WO2002012344A2 - Methods for using 14266, a human g protein-coupled receptor - Google Patents

Methods for using 14266, a human g protein-coupled receptor Download PDF

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WO2002012344A2
WO2002012344A2 PCT/US2001/024835 US0124835W WO0212344A2 WO 2002012344 A2 WO2002012344 A2 WO 2002012344A2 US 0124835 W US0124835 W US 0124835W WO 0212344 A2 WO0212344 A2 WO 0212344A2
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cells
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amino acid
sequence
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PCT/US2001/024835
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WO2002012344A3 (en
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Nadine S. Weich
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Millennium Pharmaceuticals, Inc.
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Priority to AU2001279232A priority Critical patent/AU2001279232A1/en
Priority to EP01957494A priority patent/EP1311546A2/en
Publication of WO2002012344A2 publication Critical patent/WO2002012344A2/en
Publication of WO2002012344A3 publication Critical patent/WO2002012344A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention relates to methods of using 14266, a G protein-coupled receptor. Methods for identifying agents that modulate or interact with 14266 polypeptides and nucleic acid molecules, and methods for detecting or modulating 14266 polypeptides and nucleic acid molecules are provided.
  • GPCRs G-protein coupled receptors
  • GPCRs constitute a major class of proteins responsible for transducing a signal within a cell. GPCRs share three structural features: an amino terminal extracellular domain, a transmembrane region containing seven transmembrane domains, three extracellular loops, and three intracellular loops, and a carboxy terminal intracellular domain.
  • a signal is transduced within the cell that results in a change in a biological or physiological property ofthe cell.
  • GPCRs, along with G- proteins and effectors are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs.
  • GPCR genes and gene-products are potential causative agents of disease (Spiegel et al., J. Clin. Invest. 92:1119-1125 (1993); McKusick et al, J. Med. Genet. 30:1-26 (1993)).
  • Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of retinitis pigmentosum (Nathans et al., Annu. Rev. Genet. 26:403-424(1992)), and nephrogenic diabetes insipidus (Holtzman et al., Hum. Mol. Genet. 2:1201-1204 (1993)).
  • These receptors are of critical importance to both the central nervous system and peripheral physiological processes. Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous systems.
  • the GPCR protein superfamily can be divided into five families: Family I, which contains receptors typified by rhodopsin and the ⁇ 2-adrenergic receptor and currently represented by over 200 unique members (Dohlman et al., Annu. Rev. Biochem.
  • Family II which contains the parathyroid hormone/calcitonin/secretin receptor family (Juppner et al., Science 254:1024-1026 (1991); Lin et al, Science 254:1022-1024 (1991)); Family III, which contains the metabotropic glutamate receptor family (Nakanishi, Science 258 597:603 (1992)); Family IV, which contains the cAMP receptor family, important in the chemotaxis and development of D. discoideum (Klein et al., Science 241:1467-1472 (1988)); and Family V, the fungal mating pheromone receptors such as STE2 (Kurjan, Annu. Rev. Biochem.
  • G-proteins represent a family of heterotrimeric proteins composed of ⁇ , ⁇ , and ⁇ subunits. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane segments. Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the ⁇ -subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the ⁇ - subunits.
  • the GTP-bound form ofthe ⁇ -subunit typically functions as an effector- modulating moiety, leading to the production of second messengers, such as cAMP (e.g., by activation of adenyl cyclase), diacylglycerol or inositol phosphates.
  • second messengers such as cAMP (e.g., by activation of adenyl cyclase), diacylglycerol or inositol phosphates.
  • cAMP e.g., by activation of adenyl cyclase
  • diacylglycerol diacylglycerol
  • inositol phosphates inositol phosphates.
  • G proteins include Gi (inhibitory G protein), Go , Gq, Gs (stimulatory G protein) and Gt (transducin).
  • G proteins are described extensively in Lodish et al., Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference. GPCRs, G proteins and G protein-linked effector and second messenger systems have been reviewed in The G-Protein Linked Receptor Fact Book, Watson et al., eds., Academic Press (1994).
  • GPCRs are a major target for drug development. Accordingly, it is valuable to the field of pharmaceutical development to identify methods using GPCRs and tissues and disorders in which GPCRs are differentially expressed.
  • a specific object ofthe invention is to identify compounds that act as agonists and antagonists and modulate the expression of 14266 in specific tissues and disorders.
  • a further specific object ofthe invention is to provide compounds that modulate expression of 14266 for diagnosis and treatment of 14266-mediated or related disorders.
  • the invention provides methods of screening, for compounds that modulate expression or activity of 14266 polypeptides or nucleic acid molecules (RNA or DNA) in the specific tissues or disorders.
  • the invention also provides a process for modulating 14266 polypeptide or nucleic acid molecule expression or activity, especially using the screened compounds. Modulation may be used to treat conditions related to abe ⁇ ant activity or expression of 14266 polypeptides or nucleic acids.
  • the invention also provides assays for detecting 14266 polypeptides or nucleic acid molecules in specific biological samples, including for disease diagnosis.
  • the invention further comprises kits comprising reagents for the detection of 14266 polypeptides or 14266 polynucleotides.
  • the invention also provides assays for determining the presence of a mutation in the polypeptides or nucleic acid molecules, including for disease diagnosis.
  • the invention utilizes isolated 14266 polypeptides, including a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 , and variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO: 1.
  • the invention also utilizes an isolated 14266 nucleic acid molecule having the sequence shown in SEQ ID NO:2, and variant nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in SEQ ID NO:2.
  • the invention also utilizes fragments ofthe polypeptide shown in SEQ ID NO:l and nucleotide sequence shown in SEQ ID NO:2, as well as substantially homologous fragments ofthe polypeptide or nucleic acid.
  • the invention further utilizes nucleic acid constructs comprising the nucleic acid molecules described herein. In a prefe ⁇ ed embodiment, the nucleic acid molecules of the invention are operatively linked to a regulatory sequence.
  • the invention also utilizes vectors and host cells that express 14266 and provides methods for expressing 14266 nucleic acid molecules and polypeptides in specific cell types and disorders.
  • the invention also utilizes methods of making the vectors and host cells and provides methods for using them to assay expression and cellular effects of expression of the 14266 nucleic acid molecules and polypeptides in specific cell types and disorders.
  • tissues and cell lines where 14266 is highly or differentially expressed include, but are not limited to, spinal chord, brain cortex, hypothalamus, aorta, heart, fetal heart, vein, astrocytes, glioblastoma, breast, breast interductal carcinoma, ovary, ovary tumor, pancreas, prostate, prostate tumor, colon cells, colon tumor, bone marrow mononuclear cells, kidney, CD34 positive haematopoietic progenitor cells, neutrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
  • Figure 1 shows the 14266 nucleotide sequence (SEQ ID NO:2).
  • Figure 2 shows the amino acid sequence of 14266 (SEQ ID NO: 1).
  • Figure 3 shows an alignment ofthe 7 transmembrane rhodopsin-like G protein- coupled receptor domain of human 14266 with a consensus amino acid sequence (SEQ ID NO:3) derived from a hidden Markov model, while the lower amino acid sequence co ⁇ esponds to amino acids 35 to 338 of SEQ ID NO: 1.
  • Figure 4 depicts a hydropathy plot of human 14266. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues 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 numbers co ⁇ esponding to the amino acid sequence (shown in SEQ ID NO:l) of human 14266 are indicated .
  • Polypeptides ofthe invention include fragments which include: all or a part of a hydrophobic sequence (a sequence above the dashed line); or all or part of a hydrophilic fragment (a sequence below the dashed line). Other fragments include a cysteine residue or an N-glycosylation site.
  • the present invention is based on methods of using molecules refe ⁇ ed to herein as 14266, 14266 GPCRs, 14266 receptors, or 14266 nucleic acid or polypeptide molecules.
  • the 14266 receptor shares sequence similarity with the norepipniephrine ⁇ 3 receptor and the serotonin 5HT-2C receptor, and there are 14266 receptor orthologs in rat and zebrafish, suggesting that the 14266 gene has been highly conserved in vertebrate evolution (Matsumoto et al. (2000) Biochem. Biophys. Res. Comm. 272: 576-582)
  • the human 14266 sequence ( Figure 2; SEQ ID NO:2), which is approximately 1128 nucleotides long, contains a predicted methionine-initiated coding sequence of about 1128 nucleotides (nucleotides 1-1128 of SEQ ID NO:2).
  • the coding sequence encodes a 375 amino acid protein (SEQ ID NO:l).
  • the mature protein form is approximately 350 amino acid residues in length (from about amino acid 35-375 of SEQ ID NO:l).
  • Human 14266 also contains a predicted G-protein coupled receptor domain (PFAM Accession PF00001) located at about amino acid 35 to 338 of SEQ ID NO : 1 , and predicted transmembrane domains at amino acids 22-44, 54-78, 97-118, 134-156, 184-208, 286-306, and 318-341 of SEQ ID NO:l.
  • PFAM Accession PF00001 located at about amino acid 35 to 338 of SEQ ID NO : 1
  • predicted transmembrane domains at amino acids 22-44, 54-78, 97-118, 134-156, 184-208, 286-306, and 318-341 of SEQ ID NO:l.
  • G-protein coupled receptor domain includes an amino acid sequence of about 250-350 amino acid residues in length and having a bit score for the alignment ofthe sequence to the G-protein coupled receptor domain (HMM) of at least 8.
  • a G-protein coupled receptor domain includes at least about 200-400 amino acids, more preferably about 250-350 amino acid residues, or about 275-325 amino acids and has a bit score for the alignment ofthe sequence to the G-protein coupled receptor domain (HMM) of at least 16 or greater.
  • the G- protein coupled receptor domain (HMM) has been assigned the PFAM Accession PF00001.
  • An alignment ofthe G-protein coupled receptor domain (amino acids 35- 338 of SEQ ID NO:l) of human 14266 with a consensus amino acid sequence derived from a hidden Markov model is depicted in Figure 3.
  • 14266 polypeptide or protein has a "G-protein coupled receptor domain” or a region which includes at least about 100-250 more preferably about 130-200 or 160-200 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with an "G-protein coupled receptor domain,” e.g., the G-protein coupled receptor domain of human 14226 (e.g., amino acid residues 35-338 of SEQ ID NO:l).
  • a 14266 protein includes at least one transmembrane domain.
  • transmembrane domain includes an amino acid sequence of about 15 amino acid residues in length that spans a phospholipid membrane. More preferably, a transmembrane domain includes about at least 18, 20, 22, 24, or 25 amino acid residues and spans a phospholipid membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an ⁇ - helical structure.
  • At least 50%, 60%, 70%, 80%, 90%, 95% or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans.
  • a 14266 polypeptide or protein has at least one transmembrane domain or a region which includes at least 18, 20, 22, 24, 25, or 30 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity with a "transmembrane domain,” e.g., at least one transmembrane domain of human 14266 (e.g., amino acid residues 22-44, 54-78, 97-118, 134-156, 184-208, 286-306, and 318-341 of SEQ ID NO:l).
  • a transmembrane domain e.g., at least one transmembrane domain of human 14266 (e.g., amino acid residues 22-44, 54-78, 97-118, 134-156, 184-208, 286-306, and 318-341 of SEQ ID NO:l).
  • a 14266 protein in another embodiment, includes at least one "non- transmembrane domain.”
  • non-transmembrane domains are domains that reside outside ofthe membrane. When refe ⁇ ing to plasma membranes, non-transmembrane domains include extracellular domains (i.e., outside ofthe cell) and intracellular domains (i.e., within the cell).
  • non-transmembrane domains include those domains of the protein that reside in the cytosol (i.e., the cytoplasm), the lumen ofthe organelle, or the matrix or the intermembrane space (the latter two relate specifically to mitochondria organelles).
  • the C-terminal amino acid residue of a non- transmembrane domain is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally occurring 14226, or 14266-like protein.
  • a 14226 polypeptide or protein has a "non- transmembrane domain” or a region which includes at least about 5-100, preferably about 8-80 acid residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100% sequence identity with a "non-transmembrane domain", e.g., a non- transmembrane domain of human 14226 (e.g., residues 45-53, 79-96, 119-133; 157- 183, 209-285, 307-317, 342-375 of SEQ ID NO:2).
  • a non-transmembrane domain e.g., a non- transmembrane domain of human 14226 (e.g., residues 45-53, 79-96, 119-133; 157- 183, 209-285, 307-317, 342-375 of SEQ ID NO:2).
  • a non-transmembrane domain located at the N-terminus of a 14226 protein or polypeptide is refe ⁇ ed to herein as an "N-terminal non-transmembrane domain.”
  • an "N-terminal non-transmembrane domain” includes an amino acid sequence having about 1-50, preferably about 18-22 amino acid residues in length and is located outside the boundaries of a membrane.
  • an N-terminal non- transmembrane domain is located at about amino acid residues 1-22 of SEQ ID NO:2.
  • a non-transmembrane domain located at the C-terminus of a 14266 protein or polypeptide is refe ⁇ ed to herein as a "C-terminal non-transmembrane domain.”
  • an "C-terminal non-transmembrane domain” includes an amino acid sequence having about 20-40, preferably about 25-35 amino acid residues in length and is located outside the boundaries of a membrane.
  • an C- terminal non-transmembrane domain is located at about amino acid residues 342-375 of SEQ ID NO:2.
  • a 14266 molecule can further include a signal sequence.
  • a signal sequence refers to a peptide of about 20-80 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains a majority of hydrophobic amino acid residues.
  • a signal sequence contains at least about 12-25 amino acid residues, preferably about 30-70 amino acid residues, more preferably about 61 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g., alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline).
  • a signal sequence serves to direct a protein containing such a sequence to a lipid bilayer.
  • a 14266 protein contains a signal sequence of about amino acids 1-34 of SEQ ID NO:2.
  • the “signal sequence” is cleaved during processing ofthe mature protein.
  • the mature 14266 protein co ⁇ esponds to amino acids 35-375 of SEQ ID NO:2.
  • 14266 expression is highest in spinal cord and brain (particularly the cortex and hypothalamus) ( Figure 4).
  • 14266 expression is also detectable in aorta, heart, fetal heart, vein, astrocytes (normal cells and glioblastoma tissue), breast (normal tissue and interductal carcinoma tissue), ovary (normal tissue and ovary tumor tissue), pancreas, prostate (normal tissue and prostate tumor cells), and colon (normal tissue, tumor tissue, and inflammatory bowel disease tissue).
  • CD34 + haematopoietic progenitor cells from bone ma ⁇ ow from volunteers treated with G-CSF showed significant levels of 14266 expression, as did both early stage and more mature embark lineage cells isolated from bone marrow from both normal and G-CSF treated volunteers.
  • Expression of 14266 was regulated during both in vivo and in vitro generation of blood cells. It was down-regulated in both megakaryocytes and erythroid cells during differentiation, and up-regulated during embarkophil differentiation.
  • Neutrophils are a special class of granulocytes that are derived from the granulocyte/macrophage progenitor cells (colony-forming cells) which arise from the division and differentiation of myeloid stem cells. Neutrophils play a key role in the nonspecific immune response, and they are recruited rapidly to sites of inflammation. Neutrophils are required for host defense against invading micro-organisms, and they respond to injurious agents by the release of granular enzymes and proteins, the production of reactive oxygen intermediates, and by phagocytosis. Patients with embarkophil deficiency disorders, including neutropenia, chronic granulomatous disease, and leukocyte adhesion deficiency, have a tendency to develop recurrent and overwhelming infections.
  • Inadequate or ineffective granulopoiesis can result from suppression of myeloid stem cells (as occurs in aplastic anemia and a variety of infiltrative ma ⁇ ow disorders), suppression ofthe committed granulocytic precursors (which often occurs after exposure to certain drugs, including alkylating agents and antimetabolites used in cancer treatment), disease states characterized by ineffective granulopoiesis (such as megaloblastic anemias caused by vitamin B 1 or folate deficiency and myelodysplastic syndromes) and rare inherited conditions (such as Kostmann syndrome).
  • myeloid stem cells as occurs in aplastic anemia and a variety of infiltrative ma ⁇ ow disorders
  • suppression ofthe committed granulocytic precursors which often occurs after exposure to certain drugs, including alkylating agents and antimetabolites used in cancer treatment
  • disease states characterized by ineffective granulopoiesis such as megaloblastic anemias caused by vitamin B 1 or folate deficiency and myelodysplastic
  • G protein-coupled receptors including 14266 receptors, use one of several signaling pathways to relay their intracellular signal
  • a “signaling pathway” refers to one or more signaling steps that lead to the modulation (e.g., stimulation or inhibition) of a cellular function/activity upon the binding of a ligand to the 14266 receptors.
  • phosphatidylinositol turnover and metabolism refers to the molecules involved in the turnover and metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2) as well as to the activities of these molecules.
  • PIP2 is a phospholipid found in the cytosolic leaflet ofthe plasma membrane.
  • Binding of ligand to the 14266 receptor may activate, in some cells, the plasma-membrane enzyme phospholipase C that, in turn, can hydrolyze PIP2 to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3).
  • DAG 1,2-diacylglycerol
  • IP3 inositol 1,4,5-triphosphate
  • IP3 can diffuse to the endoplasmic reticulum surface where it can bind an IP3 receptor, e.g., a calcium channel protein containing an IP3 binding site. IP3 binding can induce opening ofthe channel, allowing calcium ions to be released into the cytoplasm.
  • IP3 can also be phosphorylated by a specific kinase to form inositol 1,3,4,5-tetraphosphate (IP4), a molecule which can cause calcium entry into the cytoplasm from the extracellular medium.
  • IP4 and IP4 can subsequently be hydrolyzed very rapidly to the inactive products inositol 1,4-biphosphate (IP2) and inositol 1,3,4-triphosphate, respectively.
  • IP2 inositol 1,4-biphosphate
  • IP2 inositol 1,3,4-triphosphate
  • Protein kinase C is usually found soluble in the cytoplasm ofthe cell, but upon an increase in the intracellular calcium concentration, this enzyme can move to the plasma membrane where it can be activated by DAG.
  • the activation of protein kinase C in different cells results in various cellular responses such as the phosphorylation of glycogen synthase, or the phosphorylation of various transcription factors, e.g., NF-kB.
  • phosphatidylinositol activity refers to an activity of PIP2 or one of its metabolites.
  • cAMP turnover and metabolism refers to the molecules involved in the turnover and metabolism of cAMP as well as to the activities of these molecules.
  • Cyclic AMP is a second messenger produced in response to ligand- induced stimulation of certain G protein coupled receptors.
  • binding of a ligand to a GPCR can lead to the activation ofthe enzyme adenyl cyclase, which catalyzes the synthesis of cAMP.
  • the newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase.
  • This activated kinase can phosphorylate a voltage-gated potassium channel protein, or an associated protein, and lead to the inability ofthe potassium channel to open during an action potential.
  • the inability ofthe potassium channel to open results in a decrease in the outward flow of potassium, which normally repolarizes the membrane of a neuron, leading to prolonged membrane depolarization.
  • the disclosed invention relates to methods and compositions for the modulation, diagnosis, and treatment of diseases related to 14266 receptor malfunction.
  • diseases related to 14266 receptor malfunction In addition to variability among individuals in their responses to drugs, several definable diseases arise from disorders in receptors or receptor-effector systems.
  • the loss of a receptor in a highly specialized signaling system may cause a relatively limited phenotypic disorder, such as the genetic deficiency ofthe androgen receptor in the testicular feminization syndrome (Griffin et al. (1995) The Metabolic and Molecular Bases of Inherited Diseases 7:2967-2998).
  • Deficiencies of more widely used signaling systems have a broader spectrum of effects, as are seen in myasthenia gravis or some forms of insulin- resistant diabetes mellitus, which result from autoimmune depletion of nicotinic cholinergic receptors or insulin receptors, respectively.
  • a lesion in a component of a signaling pathway that is used by many receptors can cause a generalized endocrinopathy.
  • Heterozygous deficiency for G5 the G protein that activates adenyl cyclase in all cells, causes multiple endocrine disorders; the disease is termed pseudohpoparathyroidism type la (Spiegel et al.
  • the erbA oncogene product is an altered form of a receptor for thyroid honnone, constitutively active because ofthe loss of its ligand-binding domain (Evans (1988) Science 240:889-895).
  • the ros and erbB oncogene products are activated, uncontrolled forms ofthe receptors for insulin and epidermal growth factor, both known to enhance cellular proliferation (Yarden et al. (1988) Annu. Rev. Biochem. 57:443-478).
  • the mas oncogene product (Young et al. (1986) Cell. 4:711-719) is a G protein-coupled receptor, probably the receptor for a peptide hormone.
  • G protein-coup receptors due to subtle mutations in receptor structure has been shown to give rise to retinitis pigmentosa, precocious puberty, and malignant hyperthyroidism (Clapham (1993) Cell. 75:1237-1239).
  • G proteins can themselves be oncogenic when either overexpressed or constitutively activated by mutation (Lyons et al (1990) Science 249:655-659).
  • Acetylcholine is implicated in higher functions ofthe brain, notably memory and cognition. Consistent with this there is a cholinergic deficiency in Alzheimer' s Disease, an illness associated with a severe impairment of cognitive function. Agonists ofthe acetylcholine receptor have been used clinically in the treatment of glaucoma. Minor uses include suppression of atrial tachycardias, stimulation of intestinal motility and bladder emptying. Antagonists have been used as a premedication in general anesthesia to reduce bronchial and salivary secretions and in the prevention of motion sickness. They have also been used to a limited extent in the treatment of peptic ulcer, to induce pupillary vasodilitation, to aid examination ofthe eye and in the treatment of certain inflammatory conditions.
  • Adrenoceptors are affected by clinically important drugs used for asthma, as an anesthetic, for nasal decongestion, for hypertension, for other cardiovascular disorders, for example, angina, certain cardiac dysrhythmias and cardiac infarction, and for the freatment of anxiety and glaucoma.
  • the angiotensin receptor is the target for compounds effective in the freatment of hypertension.
  • the bradykinin receptor is a target for treatment of inflammation, asthma, mild pain, and endotoxic shock.
  • the calcitonin receptor is a target for treatment of Paget's disease ofthe bone.
  • the cannabinoid receptor is a potential therapeutic target as an analgesic or antiemetic agent.
  • cholecystokinin and gastrin receptors are implicated in the pathogenesis of schizophrenia, Parkinson's disease, drug addiction, and feeding disorders.
  • Dopamine receptors have been implicated in Parkinson's disease, Huntington's disease and schizophrenia.
  • the endothelin receptor is a target for several pathophysiological conditions associated with stress including hypertension, myocardial infarction, subarachnoid hemo ⁇ hage and renal failure.
  • the galanin receptor is involved in insulin release induced by glucose and may be the sympathetic mediator of this effect during stress. It is synergistic with opiates in inducing analgesia. It stimulates feeding behavior and release of growth hormone. It may be of use in the treatment of Alzheimer's disease.
  • Galanin agonists may be novel analgesics.
  • the glucagon receptor is involved in the pathogenesis of diabetes. It is also been implicated in increasing the rate and force of contraction in acute cardiac failure. (12) The receptors for glucagon-like peptides 1 and 2. These receptors could serve as a target for non-insulin dependent diabetes mellitus and intestinal disorders, respectively.
  • Glutamate receptors may be important in neuronal plasticity, cognition, memory, learning and some neurological disorders such as epilepsy, stroke and neurodengeneration.
  • Glycoprotein hormone receptors FSH, LH/hCG, TSH
  • FSH Glycoprotein hormone receptors
  • TSH Graves disease
  • Gonadotropin-releasing hormone receptor is a potential target in therapeutic use in the suppression of prostrate cancer, precocious puberty, and endometriosis.
  • Histamine receptors may be target for a variety of CNS functions including sexual behavior and analgesia. It may also be useful clinically in the treatment of allergic and anaphylactic reactions and various inflammatory conditions for example, hay fever and itching. It may also be useful in treatment of motion sickness.
  • the H2 receptors are found in high levels in stomach and heart. H2 antagonists are used clinically in the treatment of peptic ulceration.
  • 5-hydroxytryptamine receptor may be involved in a vast array of physiological and pathophysiological pathways. It is a mediator of peristalsis and may be involved in platelet aggregation and haemostasis. It may also have a role as an inflammatory mediator and involvement in microvascular control. It could be useful in a wide range of functions including control of appetite, mood, anxiety, hallucination, sleep, vomiting and pain perception and may have clinical use in the freatment of depression, migraine and post-operative vomiting.
  • the 5-Htlb/5-Htld receptor may be the therapeutic substrate ofthe anti-migraine drug sumitriptan. These sites are also implicated in feeding, behavior, anxiety, depression, cardiac function and movement. Clinically, 5-Htl a receptors represent potential anxiolytic and anti-hypertensive targets.
  • Leukotrienes have important physiological roles in the cardiovascular respiratory and immune systems. Some of these are found in high levels in inflammatory conditions, for example, septic shock, inflammatory bowel disease and allergic asthma. They can be found in high levels in bronchial tissue and lung where they may have a pathological role in allergic asthma and respiratory distress syndrome. Accordingly, leukotriene receptors may be useful as targets in these areas. The receptors have been involved in inducing chemostasis and adhesion of neutrophils to vascular endothelium, inducing contraction of gastrointestinal, pulmonary, reproductive, and vascular smooth muscles, and stimulating mucus secretion in bronchial tissue.
  • Melanocortins include ACTH, ⁇ -, ⁇ - and ⁇ -melanocyte-stimulating hormones (MSH), and ⁇ -endorphin.
  • ACTH and ⁇ -endorphin are synthesized and released at times of stress, i.e. cold, infections, etc. and their release leads to metabolism and analgesia.
  • ACTH is used clinically to diagnose adrenocorticol insufficiency and to stimulate adrenocortex function or as an alternative to glucocorticoids to treat inflammatory disorders.
