 128 which is guanine (G)(SEQ ID NO: 165). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 25 that is arginine (R)(SEQ ID NO: 166), i.e., a conservative substitution.
In another embodiment a cDNA sequence of mouse TANGO 509 has a nucleotide at position 132 which is cytosine (C)(SEQ ID NO:76). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position
26 that is aspartate (D)(SEQ ID NO:78). In an alternative embodiment, a species variant cDNA sequence of mouse TANGO 509 has a nucleotide at position 132 which is adenine (A)(SEQ ID NO: 167). In this embodiment, the cDNA contains an open reading frame encoding a polypeptide having an amino acid at position 45 that is glutamate (E)(SEQ ID NO: 168), i.e., a conservative substitution.
Mouse TANGO 509 has six N-glycosylation sites with the first sequence NVTM (at amino acid residues 35 to 38 of SEQ ID NO:78), the second has the sequence NVTS (at amino acid residues 191 to 194 SEQ ID NO: 78), the third has the sequence NAT A (at amino acid residues 199 to 202 SEQ ID NO:78), the fourth has the sequence NHTA (at amino acid residues 218 to 221 SEQ ID NO:78), the fifth has the sequence NRTH (at amino acid residues 236 to 239 SEQ ID NO:78), and the sixth has the sequence NDTQ (at amino acid residues 283 to 286 SEQ ID NO:78).
Mouse TANGO 509 has one cAMP and cGMP-dependent protein kinase phosphorylation site, having the sequence KRIT (at amino acid residues 124 to 127 SEQ ID NO:78).
Mouse TANGO 509 has five protein kinase C phosphorylation sites. The first has the sequence TLK (at amino acid residues 127 to 129 of SEQ ID NO:78), the second has the sequence SGK (at amino acid residues 175 to 177 of SEQ ID NO:78), the third has the sequence TSR (at amino acid residues 182 to 184 SEQ ID NO:78), the fourth has the sequence SLR (at amino acid residues 195 to 197 SEQ ID NO:78), and the fifth has the sequence SSK (at amino acid residues 278 to 280 SEQ ID NO:78).
Mouse TANGO 509 has five casein kinase II phosphorylation sites. The first has the sequence SEHE (at amino acid residues 148 to 151 of SEQ ID NO:78), the second has the sequence TNSD (at amino acid residues 167 to 170 of SEQ ID NO:78), the third has the sequence SRTE (at amino acid residues 183 to 186 of SEQ ID NO:78), the fourth has the sequence TAND (at amino acid residues 201 to 204 SEQ ID NO:78), and the fifth has the sequence TQFE (at amino acid residues 285 to 288 SEQ ID NO:78).
Mouse TANGO 509 has a tyrosine kinase phosphorylation site with the sequence KLQDAGVY (at amino acid residues 105 to 112 of SEQ ID NO:78).
Mouse TANGO 509 has five N-myristoylation sites. The first has the sequence GIIFTA (at amino acid residues 6 to 11 of SEQ ID NO:78), the second has the sequence GSNVTM (at amino acid residues 33 to 38 of SEQ ID NO:78), the third has the sequence GVYCCI (at amino acid residues 110 to 115 SEQ ID NO:78), the fourth has the sequence GMLLNV (at amino acid residues 187 to 192 of SEQ ID NO:78), the fifth has the sequence GQNHTA (at amino acid residues 216 to 221 of SEQ ID NO:78), and the sixth has the sequence GVEDTS (at amino acid residues 273 to 278 of SEQ ID NO:78).
Uses of TANGO 509 Nucleic acids. Polypeptides. and Modulators Thereof
As human TANGO 509 was originally found in a mammary epithelial library, TANGO 509 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, activation, development, differentiation, and/or function of mammary cells, tissues and/or organs, e.g., tissues and cells of mammary epithelium origin. TANGO 509 nucleic acids, proteins and modulators thereof can be used to treat mammary-related disorders, e.g. , breast cancer.
TANGO 509 exhibits homology to butyrophilin (BTN). BTN is the major protein associated with fat droplets in the milk of many species. BTN has immunoglobulin-like domains and is specifically expressed on the apical surface of mammary epithelial cells during lactation and becomes incorporated as an integral protein into the membrane of the milk fat globule during the budding and secretion of fat droplets into milk. As such, TANGO 509 nucleic acids, proteins and modulators thereof can be utilized to modulate fat secretion, e.g., fat secretion by the mammary epithelium, and milk secretion. In addition, such TANGO 509 compositions and modulators thereof can be used to bind to and, e.g., enhance, deplete or purify milk- associated factors. Further, TANGO 509 nucleic acids, proteins and modulators thereof can be utilized to treat mammary epithelium secretory diseases and/or disorders.
As mouse TANGO 509 was isolated from an alveolar macrophage library, and in light of the fact that TANGO 509 family members have characteristics of immunoglobulin superfamily proteins which are cell surface molecules involved in signal transduction and cellular proliferation, TANGO 509 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and progression of cancerous and non-cancerous cell proliferative disorders, such as deregulated proliferation (such as hyperdysplasia, hyper- IgM syndrome, or lymphoproliferative disorders), cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hyperplasia), cancers such as neoplasms or tumors (such as carcinomas, sarcomas, adenomas or myeloid lymphoma tumors, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, colon sarcoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hematoma, bile duct carcinoma, melanoma, choriocarcinoma, semicoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependynoma, pinealoma, hemangioblastoma, retinoblastoma), leukemias, (e.g. acute lymphocytic leukemia), acute myelocytic leukemia (myelolastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), or polycythemia vera, or lymphomas (Hodgkin's disease and non-Hodgkin's diseases), multiple myelomas and Waldenstrδm's macroglobulinemia.
