WO2002020758A2 - Nouvelles proteines et acides nucleiques codant pour ces proteines - Google Patents

Nouvelles proteines et acides nucleiques codant pour ces proteines Download PDF

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WO2002020758A2
WO2002020758A2 PCT/US2001/027435 US0127435W WO0220758A2 WO 2002020758 A2 WO2002020758 A2 WO 2002020758A2 US 0127435 W US0127435 W US 0127435W WO 0220758 A2 WO0220758 A2 WO 0220758A2
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ofthe
nucleic acid
polypeptide
amino acid
protein
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WO2002020758A3 (fr
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Muralidhara Padigaru
Esha A. Gangolli
Suresh Shenoy
Valerie L. Gerlach
John Macdougall
Glennda Smithson
David Stone
Karen Ellerman
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Curagen Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/70503—Immunoglobulin superfamily

Definitions

  • the invention generally relates to nucleic acids and polypeptides encoded thereby.
  • the invention generally relates to nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as NONX, or ⁇ ON1 and ⁇ ON2 nucleic acids and polypeptides.
  • ⁇ OVX nucleic acid or polypeptide sequences.
  • the invention provides an isolated ⁇ ONX nucleic acid molecule encoding a ⁇ ONX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID ⁇ OS: 1 and 3.
  • the ⁇ ONX nucleic - acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a ⁇ ONX nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes a ⁇ ONX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID ⁇ OS:2 and 4.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS: 1 and 3.
  • an oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NONX nucleic acid (e.g., SEQ ID ⁇ OS: 1 and 3) or a complement of said oligonucleotide.
  • substantially purified NONX polypeptides SEQ ID ⁇ OS: 2 and 4.
  • the ⁇ ONX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human ⁇ ONX polypeptide.
  • the invention also features antibodies that immunoselectively bind to ⁇ ONX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic can be, e.g., a ⁇ ONX nucleic acid, a ⁇ ONX polypeptide, or an antibody specific for a ⁇ ONX 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 a ⁇ ONX nucleic acid, under conditions allowing for expression of the ⁇ ONX polypeptide encoded by the D ⁇ A. If desired, the ⁇ ONX polypeptide can then be recovered.
  • the invention includes a method of detecting the presence of a ⁇ ONX 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 ⁇ ONX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a ⁇ ONX.
  • Also included in the invention is a method of detecting the presence of a ⁇ ONX nucleic acid molecule in a sample by contacting the sample with a ⁇ ONX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a ⁇ ONX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a ⁇ ONX polypeptide by contacting a cell sample that includes the ⁇ ONX polypeptide with a compound that binds to the ⁇ ONX polypeptide in an amount sufficient to modulate the activity of said 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 disorders or syndromes including, e.g., various tissue/organ inflammation, muscular dystrophy, neurological and neurodegenerative disorders, Duchenne muscular dystrophy (DMD), cardiovascular diseases and disorders, coagulation disorders, Mediterranean fever, various cancers including but not limited to meningiomas, breast, lung, colorectal cancers, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, infertility, reproductive disorders, birth control, developmental disorders, seizures, Alzheimer's disease, sleep disorders, appetite disorders, thermoregulation, pain perception, hormone secretion, sexual behavior, mental depression, migraine, epilepsy, obsessive- compulsive behavior (schizophrenia), vertex balding (hair loss), muscle fibre atrophy (motor neuron disease), Infantile neuronal ceroid lipofuscinosis (INCL), smooth muscle disorder, immunological disorder, Add
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cD ⁇ A encoding ⁇ ONX may be useful in gene therapy, and ⁇ ONX may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the method includes contacting a test compound with a NONX polypeptide and determining ifthe test compound binds to said ⁇ ONX polypeptide. Binding ofthe test compound to the ⁇ ONX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope ofthe invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a ⁇ OVX nucleic acid. Expression or activity of ⁇ ONX polypeptide is then measured in the test animal, as is expression or activity ofthe protein in a control animal which recombinantly- expresses ⁇ ONX polypeptide and is not at increased risk for the disorder or syndrome.
  • NONX polypeptide in both the test animal and the control animal is compared.
  • a change in the activity of ⁇ ONX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency ofthe disorder or syndrome.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a ⁇ ONX polypeptide, a ⁇ ONX nucleic acid, or both, in a subject (e.g., a human subject).
  • the method includes measuring the ' amount ofthe ⁇ ONX polypeptide in a test sample from the subject and comparing the amount ofthe polypeptide in the test sample to the amount ofthe ⁇ ONX polypeptide present in a control sample.
  • An alteration in the level ofthe ⁇ ONX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the expression levels ofthe new polypeptides ofthe invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a ⁇ ONX polypeptide, a ⁇ ONX nucleic acid, or a ⁇ ONX-specific antibody to a subject (e.g. , a human subject), in an amount sufficient to alleviate or prevent the pathological condition, preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the invention can be used in a method to identity the cellular receptors and downstream effectors ofthe invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.
  • the present invention provides novel nucleotides and polypeptides encoded thereby.
  • nucleic acid sequences and their polypeptides.
  • the sequences are collectively referred to as “NONX nucleic acids” or “ ⁇ ONX polynucleotides” and the corresponding encoded polypeptides are referred to as “ ⁇ ONX polypeptides” or “ ⁇ ONX proteins.” Unless indicated otherwise, " ⁇ ONX” is meant to refer to any ofthe novel sequences disclosed herein. Table A provides a summary ofthe ⁇ ONX nucleic acids and their encoded polypeptides.