  • Neuropeptide Y is one of the most abundant peptides in the mammalian brain, inducing a variety of behavior effects, stimulation of food intake, anxiety, facilitation of learning and memory, and regulation ofthe cardiovascular and neuroendocrine systems. It has been implicated in the pathophysiology of hypertension, congestive heart failure, affective disorders, and appetite regulation.
  • Neurotensin induces a variety of effects including antinoception, hypothermia and increased locomotor activity.
  • Opioid peptides have important roles in the regulation of sensory function (including pain), neuroendocrine activity, the central control of respiration and mood, and the regulation of gut motility.
  • Non-peptide agonists at opioid receptors include codeine, morphine and related substances. Many of these are used clinically in the treatment of pain and constipation. Some opioid receptors are believe to mediate analgesia sedation, mitosis and diuresis.
  • Parathyroid hormone is involved in calcium homeostasis. Antagonists at the parathyroid hormone receptor are of potential clinical use in the treatment of hyperparathyroidism and short-term hypercalcemic states.
  • Platelet activating factor is an important mediator in allergic and inflammatory conditions. Platelet activating factor antagonists are potential anti- inflammatory and anti-asthmatic agents.
  • Prostanoids prostiglandins and thromboxanes
  • They mediate relaxation in vascular, gastrointestinal, and uterine smooth muscle in human, inhibit platelet activation, and modify release of hypothalamic and pituitary hormones. Some also inhibit neurotransmitter release in central and autonomic nerves and inhibit secretion in glandular tissues, i.e. acid secretion from gastric mucosa and sodium and water reabsorption in kidney.
  • Somatostatin is a neurotransmitter/hormone with a wide spectrum of biological actions. It has been used clinically in the treatment of certain tumors, carcinoid syndrome and glucagonoma. A reduction in cortical somatostatin levels has been reported in Alzheimer's disease and Parkinson's disease.
  • Tachykinins are a family of peptide neurotransmitters. They can stimulate smooth muscle contraction, glandular secretion, induce activation of cells of the immune system, and activate peripheral nerves. They can also regulate dopaminergic neurons and are involved in the transmission of sensory information, including noxious stimuli.
  • Thrombin has a role in blood clotting.
  • the thrombin receptor is a target for the treatment of clotting disorders, and inflammatory disorders.
  • Thyrotrophin releasing hormone releases thyroid stimulating hormone and stimulates the synthesis and release of prolactin.
  • Vasoactive intestinal polypeptide family is grouped with the number of structurally related peptides that share an overlapping profile of biological activity. It induces relaxation in smooth muscle, for example, intestine, blood vessels and trachea. It inhibits secretion in certain tissues for example, stomach. It stimulates secretion in others for example, intestinal epithelium, pancreas, and gall bladder. It modulates activity of cells in the immune system. In the central nervous system it has a wide range of excitatory and inhibitory actions. Some members ofthe family are involved in secretion of enzymes and ions in pancreas and intestine (secretin) and regulating synthesis and release of growth hormone (growth hormone releasing factor).
  • Vasopressin and oxytocin are members of a family of peptides found in all mammalian species. Vasopressin controls the water content ofthe body and acts in the kidney to increase water and sodium absorption. It can stimulate the contraction of vascular smooth muscle, stimulate glycogen breakdown in liver, induce platelet activation, or evoke release or corticotrophin. Vasopressin is used clinically to treat diabetes insipidus. Oxytocin stimulates contraction of uterine smooth muscle, and stimulates milk secretion. It is used clinically to induce labor and to promote lactation.
  • the disclosed invention further relates to the modulation, diagnosis, and treatment of various other disorders.
  • Disorders involving the spleen include, but are not limited to, splenomegaly, including nonspecific acute splenitis, congestive spenomegaly, and spenic infarcts; neoplasms, congenital anomalies, and rupture.
  • disorders associated with splenomegaly include infections, such as nonspecific splenitis, infectious mononucleosis, tuberculosis, typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and echinococcosis; congestive states related to partial hypertension, such as ci ⁇ hosis ofthe liver, portal or splenic vein thrombosis, and cardiac failure; lymphohematogenous disorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia, multiple myeloma, myeloproliferative disorders, hemolytic anemias, and thrombocytopenic purpura; immunologic-inflammatory conditions, such as rheumatoid arthritis and systemic lupus erythe
  • disorders involving the lung include, but are not limited to, congenital anomalies; atelectasis; diseases of vascular origin, such as pulmonary congestion and edema, including hemodynamic pulmonary edema and edema caused by microvascular injury, adult respiratory distress syndrome (diffuse alveolar damage), pulmonary embolism, hemo ⁇ hage, and infarction, and pulmonary hypertension and vascular sclerosis; chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis; diffuse interstitial (infiltrative, restrictive) diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia (pulmonary infiltration with eosinophilia), Bronchiolitis obliterans- organizing pneumonia, diffuse pulmonary hemo ⁇ h
  • disorders involving the colon include, but are not limited to, congenital anomalies, such as atresia and stenosis, Meckel diverticulum, congenital aganglionic megacolon-Hirschsprung disease; enterocolitis, such as diarrhea and dysentery, infectious enterocolitis, including viral gastroenteritis, bacterial enterocolitis, necrotizing enterocolitis, antibiotic-associated colitis (pseudomembranous colitis), and collagenous and lymphocytic colitis, miscellaneous intestinal inflammatory disorders, including parasites and protozoa, acquired immunodeficiency syndrome, transplantation, drug- induced intestinal injury, radiation enterocolitis, neutropenic colitis (typhlitis), and diversion colitis; idiopathic inflammatory bowel disease, such as Crohn disease and ulcerative colitis; tumors ofthe colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and
  • disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and ci ⁇ hosis, such as ci ⁇ hosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and toxin-induced liver disease, such as alcoholic liver disease; inborn e ⁇ ors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, al-antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease,
  • disorders involving the uterus and endometrium include, but are not limited to, endometrial histology in the menstrual cycle; functional endometrial disorders, such as anovulatory cycle, inadequate luteal phase, oral contraceptives and induced endometrial changes, and menopausal and postmenopausal changes; inflammations, such as chronic endometritis; adenomyosis; endometriosis; endometrial polyps; endometrial hyperplasia; malignant tumors, such as carcinoma ofthe endometrium; mixed Mullerian and mesenchymal tumors, such as malignant mixed Mullerian tumors; tumors ofthe myometrium, including leiomyomas, leiomyosarcomas, and endometrial stromal tumors.
  • Disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and hemiation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states-- global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemo ⁇ hage, including intracerebral (intraparenchymal) hemo ⁇ hage, subarachnoid hemo ⁇ hage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar in
  • T-cells disorders involving T-cells include, but are not limited to, cell-mediated hypersensitivity, such as delayed type hypersensitivity and T-cell-mediated cytotoxicity, and transplant rejection; autoimmune diseases, such as systemic lupus erythematosus, Sj ⁇ gren syndrome, systemic sclerosis, inflammatory myopathies, mixed connective tissue disease, and polyarteritis nodosa and other vasculitides; immunologic deficiency syndromes, including but not limited to, primary immunodeficiencies, such as thymic hypoplasia, severe combined immunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory) proliferations of white cells, including but not limited to, leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecific lymphadenitis;
  • autoimmune diseases such as systemic lupus erythematosus, Sj ⁇ gren syndrome, systemic sclerosis, inflammatory myopathies, mixed connective tissue disease, and polyarteritis no
  • lymphoid neoplasms such as precursor T-cell neoplasms, such as acute lymphoblastic leukemia/lymphoma, peripheral T-cell and natural killer cell neoplasms that include peripheral T-cell lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides and Sezary syndrome, and Hodgkin disease.
  • Diseases ofthe skin include but are not limited to, disorders of pigmentation and melanocytes, including but not limited to, vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, and malignant melanoma; benign epithelial tumors, including but not limited to, sebo ⁇ heic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors ofthe dermis, including but not limited to, benign fibrous histiocytoma, dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of the
  • the myelocytic series (polymorphoneuclear cells) make up approximately 60% ofthe cellular elements, and the erythrocytic series, 20-30%. Lymphocytes, monocytes, reticular cells, plasma cells and megakaryocytes together constitute 10-20%. Lymphocytes make up 5-15% of normal adult ma ⁇ ow.
  • cell types are add mixed so that precursors of red blood cells (erythroblasts), macrophages (monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes (myeloblasts), and lymphocytes (lymphoblasts) can be visible in one microscopic field.
  • the invention is directed to disorders arising from these cells. These disorders include but are not limited to the following: diseases involving hematopoeitic stem cells; committed lymphoid progenitor cells; lymphoid cells, including B and T- cells; committed myeloid progenitors, including monocytes, granulocytes, and megakaryocytes; and committed erythroid progenitors.
  • leukemias include B-lymphoid leukemias, T-lymphoid leukemias, ⁇ differentiated leukemias; erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias are encompassed with and without differentiation]; chronic and acute lymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronic and acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic and acute myeloblastic leukemia, chronic and acute myelogenous leukemia, chronic and acute promyelocytic leukemia, chronic and acute myelocytic leukemia, hematologic malignancies of monocyte- macrophage lineage, such as juvenile chronic myelogenous leukemia; secondary AML, antecedent hematological disorder; refractory anemia;
  • disorders involving the heart include but are not limited to, heart failure, including but not limited to, cardiac hypertrophy, left-sided heart failure, and right-sided heart failure; ischemic heart disease, including but not limited to angina pectoris, myocardial infarction, chronic ischemic heart disease, and sudden cardiac death; hypertensive heart disease, including but not limited to, systemic (left-sided) hypertensive heart disease and pulmonary (right-sided) hypertensive heart disease; valvular heart disease, including but not limited to, valvular degeneration caused by calcification, such as calcific aortic stenosis, calcification of a congenitally bicuspid aortic valve, and mitral annular calcification, and myxomatous degeneration ofthe mitral valve (mitral valve prolapse), rheumatic fever and rheumatic heart disease, infective endocarditis, and noninfected vegetations, such as nonbacterial thrombotic endocarditis and endo
  • disorders involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease-the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis
  • tumors including benign tumors and tumor-like conditions, such as hemangioma, lymphangioma, glomus tumor (glomangiom
  • disorders involving red cells include, but are not limited to, anemias, such as hemolytic anemias, including hereditary spherocytosis, hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphate dehydrogenase deficiency, sickle cell disease, thalassemia syndromes, paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, and hemolytic anemia resulting from trauma to red cells; and anemias of diminished erythropoiesis, including megaloblastic anemias, such as anemias of vitamin B12 deficiency: pernicious anemia, and anemia of folate deficiency, iron deficiency anemia, anemia of chronic disease, aplastic anemia, pure red cell aplasia, and other forms of ma ⁇ ow failure.
  • anemias such as hemolytic anemias, including hereditary spherocytosis, hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphate dehydrogena
  • Thamomas can include benign or encapsulated thymoma, and malignant thymoma Type I (invasive thymoma) or Type II, designated thymic carcinoma.
  • B-cells include, but are not limited to precursor B-cell neoplasms, such as lymphoblastic leukemia lymphoma.
  • Peripheral B-cell neoplasms include, but are not limited to, chronic lymphocytic leukemia small lymphocytic lymphoma, foUicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and related entities, lymphoplasmacytic lymphoma (Waldensfrom macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma (MALToma), and hairy cell leukemia.
  • lymphoplasmacytic lymphoma (Waldensfrom macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma (MALToma), and hairy cell leukemia.
  • disorders involving the kidney include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia, autosomal dominant (adult) polycystic kidney disease, autosomal recessive (childhood) polycystic kidney disease, and cystic diseases of renal medulla, which include, but are not limited to, medullary sponge kidney, and nephronophthisis-uremic medullary cystic disease complex, acquired (dialysis- associated) cystic disease, such as simple cysts; glomerular diseases including pathologies of glomerular injury that include, but are not limited to, in situ immune complex deposition, that includes, but is not limited to, anti-GBM nephritis, Heymann nephritis, and antibodies against planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative
  • HUS/TTP and other vascular disorders including, but not limited to, atherosclerotic ischemic renal disease, atheroembolic renal disease, sickle cell disease nephropathy, diffuse cortical necrosis, and renal infarcts; urinary tract obstruction (obstructive uropathy); urolithiasis (renal calculi, stones); and tumors ofthe kidney including, but not limited to, benign tumors, such as renal papillary adenoma, renal fibroma or hamartoma (renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma, and malignant tumors, including renal cell carcinoma (hypemephroma, adenocarcinoma of kidney), which includes urothelial carcinomas of renal pelvis.
  • benign tumors such as renal papillary adenoma, renal fibroma or hamartoma (renomedullary interstitial cell tumor), angiomyolipoma, and oncocyto
  • disorders ofthe breast include, but are not limited to, disorders of development; inflammations, including but not limited to, acute mastitis, periductal mastitis, periductal mastitis (recu ⁇ ent subareolar abscess, squamous metaplasia of lactiferous ducts), mammary duct ectasia, fat necrosis, granulomatous mastitis, and pathologies associated with silicone breast implants; fibrocystic changes; proliferative breast disease including, but not limited to, epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors including, but not limited to, sfromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papiUoma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's
  • Testis and epididymis disorders involving the testis and epididymis include, but are not limited to, congenital anomalies such as cryptorchidism, regressive changes such as atrophy, inflammations such as nonspecific epididymitis and orchitis, granulomatous (autoimmune) orchitis, and specific inflammations including, but not limited to, gono ⁇ hea, mumps, tuberculosis, and syphilis, vascular disturbances including torsion, testicular tumors including germ cell tumors that include, but are not limited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolk sac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sex cord-gonadal sfroma including, but not limited to, Ley dig (interstitial) cell tumors and sertoli cell tumors
  • congenital anomalies such as cryptorchidism
  • regressive changes such as atrophy
  • carcinoma a malignant neoplasm originating from adenosar cells.
  • androblastoma and testicular lymphoma, and miscellaneous lesions of tunica vaginalis.
  • disorders involving the prostate include, but are not limited to, inflammations, benign enlargement, for example, nodular hyperplasia (benign prostatic hypertrophy or hyperplasia), and tumors such as carcinoma.
  • disorders involving the thyroid include, but are not limited to, hyperthyroidism; hypothyroidism including, but not limited to, cretinism and myxedema; thyroiditis including, but not limited to, hashimoto thyroiditis, subacute (granulomatous) thyroiditis, and subacute lymphocytic (painless) thyroiditis; Graves disease; diffuse and multinodular goiter including, but not limited to, diffuse nontoxic (simple) goiter and multinodular goiter; neoplasms ofthe thyroid including, but not limited to, adenomas, other benign tumors, and carcinomas, which include, but are not limited to, papillary carcinoma, foUicular carcinoma, medullary carcinoma, and anaplastic carcinoma; and cogenital anomalies.
  • Disorders involving the skeletal muscle include tumors such as rhabdomyosarcoma.
  • Disorders involving the pancreas include those ofthe exocrine pancreas such as congenital anomalies, including but not limited to, ectopic pancreas; pancreatitis, including but not limited to, acute pancreatitis; cysts, including but not limited to, pseudocysts; tumors, including but not limited to, cystic tumors and carcinoma ofthe pancreas; and disorders ofthe endocrine pancreas such as, diabetes mellitus; islet cell tumors, including but not limited to, insulinomas, gastrinomas, and other rare islet cell tumors.
  • disorders involving the small intestine include the malabsorption syndromes such as, celiac sprue, tropical sprue (postinfectious sprue), whipple disease, disaccharidase (lactase) deficiency, abetalipoproteinemia, and tumors ofthe small intestine including adenomas and adenocarcinoma.
  • malabsorption syndromes such as, celiac sprue, tropical sprue (postinfectious sprue), whipple disease, disaccharidase (lactase) deficiency, abetalipoproteinemia, and tumors ofthe small intestine including adenomas and adenocarcinoma.
  • thrombocytopenia disorders related to reduced platelet number, thrombocytopenia, include idiopathic thrombocytopenic purpura, including acute idiopathic thrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic microangiopathies: thrombotic thrombocytopenic purpura and hemolytic- uremic syndrome.
  • disorders involving precursor T-cell neoplasms include precursor T lymphoblastic leukemia/lymphoma.
  • Disorders involving peripheral T-cell and natural killer cell neoplasms include T-cell chronic lymphocytic leukemia, large granular lymphocytic leukemia, mycosis fungoides and Sezary syndrome, peripheral T-cell lymphoma, unspecified, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma (NK/T-cell lymphoma4a), intestinal T-cell lymphoma, adult T-cell leukemia/lymphoma, and anaplastic large cell lymphoma.
  • ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors. Bone-forming
  • disorders ofthe bone are complex because they may have an impact on the skeleton during any of its stages of development. Hence, the disorders may have variable manifestations and may involve one, multiple or all bones ofthe body.
  • Such disorders include, congenital malformations, achondroplasia and thanatophoric dwarfism, diseases associated with abnormal matix such as type 1 collagen disease, osteoporosis, Paget disease, rickets, osteomalacia, high-turnover osteodystrophy, low-turnover of aplastic disease, osteonecrosis, pyogenic osteomyelitis, tuberculous osteomyelitism, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous cortical defects, fibrous dysplasia, fibrosarcoma, malignant fibrous histiocytoma,
  • the 14266 sequences ofthe invention are members of a family of molecules (the "G-protein coupled receptors" or "GPCRs") having conserved functional features.
  • the term "family" when refe ⁇ ing to the proteins and nucleic acid molecules ofthe invention is intended to mean two or more proteins or nucleic acid molecules having sufficient amino acid or nucleotide sequence identity as defined herein.
  • Such family members can be naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of murine origin and a homologue of that protein of human origin, as well as a second, distinct protein of human origin and a murine homologue of that protein.
  • Members of a family may also have common functional characteristics.
  • the invention provides methods using the 14266 variants, or fragments, including but not limited to use in the cells, tissues, and disorders as disclosed herein.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17) :3389-3402.
  • the default parameters ofthe respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See http://www.ncbi.nlm.nih.gov.
  • the 14266 polypeptides are useful for producing antibodies specific for the 14266, regions, or fragments.
  • the invention provides a method (also refe ⁇ ed 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) that bind to 14266 receptors or have a stimulatory or inhibitory effect on, for example, 14266 receptor expression or 14266 receptor activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules, or other drugs) that bind to 14266 receptors or have a stimulatory or inhibitory effect on, for example, 14266 receptor expression or 14266 receptor activity.
  • the invention provides screening assays, in cell-based or cell-free systems.
  • Cell-based systems can be native, i.e., cells that normally express the 14266 receptor, as a biopsy, or expanded in cell culture.
  • cell-based assays involve recombinant host cells expressing the 14266 receptor.
  • cells that are useful in this regard include, but are not limited to, those disclosed herein as expressing 1466.
  • Cells containing one or more copies of exogenously-introduced 14266 sequences or cells genetically modified to modulate expression ofthe endogenous 14266 sequence may also be used.
  • test compounds ofthe present invention can be obtained using any ofthe 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.
  • biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, nonpeptide ohgomer, or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).
  • Determining the ability ofthe test compound to bind to the 14266 receptor can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the 14266 receptor or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, ! C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • target molecule is intended a molecule with which a 14266 receptor binds or interacts in nature.
  • the ability ofthe 14266 receptor to bind to or interact with a 14266 target molecule can be determined by monitoring the activity ofthe target molecule.
  • the activity ofthe target molecule can be monitored by detecting induction of a cellular second messenger ofthe target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a 14266 -responsive regulatory element operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cellular differentiation or cell proliferation.
  • a reporter gene e.g., a 14266 -responsive regulatory element operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase
  • detecting a cellular response for example, cellular differentiation or cell proliferation.
  • an assay ofthe present invention is a cell-free assay comprising contacting a 14266 receptor or biologically active portion thereof with a test compound and determining the ability ofthe test compound to bind to the 14266 receptor or biologically active portion thereof. Binding ofthe test compound to the 14266 receptor can be determined either directly or indirectly as described above.
  • the assay includes contacting the 14266 receptor or biologically active portion thereof with a known compound that binds the 14266 receptor to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to preferentially bind to the 14266 receptor or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting the 14266 receptor or biologically active portion thereof with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe 14266 receptor or biologically active portion thereof. Determining the ability ofthe test compound to modulate the activity of a 14266 receptor can be accomplished, for example, by determining the ability ofthe 14266 receptor to bind to a 14266 target molecule as described above for determining direct binding. In an alternative embodiment, determining the ability ofthe test compound to modulate the activity of a 14266 receptor can be accomplished by determining the ability ofthe 14266 receptor to further modulate a 14266 target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay comprises contacting the 14266 receptor or biologically active portion thereof with a known compound that binds a 14266 receptor to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to preferentially bind to or modulate the activity of a 14266 target molecule.
  • a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • glutathione-S- fransferase/14266 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the complexes can be dissociated from the matrix, and the level of 14266 binding or activity determined using standard techniques.
  • immobilizing proteins on matrices can also be used in the screening assays ofthe invention.
  • either the 14266 receptor or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated 14266 molecules 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 Chemicals).
  • biotinylation kit Pierce Chemicals, Rockford, IL
  • streptavidin-coated 96- well plates Piereptavidin-coated 96- well plates
  • antibodies reactive with a 14266 receptor or target molecules but which do not interfere with binding ofthe 14266 receptor to its target molecule can be derivatized to the wells ofthe plate, and unbound target or 14266 receptor trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the 14266 receptor or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the 14266 receptor or target molecule.
  • modulators of 14266 expression are identified in a method in which a cell is contacted with a candidate compound and the expression of 14266 mRNA or protein in the cell is determined relative to expression of 14266 mRNA or protein in a cell in the absence ofthe candidate compound.
  • the candidate compound When expression is greater (statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of 14266 mRNA or protein expression. Alternatively, when expression is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of 14266 mRNA or protein expression.
  • the level of 14266 mRNA or protein expression in the cells can be determined by methods described herein for detecting 14266 mRNA or protein.
  • thel4266 receptors 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.
  • 14266-binding proteins bind to or interact with 14266 receptor
  • 14266-binding proteins bind to or interact with 14266 receptor
  • Such 14266-binding proteins are also likely to be involved in the propagation of signals by the 14266 receptors as, for example, upstream or downstream elements ofthe signal transduction pathway.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • One aspect ofthe present invention relates to diagnostic assays for detecting 14266 receptor and/or nucleic acid expression as well as 14266 activity, in the context of a biological sample.
  • 14266 receptors 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 14266 receptor or nucleic acid (e.g., mRNA, genomic DNA) that encodes 14266 receptor such that the presence of 14266 receptor is detected in the biological sample.
  • Results obtained with a biological sample from the test subject may be compared to results obtained with a biological sample from a confrol subject.
  • “Misexpression or abe ⁇ ant expression”, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non- wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms ofthe time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms ofthe splicing size, amino acid sequence, post-transitional modification, or biological activity ofthe expressed polypeptide; a pattern of expression that differs from wild type in terms ofthe effect of an environmental stimulus or extracellular stimulus on expression ofthe gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in
  • a prefe ⁇ ed agent for detecting 14266 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to 14266 mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length nucleic acid of SEQ ID NO:2, or a portion thereof, such as a nucleic acid molecule of at least 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 14266 mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays ofthe invention are described herein.
  • a prefe ⁇ ed agent for detecting 14266 receptor is an antibody capable of binding to 14266 receptor, 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(abN) )can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe 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 ofthe invention can be used to detect 14266 mRNA, protein, or genomic DNA in a biological sample in vifro as well as in vivo.
  • vifro techniques for detection of 14266 mRNA include Northern hybridizations and in situ hybridizations.
  • vifro techniques for detection ofthe 14266 receptor include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • vifro techniques for detection of 14266 genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of 14266 receptor include introducing into a subject a labeled anti- 14266 antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • kits for detecting the presence of 14266 receptors 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 14266 receptor.
  • the kit can comprise a labeled compound or agent capable of detecting 14266 receptor or mRNA in a biological sample and means for determining the amount of a 14266 receptor in the sample (e.g., an anti- 14266 antibody or an oligonucleotide probe that binds to DNA encoding a 14266 receptor, e.g., encoded by the nucleic acid sequences of SEQ ID NO:2).
  • 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 14266 sequences if the amount of 14266 receptor or mRNA is above or below a normal level.
  • the kit can comprise, for example: (1) a first antibody
  • the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, that hybridizes to a 14266 nucleic acid sequence or (2) a pair of primers useful for amplifying a 14266 nucleic acid molecule.
  • an oligonucleotide e.g., a detectably labeled oligonucleotide, that hybridizes to a 14266 nucleic acid sequence or (2) a pair of primers useful for amplifying a 14266 nucleic acid molecule.
  • 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 that can be assayed and compared to the test sample contained.
  • Each component ofthe kit is usually enclosed within an individual container, and all ofthe 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 abe ⁇ ant expression of 14266 receptors.
  • the invention features a method of analyzing a plurality of capture probes.
  • the method can be used, e.g., to analyze gene expression.
  • the method includes: providing a two dimensional a ⁇ ay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence; contacting the a ⁇ ay with a 14266 nucleic acid, preferably purified, polypeptide, preferably purified, or antibody, and thereby evaluating the plurality of capture probes.
  • Binding e.g., in the case of a nucleic acid, hybridization, with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a label attached to the 14266 nucleic acid, polypeptide, or antibody.
  • the capture probes can be a set of nucleic acids from a selected sample, e.g., a sample of nucleic acids derived from a confrol or non-stimulated tissue or cell.
  • the method can include contacting the 14266 nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second a ⁇ ay having a different plurality of capture probes.
  • the results of each hybridization can be compared, e.g., to analyze differences in expression between a first and second sample.
  • the first plurality of capture probes can be from a control sample, e.g., a wild type, normal, or non-diseased, non-stimulated, sample, e.g., a biological fluid, tissue, or cell sample.
  • the second plurality of capture probes can be from an experimental sample, e.g., a mutant type, at risk, disease-state or disorder-state, or stimulated, sample, e.g., a biological fluid, tissue, or cell sample.