As TANGO 509 proteins exhibit similarity to immunoglobulin domains, TANGO 509 nucleic acids, proteins and modulators thereof can be utilized to modulate immune activation. For example, antagonists to TANGO 509 action, such as peptides, antibodies or small molecules that decrease or block TANGO 509 activity, e.g., binding to extracellular matrix components, e.g., integrins, or that prevent TANGO 509 signaling, can be used as immune system activation blockers. In another example, agonists that mimic TANGO 509 activity, such as peptides, antibodies or small molecules, can be used to induce immune system activation. Antibodies may activate or inhibit the cell adhesion, proliferation and activation, and may help in treating infection, autoimmunity, inflammation, and cancer by affecting these cellular processes. TANGO 509 nucleic acids, proteins and modulators thereof can also be utilized to modulate intercellular signaling in the immune system, e.g., modulate intercellular signal transduction in immune stimulation or suppression and modulate immune cell membrane adhesion to ECM components, during development, e.g., late stages of development.
As TANGO 509 family members exhibit homology with the immune co- stimulatory molecules, CD80 and CD86, TANGO 509 nucleic acids, proteins and modulators thereof can be used for modulation of lymphocyte activation, cytokine secretion, e.g., IL-2, B-cell selection and maturation, as well as T-cell selection and maturation. TANGO 509 nucleic acids, proteins and modulators thereof can also be used to treat subjects infected with a pathogen, or to modulate autoimmune diseases, e.g., rheumatoid arthritis, Morbus Bechterew, Sjogren's Syndrome, and ulcerative colitis.
Furthermore, TANGO 509 nucleic acids, proteins and modulators thereof can be used for immune cell receptor co-stimulation via CD28 to modulate IL-2 expression in addition to modulating the expression of other lymphokines. Moreover, TANGO 509 nucleic acids, proteins and modulators thereof can be used to modulate diseases of the immune system, in particular AIDS, asthma or chronic viral diseases such as hepatitis C virus or hepatitis B virus infections, or to modulate the immune system in cancer patients, or patients undergoing organ or tissue transplantation procedures, or inflammatory disorders, e.g. , bacterial or viral infection, psoriasis, septicemia, arthritis, allergic reactions.
TANGO 509 expression can be utilized as a marker (e.g., an in situ marker) for specific tissues (e.g., the mammary glands) and/or cells (e.g., mammary epithelial cells) in which TANGO 509 is expressed. TANGO 509 nucleic acids can also be utilized for chromosomal mapping, or as chromosomal markers, e.g., in radiation hybrid mapping. Tables 1 provides a summary of the nucleotide sequence information for INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509.
Table 2 provides a summary of the domains of human INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509.
TABLE 1 : Summary of Nucleotide Sequence Information of INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 Nucleic Acids.
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Various aspects of the invention are described in further detail in the following subsections:
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a biologically active portion thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g. , cDNA or genomic DNA) and RNA molecules (e.g. , mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. As used herein, the term "isolated"when referring to a nucleic acid molecule does not include an isolated chromosome.
In instances wherein the nucleic acid molecule is a cDNA or RNA, e.g., mRNA, molecule, such molecules can include a poly A "tail", or, alternatively, can lack such a 3' tail. Although cDNA or RNA nucleotide sequences may be depicted herein with such tail sequences, it is to be understood that cDNA nucleic acid molecules of the invention are also intended to include such sequences lacking the depicted poly A tails.
All or a portion of the nucleic acid sequences of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62 66, 67, 76, or 77 or a complement thereof, can be used as molecular weight markers when compared to a comparably sized nucleic acid sequence. Likewise, all or a portion of the amino acid sequences encoded by SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62 66, 67, 76, or 77 or a complement thereof, can be used as molecular weight markers, in particular as molecular weight markers on SDS-PAGE electrophoresis. A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62 66, 67, 76, or 77 or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77 as a hybridization probe, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77, 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 under the conditions set forth herein, 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 homologs in other cell types, e.g., from other tissues, as well as homologs from other mammals. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77 of a naturally occurring mutant of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 66, 67, 76, or 77. In another embodiment, the oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least 400, preferably 450, 500, 530, 550, 600, 700,800, 900, 1000 or 1150 consecutive oligonucleotides of the sense or antisense sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 66, 67, 76, or 77, of a naturally occurring mutant of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77.
Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
A nucleic acid fragment encoding a biologically active portion of a polypeptide of the invention can be prepared by isolating a portion of any of SEQ ID 1, 2, 23, 24, 34, 35,
42, 43, 54, 55, 61, 62, 66, 67, 76, or 77 expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the polypeptide.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO: SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77 due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42,
43, 54, 55, 61, 62, 66, 67, 76, or 77. In addition to the nucleotide sequences of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43,
54, 55, 61, 62, 66, 67, 76, or 77, it will be appreciated by those skilled in the art 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 allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. For example, INTERCEPT 307 has been mapped to chromosome 11, and therefore INTERCEPT 307 family members can include nucleotide sequence polymorphisms (e.g., nucleotide sequences that vary from SEQ ID NO: 1 and SEQ ID NO:2) that map to this chromosome 11 region (i.e., between markers Dl 1S1357 and Dl 1 SI 765), and such sequences represent INTERCEPT 307 allelic variants. 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. In one embodiment, polymorphisms that are associated with a particular disease and/or disorder are used as markers to diagnose said disease or disorder. In a preferred embodiment, polymorphisms are used as a marker to diagnose abnormal coronary function such as atherosclerosis.
