EP1204754A2 - Polynucleotides exprimes dans les lymphocytes t actives et proteines codees par ces polynucleotides - Google Patents

Polynucleotides exprimes dans les lymphocytes t actives et proteines codees par ces polynucleotides

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Publication number
EP1204754A2
EP1204754A2 EP00955700A EP00955700A EP1204754A2 EP 1204754 A2 EP1204754 A2 EP 1204754A2 EP 00955700 A EP00955700 A EP 00955700A EP 00955700 A EP00955700 A EP 00955700A EP 1204754 A2 EP1204754 A2 EP 1204754A2
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EP
European Patent Office
Prior art keywords
atlas
polypeptide
nucleic acid
seq
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00955700A
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German (de)
English (en)
Inventor
John A. Peyman
Cynthia D. Green
Andro Hsu
Jeffrey A. Browning
John Carulli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biogen Inc
CuraGen Corp
Original Assignee
Biogen Inc
CuraGen Corp
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Filing date
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Application filed by Biogen Inc, CuraGen Corp filed Critical Biogen Inc
Publication of EP1204754A2 publication Critical patent/EP1204754A2/fr
Withdrawn legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates in general to nucleic acids and polypeptides and more particularly to polynucleotides expressed in activated T-lymphocytes, and polypeptides encoded by such polynucleotides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides.
  • the mammalian immune system can be characterized by cell-mediated immune responses and antibody mediated immune responses.
  • Cell-mediated immune responses are effected by a type of lymphocyte known as a T lymphocyte.
  • T lymphocyte For a T lymphocyte to mount a productive response to an antigen, it must recognize an antigen that is presented by an MHC class I or class Il-expressing cell. T cells typically do not respond to an antigen until they are activated. A failure of a T cell to be properly activated, or, conversely, inappropriate activation of a T cell, can result in deleterious consequences to an individual.
  • the present invention is based, in part, on the discovery of novel polynucleotide sequences expressed in activated T lymphocytes.
  • activated T lymphocyte associated sequences include ATLAS-1, a novel phosphatase regulator, ATLAS-2, A novel cytokine receptor, ATLAS-3, a novel member of the thioredoxin and protein disulfide isomerase family, and ATLAS-4, a novel protein with homology to a putative multiple sclerosis aetiologic agent.
  • ATLAS-1, ATLAS-2, ATLAS-3, and ATLAS-4 nucleotide sequences are referred to herein as "ATLAS-X”.
  • the invention provides an isolated nucleic acid molecule encoding a polypeptide that includes an amino acid sequence of a polypeptide that is at least 90% identical to an ATLAS-X polypeptide.
  • the nucleic acid molecule can hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of an ALTAS-X nucleic acid sequence.
  • an oligonucleotide e.g., an oligonucleotide less than 100 nucleotides in length that includes at least 6 contiguous nucleotides of an ATLAS-X nucleic acid, e.g., SEQ ID NO:l, or a complement of the oligonucleotide.
  • the ATLAS-X polypeptide includes an amino acid sequence at least 80% identical to a polypeptide that includes the amino acid sequence of SEQ ID NO:2, 4, 6, or 8.
  • the invention also features an antibody that selectively binds to an ATLAS-X polypeptide.
  • the invention includes a pharmaceutical composition which includes a therapeutically or prophylactically effective amount of a therapeutic and a pharmaceutically acceptable carrier.
  • the therapeutic can be, e.g., an ATLAS-X nucleic acid, and ATLAS-X polypeptide, or an antibody to an ATLAS-X polypeptide.
  • the invention includes, in one or more containers, a therapeutically or prophylactically effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes an ATLAS-X nucleic acid, e.g., an ATLAS-X DNA, under conditions allowing for expression of the ATLAS-X polypeptide encoded by the DNA. If desired, the ATLAS-X polypeptide can then be recovered.
  • an ATLAS-X nucleic acid e.g., an ATLAS-X DNA
  • the invention includes a method of detecting the presence of an ATLAS-X polypeptide in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the polypeptide in the sample.
  • Also included in the invention is a method of detecting the presence of an ATLAS-X nucleic acid molecule in a sample by contacting the sample with an ATLAS-X nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to an ATLAS- X nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of an
  • ATLAS-X polypeptide by contacting a cell sample that includes an ATLAS-X polypeptide with a compound that binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic in the manufacture of a medicament for treating or preventing a syndrome associated with a human immune system disorder.
  • the therapeutic can be, e.g., an ATLAS-X nucleic acid, and ATLAS-X polypeptide, or an ATLAS-X antibody.
  • the invention further includes a method for screening for a modulator of an immune disorder.
  • the method includes contacting a test compound with an ATLAS-X polypeptide and determining if the test compound binds to the polypeptide. Binding of the test compound to the polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to an immune disorder.
  • Also within the invention is a method for screening for a modulator of activity, or of latency or predisposition to an immune disorder by administering a test compound to a test animal at increased risk for the disorder.
  • the test animal expresses a recombinant polypeptide encoded by an ATLAS-X nucleic acid. Expression or activity of the ATLAS-X polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal that recombinantly expresses the protein and is not at increased risk for the disorder.
  • the expression of the protein in the test animal and the control animal is compared.
  • a change in the activity of the protein in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder.
  • the disorder is an autoimmune disorder, an immune disorder, a T-lymphocyte-associated disorder, a cell-proliferation disorder, a cell differentiation disorder, or an immune deficiency order.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of an ATLAS-X polypeptide, an ATLAS-X nucleic acid, or both, in a subject, e.g., a human subject.
  • the method includes measuring the amount of the polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the polypeptide present in a control sample. An alteration in the level of the polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the invention includes a method of treating or preventing a pathological condition associated with an immune system disorder in a mammal by administering to the subject an ATLAS-X polypeptide, an ATLAS-X nucleic acid, or an ATLAS-X antibody to a subject, e.g., a human subject, in an amount sufficient to alleviate or prevent the pathological condition.
  • the immune system associated disorder is an autoimmune disorder, an immune disorder, a T-lymphocyte-associated disorder, a cell-proliferation disorder, a cell differentiation disorder, or an immune deficiency disorder.
  • Figures 1A-1D are a representation of a nucleic acid sequence (SEQ ID NO:l) and an encoded amino acid sequence (SEQ ID NO:2) of ATLAS-1 nucleic acids and polypeptides according to the invention.
  • Figures 2A-2H are a representation of a nucleic acid sequence (SEQ ID NO:3) and an encoded amino acid sequence (SEQ ID NO:4) of ATLAS-2 nucleic acids and polypeptides according to the invention.
  • Figures 3A-3B are a representation of a nucleic acid sequence (SEQ ID NO:5) and an encoded amino acid sequence (SEQ ID NO:6) of ATLAS-3 nucleic acids and polypeptides according to the invention.
  • Figures 4A-4B are a representation of a nucleic acid sequence (SEQ ID NO:7) and an encoded amino acid sequence (SEQ ID NO:8) of ATLAS-4 nucleic acids and polypeptides according to the invention.
  • the present invention is based, in part, upon the discovery of nucleic acids encoded in activated T lymphocytes and of polypeptides encoded by these nucleic acids.
  • the nucleic acids have been named "Activated T Lymphocyte Associated Sequences 1-4", or collectively, "ATLAS-X”. Representative ATLAS-X sequences, and examples of uses of these sequences, are next briefly discussed.
  • ATLAS-1 A novel phosphatase regulator
  • An ATLAS-1 sequence according to the invention includes a nucleotide sequence encoding a polypeptide related to previously described phosphatase regulators.
  • a representation of an ATLAS-1 nucleic acid sequence according to the invention, and a polypeptide sequence encoded by this nucleic acid sequence, is shown in Figures 1 A-1D.
  • the sequence in Figures 1A-1D includes a nucleotide sequence of 3290 nucleotides (SEQ ID NO:l).
  • Nucleotides 1 to 2586 define an open reading frame encoding a polypeptide of 862 amino acid residues (SEQ ID NO:2).
  • An ATLAS-1 nucleotide sequence according to the invention is also present in clone 5.02w0c0-60.3.
  • An ATLAS-1 gene is localized to human chromosome 17.
  • the calculated molecular weight of the ATLAS-1 protein is 94,019.9 daltons.
  • the protein contains four to six hydrophobic regions.
  • the predicted protein is 89% homologous (266 aa 296 aa) to rat neurabin II (see, e.g., J. Biol. Chem. 273 (6), 3470-3475 (1998)) and 68% homologous (407 of 594 aa) to rat spinophilin (see, e.g., Proc. Natl. Acad. Sci. U.S.A. 94 (18), 9956-9961 (1997).