  • NONX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
  • the various ⁇ ONX nucleic acids and polypeptides according to the invention are useful as novel members ofthe protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, ⁇ ONX nucleic acids and polypeptides can also be used to identify proteins that are members ofthe family to which the ⁇ ONX polypeptides belong.
  • ⁇ ON1 is homologous to a D-dopachrome tautomerase/macrophage migration inhibitory factor-like family of proteins.
  • the ⁇ ON1 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; from endotoxaemia; septic shock; ocular inflammation; Persistent Mullerian Duct Syndrome, type I; prostatic adenocarcinoma; cardiopulmonary Inflammation; proliferative diabetic retinopathy; Alzheimer disease; asthma; arthritis; sepsis; glomerulonephritis; atopic dermatitis; lymphoma and related various immunological and cancer disorders.
  • NOV2 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; neurological disorders, Alzheimer's disease, Parkinson's disease, spinal cord injury, and cancer such as neuroblastoma and related disorders.
  • the NONX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ONX activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
  • ⁇ OV1 A ⁇ ON1 nucleic acid of 458 nucleotides also referred to as SC132519848_A
  • Table 1 A An open reading frame was identified beginning with ah ' TG ' initiation codon at nucleotides 24-6 and ending with a TGA codon at nucleotides 435-437.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 1 A, and the start and stop codons are in bold letters.
  • Table 1A NOV1 nucleotide sequence (SEQ ID NO:l).
  • a NON1 nucleic acid was identified on chromosome 22 by Tblast ⁇ using CuraGen Corporation's sequence file for D-dopachrome tautomerase/macrophage migration inhibitory factor or homolog as run against the Genomic Daily Files made available by GenBank or from files downloaded from the individual sequencing centers. Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlast ⁇ , BlastX, and Blast ⁇ ) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The D ⁇ A sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
  • BLAST for example, tBlast ⁇ , BlastX, and Blast ⁇
  • the ⁇ ON1 nucleic acid sequence disclosed in this invention has 356 of 421 bases (84 %) identical to one region of a Homo Sapiens D-dopachrome tautomerase mR ⁇ A, with an E- value of 5.7e "
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • the "E- value” or “Expect” value is a numeric indication ofthe probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • the probability that the subject ("Sbjct") retrieved from the NONl BLAST analysis, e.g., Homo Sapiens D-dopachrome tautomerase rnR ⁇ A, matched the Query ⁇ ON1 sequence purely by chance is 5.7e "58 .
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
  • the Expect value is used as a convenient way to create a significance threshold for reporting results.
  • the default value used for blasting is typically set to 0.0001.
  • h BLAST 2.0 the Expect value is also used instead ofthe P value (probability) to report the significance of matches.
  • P value probability
  • an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance.
  • An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/.
  • a disclosed ⁇ ON1 protein encoding a polypeptide with 137 amino acid residues is presented in Table IB using the one-letter amino acid code.
  • the disclosed ⁇ ON1 protein was analyzed for signal peptide prediction and cellular localization.
  • the SignalP and Psort results predict that ⁇ ON1 has signal peptide, with the most likely cleavage site between positions 35 and 36 of SEQ ED NO: 2, and is likely to be localized to the cytoplasm with a certainty of 0.6500.
  • Table IB Encoded NOV1 protein sequence (SEQ ID NO:2).
  • Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
  • the global sequence homology (as defined by FASTA alignment with the full length sequence of this protein) is 87%> amino acid homology and 85%> amino acid identity.
  • NONl had homology to the amino acid sequences shown in the BLASTP data listed in Table IC.
  • ⁇ OV1 ⁇ ovel_Human_DOPD; 137 aa; SEQ ID NO. 5
  • 2 DOPDJHUMAN; Homo sapiens; ACC: P30046; 117 aa (SEQ ID NO. 6)
  • 3 DOPD_RAT; Rattus spp; ACC: S68237; 118 aa (SEQ ID NO. 7)
  • 4 DOPD_MOUSE; Mus musculus; ACC: 035215; 117 aa (SEQ ID NO. 8)
  • the "strong” group of conserved amino acid residues may be any one ofthe following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILN, MILF, HY, FYW.
  • the disclosed ⁇ ON1 protein contains following protein domains (as defined by Inte ⁇ ro) at the indicated nucleotide positions: Macrophage migration inhibitory factor (MIF) (IPR001398) at amino acid positions 2 to 114 of SEQ ID NO: 2.
  • MIF Macrophage migration inhibitory factor
  • Patp alignments of NO VI include sequences from the Patp database, which is a proprietary database that contains sequences published in patents and patent publications. Patp results include those listed in Table IE. Table IE. Patp alignments of NO VI
  • Macrophage migration inhibitory factor seems to play an important role in host inflammatory responses where it is involved in the host response to endotoxic shock probably serving as a pituitary "stress" hormone that regulates systemic inflammatory responses.
  • MIF is a secreted protein that is not processed from a larger precursor.
  • D-dopachrome tautomerase related to MIF, is a mammalian cytoplasmic enzyme involved in melanin biosynthesis that tautomerizes D-dopachrome with concomitant decarboxylation to give 5,6-dihydroxyindole (DHI).
  • DHI 5,6-dihydroxyindole
  • MMIF activity was found to correlate well with delayed hypersensitivity and cellular immunity in humans. MMIF activity could be detected in the synovia of patients with rheumatoid arthritis. The expression of MMIF at sites of inflammation suggested a role for the mediator in regulating the function of macrophages in host defense. MMIF is a major secreted protein released by anterior pituitary cells in culture and in vivo in response to stimulation with bacterial lipopolysaccharide. It plays a central role in the toxic response to endotoxemia and possibly septic shock.