Moreover, nucleic acid molecules encoding proteins of the invention from other species (homologs), which have a nucleotide sequence which differs from that of the human or mouse protein described herein are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologs 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 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 contiguous nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO:l, 23, 34, 42, 54, 61, 66, or 76 or a complement thereof.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 25, 50, 100, 200, 300, 400, 500, 600, 700, 800 or 900 contiguous nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO: 1, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77, or a complement thereof.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45° C followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, or 77, 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 homologs of various species may be non-essential for activity and thus would be likely targets for alteration. Specific examples of conservative amino acid alterations from the original sequence are shown in SEQ ID NOs:114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168. Alternatively, amino acid residues that are conserved among the homologs of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from SEQ ID NO:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168 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 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.
An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, 77, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 or 167, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, asparagine, glutamine), uncharged polar side chains (e.g., glycine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form protein-protein interactions with proteins in a signaling pathway of the polypeptide of the invention such as in central nervous system cells, lymphoid cells, hypothalamus cells, or prostate cells with the proteins encoded by the genes of the present invention; (2) the ability to bind a ligand of the polypeptide of the invention (i.e., in transmembrane proteins of the invention or alternatively, secreted proteins which are the ligand for a cellular receptor); 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, motility or chemotaxis, or cellular differentiation.
The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.
An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-mefhoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g. , by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, (1988), Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be designed based upon the nucleotide sequence of a cDNA disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel and Szostak (1993) Science 261 :1411-1418.
The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.
In various embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g. , DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.
PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).
In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al. (1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124). In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g. , for targeting host cell receptors in vivo ), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
In still other embodiments, the nucleotides of the invention including variants and 0 derivatives can be used as vaccines, for example by genetic immunization. Genetic immunization is particularly advantageous as it stimulates a cytotoxic T-cell response but does not utilize live attenuated vaccines, which can revert to a virulent form and infect the host causing the very infection sought to be prevented. As used herein, genetic immunization comprises inserting the nucleotides of the invention into a host, such that 5 the nucleotides are taken up by cells of the host and the proteins encoded by the nucleotides are translated. These translated proteins are then either secreted or processed by the host cell for presentation to immune cells and an immune reaction is stimulated. Preferably, the immune reaction is a cytotoxic T cell response, however, a humoral response or macrophage stimulation is also useful in preventing future infections. The 0 skilled artisan will appreciate that there are various methods for introducing foreign nucleotides into a host animal and subsequently into cells for genetic immunization, for example, by intramuscular injection of about 50 μg of plasmid DNA encoding the proteins of the invention solubilized in 50 μl of sterile saline solution, with a suitable adjuvant <- (Weiner and Kennedy (1999) Scientific American 7:50-57; Lowrie et al., (1999) Nature 400:269-271).
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 c 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%ι, 20%, 10%, or 5%> (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
Biologically active portions of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein (e.g., the amino acid sequence shown in any of SEQ ID NO:4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 26, 28, 29, 30, 31, 37, 39, 40, 41, 45, 47, 48, 49, 50, 51, 52, 53, 57, 59, 60, 64, 69, 71, 72, 73, 74, 75, 79, 81, 82, 83, 84, or 85, 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 NO:4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 26, 28, 29, 30, 31, 37, 39, 40, 41, 45, 47, 48, 49, 50, 51, 52, 53, 57, 59, 60, 64, 69, 71, 72, 73, 74, 75, 79, 81, 82, 83, 84, or 85. Other useful proteins are substantially identical (e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ ID NO:4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 26, 28, 29, 30, 31, 37, 39, 40, 41, 45, 47, 48, 49, 50, 51, 52, 53, 57, 59, 60, 64, 69, 71, 72, 73, 74, 75, 79, 81, 82, 83, 84, or 85, 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 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. 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. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 55:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If krup=2, similar regions in the two sequences being compared are found by looking at pairs of aligned residues; if ktup=l, single aligned amino acids are examined, ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 for DNA. For a further description of FASTA parameters, see http://bioweb.pasteur.fr/docs/man/man/fasta.l.html#sect2, the contents of which are incorporated herein by reference.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only 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 term "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the invention.
One useful fusion protein is a GST fusion protein in which the polypeptide of the invention is fused to the C-terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.
In another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be 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 sequence of a polypeptide of the invention SEQ ID NO: 4, 26, 37, 45, 57, 64, 69 or 79 can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to the described polypeptides having a signal sequence, as well as to the signal sequence itself and to the polypeptide in the absence of the signal sequence (i.e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence of the invention can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
In another embodiment, the signal sequences of the present invention can be used to identify regulatory sequences, e.g., promoters, enhancers, repressors. Since signal sequences are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal sequence on its amino-terminal side will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal sequence can be used as a probe to identify and isolate signal sequences and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein.
The present invention also pertains to variants of the polypeptides of the invention. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e.g., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of rhe protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.
Variants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477).
In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
The polypeptides of the invention can exhibit post-translational modifications, including, but not limited to glycosylations, (e.g., N-linked or O-linked glycosylations), myristylations, palmitylations, acetylations and phosphorylations (e.g., serine/threonine or tyrosine). In one embodiment, the INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509, polypeptides of the invention exhibit reduced levels of O-linked glycosylation and/or N-linked glycosylation relative to endogenously expressed INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 polypeptides. In another embodiment, the INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 polypeptides of the invention do not exhibit O-linked glycosylation or N-linked glycosylation.
The polypeptides of the invention can, for example, include modifications that can increase such attributes as stability, half-life, ability to enter cells and aid in administration, e.g., in vivo administration of the polypeptides of the invention. For example, polypeptides of the invention can comprise a protein transduction domain of the HIV TAT protein as described in Schwarze, et al. (1999 Science 285:1569-1572), thereby facilitating delivery of polypeptides of the invention into cells.