  • Rat spinophilin is reported to modulate the activity of protein phosphatase- 1 in neurons.
  • an ATLAS-1 protein of the invention is as a novel phosphatase regulator expressed in activated T lymphocytes.
  • the ATLAS-1 protein may modulate, e.g., stimulate or suppress, activation of T-lymphocytes.
  • the ATLAS-1 polypeptide, or nucleic acids encoding the ATLAS-1 polypeptide can be used to identify small molecule agonists and antagonists.
  • the natural substrate(s) of this novel phosphatase regulator may likewise be used in methods of modulating, e.g., stimulating or suppressing the activation of T lymphocytes, and may similarly be used to identify compounds, e.g., small molecule agonists and antagonists of immune responses.
  • ATLAS-2 A novel cytokine receptor
  • An ATLAS-2 nucleic acid sequence according to the invention includes a nucleotide sequence that encodes a polypeptide related to previously described cytokine receptors.
  • Figures 2A-2H include a representation of an ATLAS-2 nucleic acid sequence of the invention, and a polypeptide sequence encoded by this polypeptide.
  • the disclosed nucleic acid sequence is 6461 nucleotides in length (SEQ ID NO:3), of which nucleotides 1 to 5553 define an open reading frame encoding a polypeptide of 1851 amino acids (SEQ ID NO:4).
  • An ATLAS-2 nucleotide sequence according to the invention is also present in clone 5.02r011 - 102.5. The sequences localize to human chromosome 1 lpl5.5.
  • the calculated molecular weight of the protein in Figures 2A-2H is 202,523 daltons.
  • the protein includes about 20 hydrophobic regions, which may be transmembrane segments.
  • the predicted protein is 52% identical (250 aa/ 458 aa) to rat vasopressin receptor (GenBank Accession Number q63035). It is 37% identical (98 aa/ 263 aa) to human angiotensin/vasopressin receptor (GenBank Accession Number o75434).
  • the ATLAS-2 protein is likely a novel member of the cytokine receptor family. Blocking antibodies raised to this putative receptor, which is expressed in activated T lymphocytes, are useful for modulating, i.e., stimulating or suppressing, T lymphocyte effector functions.
  • the receptor can also be used to identify ligand(s) which may be immunosuppressive or immunostimulatory.
  • the receptor may be also be used to identify compounds, e.g., small molecule agonists or antagonists, which may be immunosuppressive or immunostimulatory.
  • ATLAS-3 A novel member of the thioredoxin and protein disulflde isomerase family
  • An ATLAS-3 nucleic acid according to the invention includes nucleotide sequence of 2564 base pairs in length (SEQ ID NO:5) shown in Figures 3A-3B. Also shown in Figures 3A-3B is a polypeptide of 269 amino acid residues (SEQ ID NO:6) that is translated from nucleotides 98 to 904 of SEQ ID NO:5. An ATLAS-3 nucleic acid sequence is also present in clone 5.02r011-149.
  • the calculated molecular weight of the predicted protein is 30,045 daltons.
  • the protein has an endoplasmic reticulum retention signal and the subcellular localization is probably in the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • the predicted protein is 43% identical to mouse and human protein disulflde isomerase (GenBank Accession Numbers p27773 [mouse] and p30101 [human]). Protein disulflde isomerases are endoplasmic reticulum resident proteins with KDEL targeting signals.
  • the disclosed ATLAS-3 protein is a novel member of the thioredoxin or protein disulflde isomerase family.
  • Two domains in the disclosed ATLAS-3 protein additionally resemble thioredoxin.
  • the thioredoxins act in DNA synthesis as dithiol hydrogen donors.
  • Thioredoxins are involved in the regulation of metabolic processes, such as growth regulation, enzyme modulation, receptor activity, or transcriptional regulation.
  • An ATLAS-3 protein may act as a protein- folding chaperone to modulate T lymphocyte activation.
  • the ATLAS-3 protein may act as a growth regulator similar to thioredoxin.
  • the ATLAS-3 protein may act on T lymphocyte substrates.
  • the ATLAS-3 protein may also be used to identify compounds, e.g., small molecules, which can regulate (stimulate or suppress) an immune response.
  • the ATLAS-3 protein can be used to identify secreted substrate protein or proteins in T lymphocytes, which themselves may be immunoregulatory.
  • ATLAS-4 A novel protein with homology to a putative multiple sclerosis aetiologic agent
  • An ATLAS-4 nucleic acid according to the invention includes a nucleotide sequence of 1828 nucleotides (SEQ ID NO: 7), as shown in Figures 4A-4B.
  • Figures 4A-4B also show a polypeptide of 358 amino acid residues (SEQ ID NO:8) that is translated from nucleotides 1 to 1074 of SEQ ID NO:7.
  • An ATLAS-4 nucleic acid sequence according to the invention is also present as clone 5.02h0n0-103.1.
  • the calculated molecular weight of the protein is 38,133 daltons.
  • a putative signal sequence of 28 hydrophobic residues is present at amino acids 1 to 128 with one hydrophobic residue.
  • the 80 residues are at the amino- and carboxy-terminus of the protein are similar. These domains contain five evenly spaced charged residues as well as two cysteine residues.
  • BLASTP sequence analysis indicates that the predicted protein is 69% (65 aa/ 94 aa) similar to a portion of sequence for multiple sclerosis associated retro virus- 1 (MSRV-1, described on WO9823755-A1).
  • ClustalW analysis indicates that the ATLAS-4 protein is also similar to portions of seven related peptides encoded by MSRV-1 (which is discussed in WO9823755-A1, FR2762601-A1 and WO9706260).
  • the disclosed ATLAS-4 nucleic acid sequence localizes to human chromosome lq23.3-24.3.
  • an ATLAS-4 protein of the invention may act in T lymphocytes to modulate immune function, e.g., in stimulation or suppression of immune responses.
  • ATLAS-X corresponds to any of ATLAS-1, ATLAS-2, ATLAS-3 or ATLAS-4.
  • Table 1 Sequences and Corresponding SEQ ID Numbers of Disclosed Sequences
  • One aspect of the invention pertains to isolated nucleic acid molecules that encode ATLAS-X polypeptides or biologically active portions thereof Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify ATLAS- X-encoding nucleic acids (e g , ATLAS-X mRNA) and fragments for use as PCR p ⁇ mers for the amplification or mutation of ATLAS-X nucleic acid molecules.
  • nucleic acid fragments sufficient for use as hybridization probes to identify ATLAS- X-encoding nucleic acids (e g , ATLAS-X mRNA) and fragments for use as PCR p ⁇ mers for the amplification or mutation of ATLAS-X nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e g , cDNA or genomic DNA), RNA molecules (e g , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA
  • Probes refer to nucleic acid sequences of va ⁇ able length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e g , 6,000 nt, depending on use Probes are used in the detection of identical, similar, or complementary nucleic acid sequences Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double- stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • an “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated ATLAS-X nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., lymphocytes, e.g., activated T lymphocytes).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 3, 5, 7, 9, 10, 11, 12, or a complement of any of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • ATLAS-X molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to ATLAS-X nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • the term "oligonucleotide” refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO:l, 3, 5, 7, or a complement thereof.
  • Oligonucleotides may be chemically synthesized and may be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:l, 3, 5, or 7, or a portion of this nucleotide sequence, e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of ATLAS-X.
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO:l, 3, 5, or 7 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l, 3, 5, or 7 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO:l, 3, 5, or 7, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 30%, 50%, 70%>, 80%, or 95%> identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of ATLAS-X polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.
  • homologous nucleotide sequences include nucleotide sequences encoding for an ATLAS-X polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human ATLAS-X protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:l, 3, 5, or 7, as well as a polypeptide having ATLAS-X activity. Biological activities of the ATLAS-X proteins are described below. A homologous amino acid sequence does not encode the amino acid sequence of a human ATLAS-X polypeptide.
  • ATLAS-X polypeptide is encoded by the open reading frame ("ORF") of an ATLAS-X nucleic acid.
  • the invention includes the nucleic acid sequence comprising the stretch of nucleic acid sequences of SEQ ID NOs:l, 3, 5, 7, 9, 10, 11, or 12 that comprises the ORF of that nucleic acid sequence and encodes a polypeptide of SEQ ID NOs:2, 4, 6, or 8.
  • An "open reading frame” corresponds to a nucleotide sequence that could potentially be translated into a polypeptide.
  • a stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.
  • An ORF that represents the coding sequence for a full protein begins with an ATG "start” codon and terminates with one of the three “stop” codons, namely, TAA, TAG, or TGA.
  • an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both.
  • a minimum size requirement is often set, for example, a stretch of DNA that would encode a protein of 50 amino acids or more.