  • MIF has the unique property of being released from macrophages and T cells in response to physiologic concentrations of glucocorticoids.
  • the secretion of MIF is tightly regulated and decreases at high, antiinflammatory steroid concentrations.
  • MIF 'overrides' or counter-regulates the immunosuppressive effects of steroids on immune cell activation and cytokine production.
  • glucocorticoids are an integral part ofthe host's global response to infection or tissue invasion, the physiologic role of MIF is to act at an inflammatory site or lymph node to counterbalance the profound inhibitory effect of steroids on the immune response.
  • the exon/intron structure ofthe mouse MIF gene (Mif) resembles that ofthe human gene.
  • mouse chromosome 10 By interspecific backcross analyses, they showed that the gene maps to mouse chromosome 10. They mapped 9 additional loci containing related sequences, apparently all processed pseudogenes, to mouse chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19.
  • the mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of 3 exons.
  • DDT D-dopachrome tautomerase
  • D-dopachrome tautomerase/macrophage migration inhibitory factor-like family of proteins, the ⁇ ON1 nucleic acids and proteins are useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • a cD ⁇ A encoding the D-dopachrome tautomerase/macrophage migration inhibitory factor-like protein may be useful in gene therapy, and the D-dopachrome tautomerase/macrophage migration inhibitory factor -like protein may be useful when administered to a subject in need thereof.
  • novel nucleic acid encoding ⁇ ON1 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • the ⁇ ON1 nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in various diseases and disorders described below and/or other pathologies and disorders.
  • the potential therapeutic applications for this invention include, but are not limited to; protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • the NONl nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in various neurological disorders, neurodegenerative disorders, olfactory disoders, and cancer such as neuroblastoma and/or other pathologies and disorders.
  • a cD ⁇ A encoding the D-dopachrome tautomerase/macrophage migration inhibitory factor -like protein may be useful in gene therapy, and the D-dopachrome tautomerase/macrophage migration inhibitory factor -like protein may be useful when administered to a subject in need thereof.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from neurological disorders, neurodegenerative disorders, olfactory disoders, and cancer such as neuroblastoma.
  • the ⁇ ON1 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • ⁇ ON1 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel ⁇ ON1 substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti- ⁇ OVX Antibodies" section below.
  • ⁇ ON1 proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated ⁇ ON1 epitope is from about amino acids 20 to 40.
  • a ⁇ ONl epitope is from about amino acids 50 to 70.
  • ⁇ ON1 epitopes are from amino acids 85 to 110 and from amino acids 115 to 130.
  • a NOV2 nucleic acid of 3284 nucleotides (also referred to as 30675585_EXT3) encoding a novel BIG-2/Neural Adhesion Molecule-like protein is shown in Table 2A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 61- 63 and ending with a TGA codon at nucleotides 3139-3141.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters.
  • NON2 nucleic acid was identified on chromosome 3 by Tblast ⁇ using CuraGen Co ⁇ oration's sequence file for BIG-2/ ⁇ eural Adhesion Molecule or homolog as run against the Genomic Daily Files made available by GenBank or from files downloaded from the individual sequencing centers.
  • Genomic file Genbank accession numbers: gb_AC018496 (version AC018496.2 G 6684201), gb_AC022002 (version AC022002.2 G 6862673), gb_AC026198 (version AC026198.2 GI:8101236), gb_AC034196 (version AC034196.2 G 8101269 ) and gb_AC018842 (version AC018842.3 GL6862637) are included in the invention by homology to a known BIG-2/Neural Adhesion Molecule or homolog.
  • the NON2 cD ⁇ A was cloned by exon linking; polymerase chain reaction (PCR) using the following primers: 5'-ATGAGGTTGCCATGGGAACTGCTA-3' (SEQ ID NO: 9) and 5'- GTCATAAACTGGACCTAGCTGTGAGGG-3' (SEQ ID NO: 10) on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • PCR polymerase
  • Primers were designed based on in silico predictions for the full length or part (one or more exons) ofthe DNA/Protein sequence ofthe invention or by translated homology ofthe predicted exons to closely related human sequences or to sequences from other species. Usually multiple clones were sequenced to derive the sequence which was then assembled similar to the SeqCalling process. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlasfN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full- length protein.
  • BLAST for example, tBlastN, BlastX, and BlasfN
  • a disclosed NON2 polypeptide (SEQ ID ⁇ O:8) encoded by SEQ ID NO:7 is 1026 amino acid residues and is presented using the one-letter code in Table 2B.
  • the NON2 protein was analyzed for signal peptide prediction and cellular localization. SignalP, Psort and Hydropathy results predict that ⁇ ON2 contains a predicted signal peptide, with the most likely cleavage position between residues 18 and 19 of SEQ ID NO: 8 and that NON2 is likely to be localized outside the cell with a certainty of 0.3700.
  • Table 2B Encoded ⁇ OV2 protein sequence (SEQ ID NO:4).
  • the full amino acid sequence of a NOV2 protein ofthe invention was found to have 934 of 1026 amino acid residues (91%) identical to, and 982 of 1026 amino acid residues
  • NOV2 is expressed in at least the following tissues: brain, testis, fetal lung, fetal liver, B cells and in oligodendrogliomas.
  • the sequence is predicted to be expressed in the following tissues because ofthe expression pattern of GENBANK-ID: RNU35371, a closely related homolog in species Rattus norvegicus : developing olfactory neurons, vomeronasal epithelium, and hippocampus. It was also found that NON2 had homology to the amino acid sequences shown in the BLASTP data listed in Table 2C.