  • the plurality of capture probes can be a plurality of nucleic acid probes each of which specifically hybridizes, with an allele of a 14266 sequence ofthe invention.
  • Such methods can be used to diagnose a subject, e.g., to evaluate risk for a disease or disorder, to evaluate suitability of a selected treatment for a subject, to evaluate whether a subject has a disease or disorder.
  • the method can be used to detect single nucleotide polymo ⁇ hisms (SNPs), as described below.
  • SNPs single nucleotide polymo ⁇ hisms
  • the invention features a method of analyzing a plurality of probes.
  • the method is useful, e.g., for analyzing gene expression.
  • the method includes: providing a two dimensional a ⁇ ay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express a 14266 polypeptide ofthe invention or from a cell or subject in which a 14266-mediated response has been elicited, e.g., by contact ofthe cell with a 14266 nucleic acid or protein ofthe invention, or adminisfration to the cell or subject a 14266 nucleic acid or protein ofthe invention; contacting the a ⁇ ay with one or more inquiry probes, wherein an inquiry probe can be a nucleic acid, polypeptide, or antibody (which is preferably other than a 14266 nucleic acid, polypeptide, or
  • Binding e.g., in the case of a nucleic acid, hybridization, with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.
  • the invention features a method of analyzing a 14266 sequence ofthe invention, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences.
  • the method includes: providing a 14266 nucleic acid or amino acid sequence, e.g., the sequence set forth in SEQ ID NO:2 (nucleic acid) or SEQ ID NO: 1 (amino acid) or a portion thereof; comparing the 14266 sequence with one or more, preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze the 14266 sequence ofthe invention.
  • the method can include evaluating the sequence identity between a 14266 sequence ofthe invention and a database sequence.
  • the method can be performed by accessing the database at a second site, e.g., over the internet.
  • the invention features, a set of oligonucleotides, useful, e.g., for identifying SNP's, or identifying specific alleles of a 14266 sequence ofthe invention.
  • the set includes a plurality of oligonucleotides, each of which has a different nucleotide at an inte ⁇ ogation position, e.g., an SNP or the site of a mutation.
  • the oligonucleotides can be provided with differential labels, such that an oligonucleotides which hybridizes to one allele provides a signal that is distinguishable from an oligonucleotides which hybridizes to a second allele.
  • 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 14266 receptor, 14266 nucleic acid expression, or 14266 activity. Prognostic assays can be used for prognostic or predictive purposes to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with 14266 receptor, 14266 nucleic acid expression, or 14266 activity.
  • test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the present invention provides methods for determining whether a subject can be administered a specific agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, antibody, nucleic acid (including an antisense nucleic acid or a ribozyme), small molecule, or other drug candidate) or class of agents (e.g., agents of a type that decrease 14266 activity) to effectively treat a disease or disorder associated with abe ⁇ ant 14266 expression or activity.
  • a test sample is obtained and 14266 receptor or nucleic acid is detected.
  • the presence of 14266 receptor or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with abe ⁇ ant 14266 expression or activity.
  • the methods ofthe invention can also be used to detect genetic lesions or mutations in a 14266 gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by abe ⁇ ant cell proliferation and/or differentiation.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding a 14266 protein, or the misexpression ofthe 14266 gene.
  • such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: (1) a deletion of one or more nucleotides from a 14266 gene; (2) an addition of one or more nucleotides to a 14266 gene; (3) a substitution of one or more nucleotides of a 14266 gene; (4) a chromosomal rearrangement of a 14266 gene; (5) an alteration in the level of a messenger RNA transcript of a 14266 gene; (6) an abe ⁇ ant modification of a 14266 gene, such as ofthe methylation pattern ofthe genomic DNA; (7) the presence of a non- wild-type splicing pattern of a messenger RNA transcript of a 14266 gene; (8) a non- wild-type level of a 14266-protein; (9) an allelic loss of a 14266 gene; and (10) an inappropriate post- translational modification of a 14266-protein.
  • detection ofthe 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 ⁇ seful for detecting point mutations in the 14266-gene (see, e.g.,
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe 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 ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a 14266 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns of isolated test sample and confrol DNA digested with one or more restriction endonucleases.
  • sequence specific ribozymes see, e.g., U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in a 14266 molecule 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).
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the 14266 gene and detect mutations by comparing the sequence ofthe sample 14266 gene 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.
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the 14266 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). See, also 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 confrol DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more "DNA mismatch repair" enzymes that recognize mismatched base pairs in double- stranded DNA in defined systems for detecting and mapping point mutations in 14266 cDNAs obtained from samples of cells. See, e.g., Hsu et al. (1994) Carcinogenesis 15 : 1657- 1662.
  • a probe based on a 14266 sequence e.g., a wild-type 14266 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 14266 genes.
  • single-strand conformation polymorphism SSCP
  • SSCP single-strand conformation polymorphism
  • the sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of confrol 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 that 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).
  • 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.
  • 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 ofthe molecule so that amplification depends on differential hybridization (Gibbs et al. (1989) Nucleic Acids Res.
  • ligation will occur only if there is a perfect match at the 3' end ofthe 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnosed patients exhibiting symptoms or family history of a disease or illness involving a 14266 gene.
  • Agents, or modulators that have a stimulatory or inhibitory effect on 14266 activity can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with abe ⁇ ant 14266 activity as well as to modulate the phenotype of a differentiative or cell proliferation disorder.
  • the pharmacogenomics i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active drug.
  • the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of 14266 receptor, expression of 14266 nucleic acid, or mutation content of 14266 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual..
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are 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 antimalarials, sulfonamides, analgesics, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map ofthe human genome consisting of already known gene-related markers (e.g., a "bi- allelic” gene marker map which consists of 60,000- 100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g., a "bi- allelic” gene marker map which consists of 60,000- 100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymo ⁇ hisms (SNPs) in the human genome.
  • SNP single nucleotide polymo ⁇ hisms
  • an "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, freatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 14266 receptor ofthe present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version ofthe gene versus another is associated with a particular drug response.
  • a gene that encodes a drug's target e.g., a 14266 receptor ofthe present invention
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., a 14266 molecule or 14266 modulator of the present invention
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one ofthe above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual.
  • 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 14266 molecule or 14266 modulator ofthe invention, such as a modulator identified by one of the exemplary screening assays described herein.
  • the present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more ofthe gene products encoded by one or more ofthe 14266 genes ofthe present invention, wherein these products may be associated with resistance ofthe cells to a therapeutic agent.
  • the activity ofthe proteins encoded by the 14266 genes ofthe present invention can be used as a basis for identifying agents for overcoming agent resistance.
  • By blocking the activity of one or more ofthe resistance proteins, target cells will become sensitive to treatment with an agent that the unmodified target cells were resistant to.
  • Agents ofthe prsent invention include small molecule modulators, antibodies, ribozymes, peptides, and antisense nucleic acid molecules. Monitoring the influence of agents (e.g., drugs) on the expression or activity of a
  • 14266 receptor can be applied in clinical trials.
  • the effectiveness of an agent determined by a screening assay as described herein to increase 14266 gene expression, protein levels, or upregulate 14266 activity can be monitored in clinical trials of subjects exhibiting decreased 14266 gene expression, protein levels, or downregulated 14266 activity.
  • the effectiveness of an agent determined by a screening assay to decrease 14266 gene expression, protein levels, or downregulate 14266 activity can be monitored in clinical trials of subjects exhibiting increased 14266 gene expression, protein levels, or upregulated 14266 activity.
  • the expression or activity of a 14266 gene and preferably, other genes that have been implicated in, for example, a 14266-associated disorder can be used as a "read out" or markers ofthe phenotype of a particular cell.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • the activity of 14266 receptor, expression of 14266 nucleic acid, or mutation content of 14266 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 14266 modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.
  • Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of 14266 genes can be applied not only in basic drug screening but also in clinical trials.
  • agents e.g., drugs, compounds
  • the effectiveness of an agent, as determined by a screening assay as described herein, to increase or decrease 14266 gene expression, protein levels, or protein activity can be monitored in clinical trials of subjects exhibiting decreased or increased 14266 gene expression, protein levels, or protein activity.
  • 14266 expression or activity and preferably that of other genes that have been implicated in for example, a cellular proliferation disorder can be used as a marker of the immune responsiveness of a particular cell.
  • genes that are modulated in cells by treatment with an agent e.g., compound, drug, or small molecule
  • an agent e.g., compound, drug, or small molecule
  • 14266 activity e.g., as identified in a screening assay described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of 14266 genes 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 ofthe methods as described herein, or by measuring the levels of activity of 14266 genes or other genes.
  • the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during, freatment ofthe individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of freatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, antibody, nucleic acid (including an antisense oligonucleotide or a ribozyme), small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (1) obtaining a preadministration sample from a subject prior to administration ofthe agent; (2) detecting the level of expression of a 14266 receptor, mRNA, or genomic DNA in the preadministration sample; (3) obtaining one or more postadministration samples from the subject; (4) detecting the level of expression or activity ofthe 14266 receptor, mRNA, or genomic DNA in the postadministration samples; (5) comparing the level of expression or activity ofthe 14266 receptor, mRNA, or genomic DNA in the preadministration sample with the 14266 receptor, mRNA, or genomic DNA in the postadministration sample or samples; and (vi)
  • the present invention provides for both prophylactic and therapeutic methods of freating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abe ⁇ ant 14266 expression or activity.
  • Subject can refer to a mammal, e.g. a human, or to an experimental or animal or disease model.
  • the subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.
  • the compositions ofthe invention find use in the treatment of disorders described herein.
  • therapies for disorders associated with CCC are encompassed herein.
  • Treatment is herein defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the pu ⁇ ose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
  • a “therapeutic agent” includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.
  • the invention provides a method for preventing in a subject a disease or condition associated with an abe ⁇ ant 14266 expression or activity by administering to the subject an agent that modulates 14266 expression or at least one 14266 gene activity.
  • Subjects at risk for a disease that is caused, or contributed to, by abe ⁇ ant 14266 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe 14266 abe ⁇ ancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a 14266 agonist or 14266 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • the modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of 14266 receptor activity associated with the cell.
  • An agent that modulates 14266 receptor activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a 14266 receptor, a peptide, a 14266 peptidomimetic, or other small molecule.
  • the agent stimulates one or more ofthe biological activities of 14266 receptor.
  • stimulatory agents include active 14266 receptor and a nucleic acid molecule encoding a 14266 receptor that has been introduced into the cell.
  • the agent inhibits one or more ofthe biological activities of 14266 receptor.
  • inhibitory agents include antisense 14266 nucleic acid molecules and anti- 14266 antibodies.
  • modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by abe ⁇ ant expression or activity of a 14266 receptor or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or a combination of agents, that modulates (e.g., upregulates or downregulates) 14266 expression or activity.
  • the method involves administering a 14266 receptor or nucleic acid molecule as therapy to compensate for reduced or abe ⁇ ant 14266 expression or activity.
  • Stimulation of 14266 activity is desirable in situations in which a 14266 receptor is abnormally downregulated and/or in which increased 14266 activity is likely to have a beneficial effect. Conversely, inhibition of 14266 activity is desirable in situations in which 14266 activity is abnormally upregulated and/or in which decreased 14266 activity is likely to have a beneficial effect.
  • the invention thus relates to a human 14266 and to the expression of a 14266 having the deduced amino acid sequence shown in Figure 1 (SEQ ID NO:l).
  • 14266 polypeptide or " 14266 protein” refers to the polypeptide in SEQ ID NO:l
  • 14266 protein or “14266 polypeptide,” however, further includes the numerous variants described herein, as well as fragments derived from the full-length 14266 and variants.
  • Prefe ⁇ ed 14266 polypeptides ofthe present invention have an amino acid sequence sufficiently identical to the amino acid sequence encoded by the nucleic acid sequences of SEQ ID NO:2.
  • the term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
  • amino acid or nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently identical.
  • the percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970) J Mol. Biol. 48:AAA-A53 algorithm which has been inco ⁇ orated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly prefe ⁇ ed set of parameters is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
  • the default parameters ofthe respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • a "14266 protein activity”, “biological activity of a 14266 protein”, or “functional activity of a 14266 protein” refers to an activity exerted by a 14266 protein, polypeptide, or nucleic acid molecule on a 14266 responsive cell as determined in vivo, or in vitro, according to standard assay techniques.
  • a 14266 activity can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction ofthe 14266 protein with a second protein.
  • a 14266 activity includes at least one or more ofthe following activities: (1) modulating (stimulating and/or enhancing or inhibiting) cellular proliferation, differentiation, and/or function; (2) mobilization of intracellular molecules that participate in a signal fransduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP 2 ), inositol 1,4,5-triphosphate (IP 3 ) and adenylate cyclase; (3) polarization ofthe plasma membrane; (4) production or secretion of molecules; (5) alteration in the structure of a cellular component; (6) cell proliferation, e.g., synthesis of DNA; (7) cell migration; (8) cell differentiation (including durophil differentiation); (9) cell survival and (10) ligand-binding.
  • a signal fransduction pathway e.g., phosphatidylinositol 4,5-bisphosphate (PIP 2 ), inositol 1,4,5-triphosphate (IP 3
  • an “isolated” or “purified” 14266 nucleic acid molecule or protein, or biologically active portion thereof is 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.
  • an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • isolated when used to refer to nucleic acid molecules excludes isolated chromosomes.
  • the isolated 14266 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 that naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived.
  • a 14266 protein that is substantially free of cellular material includes preparations of 14266 protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non- 14266 protein (also refe ⁇ ed to herein as a "contaminating protein").
  • culture medium represents less than about 30%, 20%, 10%, or 5% ofthe volume of the protein preparation.
  • 14266 protein is produced by chemical synthesis, preferably the protein preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non- 14266 chemicals.
  • 14266 receptor is intended a protein having the amino acid sequence encoded by the amino acid sequence set forth in SEQ ID NOS: as well as fragments, biologically active portions, and variants thereof.
  • Fragments or “biologically active portions” include polypeptide fragments suitable for use as immunogens to raise anti- 14266 antibodies. Fragments include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a 14266 protein, or a fragment thereof, ofthe invention and exhibiting at least one activity of a 14266 protein, but which include fewer amino acids than the 14266 protein encoded by the nucleic acid sequences disclosed herein. Typically, biologically active portions comprise a domain or motif with at least one activity ofthe 14266 protein.
  • a biologically active portion of a 14266 protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • Such biologically active portions can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 14266 protein.
  • a fragment comprises at least 5 contiguous amino acids of an amino acid sequence set forth in SEQ ID NO:l.
  • the invention encompasses other fragments, however, such as any fragment in the protein greater than 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.
  • variants proteins or polypeptides having an amino acid sequence that is at least about 45%, 55%, 65%, preferably about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NO: 1.
  • Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO:2, or a complement thereof, under stringent conditions. Such variants generally retain the functional activity ofthe 14266 proteins ofthe invention.
  • variants include polypeptides that differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • the invention also provides 14266 chimeric or fusion proteins.
  • a 14266 "chimeric protein” or “fusion protein” comprises a 14266 polypeptide operably linked to a non- 14266 polypeptide.
  • a “14266 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a 14266 protein
  • a “non- 14266 polypeptide” refers to a polypeptide having an amino acid sequence co ⁇ esponding to a protein that is not substantially identical to the 14266 protein, e.g., a protein that is different from the 14266 protein and which is derived from the same or a different organism.
  • the 14266 polypeptide can co ⁇ espond to all or a portion of a 14266 protein, preferably at least one biologically active portion of a 14266 protein.
  • the term "operably linked" is intended to indicate that the 14266 polypeptide and the non- 14266 polypeptide are fused in-frame to each other.
  • the non-14266 polypeptide can be fused to the N-terminus or C-terminus ofthe 14266 polypeptide.
  • One useful fusion protein is a GST- 14266 fusion protein in which the 14266 sequences are fused to the C-terminus ofthe GST sequences. Such fusion proteins can facilitate the purification of recombinant 14266 proteins.
  • the fusion protein is a 14266-immunoglobulin fusion protein in which all or part of a 14266 protein is fused to sequences derived from a member ofthe immunoglobulin protein family.
  • the 14266-immunoglobulin fusion proteins ofthe invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a 14266 ligand and a 14266 protein on the surface of a cell, thereby suppressing 14266-mediated signal fransduction in vivo.
  • the 14266-immunoglobulin fusion proteins can be used to affect the bioavailability of a 14266 cognate ligand.
  • Inhibition ofthe 14266 ligand/ 14266 interaction may be useful therapeutically, both for treating proliferative, differentiative, developmental and hemOpoietic disorders and for modulating (e.g., promoting or inhibiting) cell survival.
  • the 14266 -immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti- 14266 antibodies in a subject, to purify 14266 ligands, and in screening assays to identify molecules that inhibit the interaction of a 14266 protein with a 14266 ligand.
  • a 14266 chimeric or fusion protein ofthe invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences may be ligated together in-frame, or the fusion gene can be synthesized, such as with automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and reamplif ⁇ ed to generate a chimeric gene sequence (see, e.g., Ausubel et ah, eds. (1995) Current Protocols in Molecular Biology) (Greene Publishing and Wiley-Interscience, NY).
  • a 14266-encoding nucleic acid can be cloned into a commercially available expression vector such that it is linked in- frame to an existing fusion moiety.
  • Variants ofthe 14266 proteins can function as either 14266 agonists (mimetics) or as 14266 antagonists. Variants ofthe 14266 protein can be generated by mutagenesis, e.g., discrete point mutation or truncation ofthe 14266 protein. An agonist ofthe 14266 protein can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occu ⁇ ing form ofthe 14266 protein. An antagonist ofthe 14266 protein can inhibit one or more ofthe activities ofthe naturally occu ⁇ ing form ofthe 14266 protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade that includes the 14266 protein.
  • variants of limited function Treatment of a subject with a variant having a subset of the biological activities ofthe naturally occurring form ofthe protein can have fewer side effects in a subject relative to treatment with the naturally occu ⁇ ing form ofthe 14266 proteins.
  • Variants of a 14266 protein that function as either 14266 agonists or as 14266 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 14266 protein for 14266 protein agonist or antagonist activity.
  • a variegated library of 14266 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of 14266 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential 14266 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of 14266 sequences therein.
  • libraries of fragments of a 14266 protein coding sequence can be used to generate a variegated population of 14266 fragments for screening and subsequent selection of variants of a 14266 protein.
  • a library of coding sequence fragments can be generated by freating a double-stranded PCR fragment of a 14266 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA which can include sense/antisense pairs from different nicked products, removing single-stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library that encodes N-terminal and internal fragments of various sizes ofthe 14266 protein.
  • REM Recursive ensemble mutagenesis
  • An isolated 14266 polypeptide ofthe invention can be used as an immunogen to generate antibodies that bind 14266 proteins using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length 14266 protein can be used or, alternatively, the invention provides antigenic peptide fragments of 14266 proteins for use as immunogens.
  • the antigenic peptide of a 14266 protein comprises at least 8, preferably 10, 15, 20, or 30 amino acid residues ofthe amino acid sequence set forth in SEQ ID NO:l and encompasses an epitope of a 14266 protein such that an antibody raised against the peptide forms a specific immune complex with the 14266 protein.
  • Prefe ⁇ ed epitopes encompassed by the antigenic peptide are regions of a 14266 protein that are located on the surface ofthe protein, e.g., hydrophilic regions. Accordingly, another aspect ofthe invention pertains to anti- 14266 polyclonal and monoclonal antibodies that bind a 14266 protein.
  • Polyclonal anti- 14266 antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with a 14266 immunogen.
  • the anti- 14266 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 14266 protein.
  • ELISA enzyme linked immunosorbent assay
  • 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) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York, NY), pp. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B. cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed
  • hybridomas The technology for producing hybridomas is well known (see generally Coligan et al, eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, NY); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) in Monoclonal Antibodies: A New Dimension In Biological Analyses (Plenum Publishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).
  • a monoclonal anti-14266 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a 14266 protein to thereby isolate immunoglobulin library members that bind the 14266 protein.
  • 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 3 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.
  • recombinant anti-14266 antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and nonhuman portions, which can be made using standard recombinant DNA techniques, are within the scope ofthe invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication Nos. WO 86/101533 and WO 87/02671; European Patent Application Nos. 184,187, 171,496, 125,023, and 173,494; U.S. Patent Nos. 4,816,567 and 5,225,539; European Patent Application 125,023; Better et al.
  • An anti-14266 antibody (e.g., monoclonal antibody) can be used to isolate 14266 proteins by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-14266 antibody can facilitate the purification of natural 14266 protein from cells and of recombinantly produced 14266 protein expressed in host cells.
  • an anti-14266 antibody can be used to detect 14266 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe 14266 protein.
  • Anti-14266 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotiiazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 1, 131 1, 35 S, or 3 H
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxanfrone, 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.,
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta- interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 ("IX-l”.), interleukin-2 ("IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IX-l interleukin-2
  • IL-6 interleukin-6
  • 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.
  • 14266 polynucleotide are particularly applicable to the cells and tissues that contain detectable levels of 14266 expression as described above. These methods pertain to isolated nucleic acid molecules comprising nucleotide sequences encoding 14266 proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify 14266-encoding nucleic acids (e.g., 14266 mRNA) and fragments for use as PCR primers for the amplification or mutation of 14266 nucleic acid molecules.
  • 14266 mRNA fragments for use as PCR primers for the amplification or mutation of 14266 nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs ofthe DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double- stranded DNA.
  • Nucleotide sequences encoding the 14266 proteins ofthe present invention include sequence set forth in SEQ ID NO:2 and complements thereof.
  • complement is intended a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex.
  • the co ⁇ esponding amino acid sequence for the 14266 protein encoded by these nucleotide sequences are also encompassed by the present invention.
  • the invention also encompasses nucleic acid molecules comprising nucleotide sequences encoding partial-length 14266 proteins, including the sequence set forth in SEQ ID NO:2, and complements thereof.
  • fragments are also encompassed by the present invention.
  • fragment is intended a portion ofthe nucleotide sequence encoding a 14266 protein.
  • a fragment of a 14266 nucleotide sequence may encode a biologically active portion of a 14266 protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • a biologically active portion of a 14266 protein can be prepared by isolating a portion of one ofthe nucleotide sequences of the invention, expressing the encoded portion ofthe 14266 protein (e.g., by recombinant expression in vitro), and assessing the activity ofthe encoded portion of the 14266 protein.
  • Nucleic acid molecules that are fragments of a 14266 nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, nucleotides, or up to the number of nucleotides present in the 14266 nucleotide sequence disclosed herein depending upon the intended use.
  • isolated fragments include any contiguous sequence not disclosed prior to the invention as well as sequences that are substantially the same and which are not disclosed. Accordingly, if an isolated fragment is disclosed prior to the present invention, that fragment is not intended to be encompassed by the invention.
  • an isolated nucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguous nucleotides. Other regions ofthe nucleotide sequence may comprise fragments of various sizes, depending upon potential homology with previously disclosed sequences.
  • a fragment of a 14266 nucleotide sequence that encodes a biologically active portion of a 14266 protein ofthe invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acids, or up to the total number of amino acids present in a full-length 14266 protein ofthe invention.
  • Fragments of a 14266 nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of a 14266 protein.
  • Nucleic acid molecules that are variants ofthe 14266 nucleotide sequences disclosed herein are also encompassed by the present invention.
  • "Variants" ofthe 14266 nucleotide sequences include those sequences that encode the 14266 proteins disclosed herein but that differ conservatively because ofthe degeneracy ofthe genetic code. These naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as the polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the 14266 proteins disclosed in the present invention as discussed below.
  • nucleotide sequence variants ofthe invention will have at least about 45%, 55%, 65%, 75%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a particular nucleotide sequence disclosed herein.
  • a variant 14266 nucleotide sequence will encode a 14266 protein that has an amino acid sequence having at least about 45%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, 98%, or 99% identity to the amino acid sequence of a 14266 protein disclosed herein.
  • DNA sequence polymo ⁇ hisms that lead to changes in the amino acid sequences of 14266 proteins may exist within a population (e.g., the human population).
  • Such genetic polymo ⁇ hism in a 14266 gene may exist among individuals within a population due to natural allelic variation.
  • An allele is one of a group of genes that occur alternatively at a given genetic locus.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a 14266 protein, preferably a mammalia 14266 protein.
  • allelic variant refers to a nucleotide sequence that occurs at a 14266 locus or to a polypeptide encoded by the nucleotide sequence.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe 14266 gene. Any and all such nucleotide variations and resulting amino acid polymo ⁇ hisms or variations in a 14266 sequence that are the result of natural allelic variation and that do not alter the functional activity of 14266 proteins are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding 14266 proteins from other species which have a nucleotide sequence differing from that ofthe 14266 sequences disclosed herein, are intended to be within the scope ofthe invention.
  • nucleic acid molecules co ⁇ esponding to natural allelic variants and homologues ofthe human 14266 cDNA.of the invention can be isolated based on their identity to the human 14266 nucleic acid disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions as disclosed below.
  • allelic variants ofthe 14266 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences ofthe invention thereby leading to changes in the amino acid sequence ofthe encoded 14266 proteins, without altering the biological activity ofthe 14266 proteins.
  • an isolated nucleic acid molecule encoding a 14266 protein having a sequence that differs from the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the co ⁇ esponding nucleotide sequence disclosed herein, 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. Such variant nucleotide sequences are also encompassed by the present invention.
  • conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues.
  • amino acid residue is a residue that can be altered from the wild-type sequence of a 14266 protein without altering the biological activity, whereas an "essential” amino acid residue is required for biological activity.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • variant 14266 nucleotide sequences can be made by introducing mutations randomly along all or part of a 14266 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 14266 biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity ofthe protein can be determined using standard assay techniques.
  • nucleotide sequences ofthe invention include the sequences disclosed herein as well as fragments and variants thereof.