  • the nucleotide sequence determined from the cloning of the human ATLAS-X gene allows for the generation of probes and primers designed for use in identifying and/or cloning ATLAS-X homologues in other cell types, e.g. from other tissues, as well as ATLAS-X homologues from other mammals.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NO:l, 3, 5, or 7, or an anti-sense strand nucleotide sequence of SEQ ID NO:l, 3, 5, or 7, or of a naturally occurring mutant of SEQ ID NO:l, 3, 5 or 7.
  • Probes based on the human ATLAS-X nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an ATLAS-X protein, such as by measuring a level of an ATLAS-X-encoding nucleic acid in a sample of cells from a subject e.g., detecting ATLAS-X mRNA levels or determining whether a genomic ATLAS-X gene has been mutated or deleted.
  • a polypeptide having a biologically active portion of ATLAS-X refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically active portion of ATLAS-X” can be prepared by isolating a portion of SEQ ID NO:l, 3, 5, or 7 that encodes a polypeptide having an ATLAS-X biological activity (the biological activities of the ATLAS-X proteins are described below), expressing the encoded portion of ATLAS-X protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of ATLAS-X
  • a nucleic acid fragment can encode a biologically active portion of ATLAS-3 includes a thioredoxin domain of SEQ ID NO:6.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l, 3, 5, or 7 due to degeneracy of the genetic code and thus encode the same ATLAS-X protein as that encoded by the nucleotide sequence shown in SEQ ID NO:l, 3, 5, or 7.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2, 4, 6, or 8.
  • ATLAS-X nucleotide sequence shown in SEQ ID NO:l, 3, 5, 7, 9, 10, 11, or 12
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of ATLAS-X may exist within a population (e.g., the human population).
  • Such genetic polymorphism in the ATLAS- X gene may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding an ATLAS-X protein, preferably a mammalian ATLAS-X protein.
  • Such natural allelic variations can typically result in l-5%> variance in the nucleotide sequence of the ATLAS-X gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in ATLAS-X that are the result of natural allelic variation and that do not alter the functional activity of ATLAS-X are intended to be within the scope of the invention.
  • nucleic acid molecules encoding ATLAS-X proteins from other species and thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NO:l, 3, 5, 7, 9, 10, 11, or 12 are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the ATLAS-X cDNAs of the invention can be isolated based on their homology to the human ATLAS-X nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • a soluble human ATLAS-X cDNA can be isolated based on its homology to human membrane-bound ATLAS-X.
  • a membrane-bound human ATLAS-X cDNA can be isolated based on its homology to soluble human ATLAS-X.
  • an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: l , 3, 5, or 7.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.
  • an isolated nucleic acid molecule of the invention hybridizes to the coding region.
  • the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding ATLAS-X proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% > of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50%> of the probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in Ausubel et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%o, 10%, 75%, 85%), 90%, 95%, 98%), or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l, 3, 5, or 7 corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 5, or 7 or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5%> SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well-known in the art. See, e.g., Ausubel et al.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 5, or 7 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%> (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g. , as employed for cross-species hybridizations).
  • allelic variants of a ATLAS-X sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO:l, 3, 5, or 7, thereby leading to changes in the amino acid sequence of the encoded ATLAS-X protein, without altering the functional ability of the ATLAS-X protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:l, 3, 5, or 7.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of ATLAS-X without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the ATLAS-X proteins of the present invention are predicted to be particularly unamenable to alteration. Amino acids for which conservative substitutions can be made are known in the art.
  • nucleic acid molecules encoding ATLAS-X proteins that contain changes in amino acid residues that are not essential for activity. Such ATLAS-X proteins differ in amino acid sequence from SEQ IDs NO:2, 4, 6, and 8, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2, 4, 6, or 8.
  • the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO:2, 4, 6, or 8, more preferably at least about 70% homologous to SEQ ID NO:2, 4, 6, or 8, still more preferably at least about 80% homologous to SEQ ID NO:2, 4, 6, or 8, even more preferably at least about 90% homologous to SEQ ID NO:2, 4, 6, or 8, and most preferably at least about 95%> homologous to SEQ ID NO:2, 4, 6, or 8.
  • An isolated nucleic acid molecule encoding an ATLAS-X protein homologous to the protein of SEQ ID NO:2, 4, 6, or 8 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:2, 4, 6, or 8 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ IDs NO:2, 4, 6, and 8 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in ATLAS-X is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an ATLAS-X coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ATLAS-X biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
  • a mutant ATLAS-X protein can be assayed for (1) the ability to form proteimprotein interactions with other ATLAS-X proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant ATLAS-X protein and an ATLAS-X ligand; (3) the ability of a mutant ATLAS-X protein to bind to an intracellular target protein or biologically active portion thereof; (e.g. avidin proteins).
  • a mutant ATLAS-X can be assayed for the ability to regulate a phosphatase activity (for ATLAS-1), bind a cytokine (ATLAS-2), or act as a dithiol hydrogen donor (ATLAS-3).
  • Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 5, 7, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is 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.
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire ATLAS-X coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an ATLAS-X protein of SEQ ID NO:2, 4, 6, or 8, or antisense nucleic acids complementary to an ATLAS-X nucleic acid sequence of SEQ ID NO:l, 3, 5, or 7, are additionally provided.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding ATLAS-X.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human ATLAS-1, 2, 3 and 4 correspond to SEQ IDs NO:9, 10, 11, and 12, respectively).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding ATLAS-X.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of ATLAS-X mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of ATLAS-X mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of ATLAS-X 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 of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydro uracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5 -methylaminomethyluracil, 5 -methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an ATLAS-X protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is 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) FEBSLett 215: 327-330).
  • Nucleic acid modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes 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 ATLAS-X mRNA transcripts to thereby inhibit translation of ATLAS-X mRNA.
  • a ribozyme having specificity for an ATLAS -X-encoding nucleic acid can be designed based upon the nucleotide sequence of an ATLAS-X cDNA disclosed herein (i.e., SEQ ID NO:l, 3, 5, 7, 9, 10, 1 1 or 12).
  • a derivative of a Tetrahymena L-19 INS R ⁇ A can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an ATLAS-X-encoding mR ⁇ A. See, e.g., Cech et al U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,1 16,742.
  • ATLAS-X mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418.
  • ATLAS-X gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the ATLAS-X (e.g., the ATLAS-X promoter and/or enhancers) to form triple helical structures that prevent transcription of the ATLAS-X gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the ATLAS-X e.g., the ATLAS-X promoter and/or enhancers
  • nucleotide sequences complementary to the regulatory region of the ATLAS-X e.g., the ATLAS-X promoter and/or enhancers
  • the nucleic acids of ATLAS-X can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23).
  • 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.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
  • PNAs of ATLAS-X can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of ATLAS-X 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 B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
  • PNAs of ATLAS-X can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of ATLAS-X can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al (1996) Nucl Acids Res 24: 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
  • PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc.
  • oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • a polypeptide according to the invention includes a polypeptide including the amino acid sequence of ATLAS-X polypeptides whose sequences are provided in Figures 1A-1D, 2A-2H, 3A-3B and 4A-4B (SEQ IDs NO:2, 4, 6, and 8).
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in Figures 1 A-1D, 2A-2H, 3A-3B or 4A-4B while still encoding a protein that maintains its ATLAS-X activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to 20% or more of the residues may be so changed.
  • an ATLAS-X variant that preserves ATLAS -X-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
  • One aspect of the invention pertains to isolated ATLAS-X proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-ATLAS-X antibodies.
  • native ATLAS-X proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • ATLAS-X proteins are produced by recombinant DNA techniques.
  • an ATLAS-X protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the ATLAS-X protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of ATLAS-X protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of ATLAS-X protein having less than about 30%> (by dry weight) of non-ATLAS- X protein (also referred to herein as a "contaminating protein"), more preferably less than about 20%) of non-ATLAS-X protein, still more preferably less than about ⁇ 0% of non-ATLAS-X protein, and most preferably less than about 5%> non-ATLAS-X protein.
  • the ATLAS-X protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%), more preferably less than about 10%, and most preferably less than about 5%> of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of ATLAS-X protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of ATLAS-X protein having less than about 30%> (by dry weight) of chemical precursors or non-ATLAS-X chemicals, more preferably less than about 20%) chemical precursors or non-ATLAS-X chemicals, still more preferably less than about 10% chemical precursors or non-ATLAS-X chemicals, and most preferably less than about 5% chemical precursors or non-ATLAS-X chemicals.