  • SPTREMBL Q62845 NEURAL CELL ADHESION PROTEIN BIG-2 PRECURSOR - Rattus norvegicus (Rat), 1026 aa. (SEQ ID NO: 12)
  • SPTREMBL Q62682 BIG-1 PROTEIN PRECURSOR - Rattus norvegicus (Rat), 1028 aa (SEQ ID NO: 13) 4.
  • SPTREMBL Q07409 PLASMACYTOMA-ASSOCIATED NEURONAL GLYCOPROTEIN (NEURONAL GLYCOPROTEIN PANG)(PLASMACYTOMA- ASSOCIATED NEURONAL GLYCOPROTEIN) - Mus musculus (Mouse), 1028 aa. (SEQ ID NO: 14)
  • BLAST results include sequences from the Patp database, which is a proprietary database that contains sequences published in patents and patent publications. Patp results include those listed in Table 2E.
  • Neural adhesion molecules play a critical role in neural development, targeting and synaptogenesis. They show the presence of characteristic immunoglobulin-like domains, which impart the ability to interact with other protein and ligands. A subset of them also contain fibronectin type III repeats, which also mediate protein-protein interactions and cell- surface binding. Mutations in certain neural adhesion molecules, for example, LI, can lead to severe genetic disorders such as mental retardation and mice deficient in the neural cell adhesion molecule have impaired spatial learning ability.
  • the human ortholog of rat BIG-2 is a molecule possibly involved in synaptogenesis.
  • NON2 may have roles in learning and memory, synaptogenesis and neurite outgrowth, neuronal regeneration and repair and cancer.
  • Neural cell adhesion molecules ofthe immunoglobulin superfamily nucleate and maintain groups of cells at key sites during early development and in the adult.
  • binding of CAMs can affect intracellular signaling. Their ability to influence developmental events, including cell migration, proliferation, and differentiation can therefore result both from their adhesive as well as their signaling properties.
  • the binding of N-CAM to cells leads to a number of signaling events, some of which result in changes in gene expression.
  • Interest in LI derives from the fact that mutations in its gene lead to human genetic diseases including mental retardation. Much is known about modifications ofthe LI cytoplasmic domain and its interaction with cytoskeletal molecules.
  • CAM signaling mechanisms have been assay-dependent rather than molecule-dependent, with particular emphasis on assays of neurite outgrowth and gene expression, an emphasis that is maintained throughout the review.
  • the signals generated following CAM binding that lead to alterations in cell mo ⁇ hology and gene expression have been linked directly in only a few cases.
  • Other CAMs are anchored in the membrane by a phospholipid anchor.
  • These proteins, including a form of N-CAM, are presumed to be localized in lipid rafts, membrane substructures that include distinctive subsets of cytoplasmic signaling molecules such as members ofthe src-family of nonreceptor protein tyrosine kinases.
  • these studies may reveal that what CAMs do after they bind cells together may have as profound consequences for the cells as the adhesive interactions themselves.
  • the neural cell adhesion molecule By mediating cell adhesion and signal transduction, the neural cell adhesion molecule
  • NCAM neuropeptide kinase
  • LTP long-term potentiation
  • PSA polysialic acid
  • ISH h situ hybridization
  • Ncam expression appeared in advance ofthe olfactory receptor expression, while Big-2 appeared after olfactory receptor expression and neither were expressed in cells outside the olfactory epithelium. Both showed the highest number of positive cells in the early post-partum period when olfactory detection is functional. Ncam is known to be involved in guidance ofthe developing olfactory axons and was expressed earlier than any ofthe olfactory receptors, while Big-2 appears somewhat later (El 4) at a time when developing axons reach the olfactory bulb. Moreover the highest periods of expression occur at post-natal day 7 when a proliferation of bulbar glomeruli are observed, suggesting the role of Big-2 to be primarily concerned with synaptogenesis.
  • the cloned rat cDNA for a novel brain-derived immunoglobulin. (Ig) superfamily molecule, BIG-1 was performed using PCR based on the amino acid sequences ofthe two closely related and well-known Ig superfamily members, rat TAG-1 and mouse F3.
  • Ig immunoglobulin superfamily molecule
  • BIG-1 is a glycosylphosphatidylinositol-anchored membrane protein with six Ig-like domains and four fibronectin type III repeats, belonging to the TAG-1/F3 subgroup.
  • the expression of BIG-1 mRNA is developmentally regulated with the highest level in the adult brain.
  • a mouse cDNA encoding a novel truncated form ofthe gene BIG-2 has been cloned from the vomeronasal organ.
  • the related proteins BIG-2 and BIG-1 possess a C-terminal glycosylphosphatidylinositol anchor, six immunoglobulin domains and four fibronectin type III repeats. They are related to certain axonal-associated cell adhesion molecules (AxCAMs) exhibiting most similarity to the TAG-1/F3 subgroup of neural cell adhesion molecules.
  • AxCAMs axonal-associated cell adhesion molecules
  • BIG- 2A appears to represent a novel splice variant of BIG-2 possessing six Ig-like domains, a single fibronectin repeat and lacking the glycosylphosphatidylinositol-anchoring domain
  • NB-2 and NB-3 Two kinds of cDNAs encoding novel Contactin/F3 -subgroup adhesion molecules termed NB-2 and NB-3, have been cloned. Nucleotide sequence analyses have shown that NB- 2 and NB-3 are comprised of 1099 and 1028 amino acid residues, respectively. There was 51%) similarity in the amino acid sequence of NB-2 and NB-3. NB-2 shared 46, 43, 55 and 55% identities with ContactinF3, Tag-1, Big-1 and Big-2, respectively, at the amino acid sequence level. Likewise, the amino acid sequence of NB-3 exhibited 42, 44, 58 and 60% identities with Contactin/F3, Tag-1, Big-1 and Big-2, respectively.