  • the 14266 nucleotide sequences ofthe invention, and fragments and variants thereof, can be used as probes and/or primers to identify and/or clone 14266 homologues in other cell types, e.g., from other tissues, as well as 14266 homologues from other mammals.
  • probes can be used to detect transcripts or genomic sequences encoding the same or identical proteins.
  • probes can be used as part of a diagnostic test kit for identifying cells or tissues that misexpress a 14266 protein, such as by measuring levels of a 14266- encoding nucleic acid in a sample of cells from a subject, e.g., detecting 14266 mRNA levels or determining whether a genomic 14266 gene has been mutated or deleted. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences having substantial identity to the sequences ofthe invention. See, for example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and Innis, et al.
  • 14266 nucleotide sequences isolated based on their sequence identity to the 14266 nucleotide sequences set forth herein or to fragments and variants thereof are encompassed by the present invention.
  • hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known 14266 nucleotide sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in a known 14266 nucleotide sequence or encoded amino acid sequence can additionally be used.
  • the probe 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 a 14266 nucleotide sequence ofthe invention or a fragment or variant thereof.
  • a previously unidentified 14266 nucleic acid molecule hybridizes under stringent conditions to a probe that is a nucleic acid molecule comprising one ofthe 14266 nucleotide sequences ofthe invention or a fragment thereof.
  • the previously unknown 14266 nucleic acid molecule is at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 2,000, 3,000, 4,000 or 5,000 nucleotides in length and hybridizes under stringent conditions to a probe that is a nucleic acid molecule comprising one of the 14266 nucleotide sequences disclosed herein or a fragment thereof.
  • an isolated previously unknown 14266 nucleic acid molecule ofthe invention is at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1,100, 1,200, 1,300, or 1,400 nucleotides in length and hybridizes under stringent conditions to a probe that is a nucleic acid molecule comprising one ofthe nucleotide sequences ofthe invention, preferably the coding sequence ofthe nucleotides sequences set forth in SEQ ID NO: 2 or a complement, fragment, or variant thereof.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6.
  • a prefe ⁇ ed, 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 0.2X SSC, 0.1% SDS at 50°C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55°C.
  • a further 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 0.2X SSC, 0.1 % SDS at 60°C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
  • Particularly prefe ⁇ ed stringency conditions are 0.5M Sodium Phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.
  • an isolated nucleic acid molecule that hybridizes under stringent conditions to an 14226 sequence ofthe invention co ⁇ esponds to a naturally- occu ⁇ ing 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 molecules ofthe invention also encompass homologous DNA sequences identified and isolated from other cells and/or organisms by hybridization with entire or partial sequences obtained from the 14266 nucleotide sequences disclosed herein or variants and fragments thereof.
  • the present invention also encompasses antisense nucleic acid molecules, i.e., molecules that are complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire 14266 coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can be antisense to a noncoding region ofthe coding strand of a nucleotide sequence encoding a 14266 protein.
  • the noncoding regions are the 5' and 3' sequences that flank the coding region and are not translated into amino acids.
  • Antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of 14266 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of 14266 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 14266 mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymatic ligation procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occu ⁇ ing nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, including, but not limited to, for example e.g., phosphorothioate derivatives and acridine substituted nucleotides.
  • 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 ofthe 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 14266 protein to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation.
  • An example of a route of administration of antisense nucleic acid molecules ofthe 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 linked to peptides or antibodies to form a complex that specifically binds to receptors or antigens expressed on a selected cell surface.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are prefe ⁇ ed.
  • An antisense nucleic acid molecule ofthe invention can be an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett.
  • the invention also encompasses ribozymes, which are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • Ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591) can be used to catalytically cleave 14266 mRNA transcripts to thereby inhibit translation of 14266 mRNA.
  • a ribozyme having specificity for a 14266-encoding nucleic acid can be designed based upon the nucleotide sequence of a 14266 cDNA disclosed herein. See, e.g., Cech et al, U.S. Patent No. 4,987,071 ; and Cech et al, U.S. Patent No. 5,116,742.
  • 14266 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.
  • the invention also encompasses nucleic acid molecules that form triple helical structures.
  • 14266 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe 14266 protein (e.g., the 14266 promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe 14266 gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe 14266 protein e.g., the 14266 promoter and/or enhancers
  • the nucleic acid molecules ofthe invention can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:5).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid-phase peptide synthesis protocols as described, for example, in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl Acad. Sci. USA 93:14670.
  • PNAs of a 14266 molecule can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs ofthe invention 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), supra).
  • PNAs of a 14266 molecule can be modified, e.g., to enhance their stability, specificity, 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.
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra; Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.
  • vectors and host cells are particularly relevant where vectors are expressed in the cells and tissues with detectable levels of 14266 expression as described herein, or where the host cells are those that naturally express the gene or which may be the native or a recombinant cell expressing the gene.
  • host cells and “recombinant host cells” refer not only to the particular subject cell but also 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 ofthe term as used herein.
  • the host cells expressing the polypeptides described herein, and particularly recombinant host cells have a variety of uses. First, the cells are useful for producing 14266 proteins or polypeptides that can be further purified to produce desired amounts of 14266 protein or fragments. Thus, host cells containing expression vectors are useful for polypeptide production, as well as cells producing significant amounts ofthe polypeptide. Such cells and tissues have been described herein above.
  • Host cells are also useful for conducting cell-based assays involving the 14266 or 14266 fragments.
  • a recombinant host cell expressing a native 14266 is useful to assay for compounds that stimulate or inhibit 14266 function. This includes substrate, coenzyme, or 14266 subunit binding, and gene expression at the level of transcription or translation.
  • Host cells are also useful for identifying 14266 mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant 14266 (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native 14266. Recombinant host cells are also useful for expressing the chimeric polypeptides described herein to assess compounds that activate or suppress activation by means of a heterologous domain, segment, site, and the like, as disclosed herein.
  • mutant 14266s can be designed in which one or more ofthe various functions is engineered to be increased or decreased (e.g., substrate or coenzyme binding) and used to augment or replace 14266 proteins in an individual.
  • host cells can provide a therapeutic benefit by replacing an abe ⁇ ant 14266 or providing an abe ⁇ ant 14266 that provides a therapeutic result.
  • the cells provide 14266s that are abnormally active.
  • the cells provide a 14266 that is abnormally inactive.
  • This 14266 can compete with endogenous 14266 in the individual.
  • cells expressing 14266s that cannot be activated are introduced into an individual in order to compete with endogenous 14266 for cAMP.
  • endogenous 14266 for cAMP For example, in the case in which excessive substrates such as ⁇ -hydroxysteroid is part of a treatment modality, it may be necessary to inactivate this molecule at a specific point in treatment. Providing cells that compete for the molecule , but which cannot be affected by 14266 activation would be beneficial.
  • Homologously recombinant host cells can also be produced that allow the in situ alteration of endogenous 14266 polynucleotide sequences in a host cell genome.
  • the host cell includes, but is not limited to, a stable cell line, cell in vivo, or cloned microorganism. This technology is more fully described in WO 93/09222, WO 91/12650, WO 91/06667, U.S. 5,272,071, and U.S. 5,641,670.
  • polynucleotide sequences co ⁇ esponding to the 14266 polynucleotides or sequences proximal or distal to a 14266 gene are allowed to integrate into a host cell genome by homologous recombination where expression ofthe gene can be affected.
  • regulatory sequences are introduced that either increase or decrease expression of an endogenous sequence. Accordingly, a 14266 protein can be produced in a cell not normally producing it. Alternatively, increased expression of 14266 protein can be effected in a cell normally producing the protein at a specific level. Further, expression can be decreased or eliminated by infroducing a specific regulatory sequence.
  • the regulatory sequence can be heterologous to the 14266 protein sequence or can be a homologous sequence with a desired mutation that affects expression. Alternatively, the entire gene can be deleted.
  • the regulatory sequence can be specific to the host cell or capable of functioning in more than one cell type. Still further, specific mutations can be introduced into any desired region ofthe gene to produce mutant 14266 proteins. Such mutations could be introduced, for example, into the specific functional regions such as the cyclic nucleotide-binding site.
  • the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the altered 14266 gene.
  • the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal. See also Thomas et al, Cell 51:503 (1987) 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 14266 gene is selected (see e.g., Li, E. 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, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. 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 ofthe animal contain the homologously recombined DNA by germline . transmission ofthe transgene.
  • a transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more ofthe cells ofthe animal include a transgene.
  • 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 ofthe mature animal in one or more cell types or tissues ofthe transgenic animal. These animals are useful for studying the function of a 14266 protein and identifying and evaluating modulators of 14266 protein activity.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
  • a host cell is a fertilized oocyte or an embryonic stem cell into which 14266 polynucleotide sequences have been introduced.
  • a transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Any ofthe 14266 nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.
  • Any ofthe regulatory or other sequences useful in expression vectors can form part ofthe transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression ofthe 14266 protein 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, both by Leder et al, U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other fransgenic animals.
  • a fransgenic founder animal can be identified based upon the presence ofthe transgene in its genome and/or expression of fransgenic mRNA in tissues or cells ofthe animals. A fransgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A fransgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.
  • fransgenic non-human animals can be produced which contain selected systems, which allow for regulated expression ofthe transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system of bacteriophage PI .
  • a recombinase system is the FLP recombinase system ofS. cerevisiae (O' Gorman etal. (1991) Science 251:1351-1355).
  • cre/loxP recombinase system is used to regulate expression ofthe transgene
  • animals containing transgenes encoding both the Cre recombinase and a selected protein is 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 ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 555:810- 813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then fransfe ⁇ ed to a pseudopregnant female foster animal.
  • the offspring bom of this female foster animal will be a clone ofthe animal from which the cell, e.g., the somatic cell, is isolated.
  • Transgenic animals containing recombinant cells that express the polypeptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could affect substrate binding or coenzyme bind may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo 14266 function, including substrate interaction, the effect of specific mutant 14266s on 14266 function and interaction, and the effect of chimeric 14266s. It is also possible to assess the effect of null mutations, that is mutations that substantially Or completely eliminate one or more 14266 functions.
  • methods for producing transgenic animals include introducing a nucleic acid sequence according to the present invention, the nucleic acid sequence capable of expressing the protein in a transgenic animal, into a cell in culture or in vivo.
  • the nucleic acid is introduced into an intact organism such that one or more cell types and, accordingly, one or more tissue types, express the nucleic acid encoding the protein.
  • the nucleic acid can be introduced into virtually all cells in an organism by transfecting a cell in culture, such as an embryonic stem cell, as described herein for the production of transgenic animals, and this cell can be used to produce an entire transgenic organism.
  • the host cell can be a fertilized oocyte. Such cells are then allowed to develop in a female foster animal to produce the transgenic organism.
  • the methods using the vectors and host cells discussed above are based on the vectors and host cells including, but not limited to, those described below.
  • the invention also provides methods using vectors containing the 14266 polynucleotides.
  • the term "vector” refers to a vehicle, preferably a nucleic acid molecule that can transport the 14266 polynucleotides.
  • the vector is a nucleic acid molecule, the 14266 polynucleotides are covalently linked to the vector nucleic acid.
  • the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAG, PAC, YAC, OR MAC.
  • a vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies ofthe 14266 polynucleotides.
  • the vector may integrate into the host cell genome and produce additional copies ofthe 14266 polynucleotides when the host cell replicates.
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) ofthe 14266 polynucleotides.
  • the vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).
  • Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the 14266 polynucleotides such that transcription ofthe polynucleotides is allowed in a host cell.
  • the polynucleotides can be infroduced into the host cell with a separate polynucleotide capable of affecting transcription.
  • the second polynucleotide may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription ofthe 14266 polynucleotides from the vector.
  • a trans-acting factor may be supplied by the host cell.
  • a frans- acting factor can be produced from the vector itself.
  • transcription and/or translation ofthe 14266 polynucleotides can occur in a cell-free system.
  • the regulatory sequence to which the polynucleotides described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage ⁇ , the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
  • expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers.
  • regions that modulate transcription include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation.
  • Other regulatory confrol elements for expression include initiation and termination codons as well as polyadenylation signals.
  • the person of ordinary skill in the art would be aware ofthe numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • a variety of expression vectors can be used to express a 14266 polynucleotide.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.
  • the regulatory sequence may provide constitutive expression in one or more host cells (i.e., tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • host cells i.e., tissue specific
  • inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • a variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
  • the 14266 polynucleotides can be inserted into the vector nucleic acid by well- known methodology.
  • the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together.
  • the vector containing the appropriate polynucleotide can be introduced into an appropriate host cell for propagation or expression using well-known techniques.
  • Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium.
  • Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells. As described herein, it may be desirable to express the polypeptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production ofthe 14266 polypeptides.
  • Fusion vectors can increase the expression of a recombinant protein, increase the solubility ofthe recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification.
  • a proteolytic cleavage site may be introduced at the junction ofthe fusion moiety so that the desired polypeptide can ultimately be separated from the fusion moiety.
  • Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase.
  • Typical fusion expression vectors include pGEX (Smith etal.
  • E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69:301-315) and pET lid (Shadier et al (1990) Gene Expression Technology: Methods in Enzymology 755:60-89).
  • Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein.
  • the sequence ofthe polynucleotide of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118).
  • the 14266 polynucleotides can also be expressed by expression vectors that are operative in yeast.
  • yeast e.g., S. cerevisiae
  • vectors for expression in yeast include pYepSecl (Baldari et al. (1987) EMBOJ. (5:229-234 ), pMFa (Kurjan et al. (1982) Cell
  • the 14266 polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al.
  • the polynucleotides described herein are expressed in mammalian cells using mammalian expression vectors.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 529:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. (5:187-195).
  • the invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA.
  • an antisense transcript can be produced to all, or to a portion, ofthe polynucleotide sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each ofthe parameters described above in relation to expression ofthe sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
  • the invention also relates to recombinant host cells containing the vectors described herein.
  • Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • the recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dexfran-mediated transfection, cationic lipid-mediated transfection, electroporation, fransduction, infection, lipofection, and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold
  • Host cells can contain more than one vector. Thus, different nucleotide sequences can be infroduced on different vectors ofthe same cell.
  • the 14266 polynucleotides can be introduced either alone or with other polynucleotides that are not related to the 14266 polynucleotides such as those providing trans-acting factors for expression vectors.
  • the vectors can be introduced independently, co-introduced or joined to the 14266 polynucleotide vector.
  • bacteriophage and viral vectors these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and fransduction.
  • Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.
  • Vectors generally include selectable markers that enable the selection ofthe subpopulation of cells that contain the recombinant vector constructs.
  • the marker can be contained in the same vector that contains the polynucleotides described herein or may be on a separate vector.
  • Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.
  • RNA derived from the DNA constructs described herein can also be used to produce these proteins using RNA derived from the DNA constructs described herein.
  • secretion signals are inco ⁇ orated into the vector.
  • the signal sequence can be endogenous to the 14266 polypeptides or heterologous to these polypeptides.
  • the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like.
  • the polypeptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.
  • the polypeptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria.
  • the polypeptides may include an initial modified methionine in some cases as a result of a host-mediated process.
  • compositions suitable for administration can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable ca ⁇ ier.
  • pharmaceutically acceptable ca ⁇ ier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • compositions ofthe invention are useful to treat any ofthe disorders discussed herein.
  • the compositions are provided in therapeutically effective amounts.
  • therapeutically effective amounts is intended an amount sufficient to modulate the desired response.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • an agent may, for example, be a small molecule.
  • 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,.
  • heteroorganic and organometalhc 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.
  • doses of small molecule agents depends upon a number of factors within the knowledge ofthe ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) ofthe small molecule will vary, for example, depending upon the identity, size, and condition ofthe 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 ofthe invention.
  • Exemplary doses include milligram or microgram amounts ofthe small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.
  • appropriate doses of a small molecule depend upon the potency ofthe small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health, gender, and diet ofthe 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.
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • 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
  • 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 ca ⁇ iers include physiological saline, bacteriostatic water, Cremophor ELTM (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.
  • the ca ⁇ ier 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 ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition.
  • Prolonged abso ⁇ tion ofthe injectable compositions can be brought about by including in the composition an agent that delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a receptor-like protein or anti-receptor-like antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a receptor-like protein or anti-receptor-like antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle that 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 ofthe 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 pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum fragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum fragacanth, or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the fonn of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal admimstiation can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated with each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical ca ⁇ ier.
  • dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated with each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical ca ⁇ ier.
  • about 1 ⁇ g/kg to about 15 mg/kg (e.g., 0.1 to 20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to about 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful.
  • the progress of this therapy is easily monitored by conventional techniques and assays.
  • An exemplary dosing regimen is disclosed in WO 94/04188.
  • the specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057).
  • the pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.

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Abstract

The present invention relates to methods for using a human G protein-coupled receptor (14266) belonging to the family of mammalian G protein-coupled receptors. The invention also relates to methods for using polynucleotides encoding the 14266 receptor. The invention relates to methods for detecting the presence of a 14266 polypeptide of a 14266 polynucleotide and kits comprising reagents for detecting the presence of a 14266 polypeptide or polynucleotide. The invention further relates to methods of modulating the level or activity of a 14266 polypeptide in a cell. The invention further relates to drug-screening methods using the 14266 polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the 14266 polypeptides and polynucleotides. The invention further relates to agonists and antagonists identified by drug screening methods with the 14266 polypeptides and polynucleotides as a target.

Description

METHODS FOR USING 14266, A HUMAN G PROTEIN-COUPLED RECEPTOR
FIELD OF THE INVENTION
The invention relates to methods of using 14266, a G protein-coupled receptor. Methods for identifying agents that modulate or interact with 14266 polypeptides and nucleic acid molecules, and methods for detecting or modulating 14266 polypeptides and nucleic acid molecules are provided.
BACKGROUND OF THE INVENTION
G-protein coupled receptors (GPCRs) constitute a major class of proteins responsible for transducing a signal within a cell. GPCRs share three structural features: an amino terminal extracellular domain, a transmembrane region containing seven transmembrane domains, three extracellular loops, and three intracellular loops, and a carboxy terminal intracellular domain. Upon binding of a ligand to an extracellular portion of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property ofthe cell. GPCRs, along with G- proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs.
GPCR genes and gene-products are potential causative agents of disease (Spiegel et al., J. Clin. Invest. 92:1119-1125 (1993); McKusick et al, J. Med. Genet. 30:1-26 (1993)). Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of retinitis pigmentosum (Nathans et al., Annu. Rev. Genet. 26:403-424(1992)), and nephrogenic diabetes insipidus (Holtzman et al., Hum. Mol. Genet. 2:1201-1204 (1993)). These receptors are of critical importance to both the central nervous system and peripheral physiological processes. Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous systems.
The GPCR protein superfamily can be divided into five families: Family I, which contains receptors typified by rhodopsin and the β2-adrenergic receptor and currently represented by over 200 unique members (Dohlman et al., Annu. Rev. Biochem. 60:653-688 (1991)); Family II, which contains the parathyroid hormone/calcitonin/secretin receptor family (Juppner et al., Science 254:1024-1026 (1991); Lin et al, Science 254:1022-1024 (1991)); Family III, which contains the metabotropic glutamate receptor family (Nakanishi, Science 258 597:603 (1992)); Family IV, which contains the cAMP receptor family, important in the chemotaxis and development of D. discoideum (Klein et al., Science 241:1467-1472 (1988)); and Family V, the fungal mating pheromone receptors such as STE2 (Kurjan, Annu. Rev. Biochem. 61:1097-1129 (1992)). G-proteins represent a family of heterotrimeric proteins composed of α, β, and γ subunits. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane segments. Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the α-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ- subunits. The GTP-bound form ofthe α-subunit typically functions as an effector- modulating moiety, leading to the production of second messengers, such as cAMP (e.g., by activation of adenyl cyclase), diacylglycerol or inositol phosphates. Greater than 20 different types of α-subunits are known in humans. These subunits associate with a smaller pool of β and γ subunits. Examples of mammalian G proteins include Gi (inhibitory G protein), Go , Gq, Gs (stimulatory G protein) and Gt (transducin). G proteins are described extensively in Lodish et al., Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference. GPCRs, G proteins and G protein-linked effector and second messenger systems have been reviewed in The G-Protein Linked Receptor Fact Book, Watson et al., eds., Academic Press (1994).
GPCRs are a major target for drug development. Accordingly, it is valuable to the field of pharmaceutical development to identify methods using GPCRs and tissues and disorders in which GPCRs are differentially expressed. SUMMARY OF THE INVENTION
A specific object ofthe invention is to identify compounds that act as agonists and antagonists and modulate the expression of 14266 in specific tissues and disorders. A further specific object ofthe invention is to provide compounds that modulate expression of 14266 for diagnosis and treatment of 14266-mediated or related disorders.
The invention provides methods of screening, for compounds that modulate expression or activity of 14266 polypeptides or nucleic acid molecules (RNA or DNA) in the specific tissues or disorders. The invention also provides a process for modulating 14266 polypeptide or nucleic acid molecule expression or activity, especially using the screened compounds. Modulation may be used to treat conditions related to abeπant activity or expression of 14266 polypeptides or nucleic acids.
The invention also provides assays for detecting 14266 polypeptides or nucleic acid molecules in specific biological samples, including for disease diagnosis. The invention further comprises kits comprising reagents for the detection of 14266 polypeptides or 14266 polynucleotides.
The invention also provides assays for determining the presence of a mutation in the polypeptides or nucleic acid molecules, including for disease diagnosis. The invention utilizes isolated 14266 polypeptides, including a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 , and variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO: 1.
The invention also utilizes an isolated 14266 nucleic acid molecule having the sequence shown in SEQ ID NO:2, and variant nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in SEQ ID NO:2.
The invention also utilizes fragments ofthe polypeptide shown in SEQ ID NO:l and nucleotide sequence shown in SEQ ID NO:2, as well as substantially homologous fragments ofthe polypeptide or nucleic acid. The invention further utilizes nucleic acid constructs comprising the nucleic acid molecules described herein. In a prefeπed embodiment, the nucleic acid molecules of the invention are operatively linked to a regulatory sequence. The invention also utilizes vectors and host cells that express 14266 and provides methods for expressing 14266 nucleic acid molecules and polypeptides in specific cell types and disorders.
The invention also utilizes methods of making the vectors and host cells and provides methods for using them to assay expression and cellular effects of expression of the 14266 nucleic acid molecules and polypeptides in specific cell types and disorders.
The methods o the invention are particularly relevant to those tissues and cell lines where 14266 is highly or differentially expressed. Such tissues include, but are not limited to, spinal chord, brain cortex, hypothalamus, aorta, heart, fetal heart, vein, astrocytes, glioblastoma, breast, breast interductal carcinoma, ovary, ovary tumor, pancreas, prostate, prostate tumor, colon cells, colon tumor, bone marrow mononuclear cells, kidney, CD34 positive haematopoietic progenitor cells, neutrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the 14266 nucleotide sequence (SEQ ID NO:2).
Figure 2 shows the amino acid sequence of 14266 (SEQ ID NO: 1).
Figure 3 shows an alignment ofthe 7 transmembrane rhodopsin-like G protein- coupled receptor domain of human 14266 with a consensus amino acid sequence (SEQ ID NO:3) derived from a hidden Markov model, while the lower amino acid sequence coπesponds to amino acids 35 to 338 of SEQ ID NO: 1.
Figure 4 depicts a hydropathy plot of human 14266. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues 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 numbers coπesponding to the amino acid sequence (shown in SEQ ID NO:l) of human 14266 are indicated . Polypeptides ofthe invention include fragments which include: all or a part of a hydrophobic sequence (a sequence above the dashed line); or all or part of a hydrophilic fragment (a sequence below the dashed line). Other fragments include a cysteine residue or an N-glycosylation site.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on methods of using molecules refeπed to herein as 14266, 14266 GPCRs, 14266 receptors, or 14266 nucleic acid or polypeptide molecules. The 14266 receptor shares sequence similarity with the norepipniephrine β3 receptor and the serotonin 5HT-2C receptor, and there are 14266 receptor orthologs in rat and zebrafish, suggesting that the 14266 gene has been highly conserved in vertebrate evolution (Matsumoto et al. (2000) Biochem. Biophys. Res. Comm. 272: 576-582)
The human 14266 sequence (Figure 2; SEQ ID NO:2), which is approximately 1128 nucleotides long, contains a predicted methionine-initiated coding sequence of about 1128 nucleotides (nucleotides 1-1128 of SEQ ID NO:2). The coding sequence encodes a 375 amino acid protein (SEQ ID NO:l).
The mature protein form is approximately 350 amino acid residues in length (from about amino acid 35-375 of SEQ ID NO:l). Human 14266 also contains a predicted G-protein coupled receptor domain (PFAM Accession PF00001) located at about amino acid 35 to 338 of SEQ ID NO : 1 , and predicted transmembrane domains at amino acids 22-44, 54-78, 97-118, 134-156, 184-208, 286-306, and 318-341 of SEQ ID NO:l.
As used herein, the term "G-protein coupled receptor domain" includes an amino acid sequence of about 250-350 amino acid residues in length and having a bit score for the alignment ofthe sequence to the G-protein coupled receptor domain (HMM) of at least 8. Preferably, a G-protein coupled receptor domain includes at least about 200-400 amino acids, more preferably about 250-350 amino acid residues, or about 275-325 amino acids and has a bit score for the alignment ofthe sequence to the G-protein coupled receptor domain (HMM) of at least 16 or greater. The G- protein coupled receptor domain (HMM) has been assigned the PFAM Accession PF00001. An alignment ofthe G-protein coupled receptor domain (amino acids 35- 338 of SEQ ID NO:l) of human 14266 with a consensus amino acid sequence derived from a hidden Markov model is depicted in Figure 3.
In a preferred embodiment 14266 polypeptide or protein has a "G-protein coupled receptor domain" or a region which includes at least about 100-250 more preferably about 130-200 or 160-200 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with an "G-protein coupled receptor domain," e.g., the G-protein coupled receptor domain of human 14226 (e.g., amino acid residues 35-338 of SEQ ID NO:l).