An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Figures 2, 8, 12, 14, 16, 20, 23 and 26 and are hydropathy plots of the proteins of the invention. These plots or similar analyses can be used to identify hydrophilic regions. In certain embodiments, the nucleic acid molecules of the invention are present as part of nucleic acid molecules comprising nucleic acid sequences that contain or encode heterologous (e.g., vector, expression vector, or fusion protein) sequences. These nucleotides can then be used to express proteins which can be used as immunogens to generate an immune response, or more particularly, to generate polyclonal or monoclonal antibodies specific to the expressed protein.
An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenic preparation can contain, for example, recombinantly expressed or chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immuno logically 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 originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) 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 SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hwm. Antibod. Hybridomas 3:81-85; Ηuse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. 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 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Ins t. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J Immunol. 141:4053-4060. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced, 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).
An antibody directed against a polypeptide of the invention (e.g., monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. The antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbel liferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125^ 131^ 35§ or 3JJ
In addition, the INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 gene sequences and gene products, including peptide fragments and fusion proteins thereof, and antibodies directed against said gene products and peptide fragments thereof, have applications for purposes independent of the role of the gene products, as described above. For example, INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 gene products, including peptide fragments, as well as specific antibodies thereto, can be used for construction of fusion proteins to facilitate recovery, detection, or localization of another protein of interest. In addition, INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 genes and gene products can be used for genetic mapping, i.e., refining the genetic map of chromosome 11. Finally, INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 nucleic acids and gene products have generic uses, such as supplemental sources of nucleic acids, proteins and amino acids for food additives or cosmetic products.
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, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ('TL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), interleukin-10 ("IL-10"), interleukin-12 ("IL-12"), interferon-γ ("IFN-γ"), interferon-α ("IFN-α"), or other immune or growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243- 56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with chemotherapeutic agents.
Alternatively, an antibody of the invention can be conjugated to a second antibody to form an "antibody heteroconjugate" as described by Segal in U.S. Patent No. 4,676,980 or alternatively, the antibodies can be conjugated to form an "antibody heteropolymer" as described in Taylor et al., in U.S. Patent Nos. 5,470,570 and 5,487,890.
An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with cytotoxic factor(s) and/or cytokine(s).
In yet a further aspect, the invention provides substantially purified antibodies or fragments thereof, including human or non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide of the invention comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA- 454; a fragment of at least 15 contiguous amino acid residues of the amino acid sequence ofSEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA- 454; an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA- 454, 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, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, 77, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,
133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 or 167, or to the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA-454, 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 SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA- 438, PTA-454; a fragment of at least 15 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA- 438, PTA-454; an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA- 438, PTA-454, 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, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, 77, 113, 115, 117, 119, 121, 123, 125,
127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,
163, 165 or 167 or to the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA-454, 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 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 of the invention comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126,
128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,
164, 166 or 168, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA- 438, PTA-454; a fragment of at least 15 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs 3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA- 438, PTA-454; an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA- 438, PTA-454, 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, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, 77, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 or 167 or the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA-454, 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 prefeπed embodiment, the substantially purified antibodies or fragments thereof, the non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind to a secreted sequence, or alternatively, to an extracellular domain of the amino acid sequence of the invention. Examples of extracellular domains of the invention are shown in SEQ ID NOs6, 7, 8, 28, 39, 49, 75 or 84.
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.
Still another aspect of the invention is a method of making an antibody that specifically recognizes INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509, the method comprising immunizing a 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:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC® Accession Number Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA-454; a fragment of at least 15 contiguous amino acid residues of the amino acid sequence of any one of SEQ ED NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168, an amino acid sequence which is at least 95%> identical to the amino acid sequence of any one of SEQ ID NOs:3, 25, 36, 44, 56, 63, 68, 78, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168, 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:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76, 77, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 or 167, or the cDNA of a clone deposited as ATCC® Accession Number PTA-455, PTA-425, PTA-424, PTA-438, PTA-454, 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 the immunogen. 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 retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic (e.g., E. coli ) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors), yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non- fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three 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 69:301-315) and pET 1 Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari et al. (1987) EMBO J 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).
Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neuro filament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (Reviews - Trends in Genetics, Vol. 1(1) 1986).
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
In another embodiment, the expression characteristics of an endogenous (e.g., 339, INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509) 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 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509) and controls, modulates or activates the endogenous gene. For example, endogenous INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 which are normally "transcriptionally silent", i.e., an INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 or TANGO 509 genes which is 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 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 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 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499 and TANGO 509 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 sequence encoding a polypeptide of the invention has been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered. Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity. In addition to particular gene expression and/or polypeptide expression phenotypes, the transgenic animals of the invention can exhibit any of the phenotypes (e.g., processes, disorder symptoms and/or disorders), as are described in the sections above. 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 homolog thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986) and Wakayama et al, (1999), Proc. Natl. Acad. Sci. USA, 96:14984-14989. 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 prefeπed 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 refeπed to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi (1987) Cell 51 :503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.
In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PL For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nαtwre 385:810-813 and PCT Publication ΝOS. WO 97/07668 and WO 97/07669.
IV. Pharmaceutical Compositions
The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") of the invention can be 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 caπier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more additional active compounds.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged 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 prefeπed 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 earner 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 prefeπed 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).
Antibodies or antibodies conjugated to therapeutic moieties can be administered to an individual alone or in combination with cytotoxic factor(s), chemotherapeutic drug(s), and/or cytokine(s). If the latter, preferably, the antibodies are administered first and the cytotoxic factor(s), chemotherapeutic drug(s) and/or cytokine(s) are administered thereafter within 24 hours. The antibodies and cytotoxic factor(s), chemotherapeutic drug(s) and/or cytokine(s) can be administered by multiple cycles depending upon the clinical response of the patient. Further, the antibodies and cytotoxic factor(s), chemotherapeutic drug(s) and/or cytokine(s) can be administered by the same or separate routes, for example, by intravenous, intranasal or intramuscular administration. Cytotoxic factors include, but are not limited to, TNF-α, TNF-β, IL-1, IFN-γ and IL-2. Chemotherapeutic drugs include, but are not limited to, 5-fluorouracil (5FU), vinblastine, actinomycin D, etoposide, cisplatin, methotrexate and doxorubicin. Cytokines include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 and IL-12. 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 prefeπed 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 present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, 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 drug combination, and the degree of expression or activity to be modulated.