  • NB-2 mRNA was restricted to cerebrum, cerebellum and was hardly detectable, if any, in spinal cord.
  • high expression of NB-3 mRNA was observed in spinal cord, as well as in cerebrum and cerebellum, the other tissues, no expression of NB-2 and NB-3 mRNAs was detected.
  • Axon-associated cell adhesion molecules AxCAMs play crucial roles in the formation, maintenance, and plasticity of functional neuronal networks.
  • BIG-2 is a member of TAG-1/F3 subgroup ofthe immunoglobulin (Ig) superfamily, with six Ig-like domains, four fibronectin type Ill-like repeats, and a glycosyl phosphatidylinositol-anchoring domain, and is an AxCAM.
  • Ig immunoglobulin
  • Plasmacytomagenesis provides a murine model to decipher progressive genetic events culminating in a B-cell neoplasia.
  • Activation ofthe c-myc protooncogene by chromosomal translocation is considered an initiating event
  • h tracisternal A-type particles IAPs
  • IAPs h tracisternal A-type particles
  • PCTs plasmacytomas
  • LAP proviral insertions have been documented to engender negative or positive effects on the expression of nearby cellular genes.
  • PANG plasmacytoma-associated neuronal glycoprotein
  • LTR long terminal repeat
  • a full-length PANG cDNA was isolated from an MPC-11 plasma cell tumor cDNA library and encodes a polypeptide of about 113 kDa with six immunoglobulin C2-like and four type III fibronectin-like domains.
  • PANG bears a striking resemblance to axonal glycoproteins TAG-1 and FI 1 known to function in neuronal outgrowth.
  • An extensive survey revealed a predominant 3.6-kb PANG transcript in 60%. (30 of 50) of PCTs as well as unique smaller and- larger species. All other normal and transformed lymphoid and nonlymphoid cell lines and normal tissues were negative for PANG expression except for the brain, wherein unique 4.0- and 6.1-kb transcripts were detected.
  • Reverse transcriptase PCR analysis revealed IAP LTR fusion to PANG mRNAs in five PCTs and in a neuroblastoma line.
  • the 5' end of a mouse brain PANG cDNA was identical to the MPC-11 PANG transcript except for the precise replacement of its 5' LTR sequence.
  • the ⁇ ON2 nucleic acids and proteins are useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders such cancer, e.g. plasmacytoma, and/or other pathologies and disorders.
  • a cD ⁇ A encoding the BIG-2-like protein may be useful in gene therapy, and the BIG-2-like protein may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from cancer.
  • the ⁇ ON2 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind imtnunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • the NON2 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • ⁇ ON2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecif ⁇ cally to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti- ⁇ OVX Antibodies" section below.
  • the disclosed ⁇ ON2 proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated ⁇ ON2 epitope is from about amino acids 1 to 20.
  • a ⁇ ON2 epitope is from about amino acids 250 to 275.
  • ⁇ ON2 epitopes are from amino acids 380 to 450 and from amino acids 1000 to 1026.
  • ⁇ OVX Nucleic Acids and Polypeptides One aspect ofthe invention pertains to isolated nucleic acid molecules that encode
  • nucleic acid fragments sufficient for use as hybridization probes to identify NONX-encoding nucleic acids (e.g., ⁇ ONX mR ⁇ As) and fragments for use as PCR primers for the amplification and/or mutation of ⁇ ONX nucleic acid molecules.
  • nucleic acid molecule is intended to include D ⁇ A molecules (e.g., cD ⁇ A or genomic D ⁇ A), R A molecules (e.g., mR ⁇ A), analogs ofthe D ⁇ A or R ⁇ A generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded D ⁇ A.
  • An ⁇ ONX nucleic acid can encode a mature ⁇ ONX polypeptide. As used herein, a
  • mature form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
  • the product "mature” form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal ofthe N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules which are present in the natural source ofthe 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'-termini ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated NONX nucleic acid molecules 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 D ⁇ A ofthe cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
  • an "isolated" nucleic acid molecule such as a cD ⁇ A 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 ofthe invention e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID ⁇ OS: 1 or 3, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • ⁇ ONX 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, lohn Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid ofthe 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 NONX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated D ⁇ A synthesizer.
  • 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 cD ⁇ A sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary D ⁇ A or R ⁇ A 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, h one embodiment ofthe invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID ⁇ OS: 1 and 3, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NOS: 1 or 3, 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 an NONX polypeptide).
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ ID ⁇ OS:l or 3 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:l or 3 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS: 1 or 3, 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, and the like.
  • 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, ifthe derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs ofthe nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 10%, 80%>, or 95%.
  • 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 NONX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of R ⁇ A.
  • homologous nucleotide sequences include nucleotide sequences encoding for an ⁇ ONX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, 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 ofthe nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human ⁇ ONX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID ⁇ OS: 1 or 3, as well as a polypeptide possessing ⁇ ONX biological activity. Various biological activities ofthe ⁇ ONX proteins are described below.
  • An ⁇ ONX polypeptide is encoded by the open reading frame ("ORF") of an ⁇ ONX nucleic acid.