To identify the presence of an "G-protein coupled receptor" domain in a 14266 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (www.sanger.ac.uk Sofitware/Pfam/HMM_search). For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description ofthe Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):A05-A20 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol 183ΛA6-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 54:4355- 4358; Krogh et al. (1994) J Mol. Biol. 255:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.
In one embodiment, a 14266 protein includes at least one transmembrane domain. As used herein, the term "transmembrane domain" includes an amino acid sequence of about 15 amino acid residues in length that spans a phospholipid membrane. More preferably, a transmembrane domain includes about at least 18, 20, 22, 24, or 25 amino acid residues and spans a phospholipid membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an α- helical structure. In aprefeπed embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, pfam.wustl.edu/cgi-bin/getdesc?name=7tm-l, and Zagotta W.N. et al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of which are incorporated herein by reference.
In a preferred embodiment, a 14266 polypeptide or protein has at least one transmembrane domain or a region which includes at least 18, 20, 22, 24, 25, or 30 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity with a "transmembrane domain," e.g., at least one transmembrane domain of human 14266 (e.g., amino acid residues 22-44, 54-78, 97-118, 134-156, 184-208, 286-306, and 318-341 of SEQ ID NO:l).
In another embodiment, a 14266 protein includes at least one "non- transmembrane domain." As used herein, "non-transmembrane domains" are domains that reside outside ofthe membrane. When refeπing to plasma membranes, non-transmembrane domains include extracellular domains (i.e., outside ofthe cell) and intracellular domains (i.e., within the cell). When refeπing to membrane-bound proteins found in intracellular organelles (e.g., mitochondria, endoplasmic reticulum, peroxisomes and microsomes), non-transmembrane domains include those domains of the protein that reside in the cytosol (i.e., the cytoplasm), the lumen ofthe organelle, or the matrix or the intermembrane space (the latter two relate specifically to mitochondria organelles). The C-terminal amino acid residue of a non- transmembrane domain is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally occurring 14226, or 14266-like protein. In a prefeπed embodiment, a 14226 polypeptide or protein has a "non- transmembrane domain" or a region which includes at least about 5-100, preferably about 8-80 acid residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100% sequence identity with a "non-transmembrane domain", e.g., a non- transmembrane domain of human 14226 (e.g., residues 45-53, 79-96, 119-133; 157- 183, 209-285, 307-317, 342-375 of SEQ ID NO:2).
A non-transmembrane domain located at the N-terminus of a 14226 protein or polypeptide is refeπed to herein as an "N-terminal non-transmembrane domain." As used herein, an "N-terminal non-transmembrane domain" includes an amino acid sequence having about 1-50, preferably about 18-22 amino acid residues in length and is located outside the boundaries of a membrane. For example, an N-terminal non- transmembrane domain is located at about amino acid residues 1-22 of SEQ ID NO:2. Similarly, a non-transmembrane domain located at the C-terminus of a 14266 protein or polypeptide is refeπed to herein as a "C-terminal non-transmembrane domain." As used herein, an "C-terminal non-transmembrane domain" includes an amino acid sequence having about 20-40, preferably about 25-35 amino acid residues in length and is located outside the boundaries of a membrane. For example, an C- terminal non-transmembrane domain is located at about amino acid residues 342-375 of SEQ ID NO:2.
A 14266 molecule can further include a signal sequence. As used herein, a "signal sequence" refers to a peptide of about 20-80 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains a majority of hydrophobic amino acid residues. For example, a signal sequence contains at least about 12-25 amino acid residues, preferably about 30-70 amino acid residues, more preferably about 61 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g., alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a "signal sequence", also refeπed to in the art as a "signal peptide", serves to direct a protein containing such a sequence to a lipid bilayer. For example, in one embodiment, a 14266 protein contains a signal sequence of about amino acids 1-34 of SEQ ID NO:2. The "signal sequence" is cleaved during processing ofthe mature protein. The mature 14266 protein coπesponds to amino acids 35-375 of SEQ ID NO:2.
The uses, reagents and methods disclosed in detail herein below apply especially to tissues and cell types where 14266 expression is relevant. Analysis using the TaqMan® brand Polymerase Chain Reaction Kit (Applied Biosystems, Foster City, CA) demonstrated that 14266 expression is highest in spinal cord and brain (particularly the cortex and hypothalamus) (Figure 4). 14266 expression is also detectable in aorta, heart, fetal heart, vein, astrocytes (normal cells and glioblastoma tissue), breast (normal tissue and interductal carcinoma tissue), ovary (normal tissue and ovary tumor tissue), pancreas, prostate (normal tissue and prostate tumor cells), and colon (normal tissue, tumor tissue, and inflammatory bowel disease tissue).
Further analysis using the TaqMan® brand Polymerase Chain Reaction Kit demonstrated high 14266 expression in bone maπow mononuclear cells, granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood and adult bone maπow CD34+ haematopoietic progenitor cells, neutrophils isolated from bone maπow from normal and G-CSF treated individuals, and in mature neutrophils generated from CD34+ haematopoietic progenitor cells in vitro. CD34+ haematopoietic progenitor cells from bone maπow from volunteers treated with G-CSF showed significant levels of 14266 expression, as did both early stage and more mature neufrophil lineage cells isolated from bone marrow from both normal and G-CSF treated volunteers. Expression of 14266 was regulated during both in vivo and in vitro generation of blood cells. It was down-regulated in both megakaryocytes and erythroid cells during differentiation, and up-regulated during neufrophil differentiation.
Neutrophils are a special class of granulocytes that are derived from the granulocyte/macrophage progenitor cells (colony-forming cells) which arise from the division and differentiation of myeloid stem cells. Neutrophils play a key role in the nonspecific immune response, and they are recruited rapidly to sites of inflammation. Neutrophils are required for host defense against invading micro-organisms, and they respond to injurious agents by the release of granular enzymes and proteins, the production of reactive oxygen intermediates, and by phagocytosis. Patients with neufrophil deficiency disorders, including neutropenia, chronic granulomatous disease, and leukocyte adhesion deficiency, have a tendency to develop recurrent and overwhelming infections. Inadequate or ineffective granulopoiesis can result from suppression of myeloid stem cells (as occurs in aplastic anemia and a variety of infiltrative maπow disorders), suppression ofthe committed granulocytic precursors (which often occurs after exposure to certain drugs, including alkylating agents and antimetabolites used in cancer treatment), disease states characterized by ineffective granulopoiesis (such as megaloblastic anemias caused by vitamin B1 or folate deficiency and myelodysplastic syndromes) and rare inherited conditions (such as Kostmann syndrome). Excessive neufrophil activation is implicated in several inflammatory disorders, including acute respiratory distress syndrome (ARDS), rheumatoid arthritis, and ischaemia-reperfusion injury (reviewed in Condliffe et al. (1998) Clin. Sci. 94: 461-471. Thus, the regulation of neufrophil differentiation and activation plays a key role in determining the balance between defense and injury, making 14266 a target for the diagnosis and treatment of neufrophil deficiency disorders and disorders associated with excessive neufrophil activation.
G protein-coupled receptors, including 14266 receptors, use one of several signaling pathways to relay their intracellular signal As used herein, a "signaling pathway" refers to one or more signaling steps that lead to the modulation (e.g., stimulation or inhibition) of a cellular function/activity upon the binding of a ligand to the 14266 receptors.
One signaling pathway that may be used by the 14266 receptor is the phosphatidylinositol second messenger pathway, which involves phosphatidylinositol turnover and metabolism. As used herein, "phosphatidylinositol turnover and metabolism" refers to the molecules involved in the turnover and metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2) as well as to the activities of these molecules. PIP2 is a phospholipid found in the cytosolic leaflet ofthe plasma membrane. Binding of ligand to the 14266 receptor may activate, in some cells, the plasma-membrane enzyme phospholipase C that, in turn, can hydrolyze PIP2 to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Once formed IP3 can diffuse to the endoplasmic reticulum surface where it can bind an IP3 receptor, e.g., a calcium channel protein containing an IP3 binding site. IP3 binding can induce opening ofthe channel, allowing calcium ions to be released into the cytoplasm. IP3 can also be phosphorylated by a specific kinase to form inositol 1,3,4,5-tetraphosphate (IP4), a molecule which can cause calcium entry into the cytoplasm from the extracellular medium. IP3 and IP4 can subsequently be hydrolyzed very rapidly to the inactive products inositol 1,4-biphosphate (IP2) and inositol 1,3,4-triphosphate, respectively. These inactive products can be recycled by the cell to synthesize PIP2. The other second messenger produced by the hydrolysis of PIP2, namely 1 ,2-diacylglycerol (DAG), remains in the cell membrane where it can serve to activate the enzyme protein kinase C. Protein kinase C is usually found soluble in the cytoplasm ofthe cell, but upon an increase in the intracellular calcium concentration, this enzyme can move to the plasma membrane where it can be activated by DAG. The activation of protein kinase C in different cells results in various cellular responses such as the phosphorylation of glycogen synthase, or the phosphorylation of various transcription factors, e.g., NF-kB. The language "phosphatidylinositol activity", as used herein, refers to an activity of PIP2 or one of its metabolites.
Another signaling pathway in which the 14266 receptor may participate is the cAMP turnover pathway. As used herein, cAMP turnover and metabolism" refers to the molecules involved in the turnover and metabolism of cAMP as well as to the activities of these molecules. Cyclic AMP is a second messenger produced in response to ligand- induced stimulation of certain G protein coupled receptors. In the cAMP signaling pathway, binding of a ligand to a GPCR can lead to the activation ofthe enzyme adenyl cyclase, which catalyzes the synthesis of cAMP. The newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase. This activated kinase can phosphorylate a voltage-gated potassium channel protein, or an associated protein, and lead to the inability ofthe potassium channel to open during an action potential. The inability ofthe potassium channel to open results in a decrease in the outward flow of potassium, which normally repolarizes the membrane of a neuron, leading to prolonged membrane depolarization.
The disclosed invention relates to methods and compositions for the modulation, diagnosis, and treatment of diseases related to 14266 receptor malfunction. In addition to variability among individuals in their responses to drugs, several definable diseases arise from disorders in receptors or receptor-effector systems. The loss of a receptor in a highly specialized signaling system may cause a relatively limited phenotypic disorder, such as the genetic deficiency ofthe androgen receptor in the testicular feminization syndrome (Griffin et al. (1995) The Metabolic and Molecular Bases of Inherited Diseases 7:2967-2998). Deficiencies of more widely used signaling systems have a broader spectrum of effects, as are seen in myasthenia gravis or some forms of insulin- resistant diabetes mellitus, which result from autoimmune depletion of nicotinic cholinergic receptors or insulin receptors, respectively. A lesion in a component of a signaling pathway that is used by many receptors can cause a generalized endocrinopathy. Heterozygous deficiency for G5, the G protein that activates adenyl cyclase in all cells, causes multiple endocrine disorders; the disease is termed pseudohpoparathyroidism type la (Spiegel et al. (1995) The Metabolic and Molecular Bases of Inherited Diseases 7:3073-3089). Homozygous deficiency in G5 would presumably be lethal. The expression of abeπant or ectopic receptors, effectors, or coupling proteins potentially can lead to supersensitivity, subsensitivity, or other untoward responses. Among the most interesting and significant events is the appearance of abeπant receptors as products of oncogenes, which transform otherwise normal cells into malignant cells. Virtually any type of signaling system may have oncogenic potential. The erbA oncogene product is an altered form of a receptor for thyroid honnone, constitutively active because ofthe loss of its ligand-binding domain (Evans (1988) Science 240:889-895). The ros and erbB oncogene products are activated, uncontrolled forms ofthe receptors for insulin and epidermal growth factor, both known to enhance cellular proliferation (Yarden et al. (1988) Annu. Rev. Biochem. 57:443-478). The mas oncogene product (Young et al. (1986) Cell. 4:711-719) is a G protein-coupled receptor, probably the receptor for a peptide hormone. Constitutive activation of G protein- coupled receptors due to subtle mutations in receptor structure has been shown to give rise to retinitis pigmentosa, precocious puberty, and malignant hyperthyroidism (Clapham (1993) Cell. 75:1237-1239). G proteins can themselves be oncogenic when either overexpressed or constitutively activated by mutation (Lyons et al (1990) Science 249:655-659).
(1) Acetylcholine is implicated in higher functions ofthe brain, notably memory and cognition. Consistent with this there is a cholinergic deficiency in Alzheimer' s Disease, an illness associated with a severe impairment of cognitive function. Agonists ofthe acetylcholine receptor have been used clinically in the treatment of glaucoma. Minor uses include suppression of atrial tachycardias, stimulation of intestinal motility and bladder emptying. Antagonists have been used as a premedication in general anesthesia to reduce bronchial and salivary secretions and in the prevention of motion sickness. They have also been used to a limited extent in the treatment of peptic ulcer, to induce pupillary vasodilitation, to aid examination ofthe eye and in the treatment of certain inflammatory conditions.
(2) Adrenoceptors are affected by clinically important drugs used for asthma, as an anesthetic, for nasal decongestion, for hypertension, for other cardiovascular disorders, for example, angina, certain cardiac dysrhythmias and cardiac infarction, and for the freatment of anxiety and glaucoma. (3) The angiotensin receptor is the target for compounds effective in the freatment of hypertension.
(4) The bradykinin receptor is a target for treatment of inflammation, asthma, mild pain, and endotoxic shock. (5) The calcitonin receptor is a target for treatment of Paget's disease ofthe bone.
(6) The cannabinoid receptor is a potential therapeutic target as an analgesic or antiemetic agent.
(7) The cholecystokinin and gastrin receptors are implicated in the pathogenesis of schizophrenia, Parkinson's disease, drug addiction, and feeding disorders.
(8) Dopamine receptors have been implicated in Parkinson's disease, Huntington's disease and schizophrenia.
(9) The endothelin receptor is a target for several pathophysiological conditions associated with stress including hypertension, myocardial infarction, subarachnoid hemoπhage and renal failure.
(10) The galanin receptor is involved in insulin release induced by glucose and may be the sympathetic mediator of this effect during stress. It is synergistic with opiates in inducing analgesia. It stimulates feeding behavior and release of growth hormone. It may be of use in the treatment of Alzheimer's disease. Galanin agonists may be novel analgesics.
(11) The glucagon receptor is involved in the pathogenesis of diabetes. It is also been implicated in increasing the rate and force of contraction in acute cardiac failure. (12) The receptors for glucagon-like peptides 1 and 2. These receptors could serve as a target for non-insulin dependent diabetes mellitus and intestinal disorders, respectively.
(13) Glutamate receptors may be important in neuronal plasticity, cognition, memory, learning and some neurological disorders such as epilepsy, stroke and neurodengeneration. (14) Glycoprotein hormone receptors (FSH, LH/hCG, TSH) can be important in treating infertility in females and for some types of failure of spermatogenesis in males (FSH), and Graves disease (TSH).
(15) Gonadotropin-releasing hormone receptor is a potential target in therapeutic use in the suppression of prostrate cancer, precocious puberty, and endometriosis.
(16) Histamine receptors may be target for a variety of CNS functions including sexual behavior and analgesia. It may also be useful clinically in the treatment of allergic and anaphylactic reactions and various inflammatory conditions for example, hay fever and itching. It may also be useful in treatment of motion sickness. The H2 receptors are found in high levels in stomach and heart. H2 antagonists are used clinically in the treatment of peptic ulceration.
(17) 5-hydroxytryptamine receptor may be involved in a vast array of physiological and pathophysiological pathways. It is a mediator of peristalsis and may be involved in platelet aggregation and haemostasis. It may also have a role as an inflammatory mediator and involvement in microvascular control. It could be useful in a wide range of functions including control of appetite, mood, anxiety, hallucination, sleep, vomiting and pain perception and may have clinical use in the freatment of depression, migraine and post-operative vomiting. The 5-Htlb/5-Htld receptor may be the therapeutic substrate ofthe anti-migraine drug sumitriptan. These sites are also implicated in feeding, behavior, anxiety, depression, cardiac function and movement. Clinically, 5-Htl a receptors represent potential anxiolytic and anti-hypertensive targets.
(18) Leukotrienes have important physiological roles in the cardiovascular respiratory and immune systems. Some of these are found in high levels in inflammatory conditions, for example, septic shock, inflammatory bowel disease and allergic asthma. They can be found in high levels in bronchial tissue and lung where they may have a pathological role in allergic asthma and respiratory distress syndrome. Accordingly, leukotriene receptors may be useful as targets in these areas. The receptors have been involved in inducing chemostasis and adhesion of neutrophils to vascular endothelium, inducing contraction of gastrointestinal, pulmonary, reproductive, and vascular smooth muscles, and stimulating mucus secretion in bronchial tissue. (19) Melanocortins include ACTH, α-, β- and λ-melanocyte-stimulating hormones (MSH), and β-endorphin. ACTH and β-endorphin are synthesized and released at times of stress, i.e. cold, infections, etc. and their release leads to metabolism and analgesia. ACTH is used clinically to diagnose adrenocorticol insufficiency and to stimulate adrenocortex function or as an alternative to glucocorticoids to treat inflammatory disorders.
(20) Melatonin regulates a variety of neuroendocrine functions and is believed to have an essential role in circadian rhythms. Drugs that modify the action of melatonin are of potential clinical importance in the modification of in circadian cycles, for example, for the treatment of jet lag.
(21) Neuropeptide Y is one of the most abundant peptides in the mammalian brain, inducing a variety of behavior effects, stimulation of food intake, anxiety, facilitation of learning and memory, and regulation ofthe cardiovascular and neuroendocrine systems. It has been implicated in the pathophysiology of hypertension, congestive heart failure, affective disorders, and appetite regulation.
(22) Neurotensin induces a variety of effects including antinoception, hypothermia and increased locomotor activity.
(23) Opioid peptides have important roles in the regulation of sensory function (including pain), neuroendocrine activity, the central control of respiration and mood, and the regulation of gut motility. Non-peptide agonists at opioid receptors include codeine, morphine and related substances. Many of these are used clinically in the treatment of pain and constipation. Some opioid receptors are believe to mediate analgesia sedation, mitosis and diuresis.
(24) Parathyroid hormone is involved in calcium homeostasis. Antagonists at the parathyroid hormone receptor are of potential clinical use in the treatment of hyperparathyroidism and short-term hypercalcemic states.
(25) Platelet activating factor is an important mediator in allergic and inflammatory conditions. Platelet activating factor antagonists are potential anti- inflammatory and anti-asthmatic agents. (26) Prostanoids (prostiglandins and thromboxanes) mediate a wide variety of actions and have important physiological roles in the cardiovascular and immune systeήis and in pain sensation. At least five classes of prostanoid receptors are known to exist. They mediate relaxation in vascular, gastrointestinal, and uterine smooth muscle in human, inhibit platelet activation, and modify release of hypothalamic and pituitary hormones. Some also inhibit neurotransmitter release in central and autonomic nerves and inhibit secretion in glandular tissues, i.e. acid secretion from gastric mucosa and sodium and water reabsorption in kidney.
(27) Somatostatin is a neurotransmitter/hormone with a wide spectrum of biological actions. It has been used clinically in the treatment of certain tumors, carcinoid syndrome and glucagonoma. A reduction in cortical somatostatin levels has been reported in Alzheimer's disease and Parkinson's disease. (28) Tachykinins are a family of peptide neurotransmitters. They can stimulate smooth muscle contraction, glandular secretion, induce activation of cells of the immune system, and activate peripheral nerves. They can also regulate dopaminergic neurons and are involved in the transmission of sensory information, including noxious stimuli. (29) Thrombin has a role in blood clotting. It cleaves a number of substrates involved in coagulation and activates cell surface receptors through proteolytic action. It stimulates aggregation and secretion in blood platelets and has inflammatory and reparative actions. It activates a number of substrates that are involved in the coagulation process. Accordingly, the thrombin receptor is a target for the treatment of clotting disorders, and inflammatory disorders.
(30) Thyrotrophin releasing hormone releases thyroid stimulating hormone and stimulates the synthesis and release of prolactin.
(31) Vasoactive intestinal polypeptide family is grouped with the number of structurally related peptides that share an overlapping profile of biological activity. It induces relaxation in smooth muscle, for example, intestine, blood vessels and trachea. It inhibits secretion in certain tissues for example, stomach. It stimulates secretion in others for example, intestinal epithelium, pancreas, and gall bladder. It modulates activity of cells in the immune system. In the central nervous system it has a wide range of excitatory and inhibitory actions. Some members ofthe family are involved in secretion of enzymes and ions in pancreas and intestine (secretin) and regulating synthesis and release of growth hormone (growth hormone releasing factor). (32) Vasopressin and oxytocin are members of a family of peptides found in all mammalian species. Vasopressin controls the water content ofthe body and acts in the kidney to increase water and sodium absorption. It can stimulate the contraction of vascular smooth muscle, stimulate glycogen breakdown in liver, induce platelet activation, or evoke release or corticotrophin. Vasopressin is used clinically to treat diabetes insipidus. Oxytocin stimulates contraction of uterine smooth muscle, and stimulates milk secretion. It is used clinically to induce labor and to promote lactation.
(33) The presence of genes encoding seven transmembrane proteins in the viral genome may be relevant for virally induced cell transformation and proliferation. Ligands targeted to the polypeptides may represent a novel class of antiviral drugs.
The disclosed invention further relates to the modulation, diagnosis, and treatment of various other disorders. Disorders involving the spleen include, but are not limited to, splenomegaly, including nonspecific acute splenitis, congestive spenomegaly, and spenic infarcts; neoplasms, congenital anomalies, and rupture. Disorders associated with splenomegaly include infections, such as nonspecific splenitis, infectious mononucleosis, tuberculosis, typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and echinococcosis; congestive states related to partial hypertension, such as ciπhosis ofthe liver, portal or splenic vein thrombosis, and cardiac failure; lymphohematogenous disorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia, multiple myeloma, myeloproliferative disorders, hemolytic anemias, and thrombocytopenic purpura; immunologic-inflammatory conditions, such as rheumatoid arthritis and systemic lupus erythematosus; storage diseases such as Gaucher disease, Niemann-Pick disease, and mucopolysaccharidoses; and other conditions, such as amyloidosis, primary neoplasms and cysts, and secondary neoplasms.
Disorders involving the lung include, but are not limited to, congenital anomalies; atelectasis; diseases of vascular origin, such as pulmonary congestion and edema, including hemodynamic pulmonary edema and edema caused by microvascular injury, adult respiratory distress syndrome (diffuse alveolar damage), pulmonary embolism, hemoπhage, and infarction, and pulmonary hypertension and vascular sclerosis; chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis; diffuse interstitial (infiltrative, restrictive) diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia (pulmonary infiltration with eosinophilia), Bronchiolitis obliterans- organizing pneumonia, diffuse pulmonary hemoπhage syndromes, including Goodpasture syndrome, idiopathic pulmonary hemosiderosis and other hemoπhagic syndromes, pulmonary involvement in collagen vascular disorders, and pulmonary alveolar proteinosis; complications of therapies, such as drug-induced lung disease, radiation-induced lung disease, and lung transplantation; tumors, such as bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies ofthe pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
Disorders involving the colon include, but are not limited to, congenital anomalies, such as atresia and stenosis, Meckel diverticulum, congenital aganglionic megacolon-Hirschsprung disease; enterocolitis, such as diarrhea and dysentery, infectious enterocolitis, including viral gastroenteritis, bacterial enterocolitis, necrotizing enterocolitis, antibiotic-associated colitis (pseudomembranous colitis), and collagenous and lymphocytic colitis, miscellaneous intestinal inflammatory disorders, including parasites and protozoa, acquired immunodeficiency syndrome, transplantation, drug- induced intestinal injury, radiation enterocolitis, neutropenic colitis (typhlitis), and diversion colitis; idiopathic inflammatory bowel disease, such as Crohn disease and ulcerative colitis; tumors ofthe colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors. Disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and ciπhosis, such as ciπhosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and toxin-induced liver disease, such as alcoholic liver disease; inborn eπors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, al-antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease, such as secondary biliary ciπhosis, primary biliary ciπhosis, primary sclerosing cholangitis, and anomalies ofthe biliary tree; circulatory disorders, such as impaired blood flow into the liver, including hepatic artery compromise and portal vein obstruction and thrombosis, impaired blood flow through the liver, including passive congestion and centrilobular necrosis and peliosis hepatis, hepatic vein outflow obstruction, including hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease associated with pregnancy, such as preeclampsia and eclampsia, acute fatty liver of pregnancy, and intrehepatic cholestasis of pregnancy; hepatic complications of organ or bone marrow transplantation, such as drug toxicity after bone maπow transplantation, graft- versus- host disease and liver rejection, and nonimmunologic damage to liver allografts; tumors and tumorous conditions, such as nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma ofthe liver and metastatic tumors. Disorders involving the uterus and endometrium include, but are not limited to, endometrial histology in the menstrual cycle; functional endometrial disorders, such as anovulatory cycle, inadequate luteal phase, oral contraceptives and induced endometrial changes, and menopausal and postmenopausal changes; inflammations, such as chronic endometritis; adenomyosis; endometriosis; endometrial polyps; endometrial hyperplasia; malignant tumors, such as carcinoma ofthe endometrium; mixed Mullerian and mesenchymal tumors, such as malignant mixed Mullerian tumors; tumors ofthe myometrium, including leiomyomas, leiomyosarcomas, and endometrial stromal tumors. Disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and hemiation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states-- global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemoπhage, including intracerebral (intraparenchymal) hemoπhage, subarachnoid hemoπhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemoπhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroboπeliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases ofthe nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemoπhagic encephalomyelitis, and other diseases with demyelinatiόn; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyofrophic lateral sclerosis (motor neuron disease), bulbospinal afrophy (Kennedy syndrome), and spinal muscular afrophy; inborn eπors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin Bl) deficiency and vitamin B12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, ohgodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.