  • Biologically active portions of an ATLAS-X protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the ATLAS-X protein, e.g., the amino acid sequence shown in SEQ ID NO:2, 4, 5 or 8, that include fewer amino acids than the full length ATLAS-X proteins, and exhibit at least one activity of an ATLAS-X protein.
  • biologically active portions comprise a domain or motif with at least one activity of the ATLAS-X protein.
  • a biologically active portion of an ATLAS-X protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • the ATLAS-X protein has an amino acid sequence shown in SEQ ID NO:2, 4, 6 or 8.
  • the ATLAS-X protein is substantially homologous to SEQ ID NO:2, 4, 6 or 8 and retains the functional activity of the protein of SEQ ID NO:2, 4, 6 or 8 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below.
  • the ATLAS-X protein is a protein that comprises an amino acid sequence at least about 45%> homologous to the amino acid sequence of SEQ ID NO:2, 4, 6 or 8 and retains the functional activity of the ATLAS-X proteins of SEQ ID NO:2, 4, 6 or 8.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 1970 JMolBiol 48: 443-453.
  • the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence corresponding to nucleotides 1-2586 of SEQ ID NO: 1, nucleotides l-5553of SEQ ID NO:3, nucleotides 98-904 of SEQ ID NO:5, or nucleotides 1-1074 of SEQ ID NO:7.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • an ATLAS-X "chimeric protein” or “fusion protein” comprises an ATLAS-X polypeptide operatively linked to a non-ATLAS-X polypeptide.
  • An "ATLAS-X polypeptide” refers to a polypeptide having an amino acid sequence corresponding to ATLAS-X
  • a “non-ATLAS-X polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the ATLAS-X protein, e.g., a protein that is different from the ATLAS-X protein and that is derived from the same or a different organism.
  • an ATLAS-X fusion protein the ATLAS-X polypeptide can correspond to all or a portion of an ATLAS-X protein.
  • an ATLAS-X fusion protein comprises at least one biologically active portion of an ATLAS-X protein.
  • an ATLAS-X fusion protein comprises at least two biologically active portions of an ATLAS-X protein.
  • an ATLAS-X fusion protein comprises at least three biologically active portions of an ATLAS-X protein.
  • the term "operatively linked" is intended to indicate that the ATLAS-X polypeptide and the non-ATLAS-X polypeptide are fused in-frame to each other.
  • the non-ATLAS-X polypeptide can be fused to the N-terminus or C-terminus of the ATLAS-X polypeptide.
  • the fusion protein is a GST- ATLAS-X fusion protein in which the ATLAS-X sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.
  • GST glutathione S-transferase
  • Such fusion proteins can facilitate the purification of recombinant ATLAS-X.
  • the fusion protein is an ATLAS-X protein containing a heterologous signal sequence at its N-terminus.
  • the native ATLAS-4 signal sequence i.e., about amino acids 1 to 28 of SEQ ID NO:8
  • the fusion protein is an ATLAS-X-immunoglobulin fusion protein in which the ATLAS-X sequences are fused to sequences derived from a member of the immunoglobulin protein family.
  • the ATLAS-X-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an ATLAS-X ligand and an ATLAS-X protein on the surface of a cell, to thereby suppress ATLAS -X-mediated signal transduction in vivo.
  • the ATLAS- X-immunoglobulin fusion proteins can be used to affect the bioavailability of an ATLAS-X cognate ligand. Inhibition of the ATLAS-X ligand/ ATLAS-X interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival.
  • the ATLAS- X-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti- ATLAS-X antibodies in a subject, to purify ATLAS-X ligands, and in screening assays to identify molecules that inhibit the interaction of ATLAS-X with an ATLAS-X ligand.
  • An ATLAS-X chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An ATLAS-X-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the ATLAS-X protein.
  • the present invention also pertains to variants of the ATLAS-X proteins that function as either ATLAS-X agonists (mimetics) or as ATLAS-X antagonists.
  • Variants of the ATLAS- X protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the ATLAS-X protein.
  • An agonist of the ATLAS-X protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the ATLAS-X protein.
  • An antagonist of the ATLAS-X protein can inhibit one or more of the activities of the naturally occurring form of the ATLAS-X protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the ATLAS-X protein.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the ATLAS-X proteins.
  • Variants of the ATLAS-X protein that function as either ATLAS-X agonists (mimetics) or as ATLAS-X antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the ATLAS-X protein for ATLAS-X protein agonist or antagonist activity.
  • a variegated library of ATLAS-X variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of ATLAS-X variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential ATLAS-X sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of ATLAS-X sequences therein.
  • methods which can be used to produce libraries of potential ATLAS-X 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.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential ATLAS-X 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) Nucl Acid Res 11 :477.
  • libraries of fragments of the ATLAS-X protein coding sequence can be used to generate a variegated population of ATLAS-X fragments for screening and subsequent selection of variants of an ATLAS-X protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an ATLAS-X 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 that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the ATLAS-X protein.
  • REM Recursive ensemble mutagenesis
  • the invention encompasses antibodies and antibody fragments, such as F ab or (F ab ) 2 that bind immunospecifically to any of the polypeptides of the invention.
  • ATLAS-X protein can be used as an immunogen to generate antibodies that bind ATLAS-X using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length ATLAS-X protein can be used or, alternatively, the invention provides antigenic peptide fragments of ATLAS-X for use as immunogens.
  • the antigenic peptide of ATLAS-X comprises at least 4 amino acid residues of the amino acid sequence shown in SEQ ID NO:2, 4, 6, or 8 and encompasses an epitope of ATLAS-X such that an antibody raised against the peptide forms a specific immune complex with ATLAS-X.
  • the antigenic peptide comprises at least 6, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to someone skilled in the art.
  • At least one epitope encompassed by the antigenic peptide is a region of ATLAS-X that is located on the surface of the protein, e.g., a hydrophilic region.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety.
  • ATLAS-X protein sequence of SEQ ID NO:2, 4, 6,8, or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens in the generation of antibodies that immunospecifically-bind these protein components.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen, such as ATLAS-X.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F ab and F (ab . )2 fragments, and an F ab expression library.
  • antibodies to human ATLAS-X proteins are disclosed.
  • Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to an ATLAS-X protein sequence of SEQ ID NO:2, 4, 6, 8, or a derivative, fragment, analog or homolog thereof. Some of these proteins are discussed below.
  • an appropriate immunogenic preparation can contain, for example, recombinantly expressed ATLAS-X protein or a chemically synthesized ATLAS-X polypeptide.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • the antibody molecules directed against ATLAS-X can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of ATLAS-X.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular ATLAS- X protein with which it immunoreacts.
  • any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized.
  • Such techniques include, but are not limited to, the hybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al, 1983.
  • Non-human antibodies can be "humanized” by techniques well known in the art. See e.g., U.S. Patent No. 5,225,539.
  • Antibody fragments that contain the idiotypes to an ATLAS-X protein may be produced by techniques known in the art including, but not limited to: (/) an F (ab , )2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F (ab , )2 fragment; (Hi) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • recombinant anti -ATLAS-X antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No.
  • methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art.
  • ELISA enzyme-linked immunosorbent assay
  • selection of antibodies that are specific to a particular domain of an ATLAS-X protein is facilitated by generation of hybridomas that bind to the fragment of an ATLAS-X protein possessing such a domain.
  • antibodies that are specific for a desired domain within an ATLAS-X protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • Anti-ATLAS-X antibodies may be used in methods known within the art relating to the localization and/or quantitation of an ATLAS-X protein (e.g., for use in measuring levels of the ATLAS-X protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies for ATLAS-X proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain are utilized as pharmacologically-active compounds [hereinafter "Therapeutics"].
  • An anti-ATLAS-X antibody (e.g., monoclonal antibody) can be used to isolate ATLAS-X by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-ATLAS-X antibody can facilitate the purification of natural ATLAS-X from cells and of recombinantly produced ATLAS-X expressed in host cells.
  • an anti-ATLAS-X antibody can be used to detect ATLAS-X protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the ATLAS-X protein.
  • Anti-ATLAS-X 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 (i.e., physically linking) 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, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • vectors preferably expression vectors, containing a nucleic acid encoding ATLAS-X protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retro viruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retro viruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., ATLAS-X proteins, mutant forms of ATLAS-X, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of ATLAS-X in prokaryotic or eukaryotic cells.
  • ATLAS-X can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells.
  • telomeres Suitable host cells are discussed further in Goeddel, G ⁇ N ⁇ EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • Suitable inducible non- fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the ATLAS-X expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al, (1987) EMBO J 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:1 13-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif).
  • ATLAS-X can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al (1983) Mol Cell Biol 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al.