  • An ORF 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 ofthe 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, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
  • the nucleotide sequences determined from the cloning ofthe human NONX genes ' allows for the generation of probes and primers designed for use in identifying and/or cloning ⁇ ONX homologues in other cell types, e.g. from other tissues, as well as ⁇ ONX homologues from other vertebrates.
  • 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 SEQ ID ⁇ OS:l or 3; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l or 3; or of a naturally occurring mutant of SEQ ID NOS: 1 or 3.
  • Probes based on the human NONX nucleotide sequences 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 tissues which mis- express an ⁇ ONX protein, such as by measuring a level of an ⁇ ONX-encoding nucleic acid in a sample of cells from a subject e.g. , detecting ⁇ ONX mR ⁇ A levels or determining whether a genomic ⁇ ONX gene has been mutated or deleted.
  • a polypeptide having a biologically-active portion of an ⁇ ONX polypeptide refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe 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 ⁇ ONX” can be prepared by isolating a portion SEQ ID ⁇ OS: 1 or 3, that encodes a polypeptide having an ⁇ OVX biological activity (the biological activities ofthe ⁇ ONX proteins are described below), expressing the encoded portion of ⁇ ONX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of ⁇ ONX.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS: 1 or 3 due to degeneracy ofthe genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in ' SEQ ID NOS: 1 or 3.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2 or 4.
  • DNA sequence polymo ⁇ hisms that lead to changes in the amino acid sequences ofthe NONX polypeptides may exist within a population (e.g., the human population).
  • Such genetic polymo ⁇ hism in the ⁇ ONX genes 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 (ORF) encoding an ⁇ ONX protein, preferably a vertebrate ⁇ ONX protein.
  • Such natural allelic variations can typically result in l-5%> variance in the nucleotide sequence ofthe ⁇ ONX genes. Any and all such nucleotide variations and resulting amino acid polymo ⁇ hisms in the ⁇ ONX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe ⁇ ONX polypeptides, are intended to be within the scope ofthe invention. Moreover, nucleic acid molecules encoding ⁇ ONX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ ED ⁇ OS: 1 or 3 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe NOVX cDNAs ofthe invention can be isolated based on their homology to the human NONX nucleic acids disclosed herein using the human cD ⁇ As, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe 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 ⁇ OS: 1 or 3.
  • 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 NONX 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% ofthe 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% ofthe 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%, 70%, 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% PNP, 0.02% FicoU, 0.02% BSA, and 500 mg/ml denatured salmon sperm D ⁇ A at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences SEQ ID NOS: 1 or 3, 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 NOS: 1 or 3, 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 within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS: 1 or 3, 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% PNP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm D ⁇ A, 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).
  • nucleotide sequences SEQ ID NOS: 1 or 3 can be made in the sequence SEQ ID NOS:2 or 4.
  • a "non-essential” amino acid residue is a residue that can be altered from the wild-type sequences ofthe NONX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
  • amino acid residues that are conserved among the ⁇ ONX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
  • nucleic acid molecules encoding ⁇ ONX proteins that contain changes in amino acid residues that are not essential for activity. Such ⁇ ONX proteins differ in amino acid sequence from SEQ ID ⁇ OS: 1 or 3 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%o homologous to the amino acid sequences SEQ ID ⁇ OS: 2 and 4.
  • the protein encoded by the nucleic acid molecule is at least about 60% ⁇ homologous to SEQ ID ⁇ OS:2 and 4; more preferably at least about 70% homologous SEQ ID NOS:2 or 4; still more preferably at least about 80%> homologous to SEQ ID NOS:2 or 4; even more preferably at least about 90% homologous to SEQ ID NOS:2 or 4; and most preferably at least about 95%> homologous to SEQ ID NOS:2 or 4.
  • An isolated nucleic acid molecule encoding an NONX protein homologous to the protein of SEQ ID ⁇ OS:2 or 4 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS: 1 or 3, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NOS: 1 or 3 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 within 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 non-essential amino acid residue in the NONX protein is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an ⁇ ONX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ⁇ ONX biological activity to identify mutants that retain activity.
  • SEQ ID ⁇ OS: 1 or 3 the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • the relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak” residues.
  • the "strong” group of conserved amino acid residues may be any one ofthe following groups: STA, ⁇ EQK, ⁇ HQK, ⁇ DEQ, QHRK, MILN, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.
  • the "weak” group of conserved residues may be any one ofthe following: CSA, ATN, SAG, ST ⁇ K, STPA, SG ⁇ D, S ⁇ DEQK, ⁇ DEQHK, ⁇ EQHRK, NLIM, HFY, wherein the letters within each group represent the single letter amino acid code.
  • a mutant ⁇ ONX protein can be assayed for (i) the ability to form protei protein interactions with other ⁇ ONX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant ⁇ ONX protein and an ⁇ ONX ligand; or (iii) the ability of a mutant ⁇ ONX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
  • a mutant ⁇ ONX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
  • Another aspect ofthe 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 NOS: 1 or 3, 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 NONX coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ ID NOS:2 or 4, or antisense nucleic acids complementary to an NONX nucleic acid sequence of SEQ ID ⁇ OS: 1 or 3, are additionally provided.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an ⁇ ONX protein.
  • coding region refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid ' molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding the ⁇ ONX protein.