Disorders involving T-cells include, but are not limited to, cell-mediated hypersensitivity, such as delayed type hypersensitivity and T-cell-mediated cytotoxicity, and transplant rejection; autoimmune diseases, such as systemic lupus erythematosus, Sjόgren syndrome, systemic sclerosis, inflammatory myopathies, mixed connective tissue disease, and polyarteritis nodosa and other vasculitides; immunologic deficiency syndromes, including but not limited to, primary immunodeficiencies, such as thymic hypoplasia, severe combined immunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory) proliferations of white cells, including but not limited to, leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecific lymphadenitis;
* neoplastic proliferations of white cells, including but not limited to lymphoid neoplasms, such as precursor T-cell neoplasms, such as acute lymphoblastic leukemia/lymphoma, peripheral T-cell and natural killer cell neoplasms that include peripheral T-cell lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides and Sezary syndrome, and Hodgkin disease.
Diseases ofthe skin, include but are not limited to, disorders of pigmentation and melanocytes, including but not limited to, vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, and malignant melanoma; benign epithelial tumors, including but not limited to, seboπheic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors ofthe dermis, including but not limited to, benign fibrous histiocytoma, dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of cellular immigrants to the skin, including but not limited to, histiocytosis X, mycosis fungoides (cutaneous T- cell lymphoma), and mastocytosis; disorders of epidermal maturation, including but not limited to, ichthyosis; acute inflammatory dermatoses, including but not limited to, urticaria, acute eczematous dermatitis, and erythema multiforme; chronic inflammatory dermatoses, including but not limited to, psoriasis, lichen planus, and lupus erythematosus; blistering (bullous) diseases, including but not limited to, pemphigus, bullous pemphigoid, dermatitis herpetiformis, and noninflammatory blistering diseases: epidermolysis bullosa and porphyria; disorders of epidermal appendages, including but not limited to, acne vulgaris; panniculitis, including but not limited to, erythema nodosum and erythema indurarum; and infection and infestation, such as verrucae, molluscum contagiosum, impetigo, superficial fungal infections, and arthropod bites, stings, and infestations.
In normal bone maπow, the myelocytic series (polymorphoneuclear cells) make up approximately 60% ofthe cellular elements, and the erythrocytic series, 20-30%. Lymphocytes, monocytes, reticular cells, plasma cells and megakaryocytes together constitute 10-20%. Lymphocytes make up 5-15% of normal adult maπow. In the bone maπow, cell types are add mixed so that precursors of red blood cells (erythroblasts), macrophages (monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes (myeloblasts), and lymphocytes (lymphoblasts) can be visible in one microscopic field. The various types of cells and stages of each would be known to the person of ordinary skill in the art and are found, for example, on page 42 (Figure 2-8) of Immunology, Imunopathology and Immunity, Fifth Edition, Sell et al. Simon and Schuster (1996), incorporated by reference for its teaching of cell types found in the bone maπow. According, the invention is directed to disorders arising from these cells. These disorders include but are not limited to the following: diseases involving hematopoeitic stem cells; committed lymphoid progenitor cells; lymphoid cells, including B and T- cells; committed myeloid progenitors, including monocytes, granulocytes, and megakaryocytes; and committed erythroid progenitors. These include but are not limited to the leukemias, including B-lymphoid leukemias, T-lymphoid leukemias, ^differentiated leukemias; erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias are encompassed with and without differentiation]; chronic and acute lymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronic and acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic and acute myeloblastic leukemia, chronic and acute myelogenous leukemia, chronic and acute promyelocytic leukemia, chronic and acute myelocytic leukemia, hematologic malignancies of monocyte- macrophage lineage, such as juvenile chronic myelogenous leukemia; secondary AML, antecedent hematological disorder; refractory anemia; aplastic anemia; reactive cutaneous angioendotheliomatosis; fibrosing disorders involving altered expression in dendritic cells, disorders including systemic sclerosis, E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitis localized forms of scleroderma, keloid, and fibrosing colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma, including primary head and neck squamous cell carcinoma; sarcoma, including kaposi's sarcoma; fibroadanoma and phyllodes tumors, including mammary fibroadenoma; sfromal tumors; phyllodes tumors, including histiocytoma; erythroblastosis; neurofibromatosis; diseases ofthe vascular endothelium; demyelinating, particularly in old lesions; gliosis, vasogenic edema, vascular disease, Alzheimer's and Parkinson's disease; T-cell lymphomas; B-cell lymphomas. Disorders involving the heart, include but are not limited to, heart failure, including but not limited to, cardiac hypertrophy, left-sided heart failure, and right-sided heart failure; ischemic heart disease, including but not limited to angina pectoris, myocardial infarction, chronic ischemic heart disease, and sudden cardiac death; hypertensive heart disease, including but not limited to, systemic (left-sided) hypertensive heart disease and pulmonary (right-sided) hypertensive heart disease; valvular heart disease, including but not limited to, valvular degeneration caused by calcification, such as calcific aortic stenosis, calcification of a congenitally bicuspid aortic valve, and mitral annular calcification, and myxomatous degeneration ofthe mitral valve (mitral valve prolapse), rheumatic fever and rheumatic heart disease, infective endocarditis, and noninfected vegetations, such as nonbacterial thrombotic endocarditis and endocarditis of systemic lupus erythematosus (Libman-Sacks disease), carcinoid heart disease, and complications of artificial valves; myocardial disease, including but not limited to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and myocarditis; pericardial disease, including but not limited to, pericardial effusion and hemopericardium and pericarditis, including acute pericarditis and healed pericarditis, and rheumatoid heart disease; neoplastic heart disease, including but not limited to, primary cardiac tumors, such as myxoma, lipoma, papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms; congenital heart disease, including but not limited to, left-to-right shunts- late cyanosis, such as atrial septal defect, ventricular septal defect, patent ductus arteriosus, and atrioventricular septal defect, right-to-left shunts—early cyanosis, such as tetralogy of fallot, transposition of great arteries, truncus arteriosus, fricuspid afresia, and total anomalous pulmonary venous connection, obstructive congenital anomalies, such as coarctation of aorta, pulmonary stenosis and afresia, and aortic stenosis and atresia, and disorders involving cardiac transplantation.
Disorders involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease-the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis
(microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis, thromboanglitis obliterans (Buerger disease), vasculitis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and aortic dissection (dissecting hematoma); disorders of veins and lymphatics, such as varicose veins, thrombophlebitis and phlebothrombosis, obstruction of superior vena cava (superior vena cava syndrome), obstruction of inferior vena cava (inferior vena cava syndrome), and lymphangitis and lymphedema; tumors, including benign tumors and tumor-like conditions, such as hemangioma, lymphangioma, glomus tumor (glomangioma), vascular ectasias, and bacillary angiomatosis, and intermediate-grade (borderline low- grade malignant) tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignant tumors, such as angiosarcoma and hemangiopericytoma; and pathology of therapeutic interventions in vascular disease, such as balloon angioplasty and related techniques and vascular replacement, such as coronary artery bypass graft surgery.
Disorders involving red cells include, but are not limited to, anemias, such as hemolytic anemias, including hereditary spherocytosis, hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphate dehydrogenase deficiency, sickle cell disease, thalassemia syndromes, paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, and hemolytic anemia resulting from trauma to red cells; and anemias of diminished erythropoiesis, including megaloblastic anemias, such as anemias of vitamin B12 deficiency: pernicious anemia, and anemia of folate deficiency, iron deficiency anemia, anemia of chronic disease, aplastic anemia, pure red cell aplasia, and other forms of maπow failure.
Disorders involving the thymus include developmental disorders, such as DiGeorge syndrome with thymic hypoplasia or aplasia; thymic cysts; thymic hypoplasia, which involves the appearance of lymphoid follicles within the thymus, creating thymic foUicular hyperplasia; and thymomas, including germ cell tumors, lynphomas, Hodgkin disease, and carcinoids. Thymomas can include benign or encapsulated thymoma, and malignant thymoma Type I (invasive thymoma) or Type II, designated thymic carcinoma.
Disorders involving B-cells include, but are not limited to precursor B-cell neoplasms, such as lymphoblastic leukemia lymphoma. Peripheral B-cell neoplasms include, but are not limited to, chronic lymphocytic leukemia small lymphocytic lymphoma, foUicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and related entities, lymphoplasmacytic lymphoma (Waldensfrom macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma (MALToma), and hairy cell leukemia.
Disorders involving the kidney include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia, autosomal dominant (adult) polycystic kidney disease, autosomal recessive (childhood) polycystic kidney disease, and cystic diseases of renal medulla, which include, but are not limited to, medullary sponge kidney, and nephronophthisis-uremic medullary cystic disease complex, acquired (dialysis- associated) cystic disease, such as simple cysts; glomerular diseases including pathologies of glomerular injury that include, but are not limited to, in situ immune complex deposition, that includes, but is not limited to, anti-GBM nephritis, Heymann nephritis, and antibodies against planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative complement pathway, epithelial cell injury, and pathologies involving mediators of glomerular injury including cellular and soluble mediators, acute glomerulonephritis, such as acute proliferative (poststreptococcal, postinfectious) glomerulonephritis, including but not limited to, poststreptococcal glomerulonephritis and nonstrepfococcal acute glomerulonephritis, rapidly progressive (crescentic) glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, IgA nephropathy (Berger disease), focal proliferative and necrotizing glomerulonephritis (focal glomerulonephritis), hereditary nephritis, including but not limited to, Alport syndrome and thin membrane disease (benign familial hematuria), chronic glomerulonephritis, glomerular lesions associated with systemic disease, including but not limited to, systemic lupus erythematosus, Henoch-Schonlein purpura, bacterial endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary and immunotactoid glomerulonephritis, and other systemic disorders; diseases affecting tubules and interstitium, including acute tubular necrosis and tubulointerstitial nephritis, including but not limited to, pyelonephritis and urinary tract infection, acute pyelonephritis, chronic pyelonephritis and reflux nephropathy, and tubulointerstitial nephritis induced by drugs and toxins, including but not limited to, acute drug-induced interstitial nephritis, analgesic abuse nephropathy, nephropathy associated with nonsteroidal anti- inflammatory drugs, and other tubulointerstitial diseases including, but not limited to, urate nephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases of blood vessels including benign nephrosclerosis, malignant hypertension and accelerated nephrosclerosis, renal artery stenosis, and thrombotic microangiopathies including, but not limited to, classic (childhood) hemolytic-uremic syndrome, adult hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura, idiopathic
HUS/TTP, and other vascular disorders including, but not limited to, atherosclerotic ischemic renal disease, atheroembolic renal disease, sickle cell disease nephropathy, diffuse cortical necrosis, and renal infarcts; urinary tract obstruction (obstructive uropathy); urolithiasis (renal calculi, stones); and tumors ofthe kidney including, but not limited to, benign tumors, such as renal papillary adenoma, renal fibroma or hamartoma (renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma, and malignant tumors, including renal cell carcinoma (hypemephroma, adenocarcinoma of kidney), which includes urothelial carcinomas of renal pelvis.
Disorders ofthe breast include, but are not limited to, disorders of development; inflammations, including but not limited to, acute mastitis, periductal mastitis, periductal mastitis (recuπent subareolar abscess, squamous metaplasia of lactiferous ducts), mammary duct ectasia, fat necrosis, granulomatous mastitis, and pathologies associated with silicone breast implants; fibrocystic changes; proliferative breast disease including, but not limited to, epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors including, but not limited to, sfromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papiUoma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, no special type, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.
Disorders involving the testis and epididymis include, but are not limited to, congenital anomalies such as cryptorchidism, regressive changes such as atrophy, inflammations such as nonspecific epididymitis and orchitis, granulomatous (autoimmune) orchitis, and specific inflammations including, but not limited to, gonoπhea, mumps, tuberculosis, and syphilis, vascular disturbances including torsion, testicular tumors including germ cell tumors that include, but are not limited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolk sac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sex cord-gonadal sfroma including, but not limited to, Ley dig (interstitial) cell tumors and sertoli cell tumors
(androblastoma), and testicular lymphoma, and miscellaneous lesions of tunica vaginalis. Disorders involving the prostate include, but are not limited to, inflammations, benign enlargement, for example, nodular hyperplasia (benign prostatic hypertrophy or hyperplasia), and tumors such as carcinoma.
Disorders involving the thyroid include, but are not limited to, hyperthyroidism; hypothyroidism including, but not limited to, cretinism and myxedema; thyroiditis including, but not limited to, hashimoto thyroiditis, subacute (granulomatous) thyroiditis, and subacute lymphocytic (painless) thyroiditis; Graves disease; diffuse and multinodular goiter including, but not limited to, diffuse nontoxic (simple) goiter and multinodular goiter; neoplasms ofthe thyroid including, but not limited to, adenomas, other benign tumors, and carcinomas, which include, but are not limited to, papillary carcinoma, foUicular carcinoma, medullary carcinoma, and anaplastic carcinoma; and cogenital anomalies.
Disorders involving the skeletal muscle include tumors such as rhabdomyosarcoma. Disorders involving the pancreas include those ofthe exocrine pancreas such as congenital anomalies, including but not limited to, ectopic pancreas; pancreatitis, including but not limited to, acute pancreatitis; cysts, including but not limited to, pseudocysts; tumors, including but not limited to, cystic tumors and carcinoma ofthe pancreas; and disorders ofthe endocrine pancreas such as, diabetes mellitus; islet cell tumors, including but not limited to, insulinomas, gastrinomas, and other rare islet cell tumors.
Disorders involving the small intestine include the malabsorption syndromes such as, celiac sprue, tropical sprue (postinfectious sprue), whipple disease, disaccharidase (lactase) deficiency, abetalipoproteinemia, and tumors ofthe small intestine including adenomas and adenocarcinoma.
Disorders related to reduced platelet number, thrombocytopenia, include idiopathic thrombocytopenic purpura, including acute idiopathic thrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic microangiopathies: thrombotic thrombocytopenic purpura and hemolytic- uremic syndrome.
Disorders involving precursor T-cell neoplasms include precursor T lymphoblastic leukemia/lymphoma. Disorders involving peripheral T-cell and natural killer cell neoplasms include T-cell chronic lymphocytic leukemia, large granular lymphocytic leukemia, mycosis fungoides and Sezary syndrome, peripheral T-cell lymphoma, unspecified, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma (NK/T-cell lymphoma4a), intestinal T-cell lymphoma, adult T-cell leukemia/lymphoma, and anaplastic large cell lymphoma.
Disorders involving the ovary include, for example, polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma peritonei and sfromal hyperthecosis; ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors. Bone-forming cells include the osteoprogenitor cells, osteoblasts, and osteocytes.
The disorders ofthe bone are complex because they may have an impact on the skeleton during any of its stages of development. Hence, the disorders may have variable manifestations and may involve one, multiple or all bones ofthe body. Such disorders include, congenital malformations, achondroplasia and thanatophoric dwarfism, diseases associated with abnormal matix such as type 1 collagen disease, osteoporosis, Paget disease, rickets, osteomalacia, high-turnover osteodystrophy, low-turnover of aplastic disease, osteonecrosis, pyogenic osteomyelitis, tuberculous osteomyelitism, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous cortical defects, fibrous dysplasia, fibrosarcoma, malignant fibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal tumor, giant cell tumor, and metastatic tumors.
The 14266 sequences ofthe invention are members of a family of molecules (the "G-protein coupled receptors" or "GPCRs") having conserved functional features. The term "family" when refeπing to the proteins and nucleic acid molecules ofthe invention is intended to mean two or more proteins or nucleic acid molecules having sufficient amino acid or nucleotide sequence identity as defined herein. Such family members can be naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of murine origin and a homologue of that protein of human origin, as well as a second, distinct protein of human origin and a murine homologue of that protein. Members of a family may also have common functional characteristics.
Methods of Using 14266
The invention provides methods using the 14266 variants, or fragments, including but not limited to use in the cells, tissues, and disorders as disclosed herein. The protein sequences ofthe present invention can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J Mol. Biol. 2i5:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules ofthe invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the proteins ofthe invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17) :3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
The 14266 polypeptides are useful for producing antibodies specific for the 14266, regions, or fragments.
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) that bind to 14266 receptors or have a stimulatory or inhibitory effect on, for example, 14266 receptor expression or 14266 receptor activity.
The invention provides screening assays, in cell-based or cell-free systems. Cell- based systems can be native, i.e., cells that normally express the 14266 receptor, as a biopsy, or expanded in cell culture. In one embodiment, cell-based assays involve recombinant host cells expressing the 14266 receptor. Accordingly, cells that are useful in this regard include, but are not limited to, those disclosed herein as expressing 1466. Cells containing one or more copies of exogenously-introduced 14266 sequences or cells genetically modified to modulate expression ofthe endogenous 14266 sequence may also be used.
The test compounds ofthe present invention can be obtained using any ofthe 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, nonpeptide ohgomer, or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Caπell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (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 (U.S. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl.
Acad. Sci. USA 89:1865-1869), 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).
Determining the ability ofthe test compound to bind to the 14266 receptor can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the 14266 receptor or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 1251, 35S, ! C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
In a similar manner, one may determine the ability ofthe 14266 receptor to bind to or interact with a 14266 target molecule. By "target molecule" is intended a molecule with which a 14266 receptor binds or interacts in nature. In a prefeπed embodiment, the ability ofthe 14266 receptor to bind to or interact with a 14266 target molecule can be determined by monitoring the activity ofthe target molecule. For example, the activity ofthe target molecule can be monitored by detecting induction of a cellular second messenger ofthe target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a 14266 -responsive regulatory element 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 ofthe present invention is a cell-free assay comprising contacting a 14266 receptor or biologically active portion thereof with a test compound and determining the ability ofthe test compound to bind to the 14266 receptor or biologically active portion thereof. Binding ofthe test compound to the 14266 receptor can be determined either directly or indirectly as described above. In a prefeπed embodiment, the assay includes contacting the 14266 receptor or biologically active portion thereof with a known compound that binds the 14266 receptor to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to preferentially bind to the 14266 receptor or biologically active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-free assay comprising contacting the 14266 receptor or biologically active portion thereof with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe 14266 receptor or biologically active portion thereof. Determining the ability ofthe test compound to modulate the activity of a 14266 receptor can be accomplished, for example, by determining the ability ofthe 14266 receptor to bind to a 14266 target molecule as described above for determining direct binding. In an alternative embodiment, determining the ability ofthe test compound to modulate the activity of a 14266 receptor can be accomplished by determining the ability ofthe 14266 receptor to further modulate a 14266 target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as previously described.
In yet another embodiment, the cell-free assay comprises contacting the 14266 receptor or biologically active portion thereof with a known compound that binds a 14266 receptor to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to preferentially bind to or modulate the activity of a 14266 target molecule.
In the above-mentioned assays, it may be desirable to immobilize either a 14266 receptor or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example, glutathione-S- fransferase/14266 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtitre plates, which are then combined with the test compound or the test compound and either the nonadsorbed target protein or 14266 receptor, 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 14266 binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays ofthe invention. For example, either the 14266 receptor or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated 14266 molecules 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 Chemicals). Alternatively, antibodies reactive with a 14266 receptor or target molecules but which do not interfere with binding ofthe 14266 receptor to its target molecule can be derivatized to the wells ofthe plate, and unbound target or 14266 receptor 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 14266 receptor or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the 14266 receptor or target molecule. In another embodiment, modulators of 14266 expression are identified in a method in which a cell is contacted with a candidate compound and the expression of 14266 mRNA or protein in the cell is determined relative to expression of 14266 mRNA or protein in a cell in the absence ofthe candidate compound. When expression is greater (statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of 14266 mRNA or protein expression. Alternatively, when expression is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of 14266 mRNA or protein expression. The level of 14266 mRNA or protein expression in the cells can be determined by methods described herein for detecting 14266 mRNA or protein.
In yet another aspect ofthe invention, thel4266 receptors 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 14266 receptor (" 14266-binding proteins" or "14266-bp") and modulate 14266 activity. Such 14266-binding proteins are also likely to be involved in the propagation of signals by the 14266 receptors as, for example, upstream or downstream elements ofthe signal transduction pathway. This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein. B. 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) purposes to thereby treat an individual prophylactically. These applications are described in the subsections below.
1. Diagnostic Assays
One aspect ofthe present invention relates to diagnostic assays for detecting 14266 receptor and/or nucleic acid expression as well as 14266 activity, in the context of a biological sample. An exemplary method for detecting the presence or absence of
14266 receptors 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 14266 receptor or nucleic acid (e.g., mRNA, genomic DNA) that encodes 14266 receptor such that the presence of 14266 receptor is detected in the biological sample. Results obtained with a biological sample from the test subject may be compared to results obtained with a biological sample from a confrol subject.
"Misexpression or abeπant expression", as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non- wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms ofthe time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms ofthe splicing size, amino acid sequence, post-transitional modification, or biological activity ofthe expressed polypeptide; a pattern of expression that differs from wild type in terms ofthe effect of an environmental stimulus or extracellular stimulus on expression ofthe gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength ofthe stimulus.
A prefeπed agent for detecting 14266 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to 14266 mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length nucleic acid of SEQ ID NO:2, or a portion thereof, such as a nucleic acid molecule of at least 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 14266 mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.
A prefeπed agent for detecting 14266 receptor is an antibody capable of binding to 14266 receptor, 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(abN) )can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe 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 ofthe invention can be used to detect 14266 mRNA, protein, or genomic DNA in a biological sample in vifro as well as in vivo. For example, in vifro techniques for detection of 14266 mRNA include Northern hybridizations and in situ hybridizations. In vifro techniques for detection ofthe 14266 receptor include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vifro techniques for detection of 14266 genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of 14266 receptor include introducing into a subject a labeled anti- 14266 antibody. 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.
The invention also encompasses kits for detecting the presence of 14266 receptors 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 14266 receptor. For example, the kit can comprise a labeled compound or agent capable of detecting 14266 receptor or mRNA in a biological sample and means for determining the amount of a 14266 receptor in the sample (e.g., an anti- 14266 antibody or an oligonucleotide probe that binds to DNA encoding a 14266 receptor, e.g., encoded by the nucleic acid sequences of SEQ ID NO:2). 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 14266 sequences if the amount of 14266 receptor or mRNA 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) that binds to the 14266 receptor; and, optionally, (2) a second, different antibody that binds to the 14266 receptor 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, that hybridizes to a 14266 nucleic acid sequence or (2) a pair of primers useful for amplifying a 14266 nucleic acid molecule.
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 that can be assayed and compared to the test sample contained. Each component ofthe kit is usually enclosed within an individual container, and all ofthe 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 14266 receptors.
2. Other Diagnostic Assays
In another aspect, the invention features a method of analyzing a plurality of capture probes. The method can be used, e.g., to analyze gene expression. The method includes: providing a two dimensional aπay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence; contacting the aπay with a 14266 nucleic acid, preferably purified, polypeptide, preferably purified, or antibody, and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization, with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a label attached to the 14266 nucleic acid, polypeptide, or antibody. The capture probes can be a set of nucleic acids from a selected sample, e.g., a sample of nucleic acids derived from a confrol or non-stimulated tissue or cell.
The method can include contacting the 14266 nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second aπay having a different plurality of capture probes. The results of each hybridization can be compared, e.g., to analyze differences in expression between a first and second sample. The first plurality of capture probes can be from a control sample, e.g., a wild type, normal, or non-diseased, non-stimulated, sample, e.g., a biological fluid, tissue, or cell sample. The second plurality of capture probes can be from an experimental sample, e.g., a mutant type, at risk, disease-state or disorder-state, or stimulated, sample, e.g., a biological fluid, tissue, or cell sample.
The plurality of capture probes can be a plurality of nucleic acid probes each of which specifically hybridizes, with an allele of a 14266 sequence ofthe invention. Such methods can be used to diagnose a subject, e.g., to evaluate risk for a disease or disorder, to evaluate suitability of a selected treatment for a subject, to evaluate whether a subject has a disease or disorder.
The method can be used to detect single nucleotide polymoφhisms (SNPs), as described below.
In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional aπay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express a 14266 polypeptide ofthe invention or from a cell or subject in which a 14266-mediated response has been elicited, e.g., by contact ofthe cell with a 14266 nucleic acid or protein ofthe invention, or adminisfration to the cell or subject a 14266 nucleic acid or protein ofthe invention; contacting the aπay with one or more inquiry probes, wherein an inquiry probe can be a nucleic acid, polypeptide, or antibody (which is preferably other than a 14266 nucleic acid, polypeptide, or antibody ofthe invention); providing a two dimensional aπay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express a 14266 sequence ofthe invention (or does not express as highly as in the case ofthe 14266 positive plurality of capture probes) or from a cell or subject in which a 14266-mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the aπay with one or more inquiry probes (which is preferably other than a 14266 nucleic acid, polypeptide, or antibody ofthe invention), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization, with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.
In another aspect, the invention features a method of analyzing a 14266 sequence ofthe invention, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 14266 nucleic acid or amino acid sequence, e.g., the sequence set forth in SEQ ID NO:2 (nucleic acid) or SEQ ID NO: 1 (amino acid) or a portion thereof; comparing the 14266 sequence with one or more, preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze the 14266 sequence ofthe invention.
The method can include evaluating the sequence identity between a 14266 sequence ofthe invention and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the internet. In another aspect, the invention features, a set of oligonucleotides, useful, e.g., for identifying SNP's, or identifying specific alleles of a 14266 sequence ofthe invention. The set includes a plurality of oligonucleotides, each of which has a different nucleotide at an inteπogation position, e.g., an SNP or the site of a mutation. In a prefeπed embodiment, the oligonucleotides ofthe plurality identical in sequence with one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotides which hybridizes to one allele provides a signal that is distinguishable from an oligonucleotides which hybridizes to a second allele.
3. 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 14266 receptor, 14266 nucleic acid expression, or 14266 activity. Prognostic assays can be used for prognostic or predictive purposes to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with 14266 receptor, 14266 nucleic acid expression, or 14266 activity.