  • lymphoid-specific promoters Calame and Eaton (1988) Adv Immunol 43:235-275
  • promoters of T cell receptors Winoto and Baltimore (1989) EMBO J8:729-733
  • immunoglobulins Bonerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477
  • pancreas-specific promoters Edlund et al.
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to ATLAS-X mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • ATLAS-X protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding ATLAS-X or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) ATLAS-X protein.
  • the invention further provides methods for producing ATLAS-X protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding ATLAS-X has been introduced) in a suitable medium such that ATLAS-X protein is produced.
  • the method further comprises isolating ATLAS-X from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which ATLAS-X-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous ATLAS-X sequences have been introduced into their genome or homologous recombinant animals in which endogenous ATLAS-X sequences have been altered.
  • Such animals are useful for studying the function and/or activity of ATLAS-X and for identifying and/or evaluating modulators of ATLAS-X activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous ATLAS-X gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing ATLAS- X-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the human ATLAS-X cDNA sequence of SEQ ID NO:l, 3, 5, 7, 9, 10, 11, or 12 can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of the human ATLAS-X gene such as a mouse ATLAS-X gene, can be isolated based on hybridization to the human ATLAS-X cDNA (described further above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the ATLAS-X transgene to direct expression of ATLAS-X protein to particular cells.
  • a transgenic founder animal can be identified based upon the presence of the ATLAS-X transgene in its genome and/or expression of ATLAS-X mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding ATLAS-X can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of an ATLAS-X gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the ATLAS-X gene.
  • the ATLAS-X gene can be a human gene (e.g., the cDNA of SEQ ID NO:l, 3, 5, or 7), but more preferably, is a non-human homologue of a human ATLAS-X gene.
  • a mouse homologue of human ATLAS-X gene of SEQ ID NO:l, 3, 5, or 7 can be used to construct a homologous recombination vector suitable for altering an endogenous ATLAS-X gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous ATLAS-X gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous ATLAS-X gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous ATLAS-X protein).
  • the altered portion of the ATLAS-X gene is flanked at its 5' and 3' ends by additional nucleic acid of the ATLAS-X gene to allow for homologous recombination to occur between the exogenous ATLAS-X gene carried by the vector and an endogenous ATLAS-X gene in an embryonic stem cell.
  • flanking ATLAS-X nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced ATLAS-X gene has homologously recombined with the endogenous ATLAS-X gene are selected (see e.g., Li et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • an animal e.g., a mouse
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombmase system of bacteriophage PI.
  • cre/loxP recombinase system see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.
  • FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF,
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an ATLAS-X protein or anti-ATLAS-X 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., an ATLAS-X protein or anti-ATLAS-X antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the isolated nucleic acid molecules of the invention can be used to express ATLAS-X protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect ATLAS-X mRNA (e.g., in a biological sample) or a genetic lesion in an ATLAS-X gene, and to modulate ATLAS-X activity, as described further below.
  • the ATLAS- X proteins can be used to screen drugs or compounds that modulate the ATLAS-X activity or expression as well as to treat disorders characterized by insufficient or excessive production of ATLAS-X protein or production of ATLAS-X protein forms that have decreased or aberrant activity compared to ATLAS-X wild type protein (e.g. proliferative disorders such as cancer and immune disorders, e.g., multiple sclerosis.
  • the anti-ATLAS-X antibodies of the invention can be used to detect and isolate ATLAS-X proteins and modulate ATLAS-X activity.
  • This invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described herein. Screening Assays
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to ATLAS-X proteins or have a stimulatory or inhibitory effect on, for example, ATLAS-X expression or ATLAS-X activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to ATLAS-X proteins or have a stimulatory or inhibitory effect on, for example, ATLAS-X expression or ATLAS-X activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to ATLAS-X proteins or have a stimulatory or inhibitory effect on, for example, ATLAS-X expression or ATLAS-X activity.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an ATLAS-X protein or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des 12:145).
  • a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.
  • Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of ATLAS-X protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an ATLAS-X protein determined.
  • the cell for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the ATLAS-X protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the ATLAS-X protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of ATLAS-X protein, or a biologically active portion thereof, on the cell surface with a known compound which binds ATLAS-X to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an ATLAS-X protein, wherein determining the ability of the test compound to interact with an ATLAS-X protein comprises determining the ability of the test compound to preferentially bind to ATLAS-X or a biologically active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of ATLAS-X protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the ATLAS-X protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of ATLAS-X or a biologically active portion thereof can be accomplished, for example, by determining the ability of the ATLAS-X protein to bind to or interact with an
  • ATLAS-X target molecule is a molecule with which an
  • ATLAS-X protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an ATLAS-X interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • An ATLAS-X target molecule can be a non-ATLAS-X molecule or an ATLAS-X protein or polypeptide of the present invention.
  • an ATLAS-X target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g.
  • the target for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with ATLAS-X.
  • Determining the ability of the ATLAS-X protein to bind to or interact with an ATLAS- X target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the ATLAS-X protein to bind to or interact with an ATLAS-X target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e.
  • telomeres intracellular Ca 2+ , diacylglycerol, IP 3 , etc.
  • detecting catalytic/enzymatic activity of the target an appropriate substrate detecting the induction of a reporter gene (comprising an ATLAS-X-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
  • a reporter gene comprising an ATLAS-X-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • detecting a cellular response for example, cell survival, cellular differentiation, or cell proliferation.
  • an assay of the present invention is a cell-free assay comprising contacting an ATLAS-X protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the ATLAS-X protein or biologically active portion thereof. Binding of the test compound to the ATLAS-X protein can be determined either directly or indirectly as described above.
  • the assay comprises contacting the ATLAS-X protein or biologically active portion thereof with a known compound which binds ATLAS-X to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an ATLAS-X protein, wherein determining the ability of the test compound to interact with an ATLAS-X protein comprises determining the ability of the test compound to preferentially bind to ATLAS-X or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting ATLAS-X protein or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g.
  • Determining the ability of the test compound to modulate the activity of ATLAS-X can be accomplished, for example, by determining the ability of the ATLAS-X protein to bind to an ATLAS-X target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of ATLAS-X can be accomplished by determining the ability of the ATLAS-X protein further modulate an ATLAS-X target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay comprises contacting the ATLAS-X protein or biologically active portion thereof with a known compound which binds ATLAS-X to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an ATLAS-X protein, wherein determining the ability of the test compound to interact with an ATLAS-X protein comprises determining the ability of the ATLAS-X protein to preferentially bind to or modulate the activity of an ATLAS-X target molecule.
  • the cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form of ATLAS-X.
  • cell-free assays comprising the membrane-bound form of ATLAS-X, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of ATLAS-X is maintained in solution.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton ® X-100, Triton ® X-114, Thesit ® , Isotridecypoly(ethylene glycol ether) n , N-dodecyl-- N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl)dimethylamminiol- 1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy- 1 -propane sulfonate (CHAPSO).
  • non-ionic detergents such as n-
  • a test compound to ATLAS-X, or interaction of ATLAS-X with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
  • GST- ATLAS-X fusion proteins or GST- target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or ATLAS-X protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of ATLAS-X binding or activity determined using standard techniques.
  • ATLAS-X or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated ATLAS-X 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, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with ATLAS-X or target molecules can be derivatized to the wells of the plate, and unbound target or ATLAS-X trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the ATLAS- X or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the ATLAS-X or target molecule.
  • modulators of ATLAS-X expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of ATLAS-X mRNA or protein in the cell is determined.
  • the level of expression of ATLAS-X mRNA or protein in the presence of the candidate compound is compared to the level of expression of ATLAS-X mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of ATLAS-X expression based on this comparison. For example, when expression of ATLAS-X mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of ATLAS-X mRNA or protein expression.
  • the candidate compound when expression of ATLAS-X mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of ATLAS-X mRNA or protein expression.
  • the level of ATLAS-X mRNA or protein expression in the cells can be determined by methods described herein for detecting ATLAS-X mRNA or protein.
  • the ATLAS-X proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. 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) Biotechniques 14:920-924; Iwabuchi et al.
  • ATLAS-X-binding proteins or "ATLAS-X-bp"
  • ATLAS-X-binding proteins are also likely to be involved in the propagation of signals by the ATLAS-X proteins as, for example, upstream or downstream elements of the ATLAS-X pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for ATLAS-X is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with ATLAS-X.
  • a reporter gene e.g., LacZ
  • Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with ATLAS-X.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; and (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the ATLAS-X, sequences, described herein, can be used to map the location of the ATLAS-X genes, respectively, on a chromosome. The mapping of the ATLAS-X sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • ATLAS-X genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the ATLAS-X sequences. Computer analysis of the ATLAS-X, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the ATLAS-X sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the ATLAS-X sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the ATLAS-X gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the ATLAS-X sequences of the present invention can also be used to identify individuals from minute biological samples.