  • 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 ofthe 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 ⁇ ONX mR ⁇ A, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of ⁇ ONX mR ⁇ A.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of ⁇ ONX mR ⁇ A.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
  • 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, dihydrouracil, beta-D-galactosylqueosine, inosine, ⁇ 6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NONX protein to thereby inhibit expression ofthe 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 D ⁇ A duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be 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 ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary R ⁇ A in which, contrary to the usual ⁇ -units, the strands run parallel to each other. See, e.g., Gaultier, et al, 1987. Nucl. Acids Res. 15: - 6625-6641.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., frioue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., frioue, et al, 1987. FEBS Lett. 215: 327-330.
  • Nucleic acid modifications include, by way of non-limiting 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 ofthe 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 ofthe 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 as described in Haselhoff and Gerlach 1988. Nature 334: 585-591
  • a ribozyme having specificity for an ⁇ ONX-encoding nucleic acid can be designed based upon the nucleotide sequence of an ⁇ ONX cD ⁇ A disclosed herein (i.e., SEQ ID ⁇ OS: 1 or 3).
  • a derivative of a Tetrahymena L-19 INS R ⁇ A can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an ⁇ OVX-encoding mR ⁇ A. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent
  • 5,116,742 to Cech, et al. ⁇ ONX mR ⁇ A can also be used to select a catalytic R ⁇ A having a specific ribonuclease activity from a pool of R ⁇ A molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
  • ⁇ ONX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the ⁇ ONX nucleic acid (e.g. , the ⁇ ONX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe ⁇ ONX gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the ⁇ ONX nucleic acid e.g. , the ⁇ ONX promoter and/or enhancers
  • the ⁇ ONX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, 1996. Bioorg Med Chem 4: 5-23.
  • peptide nucleic acids refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
  • PNAs of NONX can be used in therapeutic and diagnostic applications.
  • P ⁇ As 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.
  • P ⁇ As of ⁇ ONX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., P ⁇ A directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Sj .
  • nucleases See, Hyrup, et al, 1996.supra); or as probes or primers for D ⁇ A sequence and hybridization (See, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
  • P ⁇ As of ⁇ OVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to P ⁇ A, by the formation of P ⁇ A-D ⁇ A chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • P ⁇ A-D ⁇ A chimeras of ⁇ ONX can be generated that may combine the advantageous properties of P ⁇ A and D ⁇ A.
  • Such chimeras allow D ⁇ A recognition enzymes (e.g., R ⁇ ase H and D ⁇ A polymerases) to interact with the D ⁇ A portion while the P ⁇ A portion would provide high binding affinity and specificity.
  • P ⁇ A-D ⁇ A chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra).
  • the synthesis of P ⁇ A-D ⁇ A chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363.
  • a D ⁇ A 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 P ⁇ A and the 5' end of D ⁇ A. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. P ⁇ A monomers are then coupled in a stepwise mam er to produce a chimeric molecule with a 5' P ⁇ A segment and a 3' D ⁇ A segment. See, e.g., Finn, et al, 1996. supra.
  • chimeric molecules can be synthesized with a 5' D ⁇ A segment and a 3' PNA segment. See, e.g., 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:
  • oligonucleotides can be modified with hybridization triggered cleavage agents (.see, e.g., Krol, etal, 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, and the like.
  • a polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2 or 4.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2 or 4 while still encoding a protein that maintains its NONX activities and physiological functions, or a functional fragment thereof.
  • an ⁇ ONX variant that preserves ⁇ ONX-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 ofthe 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 ofthe invention pertains to isolated ⁇ ONX 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- ⁇ ONX antibodies.
  • native ⁇ ONX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • NONX proteins are produced by recombinant D ⁇ A techniques.
  • an ⁇ ONX 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 ⁇ ONX 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 ⁇ ONX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly-produced.
  • the language "substantially free of cellular material” includes preparations of ⁇ ONX proteins having less than about 30%> (by dry weight) of non- ⁇ ONX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non- ⁇ ONX proteins, still more preferably less than about 10%> of non- ⁇ ONX proteins, and most preferably less than about 5% of non- ⁇ ONX proteins.
  • a contaminating protein 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% ofthe volume of the ⁇ ONX protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of ⁇ ONX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein, h one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of ⁇ ONX proteins having less than about 30% > (by dry weight) of chemical precursors or non- ⁇ ONX chemicals, more preferably less than about 20%> chemical precursors or non- ⁇ ONX chemicals, still more preferably less than about 10% chemical precursors or non- ⁇ ONX chemicals, and most preferably less than about 5%> chemical precursors or non- ⁇ ONX chemicals.
  • Biologically-active portions of ⁇ ONX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe ⁇ ONX proteins (e.g., the amino acid sequence shown in SEQ ID ⁇ OS:2 or 4) that include fewer amino acids than the full-length NONX proteins, and exhibit at least one activity of an ⁇ ONX protein.
  • biologically-active portions comprise a domain or motif with at least one activity ofthe NONX protein.
  • a biologically-active portion of an ⁇ ONX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
  • the ⁇ ONX protein has an amino acid sequence shown SEQ ID ⁇ OS:2 or 4.
  • the NONX protein is substantially homologous to SEQ ID ⁇ OS:2 or 4, and retains the functional activity ofthe protein of SEQ ID NOS:2 or 4, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
  • the NONX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID ⁇ OS:2 or 4, and retains the functional activity ofthe NOVX proteins of SEQ ID NOS:2 or 4.
  • the sequences are aligned for optimal comparison pu ⁇ oses (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. J Mol Biol 48: 443-453.
  • GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48: 443-453.
  • GAP creation penalty of 5.0 GAP extension penalty of 0.3
  • the coding region ofthe 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 ofthe DNA sequence shown in SEQ ID NOS: 1 or 3.