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. Nαtl. Acαd. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retro viral 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 homologs, 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, polypeptides of the invention can to used to (i) modulate cellular proliferation; (ii) modulate cellular differentiation; and/or (iii) modulate cellular adhesion. The isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in a biological sample) or a genetic lesion, and to modulate activity of a polypeptide of the invention. In addition, the polypeptides of the invention can be used to screen drugs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or abeπant activity compared to the wild type protein. In addition, the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
A. Screening Assays
The invention provides a method (also refeπed to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a polypeptide of the invention or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer DrugDes. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Caπell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (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 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent NOS. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J Mol. Biol. 222:301-310). In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the 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 125^ 35s, 14 or 3ji; 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 prefeπed embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule.
Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. As used herein, a "target molecule" is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention) binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A target molecule can be a polypeptide of the invention or some other polypeptide or protein. For example, a target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a polypeptide of the invention) through the cell membrane and into the cell or a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above. In a prefeπed 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-octylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X-l 14, Thesit, Isotridecypoly(ethylene glycol ether)n,
3-[(3-cholamidopropyl)dimethylamminio]-l-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio- 1 -propane sulfonate.
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either the polypeptide of the invention or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the polypeptide of the invention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g. , biotinylation kit, Pierce Chemicals; Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the polypeptide of the invention or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the invention or target molecule.
In another embodiment, modulators of expression of a polypeptide of the invention are identified in a method in which a cell is contacted with a candidate compound and the expression of the selected mRNA or protein (i.e., the mRNA or protein coπesponding to a polypeptide or nucleic acid of the invention) in the cell is determined. The level of expression of the selected mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of expression of the polypeptide of the invention based on this comparison. For example, when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression. Alternatively, when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression. The level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
In yet another aspect of the invention, a polypeptide of the inventions can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO 94/10300), to identify other proteins, which bind to or interact with the polypeptide of the invention and modulate activity of the polypeptide of the invention. Such binding proteins are also likely to be involved in the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
B. Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the coπesponding 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 coπesponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step in coπelating 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 coπesponding to the gene sequences will yield an amplified fragment. For a review of this technique, see D'Eustachio et al. ((1983) Science 220:919-924).
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sub localization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes (CITE), and 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 (Pergamon Press, New York, 1988)).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents coπesponding to noncoding regions of the genes actually are prefeπed 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 coπelated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland et al. (1987) Nature 325:783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from 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. 47:31-41 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 cuπent limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of coπesponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The nucleic acid sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency at about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:l, 27, 36, 44, 52, 58, 63, 73, 93, 110, 123 or 128 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:l, 27, 36, 44, 52, 58, 63, 73, 93, 110, 123 or 128 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. If a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.
3. Use of Partial Gene Sequences in Forensic Biology
DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a peφetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymoφhisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
The nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
C. Predictive Medicine
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trails are used for prognostic (predictive) puφoses to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention and/or activity of a polypeptide of the invention, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with abeπant expression or activity of a polypeptide of the invention, such as a proliferative disorder, e.g., psoriasis or cancer, or an angiogenic disorder. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with abeπant expression or activity of a polypeptide of the invention. For example, mutations in a gene of the invention can be assayed in a biological sample. Such assays can be used for prognostic or predictive puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with abeπant expression or activity of a polypeptide of the invention.
Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (refeπed to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent).
Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of a polypeptide of the invention in clinical trials. These and other agents are described in further detail in the following sections.
1. Diagnostic Assays
An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample. A prefeπed agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention. The nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NO:l, 23, 34, 42, 54, 61, 66, or 76, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 contiguous 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 prefeπed 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 immuno fluorescence. 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 prefeπed 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 abeπant expression of a polypeptide of the invention as discussed, for example, in sections above relating to uses of the sequences of the invention.
For example, kits can be used to determine if a subject is suffering from or is at increased risk of disorders such as immuno logical disorders, e.g., autoimmune disorders (e.g., arthritis, graft rejection (e.g., allograft rejection), T cell disorders (e.g., AIDS)) and inflammatory disorders (e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma, psoriasis), neurological disorders, eye disorders and embryonic disorders, which are associated with abeπant INTERCEPT 307, MANGO 511, TANGO 361, and TANGO 509 expression.
In another example, kits can be used to determine if a subject is suffering from or is at increased risk of renal disorders, e.g., glomerular disease, (e.g., acute and chronic glomerulonephritis), tubular diseases, and tubulo-interstitial diseases or alternatively, embryonic and fetal disorders, and include other disorders which are associated with abeπant TANGO 351 expression.
In another example, kits can be used to determine if a subject is suffering from or is at risk for brain-related disorders such as peripheral nerve survival, cerebral edema, hydrocephalus, brain herniations, iatrogenic 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 hemoπhage 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, which are associated with abeπant TANGO 499 activity and/or expression.