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • the sequences of the present invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Pat. No. 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the ATLAS-X sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the ATLAS-X sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
  • SNPs single nucleotide polymorphisms
  • RFLPs restriction fragment length polymorphisms
  • each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of SEQ ID NO:l, 3, 5 or 7 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases.
  • nucleotides 1-2586 of SEQ ID NO:l nucleotides l-5553of SEQ ID NO:3, nucleotides 98-904 of SEQ ID NO:5, or nucleotides 1-1074 of SEQ ID NO:7.are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • diagnostic assays for determining ATLAS-X protein and or nucleic acid expression as well as ATLAS-X activity in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant ATLAS-X expression or activity.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with ATLAS-X protein, nucleic acid expression or activity. For example, mutations in an ATLAS-X gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with ATLAS-X protein, nucleic acid expression or activity.
  • Another aspect of the invention provides methods for determining ATLAS-X protein, nucleic acid expression or ATLAS-X activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)
  • Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of ATLAS-X in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of ATLAS-X 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 ATLAS-X protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes ATLAS-X protein such that the presence of ATLAS-X is detected in the biological sample.
  • a compound or an agent capable of detecting ATLAS-X protein or nucleic acid e.g., mRNA, genomic DNA
  • An agent for detecting ATLAS-X mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to ATLAS-X mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length ATLAS-X nucleic acid, such as the nucleic acid of SEQ ID NO:l, 3, 5, 7, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to ATLAS-X mRNA or genomic DNA.
  • a full-length ATLAS-X nucleic acid such as the nucleic acid of SEQ ID NO:l, 3, 5, 7, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to ATLAS-X mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • An agent for detecting ATLAS-X protein is an antibody capable of binding to ATLAS- X protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect ATLAS- X mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of ATLAS-X mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of ATLAS-X protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immuno fluorescence.
  • In vitro techniques for detection of ATLAS-X genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of ATLAS-X protein include introducing into a subject a labeled anti-ATLAS-X antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting ATLAS-X protein, mRNA, or genomic DNA, such that the presence of ATLAS-X protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of ATLAS-X protein, mRNA or genomic DNA in the control sample with the presence of ATLAS-X protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of ATLAS-X in a biological sample can comprise: a labeled compound or agent capable of detecting ATLAS-X protein or mRNA in a biological sample; means for determining the amount of ATLAS-X in the sample; and means for comparing the amount of ATLAS-X in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect ATLAS-X protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant ATLAS- X expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with ATLAS-X protein, nucleic acid expression or activity such as cancer, immune system associated (e.g., multiple sclerosis), or fibrotic disorders.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant ATLAS-X expression or activity in which a test sample is obtained from a subject and ATLAS-X protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of ATLAS-X protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant ATLAS-X expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant ATLAS-X expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • an agent for a disorder such as cancer, immune system associated disorders, e.g., multiple sclerosis.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant ATLAS-X expression or activity in which a test sample is obtained and ATLAS-X protein or nucleic acid is detected (e.g., wherein the presence of ATLAS-X protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant ATLAS-X expression or activity.)
  • the methods of the invention can also be used to detect genetic lesions in an ATLAS-
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an ATLAS-X-protein, or the mis-expression of the ATLAS-X gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (1) a deletion of one or more nucleotides from an ATLAS-X gene; (2) an addition of one or more nucleotides to an ATLAS-X gene; (3) a substitution of one or more nucleotides of an ATLAS-
  • a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. 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) PNAS 91 :360-364), the latter of which can be particularly useful for detecting point mutations in the ATLAS-X-gene (see Abravaya et al. (1995) Nucl Acids Res 23:675-682).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an ATLAS-X gene under conditions such that hybridization and amplification of the ATLAS-X gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli et al, 1990, Proc Natl Acad Sci USA 87: 1874-1878), transcriptional amplification system (Kwoh, et al, 1989, Proc Natl Acad Sci USA 86:1173-1177), Q-Beta Replicase (Lizardi et al, 1988, BioTechnology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in an ATLAS-X gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Pat. No. 5,493,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in ATLAS-X 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 1: 244-255; Kozal et al. (1996) Nature Medicine 2: 753-759).
  • genetic mutations in ATLAS-X can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al. above. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
  • This step allows the identification of point mutations.
  • This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the ATLAS-X gene and detect mutations by comparing the sequence of the sample ATLAS-X with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (1977) PNAS 74:560 or Sanger (1977) PNAS 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve et al, (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al.
  • RNA/RNA or RNA/DNA heteroduplexes 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 methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes.
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type ATLAS-X sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in ATLAS-X cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on an ATLAS-X sequence e.g., a wild-type ATLAS-X sequence
  • a probe based on an ATLAS-X 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, for example, U.S. Pat. No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in ATLAS-X genes.
  • single strand conformation polymorphism For example, single strand conformation polymorphism
  • SSCP SSCP
  • Single-stranded DNA fragments of sample and control ATLAS-X nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265: 12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions 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.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc Natl Acad Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an ATLAS-X gene.
  • any cell type or tissue preferably peripheral blood leukocytes, in which ATLAS-X is expressed may be utilized in the prognostic assays described herein.
  • any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
  • Agents, or modulators that have a stimulatory or inhibitory effect on ATLAS-X activity can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., cancer or immune disorders associated with aberrant ATLAS-X activity.
  • disorders e.g., cancer or immune disorders associated with aberrant ATLAS-X activity.
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of ATLAS- X protein, expression of ATLAS-X nucleic acid, or mutation content of ATLAS-X genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.
  • glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymorphisms 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.
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of ATLAS-X protein, expression of ATLAS-X nucleic acid, or mutation content of ATLAS-X genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply geno typing of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an ATLAS-X modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • ATLAS-X e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • agents e.g., drugs, compounds
  • ATLAS-X e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • the effectiveness of an agent determined by a screening assay as described herein to increase ATLAS-X gene expression, protein levels, or upregulate ATLAS-X activity can be monitored in clinical trails of subjects exhibiting decreased ATLAS-X gene expression, protein levels, or downregulated ATLAS-X activity.
  • the effectiveness of an agent determined by a screening assay to decrease ATLAS-X gene expression, protein levels, or downregulate ATLAS-X activity can be monitored in clinical trails of subjects exhibiting increased ATLAS-X gene expression, protein levels, or upregulated ATLAS-X activity.
  • the expression or activity of ATLAS-X and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
  • genes including ATLAS-X, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates ATLAS-X activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • ATLAS-X activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of ATLAS-X and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of ATLAS-X or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (/) obtaining a pre- administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an ATLAS-X protein, mRNA, or genomic DNA in the preadministration sample; (Hi) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the ATLAS-X protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the ATLAS- X protein, mRNA, or genomic DNA in the pre-administration sample with the ATLAS-X protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent
  • increased administration of the agent may be desirable to increase the expression or activity of ATLAS- X to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of ATLAS-X to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant ATLAS-X expression or activity.
  • Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (Hi) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989, Science 244: 1288-1292); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.
  • modulators
  • Therapeutics that increase (i.e., are agonists to) activity may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
  • Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
  • tissue sample e.g., from biopsy tissue
  • assaying it in vitro for RNA or peptide levels, structure and or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
  • Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
  • hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant ATLAS-X expression or activity, by administering to the subject an agent that modulates ATLAS-X expression or at least one ATLAS-X activity.
  • Subjects at risk for a disease that is caused or contributed to by aberrant ATLAS-X expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the ATLAS-X aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an ATLAS-X agonist or ATLAS-X antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein.
  • the prophylactic methods of the present invention are further discussed in the following subsections. Therapeutic Methods
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of ATLAS-X protein activity associated with the cell.
  • An agent that modulates ATLAS-X protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occumng cognate ligand of an ATLAS-X protein, a peptide, an ATLAS-X peptidomimetic, or other small molecule.
  • the agent stimulates one or more ATLAS-X protein activity. Examples of such stimulatory agents include active ATLAS-X protein and a nucleic acid molecule encoding ATLAS-X that has been introduced into the cell.
  • the agent inhibits one or more ATLAS-X protein activity.
  • inhibitory agents include antisense ATLAS-X nucleic acid molecules and anti-ATLAS-X antibodies.
  • modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an ATLAS-X protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) ATLAS-X expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering an ATLAS-X protein or nucleic acid molecule as therapy to compensate for reduced or aberrant ATLAS-X expression or activity.