Thus, the present invention provides a method in which a test sample is obtained from a subject, and 14266 receptor or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of 14266 receptor or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abeπant 14266 expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, using the prognostic assays described herein, the present invention provides methods for determining whether a subject can be administered a specific agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, antibody, nucleic acid (including an antisense nucleic acid or a ribozyme), small molecule, or other drug candidate) or class of agents (e.g., agents of a type that decrease 14266 activity) to effectively treat a disease or disorder associated with abeπant 14266 expression or activity. In this manner, a test sample is obtained and 14266 receptor or nucleic acid is detected. The presence of 14266 receptor or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with abeπant 14266 expression or activity.
The methods ofthe invention can also be used to detect genetic lesions or mutations in a 14266 gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by abeπant cell proliferation and/or differentiation. 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 a 14266 protein, or the misexpression ofthe 14266 gene. 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 a 14266 gene; (2) an addition of one or more nucleotides to a 14266 gene; (3) a substitution of one or more nucleotides of a 14266 gene; (4) a chromosomal rearrangement of a 14266 gene; (5) an alteration in the level of a messenger RNA transcript of a 14266 gene; (6) an abeπant modification of a 14266 gene, such as ofthe methylation pattern ofthe genomic DNA; (7) the presence of a non- wild-type splicing pattern of a messenger RNA transcript of a 14266 gene; (8) a non- wild-type level of a 14266-protein; (9) an allelic loss of a 14266 gene; and (10) an inappropriate post- translational modification of a 14266-protein. As described herein, there are a large number of assay techniques known in the art that can be used for detecting lesions in a 14266 gene. Any cell type or tissue, in which 14266 receptors are expressed may be utilized in the prognostic assays described herein. In certain embodiments, detection ofthe 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 μseful for detecting point mutations in the 14266-gene (see, e.g.,
Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). It is anticipated that the PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe 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 ofthe 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 14266 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns of isolated test sample and confrol DNA digested with one or more restriction endonucleases. 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 in a 14266 molecule 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). In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 14266 gene and detect mutations by comparing the sequence ofthe sample 14266 gene 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 the 14266 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). See, also 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 confrol DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more "DNA mismatch repair" enzymes that recognize mismatched base pairs in double- stranded DNA in defined systems for detecting and mapping point mutations in 14266 cDNAs obtained from samples of cells. See, e.g., Hsu et al. (1994) Carcinogenesis 15 : 1657- 1662. According to an exemplary embodiment, a probe based on a 14266 sequence, e.g., a wild-type 14266 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 14266 genes. For example, single-strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayaslii (1992) Genet. Anal. Tech. Appl. 9:73-79). The sensitivity ofthe 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 confrol 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 that 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 ofthe molecule so that amplification depends on differential hybridization (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the exfreme 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 ofthe 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 ofthe 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnosed patients exhibiting symptoms or family history of a disease or illness involving a 14266 gene.
4. Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on 14266 activity (e.g., 14266 gene expression) as identified by a screening assay described herein, can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with abeπant 14266 activity as well as to modulate the phenotype of a differentiative or cell proliferation disorder. In conjunction with such treatment, the pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drug) ofthe 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 ofthe pharmacologically active drug. Thus, the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of 14266 receptor, expression of 14266 nucleic acid, or mutation content of 14266 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe 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 (antimalarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", relies primarily on a high-resolution map ofthe human genome consisting of already known gene-related markers (e.g., a "bi- allelic" gene marker map which consists of 60,000- 100,000 polymoφhic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymoφhisms (SNPs) in the human genome. As used herein, an "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease- associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, freatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
Alternatively, a method termed the "candidate gene approach", can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 14266 receptor ofthe present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version ofthe gene versus another is associated with a particular drug response.
Alternatively, a method termed the "gene expression profiling", can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 14266 molecule or 14266 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.
Information generated from more than one ofthe above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. 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 14266 molecule or 14266 modulator ofthe invention, such as a modulator identified by one of the exemplary screening assays described herein. The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more ofthe gene products encoded by one or more ofthe 14266 genes ofthe present invention, wherein these products may be associated with resistance ofthe cells to a therapeutic agent. Specifically, the activity ofthe proteins encoded by the 14266 genes ofthe present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more ofthe resistance proteins, target cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to. Agents ofthe prsent invention include small molecule modulators, antibodies, ribozymes, peptides, and antisense nucleic acid molecules. Monitoring the influence of agents (e.g., drugs) on the expression or activity of a
14266 receptor can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 14266 gene expression, protein levels, or upregulate 14266 activity, can be monitored in clinical trials of subjects exhibiting decreased 14266 gene expression, protein levels, or downregulated 14266 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 14266 gene expression, protein levels, or downregulate 14266 activity, can be monitored in clinical trials of subjects exhibiting increased 14266 gene expression, protein levels, or upregulated 14266 activity. In such clinical trials, the expression or activity of a 14266 gene, and preferably, other genes that have been implicated in, for example, a 14266-associated disorder can be used as a "read out" or markers ofthe phenotype of a particular cell. 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 metabohzers 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 exfreme are the so called ultra-rapid metabohzers 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 14266 receptor, expression of 14266 nucleic acid, or mutation content of 14266 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 14266 modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.
5. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of 14266 genes (e.g., the ability to modulate abeπant cell proliferation 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 or decrease 14266 gene expression, protein levels, or protein activity, can be monitored in clinical trials of subjects exhibiting decreased or increased 14266 gene expression, protein levels, or protein activity. In such clinical trials, 14266 expression or activity and preferably that of other genes that have been implicated in for example, a cellular proliferation disorder, can be used as a marker of the immune responsiveness of a particular cell.
For example, and not by way of limitation, genes that are modulated in cells by treatment with an agent (e.g., compound, drug, or small molecule) that modulates 14266 activity (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 14266 genes 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 ofthe methods as described herein, or by measuring the levels of activity of 14266 genes or other genes. In this way, the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during, freatment ofthe individual with the agent.
In a prefeπed embodiment, the present invention provides a method for monitoring the effectiveness of freatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, antibody, nucleic acid (including an antisense oligonucleotide or a ribozyme), small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (1) obtaining a preadministration sample from a subject prior to administration ofthe agent; (2) detecting the level of expression of a 14266 receptor, mRNA, or genomic DNA in the preadministration sample; (3) obtaining one or more postadministration samples from the subject; (4) detecting the level of expression or activity ofthe 14266 receptor, mRNA, or genomic DNA in the postadministration samples; (5) comparing the level of expression or activity ofthe 14266 receptor, mRNA, or genomic DNA in the preadministration sample with the 14266 receptor, mRNA, or genomic DNA in the postadministration sample or samples; and (vi) altering the administration ofthe agent to the subject accordingly to bring about the desired effect, i.e., for example, an increase or a decrease in the expression or activity of a 14266 receptor.
C. Methods of Treatment
The present invention provides for both prophylactic and therapeutic methods of freating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abeπant 14266 expression or activity. "Subject", as used herein, can refer to a mammal, e.g. a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal. Additionally, the compositions ofthe invention find use in the treatment of disorders described herein. Thus, therapies for disorders associated with CCC are encompassed herein.
"Treatment" is herein defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the puφose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A "therapeutic agent" includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides. 1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject a disease or condition associated with an abeπant 14266 expression or activity by administering to the subject an agent that modulates 14266 expression or at least one 14266 gene activity. Subjects at risk for a disease that is caused, or contributed to, by abeπant 14266 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe 14266 abeπancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 14266 abeπancy, for example, a 14266 agonist or 14266 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
2. Therapeutic Methods
Another aspect ofthe invention pertains to methods of modulating 14266 expression or activity for therapeutic puφoses. The modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of 14266 receptor activity associated with the cell. An agent that modulates 14266 receptor activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a 14266 receptor, a peptide, a 14266 peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more ofthe biological activities of 14266 receptor. Examples of such stimulatory agents include active 14266 receptor and a nucleic acid molecule encoding a 14266 receptor that has been introduced into the cell. In another embodiment, the agent inhibits one or more ofthe biological activities of 14266 receptor. Examples of such inhibitory agents include antisense 14266 nucleic acid molecules and anti- 14266 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 14266 receptor or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or a combination of agents, that modulates (e.g., upregulates or downregulates) 14266 expression or activity. In another embodiment, the method involves administering a 14266 receptor or nucleic acid molecule as therapy to compensate for reduced or abeπant 14266 expression or activity.
Stimulation of 14266 activity is desirable in situations in which a 14266 receptor is abnormally downregulated and/or in which increased 14266 activity is likely to have a beneficial effect. Conversely, inhibition of 14266 activity is desirable in situations in which 14266 activity is abnormally upregulated and/or in which decreased 14266 activity is likely to have a beneficial effect.
Polypeptides
The invention thus relates to a human 14266 and to the expression of a 14266 having the deduced amino acid sequence shown in Figure 1 (SEQ ID NO:l). "14266 polypeptide" or " 14266 protein" refers to the polypeptide in SEQ ID
NO:l. The term "14266 protein" or "14266 polypeptide," however, further includes the numerous variants described herein, as well as fragments derived from the full-length 14266 and variants.
Prefeπed 14266 polypeptides ofthe present invention have an amino acid sequence sufficiently identical to the amino acid sequence encoded by the nucleic acid sequences of SEQ ID NO:2. The term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently identical. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison puφoses. The percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences (i.e., percent identity = number of identical positions/total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a prefeπed embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970) J Mol. Biol. 48:AAA-A53 algorithm which has been incoφorated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another prefeπed embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly prefeπed set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation ofthe invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incoφorated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to 14266 nucleic acid molecules ofthe invention. BLAST protein searches can be performed with the XBLAST program, score — 50, wordlength = 3, to obtain amino acid sequences homologous to 14266 protein molecules ofthe invention. To obtain gapped alignments for comparison puφoses, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another prefeπed, 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 incoφorated into the ALIGN program (version 2.0), which is part ofthe 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.
Accordingly, another embodiment ofthe invention features isolated 14266 proteins and polypeptides having a 14266 protein activity. As used interchangeably herein, a "14266 protein activity", "biological activity of a 14266 protein", or "functional activity of a 14266 protein" refers to an activity exerted by a 14266 protein, polypeptide, or nucleic acid molecule on a 14266 responsive cell as determined in vivo, or in vitro, according to standard assay techniques. A 14266 activity can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction ofthe 14266 protein with a second protein. In a prefeπed embodiment, a 14266 activity includes at least one or more ofthe following activities: (1) modulating (stimulating and/or enhancing or inhibiting) cellular proliferation, differentiation, and/or function; (2) mobilization of intracellular molecules that participate in a signal fransduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5-triphosphate (IP3) and adenylate cyclase; (3) polarization ofthe plasma membrane; (4) production or secretion of molecules; (5) alteration in the structure of a cellular component; (6) cell proliferation, e.g., synthesis of DNA; (7) cell migration; (8) cell differentiation (including neufrophil differentiation); (9) cell survival and (10) ligand-binding.
An "isolated" or "purified" 14266 nucleic acid molecule or protein, or biologically active portion thereof, is 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. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For purposes ofthe invention, "isolated" when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the isolated 14266 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 that naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived. A 14266 protein that is substantially free of cellular material includes preparations of 14266 protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non- 14266 protein (also refeπed to herein as a "contaminating protein"). When the 14266 protein or biologically active portion thereof is recombinantly produced, preferably, culture medium represents less than about 30%, 20%, 10%, or 5% ofthe volume of the protein preparation. When 14266 protein is produced by chemical synthesis, preferably the protein preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non- 14266 chemicals.
Fragments or biologically active portions ofthe 14266 receptor are also encompassed within the present invention. By "14266 receptor" is intended a protein having the amino acid sequence encoded by the amino acid sequence set forth in SEQ ID NOS: as well as fragments, biologically active portions, and variants thereof.
"Fragments" or "biologically active portions" include polypeptide fragments suitable for use as immunogens to raise anti- 14266 antibodies. Fragments include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a 14266 protein, or a fragment thereof, ofthe invention and exhibiting at least one activity of a 14266 protein, but which include fewer amino acids than the 14266 protein encoded by the nucleic acid sequences disclosed herein. Typically, biologically active portions comprise a domain or motif with at least one activity ofthe 14266 protein. A biologically active portion of a 14266 protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 14266 protein. As used here, a fragment comprises at least 5 contiguous amino acids of an amino acid sequence set forth in SEQ ID NO:l. The invention encompasses other fragments, however, such as any fragment in the protein greater than 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.
By "variants" is intended proteins or polypeptides having an amino acid sequence that is at least about 45%, 55%, 65%, preferably about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NO: 1. Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO:2, or a complement thereof, under stringent conditions. Such variants generally retain the functional activity ofthe 14266 proteins ofthe invention. Variants include polypeptides that differ in amino acid sequence due to natural allelic variation or mutagenesis. The invention also provides 14266 chimeric or fusion proteins. As used herein, a 14266 "chimeric protein" or "fusion protein" comprises a 14266 polypeptide operably linked to a non- 14266 polypeptide. A "14266 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a 14266 protein, whereas a "non- 14266 polypeptide" refers to a polypeptide having an amino acid sequence coπesponding to a protein that is not substantially identical to the 14266 protein, e.g., a protein that is different from the 14266 protein and which is derived from the same or a different organism. Within a 14266 fusion protein, the 14266 polypeptide can coπespond to all or a portion of a 14266 protein, preferably at least one biologically active portion of a 14266 protein. Within the fusion protein, the term "operably linked" is intended to indicate that the 14266 polypeptide and the non- 14266 polypeptide are fused in-frame to each other. The non-14266 polypeptide can be fused to the N-terminus or C-terminus ofthe 14266 polypeptide.
One useful fusion protein is a GST- 14266 fusion protein in which the 14266 sequences are fused to the C-terminus ofthe GST sequences. Such fusion proteins can facilitate the purification of recombinant 14266 proteins.
In yet another embodiment, the fusion protein is a 14266-immunoglobulin fusion protein in which all or part of a 14266 protein is fused to sequences derived from a member ofthe immunoglobulin protein family. The 14266-immunoglobulin fusion proteins ofthe invention can be incoφorated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a 14266 ligand and a 14266 protein on the surface of a cell, thereby suppressing 14266-mediated signal fransduction in vivo. The 14266-immunoglobulin fusion proteins can be used to affect the bioavailability of a 14266 cognate ligand. Inhibition ofthe 14266 ligand/ 14266 interaction may be useful therapeutically, both for treating proliferative, differentiative, developmental and hemOpoietic disorders and for modulating (e.g., promoting or inhibiting) cell survival. Moreover, the 14266 -immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti- 14266 antibodies in a subject, to purify 14266 ligands, and in screening assays to identify molecules that inhibit the interaction of a 14266 protein with a 14266 ligand.
Preferably, a 14266 chimeric or fusion protein ofthe invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences may be ligated together in-frame, or the fusion gene can be synthesized, such as with automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and reamplifϊed to generate a chimeric gene sequence (see, e.g., Ausubel et ah, eds. (1995) Current Protocols in Molecular Biology) (Greene Publishing and Wiley-Interscience, NY). Moreover, a 14266-encoding nucleic acid can be cloned into a commercially available expression vector such that it is linked in- frame to an existing fusion moiety.
Variants ofthe 14266 proteins can function as either 14266 agonists (mimetics) or as 14266 antagonists. Variants ofthe 14266 protein can be generated by mutagenesis, e.g., discrete point mutation or truncation ofthe 14266 protein. An agonist ofthe 14266 protein can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occuπing form ofthe 14266 protein. An antagonist ofthe 14266 protein can inhibit one or more ofthe activities ofthe naturally occuπing form ofthe 14266 protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade that includes the 14266 protein. 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 ofthe naturally occurring form ofthe protein can have fewer side effects in a subject relative to treatment with the naturally occuπing form ofthe 14266 proteins. Variants of a 14266 protein that function as either 14266 agonists or as 14266 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 14266 protein for 14266 protein agonist or antagonist activity. In one embodiment, a variegated library of 14266 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of 14266 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential 14266 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of 14266 sequences therein. There are a variety of methods that can be used to produce libraries of potential 14266 variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential 14266 sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477).
In addition, libraries of fragments of a 14266 protein coding sequence can be used to generate a variegated population of 14266 fragments for screening and subsequent selection of variants of a 14266 protein. In one embodiment, a library of coding sequence fragments can be generated by freating a double-stranded PCR fragment of a 14266 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA which can include sense/antisense pairs from different nicked products, removing single-stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, one can derive an expression library that encodes N-terminal and internal fragments of various sizes ofthe 14266 protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of 14266 proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation ofthe vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 14266 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
An isolated 14266 polypeptide ofthe invention can be used as an immunogen to generate antibodies that bind 14266 proteins using standard techniques for polyclonal and monoclonal antibody preparation. The full-length 14266 protein can be used or, alternatively, the invention provides antigenic peptide fragments of 14266 proteins for use as immunogens. The antigenic peptide of a 14266 protein comprises at least 8, preferably 10, 15, 20, or 30 amino acid residues ofthe amino acid sequence set forth in SEQ ID NO:l and encompasses an epitope of a 14266 protein such that an antibody raised against the peptide forms a specific immune complex with the 14266 protein. Prefeπed epitopes encompassed by the antigenic peptide are regions of a 14266 protein that are located on the surface ofthe protein, e.g., hydrophilic regions. Accordingly, another aspect ofthe invention pertains to anti- 14266 polyclonal and monoclonal antibodies that bind a 14266 protein. Polyclonal anti- 14266 antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with a 14266 immunogen. The anti- 14266 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 14266 protein. At an appropriate time after immunization, e.g., when the anti-14266 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) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York, NY), pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Coligan et al, eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, NY); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) in Monoclonal Antibodies: A New Dimension In Biological Analyses (Plenum Publishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-14266 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a 14266 protein to thereby isolate immunoglobulin library members that bind the 14266 protein. 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 3 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 Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antϊbod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBOJ 12:725-734.
Additionally, recombinant anti-14266 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and nonhuman portions, which can be made using standard recombinant DNA techniques, are within the scope ofthe invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication Nos. WO 86/101533 and WO 87/02671; European Patent Application Nos. 184,187, 171,496, 125,023, and 173,494; U.S. Patent Nos. 4,816,567 and 5,225,539; 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 α/. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J Natl. Cancer Inst. 80:1553-1559); Moπison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93); and U.S. Patent Nos.
5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, CA), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Completely human antibodies that recognize a selected epitope can be generated using a technique refeπed 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. This technology is described by Jespers et al. (1994) Bio/Technology 12:899-903).
An anti-14266 antibody (e.g., monoclonal antibody) can be used to isolate 14266 proteins by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-14266 antibody can facilitate the purification of natural 14266 protein from cells and of recombinantly produced 14266 protein expressed in host cells. Moreover, an anti-14266 antibody can be used to detect 14266 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe 14266 protein. Anti-14266 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotiiazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 1311, 35S, or 3H.
Further, an antibody (or fragment thereof) 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, mitoxanfrone, 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 ofthe invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta- interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 ("IX-l".), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thoφe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thoφe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
Methods for Using the Polynucleotide
The methods and uses described herein below for the 14266 polynucleotide are particularly applicable to the cells and tissues that contain detectable levels of 14266 expression as described above. These methods pertain to isolated nucleic acid molecules comprising nucleotide sequences encoding 14266 proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify 14266-encoding nucleic acids (e.g., 14266 mRNA) and fragments for use as PCR primers for the amplification or mutation of 14266 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 ofthe DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double- stranded DNA.
Nucleotide sequences encoding the 14266 proteins ofthe present invention include sequence set forth in SEQ ID NO:2 and complements thereof. By "complement" is intended a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex. The coπesponding amino acid sequence for the 14266 protein encoded by these nucleotide sequences are also encompassed by the present invention. The invention also encompasses nucleic acid molecules comprising nucleotide sequences encoding partial-length 14266 proteins, including the sequence set forth in SEQ ID NO:2, and complements thereof.
Nucleic acid molecules that are fragments of these 14266 nucleotide sequences are also encompassed by the present invention. By "fragment" is intended a portion ofthe nucleotide sequence encoding a 14266 protein. A fragment of a 14266 nucleotide sequence may encode a biologically active portion of a 14266 protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a 14266 protein can be prepared by isolating a portion of one ofthe nucleotide sequences of the invention, expressing the encoded portion ofthe 14266 protein (e.g., by recombinant expression in vitro), and assessing the activity ofthe encoded portion of the 14266 protein. Nucleic acid molecules that are fragments of a 14266 nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, nucleotides, or up to the number of nucleotides present in the 14266 nucleotide sequence disclosed herein depending upon the intended use.
It is understood that isolated fragments include any contiguous sequence not disclosed prior to the invention as well as sequences that are substantially the same and which are not disclosed. Accordingly, if an isolated fragment is disclosed prior to the present invention, that fragment is not intended to be encompassed by the invention. When a sequence is not disclosed prior to the present invention, an isolated nucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguous nucleotides. Other regions ofthe nucleotide sequence may comprise fragments of various sizes, depending upon potential homology with previously disclosed sequences.
A fragment of a 14266 nucleotide sequence that encodes a biologically active portion of a 14266 protein ofthe invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acids, or up to the total number of amino acids present in a full-length 14266 protein ofthe invention.
Fragments of a 14266 nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of a 14266 protein.
Nucleic acid molecules that are variants ofthe 14266 nucleotide sequences disclosed herein are also encompassed by the present invention. "Variants" ofthe 14266 nucleotide sequences include those sequences that encode the 14266 proteins disclosed herein but that differ conservatively because ofthe degeneracy ofthe genetic code. These naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as the polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the 14266 proteins disclosed in the present invention as discussed below. Generally, nucleotide sequence variants ofthe invention will have at least about 45%, 55%, 65%, 75%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a particular nucleotide sequence disclosed herein. A variant 14266 nucleotide sequence will encode a 14266 protein that has an amino acid sequence having at least about 45%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, 98%, or 99% identity to the amino acid sequence of a 14266 protein disclosed herein. In addition to the 14266 nucleotide sequences shown in SEQ ID NOS:2, it will be appreciated by those skilled in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequences of 14266 proteins may exist within a population (e.g., the human population). Such genetic polymoφhism in a 14266 gene may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes that occur alternatively at a given genetic locus. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a 14266 protein, preferably a mammalia 14266 protein. As used herein, the phrase "allelic variant" refers to a nucleotide sequence that occurs at a 14266 locus or to a polypeptide encoded by the nucleotide sequence. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe 14266 gene. Any and all such nucleotide variations and resulting amino acid polymoφhisms or variations in a 14266 sequence that are the result of natural allelic variation and that do not alter the functional activity of 14266 proteins are intended to be within the scope ofthe invention.
Moreover, nucleic acid molecules encoding 14266 proteins from other species (14266 homologues), which have a nucleotide sequence differing from that ofthe 14266 sequences disclosed herein, are intended to be within the scope ofthe invention. For example, nucleic acid molecules coπesponding to natural allelic variants and homologues ofthe human 14266 cDNA.of the invention can be isolated based on their identity to the human 14266 nucleic acid disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions as disclosed below. In addition to naturally-occurring allelic variants ofthe 14266 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences ofthe invention thereby leading to changes in the amino acid sequence ofthe encoded 14266 proteins, without altering the biological activity ofthe 14266 proteins. Thus, an isolated nucleic acid molecule encoding a 14266 protein having a sequence that differs from the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the coπesponding nucleotide sequence disclosed herein, 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. Such variant nucleotide sequences are also encompassed by the present invention.
For example, preferably, conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues. A
"nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a 14266 protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif.
Alternatively, variant 14266 nucleotide sequences can be made by introducing mutations randomly along all or part of a 14266 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 14266 biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity ofthe protein can be determined using standard assay techniques.
Thus the nucleotide sequences ofthe invention include the sequences disclosed herein as well as fragments and variants thereof. The 14266 nucleotide sequences ofthe invention, and fragments and variants thereof, can be used as probes and/or primers to identify and/or clone 14266 homologues in other cell types, e.g., from other tissues, as well as 14266 homologues from other mammals. Such probes can be used to detect transcripts or genomic sequences encoding the same or identical proteins. These probes can be used as part of a diagnostic test kit for identifying cells or tissues that misexpress a 14266 protein, such as by measuring levels of a 14266- encoding nucleic acid in a sample of cells from a subject, e.g., detecting 14266 mRNA levels or determining whether a genomic 14266 gene has been mutated or deleted. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences having substantial identity to the sequences ofthe invention. See, for example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY). 14266 nucleotide sequences isolated based on their sequence identity to the 14266 nucleotide sequences set forth herein or to fragments and variants thereof are encompassed by the present invention.
In a hybridization method, all or part of a known 14266 nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY). The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known 14266 nucleotide sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in a known 14266 nucleotide sequence or encoded amino acid sequence can additionally be used. The probe 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 a 14266 nucleotide sequence ofthe invention or a fragment or variant thereof. Preparation of probes for hybridization is generally known in the art and is disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), herein incoφorated by reference.
For example, in one embodiment, a previously unidentified 14266 nucleic acid molecule hybridizes under stringent conditions to a probe that is a nucleic acid molecule comprising one ofthe 14266 nucleotide sequences ofthe invention or a fragment thereof. In another embodiment, the previously unknown 14266 nucleic acid molecule is at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 2,000, 3,000, 4,000 or 5,000 nucleotides in length and hybridizes under stringent conditions to a probe that is a nucleic acid molecule comprising one of the 14266 nucleotide sequences disclosed herein or a fragment thereof.