In still another example, kits can be used to determine if a subject is suffering from or is at risk for prostate-related disorders, (e.g. prostate cancer, prostatitis, benign prostatic hypertrophy, benign prostatic hypeφlasia and atypical prostatic stromal lesions) which can be associated with abeπant INTERCEPT 307 and TANGO 361 activity and/or expression. In another example, kits can be used to determine if a subject is suffering from or is at risk for pituitary-related disorders, e.g., Cushing's disease, hypeφrolactinemia, acromegaly-gigantism, and precocious and delayed puberty, or Parkinson's disease or peripheral neuropathy-related disorders (e.g., amylo-lateral sclerosis) which are associated with abeπant TANGO 499 activity and/or expression. The kit, for example, 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 instructions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with abeπant 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 instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with abeπant expression of the polypeptide.
2. Prognostic Assays
The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with abeπant 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 abeπant expression or activity of a polypeptide of the invention, e.g., an immunologic disorder, or embryonic disorders. 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 abeπant 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. The prognostic assays described herein, for example, can be used to identify a subject having or at risk of developing disorders such as disorders discussed, for example, in sections above relating to uses of the sequences of the invention. For example, prognostic assays described herein can be used to identify a subject having or at risk of developing immunological disorders, e.g., autoimmune disorders (e.g., arthritis, graft rejection (e.g., allograft rejection), T cell disorders (e.g., AIDS)) and inflammatory disorders (e.g. , bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g. , asthma, psoriasis), which are associated with abeπant INTERCEPT 307, MANGO 511 and TANGO 361 activity and/or expression.
In another example, prognostic assays described herein can be used to identify a subject having or at risk of developing renal disorders e.g., glomerular diseases, tubular diseases and tubulo-interstitial diseases, embryonic disorders and fetal disorders, and which are associated with abeπant INTERCEPT 307 activity and/or expression.
In another example, prognostic assays described herein can be used to identify a subject having or at risk of developing immunological disorders (e.g., platelet disorders), endothelial disorders and embryonic disorders, which are associated with abeπant TANGO 361 activity and/or expression. In another example, prognostic assays described herein can be used to identify a subject having or at risk of developing brain-related disorders such as cerebral edema, hydrocephalus, brain hemiations, iatrogenic 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 hemoπhage 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, which are associated with abeπant TANGO 509 and/or expression. In another example, prognostic assays described herein can be used to identify a subject having or at risk of developing prostate-related disorders (e.g, prostate cancer, prostatis, benign prostatic hypertrophy, benign prostatic hypeφlasmia and atypical prostatic stromal lesions) which are associated with abeπant INTERCEPT 307, and TANGO 361 activity and/or expression.
In another example, prognostic assays described herein can be used to identify a subject having or at risk of developing pituitary-related disorders (e.g., Cushing's disease, hypeφrolactinemia, acromegaly-gigantism, and precocious and delayed puberty disorders), or Parkinson's disease or peripheral neuropathy-related disorders (e.g., amylothrophic lateral sclerosis) which are associated with abeπant TANGO 499 activity and/or expression. 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 abeπant 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 abeπant 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 abeπant 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 abeπant expression or activity of a polypeptide of the invention. In prefeπed 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 reaπangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an abeπant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a gene.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 :1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 :360-364), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in a selected gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a 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 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations can be identified in two-dimensional aπays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization aπay 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 aπays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization aπay that allows the characterization of specific mutations by using smaller, specialized probe aπays complementary to all variants or mutations detected. Each mutation aπay 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 coπesponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Bio/Techniques 19:448), including sequencing by mass spectrometry ( see, e.g., PCT Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the technique of "mismatch cleavage" entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions.
In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a prefeπed embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a selected sequence, e.g., a wild-type sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039. In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in genes. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a prefeπed embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a "GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition, it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention. Furthermore, any cell type or tissue, e.g., preferably peripheral blood leukocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
3. Pharmacogenomics
Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with abeπant activity of the polypeptide. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g. , drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are refeπed to as "altered drug action." Genetic conditions transmitted as single factors altering the way the body acts on drugs are refeπed to as "altered drug metabolism". These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitro furans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymoφhisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These 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 polymoφhic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite moφhine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply geno typing of polymoφhic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.
4. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a polypeptide of the invention (e.g., the ability to modulate abeπant 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 prefeπed embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the post-administration samples; (v) comparing the level of the polypeptide or nucleic acid of the invention in the pre-administration sample with the level of the polypeptide or nucleic acid of the invention in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of the polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.
C. Methods of Treatment
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abeπant expression or activity of a polypeptide of the invention. For example, disorders characterized by abeπant expression or activity of the polypeptides of the invention include proliferative disorders such as cancer.
1. Prophylactic Methods In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an abeπant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide. Subjects at risk for a disease which is caused or contributed to by abeπant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the abeπancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of abeπancy, for example, an agonist or antagonist agent can be used for treating the subject. For example, an antagonist of an INTERCEPT 307, MANGO 511 or TANGO 509 protein may be used to treat an immunological disorder. The appropriate agent can be determined based on screening assays described herein.
2. Therapeutic Methods Another aspect of the invention pertains to methods of modulating expression or activity of a polypeptide of the invention for therapeutic puφoses. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide. An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of the polypeptide of the invention. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by abeπant 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 abeπant 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.
This invention is further illustrated by the following examples which should not be construed as limiting. 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 TANGO 351 (clone 351), were deposited with the American Type Culture Collection (Manassas, VA) on July 23, 1999 as Accession Number PTA-424, as part of a composite deposit representing a mixture of five 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 lOOμg/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 3.4 kb fragments that coπesponds to TANGO 351 (351). The identity of the strain containing TANGO 351 can be infeπed from the liberation of a fragment of the above identified size.
Clones containing cDNA molecules encoding MANGO 511 (clone 511), were deposited with the American Type Culture Collection (Manassas, VA) on July 23, 1999 as Accession Number PTA-425, 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 lOOμg/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 1.5 kb fragments that coπespond to MANGO 511 (511). The identity of the strain containing MANGO 511 can be infeπed from the liberation of a fragment of the above identified size.