  • Stimulation of ATLAS-X activity is desirable in situations in which ATLAS-X is abnormally downregulated and/or in which increased ATLAS-X activity is likely to have a beneficial effect.
  • a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders).
  • a gestational disease e.g., preclampsia
  • suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
  • in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s).
  • Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.
  • Therapeutics of the present invention may be useful in the therapeutic or prophylactic treatment of diseases or disorders that are associated with cell hyperproliferation and/or loss of control of cell proliferation (e.g., cancers, malignancies and tumors).
  • diseases or disorders that are associated with cell hyperproliferation and/or loss of control of cell proliferation
  • e.g., cancers, malignancies and tumors e.g., cancers, malignancies and tumors.
  • hyperproliferation disorders see e.g., Fishman, et al, 1985. MEDICINE, 2nd ed., J.B. Lippincott Co., Philadelphia, PA.
  • Therapeutics of the present invention may be assayed by any method known within the art for efficacy in treating or preventing malignancies and related disorders.
  • Such assays include, but are not limited to, in vitro assays utilizing transformed cells or cells derived from the patient's tumor, as well as in vivo assays using animal models of cancer or malignancies.
  • Potentially effective Therapeutics are those that, for example, inhibit the proliferation of tumor-derived or transformed cells in culture or cause a regression of tumors in animal models, in comparison to the controls.
  • cancer or malignancy may subsequently be treated or prevented by the administration of a Therapeutic that serves to modulate protein function.
  • the Therapeutics of the present invention that are effective in the therapeutic or prophylactic treatment of cancer or malignancies may also be administered for the treatment of pre-malignant conditions and/or to prevent the progression of a pre-malignancy to a neoplastic or malignant state.
  • Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia or, most particularly, dysplasia has occurred.
  • non-neoplastic cell growth consisting of hyperplasia, metaplasia or, most particularly, dysplasia has occurred.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in its structure or function. For example, it has been demonstrated that endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of mature or fully differentiated cell substitutes for another type of mature cell. Metaplasia may occur in epithelial or connective tissue cells. Dysplasia is generally considered a precursor of cancer, and is found mainly in the epithelia. Dysplasia is the most disorderly form of non-neoplastic cell growth, and involves a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • the presence of one or more characteristics of a transformed or malignant phenotype displayed either in vivo or in vitro within a cell sample derived from a patient is indicative of the desirability of prophylactic/therapeutic administration of a Therapeutic that possesses the ability to modulate activity of An aforementioned protein.
  • Characteristics of a transformed phenotype include, but are not limited to: (i) morphological changes; (ii) looser substratum attachment; (Hi) loss of cell-to-cell contact inhibition; (iv) loss of anchorage dependence; (v) protease release; (vi) increased sugar transport; (vii) decreased serum requirement; (viii) expression of fetal antigens, (ix) disappearance of the 250 kDal cell-surface protein, and the like. See e.g., Richards, et al, 1986. MOLECULAR PATHOLOGY, W.B. Saunders Co., Philadelphia, PA.
  • a patient that exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a Therapeutic: (i) a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome (bcrlabl) for chronic myelogenous leukemia and t(14;18) for follicular lymphoma, etc.); (ii) familial polyposis or Gardner's syndrome, (possible forerunners of colon cancer); (Hi) monoclonal gammopathy of undetermined significance (a possible precursor of multiple myeloma) and (iv) a first degree kinship with persons having a cancer or pre-cancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, Peutz-Jeghers syndrome,
  • a Therapeutic of the present invention is administered to a human patient to prevent the progression to breast, colon, lung, pancreatic, or uterine cancer, or melanoma or sarcoma.
  • a Therapeutic is administered in the therapeutic or prophylactic treatment of hyperproliferative or benign dysproliferative disorders.
  • the efficacy in treating or preventing hyperproliferative diseases or disorders of a Therapeutic of the present invention may be assayed by any method known within the art.
  • Such assays include in vitro cell proliferation assays, in vitro or in vivo assays using animal models of hyperproliferative diseases or disorders, or the like.
  • Potentially effective Therapeutics may, for example, promote cell proliferation in culture or cause growth or cell proliferation in animal models in comparison to controls.
  • Specific embodiments of the present invention are directed to the treatment or prevention of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes); treatment of keloid (hypertrophic scar) formation causing disfiguring of the skin in which the scarring process interferes with normal renewal; psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination); benign tumors; fibrocystic conditions and tissue hypertrophy (e.g., benign prostatic hypertrophy).
  • ATLAS-X protein have been implicated in the deregulation of cellular maturation and apoptosis, which are both characteristic of neurodegenerative disease. Accordingly, Therapeutics of the invention, particularly but not limited to those that modulate (or supply) activity of an aforementioned protein, may be effective in treating or preventing neurodegenerative disease. Therapeutics of the present invention that modulate the activity of an aforementioned protein involved in neurodegenerative disorders can be assayed by any method known in the art for efficacy in treating or preventing such neurodegenerative diseases and disorders. Such assays include in vitro assays for regulated cell maturation or inhibition of apoptosis or in vivo assays using animal models of neurodegenerative diseases or disorders, or any of the assays described below. Potentially effective Therapeutics, for example but not by way of limitation, promote regulated cell maturation and prevent cell apoptosis in culture, or reduce neurodegeneration in animal models in comparison to controls.
  • neurodegenerative disease or disorder Once a neurodegenerative disease or disorder has been shown to be amenable to treatment by modulation activity, that neurodegenerative disease or disorder can be treated or prevented by administration of a Therapeutic that modulates activity.
  • Such diseases include all degenerative disorders involved with aging, especially osteoarthritis and neurodegenerative disorders.
  • ATLAS-X has been implicated in disorders related to organ transplantation, in particular but not limited to organ rejection.
  • Therapeutics of the invention particularly those that modulate (or supply) activity, may be effective in treating or preventing diseases or disorders related to organ transplantation.
  • Therapeutics of the invention (particularly Therapeutics that modulate the levels or activity of an aforementioned protein) can be assayed by any method known in the art for efficacy in treating or preventing such diseases and disorders related to organ transplantation.
  • Such assays include in vitro assays for using cell culture models as described below, or in vivo assays using animal models of diseases and disorders related to organ transplantation, see e.g., below.
  • Potentially effective Therapeutics for example but not by way of limitation, reduce immune rejection responses in animal models in comparison to controls.
  • diseases and disorders related to organ transplantation are shown to be amenable to treatment by modulation of activity, such diseases or disorders can be treated or prevented by administration of a Therapeutic that modulates activity.
  • ATLAS-X has been implicated in cardiovascular disorders, including in atherosclerotic plaque formation.
  • Diseases such as cardiovascular disease, including cerebral thrombosis or hemorrhage, ischemic heart or renal disease, peripheral vascular disease, or thrombosis of other major vessel, and other diseases, including diabetes mellitus, hypertension, hypothyroidism, cholesterol ester storage disease, systemic lupus erythematosus, homocysteinemia, and familial protein or lipid processing diseases, and the like, are either directly or indirectly associated with atherosclerosis.
  • Therapeutics of the invention particularly those that modulate (or supply) activity or formation may be effective in treating or preventing atherosclerosis-associated diseases or disorders.
  • Therapeutics of the invention can be assayed by any method known in the art, including those described below, for efficacy in treating or preventing such diseases and disorders.
  • a limited and non-exclusive list of animal models includes knockout mice for premature atherosclerosis (Kurabayashi and Yazaki, 1996, Int. Angiol. 15: 187-194), transgenic mouse models of atherosclerosis (Kappel et al, 1994, FASEB J. 8: 583-592), antisense oligonucleotide treatment of animal models (Callow, 1995, Curr. Opin. Cardiol. 10: 569-576), transgenic rabbit models for atherosclerosis (Taylor, 1997, Ann. N.Y. Acad. Sci 811: 146-152), hypercholesterolemic animal models (Rosenfeld, 1996, Diabetes Res. Clin.
  • Pract. 30 Suppl.: 1 -11 hyperlipidemic mice (Paigen et al, 1994, Curr. Opin. Lipidol. 5: 258-264), and inhibition of lipoxygenase in animals (Sigal et al, 1994, Ann. N.Y. Acad. Sci. 714: 211-224).
  • in vitro cell models include but are not limited to monocytes exposed to low density lipoprotein (Frostegard et al, 1996, Atherosclerosis 121 : 93-103), cloned vascular smooth muscle cells (Suttles et al, 1995, Exp. Cell Res.