Accordingly, in another embodiment, an isolated previously unknown 14266 nucleic acid molecule ofthe invention is at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1,100, 1,200, 1,300, or 1,400 nucleotides in length and hybridizes under stringent conditions to a probe that is a nucleic acid molecule comprising one ofthe nucleotide sequences ofthe invention, preferably the coding sequence ofthe nucleotides sequences set forth in SEQ ID NO: 2 or a complement, fragment, or variant thereof.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences 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, New York (1989)), 6.3.1-6.3.6. A prefeπed, 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 0.2X SSC, 0.1% SDS at 50°C. Another 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 0.2X SSC, 0.1% SDS at 55°C. A further 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 0.2X SSC, 0.1 % SDS at 60°C. Preferably, stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. Particularly prefeπed stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation ofthe invention) are 0.5M Sodium Phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Preferably, an isolated nucleic acid molecule that hybridizes under stringent conditions to an 14226 sequence ofthe invention coπesponds to a naturally- occuπing 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).
Thus, in addition to the 14266 nucleotide sequences disclosed herein and fragments and variants thereof, the isolated nucleic acid molecules ofthe invention also encompass homologous DNA sequences identified and isolated from other cells and/or organisms by hybridization with entire or partial sequences obtained from the 14266 nucleotide sequences disclosed herein or variants and fragments thereof. The present invention also encompasses antisense nucleic acid molecules, i.e., molecules that are complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire 14266 coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to a noncoding region ofthe coding strand of a nucleotide sequence encoding a 14266 protein. The noncoding regions are the 5' and 3' sequences that flank the coding region and are not translated into amino acids. Antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of 14266 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of 14266 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 14266 mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymatic ligation procedures known in the art.
For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occuπing nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, including, but not limited to, for example e.g., phosphorothioate derivatives and acridine substituted nucleotides. 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).
When used therapeutically, the antisense nucleic acid molecules ofthe 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 14266 protein to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation. An example of a route of administration of antisense nucleic acid molecules ofthe 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, antisense molecules can be linked to peptides or antibodies to form a complex that specifically binds to receptors or antigens expressed on a selected cell surface. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations ofthe 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 prefeπed.
An antisense nucleic acid molecule ofthe 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, which are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
Ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591) can be used to catalytically cleave 14266 mRNA transcripts to thereby inhibit translation of 14266 mRNA. A ribozyme having specificity for a 14266-encoding nucleic acid can be designed based upon the nucleotide sequence of a 14266 cDNA disclosed herein. See, e.g., Cech et al, U.S. Patent No. 4,987,071 ; and Cech et al, U.S. Patent No. 5,116,742. Alternatively, 14266 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.
The invention also encompasses nucleic acid molecules that form triple helical structures. ' For example, 14266 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe 14266 protein (e.g., the 14266 promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe 14266 gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N.Y. Acad. Sci. 660:27; and Maher (1992) Bioassays 14(12):807.
In prefeπed embodiments, the nucleic acid molecules ofthe invention can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule. For example, the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:5). 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, for example, in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl Acad. Sci. USA 93:14670.
PNAs of a 14266 molecule 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 ofthe invention 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), supra). In another embodiment, PNAs of a 14266 molecule can be modified, e.g., to enhance their stability, specificity, 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. The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra; Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.
Methods Using Vectors and Host Cells
The methods using vectors and host cells are particularly relevant where vectors are expressed in the cells and tissues with detectable levels of 14266 expression as described herein, or where the host cells are those that naturally express the gene or which may be the native or a recombinant cell expressing the gene.
It is understood that "host cells" and "recombinant host cells" refer not only to the particular subject cell but also 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 ofthe term as used herein. The host cells expressing the polypeptides described herein, and particularly recombinant host cells, have a variety of uses. First, the cells are useful for producing 14266 proteins or polypeptides that can be further purified to produce desired amounts of 14266 protein or fragments. Thus, host cells containing expression vectors are useful for polypeptide production, as well as cells producing significant amounts ofthe polypeptide. Such cells and tissues have been described herein above.
Host cells are also useful for conducting cell-based assays involving the 14266 or 14266 fragments. Thus, a recombinant host cell expressing a native 14266 is useful to assay for compounds that stimulate or inhibit 14266 function. This includes substrate, coenzyme, or 14266 subunit binding, and gene expression at the level of transcription or translation.
Host cells are also useful for identifying 14266 mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant 14266 (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native 14266. Recombinant host cells are also useful for expressing the chimeric polypeptides described herein to assess compounds that activate or suppress activation by means of a heterologous domain, segment, site, and the like, as disclosed herein.
Further, mutant 14266s can be designed in which one or more ofthe various functions is engineered to be increased or decreased (e.g., substrate or coenzyme binding) and used to augment or replace 14266 proteins in an individual. Thus, host cells can provide a therapeutic benefit by replacing an abeπant 14266 or providing an abeπant 14266 that provides a therapeutic result. In one embodiment, the cells provide 14266s that are abnormally active. In another embodiment, the cells provide a 14266 that is abnormally inactive.
This 14266 can compete with endogenous 14266 in the individual.
In another embodiment, cells expressing 14266s that cannot be activated are introduced into an individual in order to compete with endogenous 14266 for cAMP. For example, in the case in which excessive substrates such as β-hydroxysteroid is part of a treatment modality, it may be necessary to inactivate this molecule at a specific point in treatment. Providing cells that compete for the molecule , but which cannot be affected by 14266 activation would be beneficial.
Homologously recombinant host cells can also be produced that allow the in situ alteration of endogenous 14266 polynucleotide sequences in a host cell genome. The host cell includes, but is not limited to, a stable cell line, cell in vivo, or cloned microorganism. This technology is more fully described in WO 93/09222, WO 91/12650, WO 91/06667, U.S. 5,272,071, and U.S. 5,641,670. Briefly, specific polynucleotide sequences coπesponding to the 14266 polynucleotides or sequences proximal or distal to a 14266 gene are allowed to integrate into a host cell genome by homologous recombination where expression ofthe gene can be affected. In one embodiment, regulatory sequences are introduced that either increase or decrease expression of an endogenous sequence. Accordingly, a 14266 protein can be produced in a cell not normally producing it. Alternatively, increased expression of 14266 protein can be effected in a cell normally producing the protein at a specific level. Further, expression can be decreased or eliminated by infroducing a specific regulatory sequence. The regulatory sequence can be heterologous to the 14266 protein sequence or can be a homologous sequence with a desired mutation that affects expression. Alternatively, the entire gene can be deleted. The regulatory sequence can be specific to the host cell or capable of functioning in more than one cell type. Still further, specific mutations can be introduced into any desired region ofthe gene to produce mutant 14266 proteins. Such mutations could be introduced, for example, into the specific functional regions such as the cyclic nucleotide-binding site.
In one embodiment, the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the altered 14266 gene. Alternatively, the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal. See also Thomas et al, Cell 51:503 (1987) 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 14266 gene is selected (see e.g., Li, E. 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, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. 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 ofthe animal contain the homologously recombined DNA by germline . transmission ofthe transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos. WO 90/11354; WO 91/01140; and WO 93/04169. The genetically engineered host cells can be used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more ofthe cells ofthe animal include a transgene. 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 ofthe mature animal in one or more cell types or tissues ofthe transgenic animal. These animals are useful for studying the function of a 14266 protein and identifying and evaluating modulators of 14266 protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
In one embodiment, a host cell is a fertilized oocyte or an embryonic stem cell into which 14266 polynucleotide sequences have been introduced. A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any ofthe 14266 nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse. Any ofthe regulatory or other sequences useful in expression vectors can form part ofthe transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression ofthe 14266 protein 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, both by Leder et al, U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other fransgenic animals. A fransgenic founder animal can be identified based upon the presence ofthe transgene in its genome and/or expression of fransgenic mRNA in tissues or cells ofthe animals. A fransgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A fransgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.
In another embodiment, fransgenic non-human animals can be produced which contain selected systems, which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236. Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O' Gorman etal. (1991) Science 251:1351-1355). If cre/loxP recombinase system is used to regulate expression ofthe transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is 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 ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 555:810- 813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then fransfeπed to a pseudopregnant female foster animal. The offspring bom of this female foster animal will be a clone ofthe animal from which the cell, e.g., the somatic cell, is isolated.
Transgenic animals containing recombinant cells that express the polypeptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could affect substrate binding or coenzyme bind may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo 14266 function, including substrate interaction, the effect of specific mutant 14266s on 14266 function and interaction, and the effect of chimeric 14266s. It is also possible to assess the effect of null mutations, that is mutations that substantially Or completely eliminate one or more 14266 functions. In general, methods for producing transgenic animals include introducing a nucleic acid sequence according to the present invention, the nucleic acid sequence capable of expressing the protein in a transgenic animal, into a cell in culture or in vivo. When introduced in vivo, the nucleic acid is introduced into an intact organism such that one or more cell types and, accordingly, one or more tissue types, express the nucleic acid encoding the protein. Alternatively, the nucleic acid can be introduced into virtually all cells in an organism by transfecting a cell in culture, such as an embryonic stem cell, as described herein for the production of transgenic animals, and this cell can be used to produce an entire transgenic organism. As described, in a further embodiment, the host cell can be a fertilized oocyte. Such cells are then allowed to develop in a female foster animal to produce the transgenic organism.
Vectors Host Cells
The methods using the vectors and host cells discussed above are based on the vectors and host cells including, but not limited to, those described below.
The invention also provides methods using vectors containing the 14266 polynucleotides. The term "vector" refers to a vehicle, preferably a nucleic acid molecule that can transport the 14266 polynucleotides. When the vector is a nucleic acid molecule, the 14266 polynucleotides are covalently linked to the vector nucleic acid. With this aspect ofthe invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAG, PAC, YAC, OR MAC.
A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies ofthe 14266 polynucleotides. Alternatively, the vector may integrate into the host cell genome and produce additional copies ofthe 14266 polynucleotides when the host cell replicates. , The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) ofthe 14266 polynucleotides. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).
Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the 14266 polynucleotides such that transcription ofthe polynucleotides is allowed in a host cell. The polynucleotides can be infroduced into the host cell with a separate polynucleotide capable of affecting transcription. Thus, the second polynucleotide may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription ofthe 14266 polynucleotides from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a frans- acting factor can be produced from the vector itself.
It is understood, however, that in some embodiments, transcription and/or translation ofthe 14266 polynucleotides can occur in a cell-free system. The regulatory sequence to which the polynucleotides described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory confrol elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware ofthe numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. A variety of expression vectors can be used to express a 14266 polynucleotide.
Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
The regulatory sequence may provide constitutive expression in one or more host cells (i.e., tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art. The 14266 polynucleotides can be inserted into the vector nucleic acid by well- known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art. The vector containing the appropriate polynucleotide can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells. As described herein, it may be desirable to express the polypeptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production ofthe 14266 polypeptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility ofthe recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction ofthe fusion moiety so that the desired polypeptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith etal. (1988) Gene (57:31-40), 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 lid (Shadier et al (1990) Gene Expression Technology: Methods in Enzymology 755:60-89). Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S. (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California 119- 128). Alternatively, the sequence ofthe polynucleotide of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118).
The 14266 polynucleotides can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBOJ. (5:229-234 ), pMFa (Kurjan et al. (1982) Cell
50:933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), and pYES2 (Invifrogen
Coφoration, San Diego, CA).
The 14266 polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.
(1983) o/. CellBiol. 5:2156-2165) and the pVL series (Lucklow et /. (1989) Virology
170:31-39).
In certain embodiments ofthe invention, the polynucleotides described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 529:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. (5:187-195).
The expression vectors listed herein are provided by way of example only ofthe well-known vectors available to those of ordinary skill in the art that would be useful to express the 14266 polynucleotides. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression ofthe polynucleotides described herein. These are found for example in Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual 2nd, ed, Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, ofthe polynucleotide sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each ofthe parameters described above in relation to expression ofthe sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression). The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells. The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dexfran-mediated transfection, cationic lipid-mediated transfection, electroporation, fransduction, infection, lipofection, and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Host cells can contain more than one vector. Thus, different nucleotide sequences can be infroduced on different vectors ofthe same cell. Similarly, the 14266 polynucleotides can be introduced either alone or with other polynucleotides that are not related to the 14266 polynucleotides such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the 14266 polynucleotide vector. In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and fransduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.
Vectors generally include selectable markers that enable the selection ofthe subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the polynucleotides described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.
While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control ofthe appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.
Where secretion ofthe polypeptide is desired, appropriate secretion signals are incoφorated into the vector. The signal sequence can be endogenous to the 14266 polypeptides or heterologous to these polypeptides.
Where the polypeptide is not secreted into the medium, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The polypeptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.
It is also understood that depending upon the host cell in recombinant production ofthe polypeptides described herein, the polypeptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the polypeptides may include an initial modified methionine in some cases as a result of a host-mediated process.
Pharmaceutical Compositions
The receptor-like nucleic acid molecules, receptor-like proteins, and anti- receptor-like antibodies (also refeπed to herein as "active compounds") ofthe invention can be incoφorated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable caπier. As used herein the language "pharmaceutically acceptable caπier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incoφorated into the compositions.
The compositions ofthe invention are useful to treat any ofthe disorders discussed herein. The compositions are provided in therapeutically effective amounts. By "therapeutically effective amounts" is intended an amount sufficient to modulate the desired response. 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 ofthe disease or disorder, previous treatments, the general health and/or age ofthe 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 organometalhc 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 knowledge ofthe ordinarily skilled physician, veterinarian, or researcher. The dose(s) ofthe small molecule will vary, for example, depending upon the identity, size, and condition ofthe 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 ofthe invention. Exemplary doses include milligram or microgram amounts ofthe 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 ofthe 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 ofthe 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 ofthe specific compound employed, the age, body weight, general health, gender, and diet ofthe 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.
A pharmaceutical composition ofthe 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 caπiers 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 caπier 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 ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absoφtion ofthe injectable compositions can be brought about by including in the composition an agent that delays absoφtion, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incoφorating the active compound (e.g., a receptor-like protein or anti-receptor-like antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the active compound into a sterile vehicle that 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 ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the puφose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum fragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com 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 fonn of an aerosol spray from a pressurized container or dispenser that 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 admimstiation can be accomplished through the use of nasal sprays or suppositories. For transdennal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated with each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical caπier. Depending on the type and severity ofthe disease, about 1 μg/kg to about 15 mg/kg (e.g., 0.1 to 20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to about 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. An exemplary dosing regimen is disclosed in WO 94/04188. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
EXPERIMENTAL
The expression of 14266 was monitored in various tissues and cell types by quantitative PCR (TaqMan® brand quantitative PCR kit, Applied Biosystems) according to the kit manufacture's instructions. The results are shown below in Tables 1-6.
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will fully convey the invention to those skilled in the art. Many modifications and other embodiments ofthe invention will come to mind in one skilled in the art to which this invention pertains having the benefit ofthe teachings presented in the foregoing description. Although specific terms are employed, they are used as in the art unless otherwise indicated.

Claims

THAT WHICH IS CLAIMED:
1. A method of detecting the presence of a polypeptide having an amino acid sequence selected from the group consisting of: a) the amino acid sequence shown in SEQ ID NO: 1 ; b) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has at least 75% sequence identity with SEQ ID NO: 1 ; c) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has at least 85% sequence identity with SEQ ID NO:l; d) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has at least 95% sequence identity with SEQ ID NO: 1 ; e) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant is encoded by a nucleotide sequence that hybridizes to SEQ ID NO:2 under stringent conditions, said stringent conditions comprising hybridization in 6X SSC at 42°C followed by at least one wash in IX SSC at 55°C; and f) the amino acid sequence of a fragment ofthe amino acid sequence set forth in SEQ ID NO:l, wherein said fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 1 ; in a sample, said method comprising contacting said sample with an agent that specifically allows detection ofthe presence ofthe polypeptide in the sample to thereby detect the presence ofthe polypeptide in the sample, wherein said sample is selected from the group consisting of spinal chord, brain cortex, hypothalamus, aorta, heart, fetal heart, vein, asfrocytes, glioblastoma tissue, breast, breast interductal carcinoma tissue, ovary, ovary tumor tissue, pancreas, prostate, prostate tumor tissue, colon, colon tumor tissue, colon inflammatory bowel disease tissue, bone marrow mononuclear cells, kidney cells, CD34 positive haematopoietic progenitor cells, neufrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
2. The method of claim 1 , wherein the agent that specifically binds to the polypeptide is an antibody.
3. A kit comprising reagents for use in the method of claim 1.
4. A method of modulating the level or activity of a polypeptide having an amino acid sequence selected from the group consisting of: a) the amino acid sequence shown in SEQ ID NO: 1 ; b) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO: 1 , wherein said sequence variant has G- protein coupled signal fransduction activity and has at least 75% sequence identity with SEQ ID NO: 1 ; c) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has G- protein coupled signal fransduction activity and has at least 85% sequence identity with SEQ ID NO: 1 ; d) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO.T, wherein said sequence variant has G- protein coupled signal fransduction activity and has at least 95% sequence identity with SEQ ID NO : 1 ; e) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has G- protein coupled signal fransduction activity and is encoded by a nucleotide sequence that hybridizes to SEQ ID NO:2 under stringent conditions, said stringent conditions comprising hybridization in 6X SSC at 42°C followed by at least one wash in IX SSC at 55°C; and f) the amino acid sequence of a fragment ofthe amino acid sequence set forth in SEQ ID NO:l, wherein said fragment comprises at least 8 contiguous amino acids of SEQ ID NO:l; in a cell, the method comprising contacting a cell expressing the polypeptide with an agent under conditions that allow the agent to modulate the level or activity ofthe polypeptide, wherein said cell is selected from the group consisting of: spinal chord cells, brain cortex cells, hypothalamus cells, aortic cells, heart cells, fetal heart cells, vein cells, asfrocytes, glioblastoma cells, breast cells, breast interductal carcinoma cells, ovary cells, ovary tumor cells, pancreatic cells, prostate cells, prostate tumor cells, colon cells, colon tumor cells, bone maπow mononuclear cells, kidney cells, CD34 positive haematopoietic progenitor cells, neufrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
5. The method of claim 4, wherein said cell is in vitro.
6. The method of claim 4, wherein said cell is in vivo.
1. The method of claim 6, wherein said modulation is in a subject having or predisposed to having a haematopoietic disorder.
8. The method of claim 6, wherein said modulation is in a subject having or predisposed to having a neufrophil deficiency disorder.
9. A method of identifying an agent that binds to a polypeptide having an amino acid sequence selected from the group consisting of: a) the amino acid sequence shown in SEQ ID NO: 1 ; b) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has at least 75% sequence identity with SEQ ID NO:l; c) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO: 1 , wherein said sequence variant has at least 85% sequence identity with SEQ ID NO:l; d) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has at least 95% sequence identity with SEQ ID NO:l; e) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant is encoded by a nucleotide sequence that hybridizes to SEQ ID NO:2 under stringent conditions, said stringent conditions comprising hybridization in 6X SSC at 42°C followed by at least one wash in IX SSC at 55°C; and f) the amino acid sequence of a fragment ofthe amino acid sequence set forth in SEQ ID NO:l, wherein said fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 1 ; said method comprising contacting an agent with a cell expressing the polypeptide under conditions suitable for binding ofthe agent thereto and detecting the formation of a complex between said agent and said polypeptide.
10. The method of claim 9, wherein said polypeptide mediates a G-protein coupled signal fransduction pathway and the step of detecting the formation of a complex between the agent and the polypeptide is performed by monitoring the G- protein coupled signal fransduction pathway.
11. The method of claim 9, wherein said polypeptide mediates a G-protein mediated cellular response and the step of detecting the formation of a complex between the agent and the polypeptide is performed by monitoring the G-protein mediated cellular response.
12. The method of claim 5, wherein said step of contacting the agent with a cell expressing the polypeptide is performed in the presence of a competing compound that can interact with said polypeptide and said step of detecting the formation of a complex between the agent and the polypeptide is performed by monitoring for a decrease in the formation of a complex between said competing compound and the polypeptide.
13. A method of identifying an agent that modulates an activity of a polypeptide having an amino acid sequence selected from the group consisting of: a) the amino acid sequence shown in SEQ ID NO: 1 ; b) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has G- protein coupled signal fransduction activity and has at least 75% sequence identity with SEQ ID NO:l; c) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has G- protein coupled signal fransduction activity and has at least 85% sequence identity with SEQ ID NO:l; d) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant has G- protein coupled signal fransduction activity and has at least 95% sequence identity with SEQ ID NO: 1 ; e) the amino acid sequence of a sequence variant ofthe amino acid sequence shown in SEQ ID NO:l, wherein said sequence variant is encoded by a nucleotide sequence that hybridizes to SEQ ID NO:2 under stringent conditions, said stringent conditions comprising hybridization in 6X SSC at 42°C followed by at least one wash in IX SSC at 55°C; and f) the amino acid sequence of a fragment ofthe amino acid sequence set forth in SEQ ID NO:l, wherein said fragment comprises at least 8 contiguous amino acids of SEQ ID NO:l; said method comprising contacting an agent to be tested with a cell expressing the polypeptide under conditions suitable for detecting the activity ofthe polypeptide and determining the ability ofthe agent to modulate the activity ofthe polypeptide, wherein said cell is selected from the group consisting of: spinal chord cells, brain cortex cells, hypothalamus cells, aortic cells, heart cells, fetal heart cells, vein cells, asfrocytes, glioblastoma cells, breast cells, breast interductal carcinoma cells, ovary cells, ovary tumor cells, pancreatic cells, prostate cells, prostate tumor cells, colon cells, colon tumor cells, bone maπow mononuclear cells, kidney cells, CD34 positive haematopoietic progenitor cells, neufrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
14. The method of claim 13, wherein the polypeptide mediates G-protein coupled signal fransduction, and the step of determining the ability ofthe agent to modulate the activity ofthe polypeptide is performed by monitoring G-protein coupled signal fransduction.
15. The method of claim 13 , wherein the polypeptide mediates a G- protein-coupled cellular response, and the step of determining the ability ofthe agent to modulate the activity ofthe polypeptide is performed by monitoring the G-protein- coupled cellular response.
16. The method of claim 13, wherein said agent inhibits the activity ofthe polypeptide and said step of determining the ability ofthe agent to modulate the activity ofthe polypeptide is performed by determining the ability ofthe agent to inhibit the activity ofthe polypeptide.
17. The method of claim 16, wherein the polypeptide mediates G-protein coupled signal fransduction, and the step of determining the ability of the agent to inhibit the activity ofthe polypeptide is performed by monitoring G-protein coupled signal fransduction.
18. The method of claim 16, wherein the polypeptide mediates a G- protein-coupled cellular response, and the step of determining the ability ofthe agent to inhibit the activity ofthe polypeptide is performed by monitoring the G-protein- coupled cellular response.
19. The method of claim 16, wherein said agent that inhibits an activity of the polypeptide inhibits binding of a compound to the polypeptide, wherein said step of contacting the agent to be tested with a cell expressing the polypeptide is conducted in the presence ofthe compound under conditions suitable for binding of said compound to the polypeptide, and said step of determining the ability ofthe agent to inhibit the activity ofthe polypeptide is performed by detecting the binding of said compound to said polypeptide.
20. The method of claim 13, wherein the agent that modulates an activity ofthe polypeptide increases an activity of said polypeptide.
21. A method of detecting the presence of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO:2; b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1; c) a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; d) a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; and e) a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; in a sample, said method comprising contacting said sample with an agent that specifically allows detection ofthe nucleic acid molecule and then detecting the presence ofthe nucleic acid molecule, wherein said sample is selected from the group consisting of spinal chord, brain cortex, hypothalamus, aorta, heart, fetal heart, vein, asfrocytes, glioblastoma tissue, breast, breast interductal carcinoma tissue, ovary, ovary tumor tissue, pancreas, prostate, prostate tumor tissue, colon, colon tumor tissue, colon inflammatory bowel disease tissue, bone marrow mononuclear cells, kidney cells, CD34 positive haematopoietic progenitor cells, neufrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
22. The method of claim 21 , wherein the nucleic acid molecule whose presence is detected is mRNA.
23. A kit comprising reagents for use in the method of claim 21.
24. A method for modulating the level of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO:2; b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1; c) a fragment of the nucleotide sequence set forth in SEQ ID NO:2, wherein said fragment comprises at least 15 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO:2; d) a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; e) a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; and f) a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; in a cell, said method comprising contacting said nucleic acid molecule under conditions that allow the agent to modulate the level ofthe nucleic acid molecule, wherein said modulation is in a cell selected from the group consisting of spinal chord cells, brain cortex cells, hypothalamus cells, aortic cells, heart cells, fetal heart cells, vein cells, astrocytes, glioblastoma cells, breast cells, breast interductal carcinoma cells, ovary cells, ovary tumor cells, pancreatic cells, prostate cells, prostate tumor cells, colon cells, colon tumor cells, bone marrow mononuclear cells, kidney cells, CD34 positive haematopoietic progenitor cells, neufrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
25. The method of claim 24, wherein said agent is an oligonucleotide that hybridizes to SEQ ID NO:2 or a complement thereof under stringent conditions, said stringent conditions comprising hybridization in 6X SSC at 42°C followed by at least one wash in IX SSC at 55°C.
26. A method identifying an agent that modulates the level of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO:2; b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1; c) a fragment ofthe nucleotide sequence set forth in SEQ ID NO:2, wherein said fragment comprises at least 15 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO:2; d) a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2; e) a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; and f) a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence set forth in SEQ ID NO:2; in a cell, said method comprising contacting an agent with a cell comprising the nucleic acid molecule under conditions suitable for the agent to modulate the level of the nucleic acid molecule and then determining the level ofthe nucleic acid molecule, wherein said cell is selected from the group consisting of spinal chord cells, brain cortex cells, hypothalamus cells, aortic cells, heart cells, fetal heart cells, vein cells, astrocytes, glioblastoma cells, breast cells, breast interductal carcinoma cells, ovary cells, ovary tumor cells, pancreatic cells, prostate cells, prostate tumor cells, colon cells, colon tumor cells, bone marrow mononuclear cells, kidney cells, CD34 positive haematopoietic progenitor cells, neufrophil precursor cells, neutrophils, megakaryocytes, and erythroid cells.
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