Clones containing cDNA molecules encoding INTERCEPT 307, TANGO 361, and TANGO 509 (clones 307, 361, and 509, respectively), were deposited with the American
10 Type Culture Collection (Manassas, VA) on July 29, 1999 as Accession Number PTA- 455, Accession Number PTA-438, and Accession Number PTA-438 respectively, as part of a composite deposit representing a mixture of five 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
15 aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with lOOμg/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 2 Not I and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
INTERCEPT 307 (307): 2.0 kb TANGO 361 (361): 5.1 kb 25 TANGO 509 (509): 3.6 kb
The identity of the strains can be infeπed from the fragments liberated. Clones containing cDNA molecules encoding TANGO 499 form 1, variant 1 (clone EpT499 form 1, variant 1), were deposited with the American Type Culture r. Collection (Manassas, VA) on August 5, 1999 as Accession Number PTA-455, 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 35 plates) supplemented with lOOμg/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 1.1 kb fragments that coπespond to TANGO 499 form 1, variant 1 (EpT499 form 1, variant 1). The identity of the strain containing TANGO 499 form 1, variant 1 can be infeπed from the liberation of a fragment of the above identified size.
Clones containing cDNA molecules encoding TANGO 499 form 2, variant 3 (clone EpT499 form 2, variant 3), were deposited with the American Type Culture Collection (Manassas, VA) on August 5, 1999 as Accession Number PTA-454, as part of a composite deposit representing a mixture of four 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 lOOμg/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 1.1 kb fragments that coπespond to TANGO 499 form 2, variant 3 (EpT499 form 2, variant 3). The identity of the strain containing TANGO 499 form 2, variant 3 can be infeπed from the liberation of a fragment of the above identified size.
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. International Application No: PCT/
MICROORGANISMS
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A. IDENTIFICATION OF DEPOSIT '
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Name of depositary institution ' American Type Culture Collection
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Date of deposit ' July 23, 1999 Accession Number ' PTA-424
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was i *■« ΠΓ F ? Λn ^ ^-
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142.1 International Application No: PCT/
Form PCT/RO/134 (cont.) American Type Culture Collection
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Accession No. Date of Deposit
PTA-425 July 23, 1999
PTA-438 July 29, 1999
PTA-454 August 5, 1999
PTA-455 August 5, 1999
142.2

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 30% identical to the nucleotide sequence of SEQ ID NO:34, 42, 43, 66 or 67, the cDNA insert of an EpT351 clone, an EpT361 clone or a human EpT509 clone deposited with the ATCC® as Accession Number PTA-424, ATCC® Accession Number PTA-438 or ATCC® Accession Number PTA-438. or a complement thereof; b) a nucleic acid molecule comprising a nucleotide sequence which is at least 30% identical to the nucleotide sequence of nucleotides 301 to 480 of SEQ ID NO: 55, or nucleotides 240 to 344 of SEQ ID NO:62, the cDNA insert of an EpT499 form 1, variant 1 or EpT499 form 2, variant 3 clone deposited with the ATCC® as Accession Number PTA-455, ATCC® Accession Number PTA-454, or a complement thereof; c) a nucleic acid molecule comprising a nucleotide sequence which is at least 45% identical to the nucleotide sequence of SEQ ID NO:23, 24 or 35, the cDNA insert of an EpM511 clone or EpT351 clone deposited with the ATCC® as Accession Number PTA-425 or Accession Number PTA-424, or a complement thereof; d) a nucleic acid molecule comprising a nucleotide sequence which is at least 58% identical to the nucleotide sequence of SEQ ID NO:l or 2, the cDNA insert of an INT307 clone deposited with the ATCC® as Accession Number PTA-455, or a complement thereof; e) a nucleic acid molecule comprising a nucleotide sequence which is at least 75% identical to the nucleotide sequence of SEQ ID NO:55 or 62, the cDNA insert of an EpT499 clone deposited with the ATCC® as Accession Number PTA-455 or Accession Number PTA-454, or a complement thereof; f) a nucleic acid molecule comprising a nucleotide sequence which is at least 80% identical to the nucleotide sequence of SEQ ID NO:54 or 61, the cDNA insert of an EpT499 clone deposited with the ATCC® as Accession Number PTA-455 or Accession Number PTA-454, or a complement thereof; g) a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to the nucleotide sequence of SEQ ID NO:76, the cDNA insert of a mouse EpT509 clone, or a complement thereof;
- 143 - h) a nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to the nucleotide sequence of SEQ ID NO:77, the cDNA insert of a mouse EpT509 clone, or a complement thereof; i) a nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:23, 24, 35 or 67, the cDNA insert of an EpM511 clone, an EpT351 clone or a human EpT509 clone deposited with the ATCC® as Accession Number PTA-425, ATCC® Accession Number PTA-424 or ATCC® Accession Number PTA-438, or a complement thereof; j) a nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of nucleotides 1 to 649 of SEQ ID NO: 1, nucleotides 1120 to 1430 of SEQ ID NO: 1, or a complement thereof; k) a nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of nucleotides 1 to 604 of SEQ ID NO:2, nucleotides 1 to 2071 of SEQ ID NO:34, or a complement thereof;
1) a nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of nucleotides 1 to 1176 of SEQ ID NO:42, nucleotides 1653 to 1821 of SEQ ID NO:42, nucleotides 2035 to 3506 of SEQ ID NO:42, nucleotides 3564 to 5058 of SEQ ID NO:42 or nucleotides 1 to 1136 of SEQ ID NO: 43, or a complement thereof; m) a nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of nucleotides 385 to 559 of SEQ ID NO:54, nucleotides 1072 of 1106 of SEQ ID NO:54 or nucleotides 301 to 480 of SEQ ID NO:55, or a complement thereof; n) a nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of nucleotides 310 to 488 of SEQ ID NO:61, nucleotides 1 to 3023 of SEQ ID NO:66, or a complement thereof; o) a nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of nucleotides 1 to 106 of SEQ ID NO:76, or nucleotides 1 to 52 of SEQ ID NO:77, or a complement thereof;