  • An ATLAS-X protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • Many protein factors discovered to date, including all known cytokines have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+ ), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: CURRENT PROTOCOLS IN IMMUNOLOGY, Ed by Coligan et al, Greene Publishing Associates and Wiley-Interscience (Chapter 3 and Chapter 7); Takai et al, J Immunol 137:3494-3500, 1986; Bertagnolli et al., J Immunol 145:1706-1712, 1990; Bertagnolli et al, Cell Immunol 133:327-341, 1991; Bertagnolli, et al, J Immunol 149:3778-3783, 1992; Bowman et al, J Immunol 152:1756-1761, 1994.
  • Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described by Kruisbeek and Shevach, In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds. Vol 1, pp. 3.12.1-14, John Wiley and Sons, Toronto 1994; and by Schreiber, In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan eds. Vol 1 pp. 6.8.1-8, John Wiley and Sons, Toronto 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described by Bottomly et al, In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto 1991; deV ⁇ es et al., JExp Med 173: 1205-1211, 1991; Moreau et al, Nature 336:690-692, 1988; Greenberger et al, Proc Natl Acad Sci U.S.A. 80:2931-2938, 1983; Nordan, In: CURRENT PROTOCOLS IN IMMUNOLOGY.
  • Assays for T-cell clone responses to antigens include, without limitation, those described In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds., Greene Publishing Associates and Wiley-Interscience (Chapter 3Chapter 6, Chapter 7); Weinberger et al.
  • An ATLAS-X protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SOD severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by vital (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by vital, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania species., malaria species, and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
  • the functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or energy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon re-exposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as, for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody
  • B7 lymphocyte antigen e.g., B7-1, B7-3 or blocking antibody
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the lack of costimulation may also be sufficient to energize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • To achieve sufficient immunosuppression or tolerance in a subject it may also be necessary to block the function of B lymphocyte antigens.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine .
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and auto- antibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of auto-antibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., FUNDAMENTAL IMMUNOLOGY, Raven Press, New York, 1989, pp. 840-856).
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic vital diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti- vital immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor- specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and/or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class II a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class II a chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds.
  • T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described in: Maliszewski, J Immunol 144:3028-3033, 1990; and Mond and Brunswick In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, (eds.) Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al. , J Immunol 134:536-544, 1995; Inaba et al, J Exp Med 173:549-559, 1991; Macatonia et al., J Immunol 154:5071-5079, 1995; Porgador et al., JExp Med 182:255-260, 1995; Nair et al, J Virol 67:4062-4069, 1993; Huang et al, Science 264:961-965, 1994; Macatonia et al, J Exp Med 169:1255-1264, 1989; Bhardwaj et al, J Clin Investig 94:797-807, 1994; and Inaba et al, JExp Med 172:631-640, 1990.
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al, Cytometry 13:795-808, 1992; Gorczyca et al, Leukemia 7:659-670, 1993; Gorczyca et al, Cancer Res 53: 1945-1951, 1993; Itoh et al, Cell 66:233-243, 1991 ; Zacharchuk, J Immunol 145:4037-4045, 1990; Zamai et al, Cytometry 14:891-897, 1993; Gorczyca et al, InternatJ Oncol 1 :639-648, 1992.
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al, Blood 84:111-117, 1994; Fine et al, Cell Immunol 155: 111-122, 1994; Galy et al, Blood 85:2770-2778, 1995; Toki et ⁇ /., Proc Nat Acad Sci USA 88:7548-7551, 1991.
  • An ATLAS-X protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al, Mol. Cell Biol. 13:473-486, 1993; McClanahan et al, Blood 81:2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, In: CULTURE OF HEMATOPOIETIC CELLS. Freshney, et al. (eds.) Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y 1994; Hirayama et al, Proc Natl Acad Sci USA 89:5907-5911 , 1992; McNiece and Briddeli, In: CULTURE OF HEMATOPOIETIC CELLS. Freshney, et al. (eds.) Vol pp.
  • An ATLAS-X protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon ligament formation.
  • a protein of the present invention which induces tendon ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament- forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendonitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and/or sequestering agent as a career as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate.
  • a protein of the invention may also exhibit angiogenic activity.
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium).
  • Assays for wound healing activity include, without limitation, those described in: Winter, EPIDERMAL WOUND HEALING, pp. 71-112 (Maibach and Rovee, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Menz, J. Invest. Dermatol 71 :382-84 (1978).
  • An ATLAS-X protein of the present invention may also exhibit activin- or inhibin-related activities.
  • Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • a protein of the present invention alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activin inhibin activity include, without limitation, those described in: Vale et al, Endocrinology 91 :562-572, 1972; Ling et al, Nature 321 :779-782, 1986; Vale et al, Nature 321 :776-779, 1986; Mason et al, Nature 318:659-663, 1985; Forage et al, Proc Natl Acad Sci USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by following methods:
  • Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al, eds. (Chapter 6.12, MEASUREMENT OF ALPHA AND BETA CHEMOKINES 6.12.1-6.12.28); Taub et al.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al, J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al, Thrombosis Res. 45:413-419, 1987; Humphrey et al, Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell — cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses).
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention may themselves be useful as inhibitors of receptor/ligand interactions.
  • Suitable assays for receptor-ligand activity include without limitation those described in: CURRENT PROTOCOLS IN IMMUNOLOGY, Ed by Coligan, et al, Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al, Proc Natl Acad Sci USA 84:6864-6868, 1987; Bierer et ⁇ /., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et ⁇ /., J Exp. Med. 169:149-160 1989; Stoltenborg et al, J Immunol Methods 175:59-68, 1994; Stitt et al, Cell 80:661-670, 1995.
  • Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell — cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting
  • a protein of the invention may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth

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Abstract

L'invention concerne quatre nouvelles séquences humaines d'acide nucléique exprimées dans les lymphocytes T activés. L'invention concerne également des polypeptides codés par ces séquences d'acide nucléique, et des anticorps qui se lient d'une manière immunospécifique à un tel polypeptide, ou un quelconque dérivé, variant, mutant ou fragment du polypeptide, du polynucléotide ou de l'anticorps. L'invention concerne en outre des méthodes thérapeutiques, diagnostiques et de recherche permettant de traiter et de diagnostiquer des troubles associés à ces nouvelles protéines des lymphocytes T humains.
EP00955700A 1999-08-20 2000-08-18 Polynucleotides exprimes dans les lymphocytes t actives et proteines codees par ces polynucleotides Withdrawn EP1204754A2 (fr)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US15010599P 1999-08-20 1999-08-20
US150105P 1999-08-20
US56094800A 2000-04-28 2000-04-28
US56010100A 2000-04-28 2000-04-28
US56153300A 2000-04-28 2000-04-28
US56036500A 2000-04-28 2000-04-28
US560948 2000-04-28
US561533 2000-04-28
US560101 2000-04-28
US560365 2000-04-28
PCT/US2000/022699 WO2001014564A2 (fr) 1999-08-20 2000-08-18 Nouveaux polynucleotides exprimes dans les lymphocytes t actives et proteines codees par ces polynucleotides

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AU (1) AU6785700A (fr)
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US6858407B2 (en) 2000-12-22 2005-02-22 Bristol-Myers Squibb Company Human leucine-rich repeat containing protein expressed predominately in small intestine, HLRRSI1
CA2353371A1 (fr) 2001-01-29 2002-07-29 Robert J. Mcleod Appareil de decapage de revetement de fibre optique
EP2075256A2 (fr) 2002-01-14 2009-07-01 William Herman Ligands ciblés
US20040002593A1 (en) * 2002-04-04 2004-01-01 Reed John C. PAAD domain-containing polypeptides, encoding nucleic acids, and methods of use
US20040203132A1 (en) 2003-02-28 2004-10-14 Cognetix, Inc. Conus protein disulfide isomerase

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WO1993005073A1 (fr) * 1991-09-11 1993-03-18 The Trustees Of Boston University RECEPTEURS D'ANGIOTENSINE IIcAMP/VASOPRESSINEV2 ET MOLECULES ET PROCEDES ASSOCIES
FR2737500B1 (fr) * 1995-08-03 1997-08-29 Bio Merieux Materiel viral et fragments nucleotidiques associes a la sclerose en plaques, a des fins de diagnostic, prophylactiques et therapeutiques
JP4226657B2 (ja) * 1996-11-26 2009-02-18 ベーイーオー メリュー 診断、予防、及び治療のための、多発性硬化症に関連するウイルス性物質及びヌクレオチド断片
FR2762601B3 (fr) * 1997-04-29 1999-06-04 Bio Merieux Polypeptide capable de reagir avec les anticorps de patients atteints de sclerose en plaques et utilisations

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JP2003507070A (ja) 2003-02-25
AU6785700A (en) 2001-03-19

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