- 144 - p) a nucleic acid molecule comprising a fragment of at least 135 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:43, the cDNA insert of an EpT361 deposited with the ATCC® as Accession Number PTA-438, or a complement thereof; q) a nucleic acid molecule comprising a fragment of at least 265 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:76 or 77, the cDNA insert of a mouse EpT509 clone, or a complement thereof; r) a nucleic acid molecule comprising a fragment of at least 350 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:42, the cDNA insert of an EpT361 clone deposited with the ATCC® as Accession Number PTA-438, or a complement thereof; s) a nucleic acid molecule comprising a fragment of at least 460 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:34, the cDNA insert of an EpT351 clone deposited with the ATCC® as Accession Number PTA-424, or a complement thereof; t) a nucleic acid molecule comprising a fragment of at least 475 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:2 or 66, the cDNA insert of an INT307 clone or a human EpT509 clone deposited with the ATCC® as Accession Number PTA-455 or Accession Number PTA-438, or a complement thereof; u) a nucleic acid molecule comprising a fragment of at least 500 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:54 or 61, the cDNA insert of an EpT499 clone deposited with the ATCC® as Accession Number PTA-455 or Accession Number PTA-454, or a complement thereof; v) a nucleic acid molecule comprising a fragment of at least 550 contiguous nucleotides of the nucleotide sequence of SEQ ED NO:l, the cDNA insert of an INT307 clone with the ATCC® as Accession Number PTA-455, or a complement thereof; w) a nucleic acid molecule comprising a fragment of at least 285 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:55, the cDNA insert of an EpT499 form 1, variant 1 clone deposited with the ATCC® as Accession Number PTA-455, or a complement thereof; x) a nucleic acid molecule comprising a fragment of at least 275 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:62, the cDNA insert of an EρT499 form 2, variant 3 clone
- 145 - deposited with the ATCC® as Accession Number PTA-454, or a complement thereof; y) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-455 or Accession Number PTA-545, or Accession Number PTA-438; and z) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA- 424, Accession Number PTA-438, Accession Number PTA-455 or PTA-454, or Accession Number PTA-438, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-455 OR PTA 454, or Accession Number PTA-438;
2. The isolated nucleic acid molecule of claim 1, which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 , 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76 or 77, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-455 or PTA-454, or Accession Number PTA-438; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone
- 146 - deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-455 or PTA- 454, or Accession Number PTA-438;
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 a recombinant nucleic acid molecule comprising 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.
An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-455 or PTA- 454, or Accession Number PTA-438, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:2, 24, 35, 43, 55, 62, 67 or 77, 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 30% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID
- 147 - NO:43 or 67, or at least 98% to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:43 or 67, or a complement thereof. d) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 58% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2, or at least 98% to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2, or a complement thereof. e) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 45% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:24 or 34, or at least 98% to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:24 or 34, or a complement thereof. f) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 75 % identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:55 or 62, or at least 98% to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:55 or 62, or a complement thereof. g) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:77, or at least 98%> to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:77, or a complement thereof.
9. The isolated polypeptide of claim 8 comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78.
10. The polypeptide of claim 8 further comprising heterologous amino acid sequences.
11. An antibody which selectively binds to a polypeptide of claim 8.
12. The antibody of claim 11, wherein the antibody is a monoclonal antibody.
13. A method for producing a polypeptide selected from the group consisting of:
- 148 - a) a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA- 424, Accession Number PTA-438, Accession Number PTA-455 or
PTA-454, or Accession Number PTA-438, b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA- 425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-455 or PTA-454, or Accession Number PTA-438, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession
Number PTA-438, Accession Number PTA-455 or PTA-454, or
Accession Number PTA-438; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 25, 36, 44, 56, 63, 68 or 78, or the amino acid sequence encoded by the cDNA insert of an INT307, EpM511, EpT351, EpT361, EpT499 or EpT509 clone deposited with the ATCC® as Accession Number PTA-455, Accession Number PTA-425, Accession Number PTA-424, Accession Number PTA-438, Accession Number PTA-455 or PTA- 454, or Accession Number PTA-438, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, 2, 23, 24, 34, 35, 42, 43, 54, 55, 61, 62, 66, 67, 76 or 77, or a complement thereof under stringent conditions; comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.
- 149 -
14. 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.
15. The method of claim 14, wherein the compound which binds to the polypeptide is an antibody.
16. A kit comprising a compound which selectively binds to a polypeptide of claim 8 and instructions for use.
17. 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.
18. The method of claim 17, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
19. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.
20. 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.
21. The method of claim 20, 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;
150 - c) detection of binding using an assay for INTERCEPT 307, MANGO 511, TANGO 351, TANGO 361, TANGO 499, and TANGO 509- mediated signal transduction.
22. 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.
23. 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.
151
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HALVORSEN Y-D C ET AL: "Adipose as an endocrine organ: The characterization of secreted proteins from human cultured adipocytes" FASEB JOURNAL, FED. OF AMERICAN SOC. FOR EXPERIMENTAL BIOLOGY, BETHESDA, MD, US, vol. 13, no. 4 part 11, 12 March 1999 (1999-03-12), page A369, XP002146933 ISSN: 0892-6638 *
See also references of WO0121631A2 *

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WO2001021631A3 (en) 2001-11-22

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