  • 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 ⁇ ONX "chimeric protein” or “fusion protein” comprises an ⁇ ONX polypeptide operatively- linked to a non- ⁇ ONX polypeptide.
  • An " ⁇ ONX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to an ⁇ ONX protein SEQ ID ⁇ OS:2 or 4), whereas a “non-NONX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the ⁇ ONX protein, e.g., a protein that is different from the ⁇ ONX protein and that is derived from the same or a different organism.
  • an ⁇ ONX fusion protein the ⁇ ONX polypeptide can correspond to all or a portion of an ⁇ ONX protein.
  • an ⁇ ONX fusion protein comprises at least one biologically-active portion of an ⁇ ONX protein.
  • an ⁇ ONX fusion protein comprises at least two biologically-active portions of an ⁇ ONX protein.
  • an ⁇ ONX fusion protein comprises at least three biologically-active portions of an ⁇ ONX protein.
  • the term "operatively-linked" is intended to indicate that the ⁇ ONX polypeptide and the non- ⁇ ONX polypeptide are fused in- frame with one another.
  • the non-NONX polypeptide can be fused to the ⁇ -terminus or C-terminus ofthe ⁇ ONX polypeptide.
  • the fusion protein is a GST- ⁇ ONX fusion protein in which the ⁇ ONX sequences are fused to the C-terminus ofthe GST (glutathione S-transferase) sequences.
  • GST glutthione S-transferase
  • Such fusion proteins can facilitate the purification of recombinant ⁇ ONX polypeptides.
  • the fusion protein is an ⁇ ONX protein containing a heterologous signal sequence at its ⁇ -terminus.
  • expression and/or secretion of ⁇ ONX can be increased through use of a heterologous signal sequence.
  • the fusion protein is an ⁇ ONX-immunoglobulin fusion protein in which the ⁇ ONX sequences are fused to sequences derived from a member ofthe immunoglobulin protein family.
  • the ⁇ ONX-immunoglobulin fusion proteins ofthe invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an ⁇ ONX ligand and an ⁇ ONX protein on the surface of a cell, to thereby suppress ⁇ ONX-mediated signal transduction in vivo.
  • the ⁇ ONX-immunoglobulin fusion proteins can be used to affect the bioavailability of an ⁇ ONX cognate ligand. Inhibition ofthe ⁇ ONX ligand/ ⁇ ONX 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 ⁇ ONX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti- ⁇ ONX antibodies in a subject, to purify ⁇ ONX ligands, and in screening assays to identify molecules that inhibit the interaction of ⁇ ONX with an ⁇ ONX ligand.
  • An ⁇ ONX chimeric or fusion protein ofthe invention can be produced by standard recombinant D ⁇ A techniques.
  • D ⁇ A 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 D ⁇ A 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, e.g., 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
  • a fusion moiety e.g., a GST polypeptide.
  • An NONX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the ⁇ OVX protein.
  • the invention also pertains to variants ofthe ⁇ OVX proteins that function as either ⁇ OVX agonists (i.e., mimetics) or as ⁇ OVX antagonists.
  • Variants ofthe ⁇ OVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation ofthe ⁇ OVX protein).
  • An agonist ofthe ⁇ OVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe ⁇ OVX protein.
  • An antagonist of the ⁇ OVX protein can inhibit one or more ofthe activities ofthe naturally occurring form of the ⁇ OVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the ⁇ OVX protein.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the ⁇ OVX proteins.
  • Variants ofthe ⁇ OVX proteins that function as either ⁇ OVX agonists (i.e., mimetics) or as ⁇ OVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) ofthe ⁇ OVX proteins for ⁇ OVX protein agonist or antagonist activity.
  • a variegated library of ⁇ OVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene
  • libraries of fragments ofthe NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX 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 ⁇ nuclease, and ligating the resulting fragment library into an expression vector.
  • expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX proteins.
  • Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of NOVX proteins.
  • the most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation ofthe vector encoding the gene whose product was detected.
  • Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331. Anti-NOVX Antibodies
  • antibodies to NOVX proteins, or fragments of NOVX proteins.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F ab , F a b> and F( a b')2 fragments, and an F ab expression library.
  • an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule.
  • Certain classes have subclasses as well, such as IgG ls IgG 2 , and others.
  • the light chain may be a kappa chain or a lambda chain.
  • Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • An isolated NOVX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens.
  • An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis ofthe human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • 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.
  • a protein ofthe invention may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein ofthe invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, inco ⁇ orated herein by reference). Some of these antibodies are discussed below.
  • an appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • 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.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffmity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, h e, Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28). Monoclonal Antibodies
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al, MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this pu ⁇ ose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells ofthe invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immuno globulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
  • Humanized Antib odies can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) that are principally comprised ofthe sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
  • Humanization can be performed following the method of Winter and co-workers (Jones et al, Nature, 321:522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) h some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, 1986; Riechmann et al, 1988; and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by 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 ofthe present invention and may be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al, 1985 h : MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • transgenic animals e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
  • This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are inco ⁇ orated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications.
  • nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F (ab') 2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F a fragment generated by reducing the disulfide bridges of an F (a ) fragment; (iii) an F ab fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Bispecific Antibodies produced by techniques known in the art including, but not limited to: (i) an F (ab') 2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F a fragment generated by reducing the disulfide bridges of an F (a ) fragment; (iii) an F ab fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one ofthe binding specificities is for an antigenic protein ofthe invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain, hi this method, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al, Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al, J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') molecule.
  • Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al, J. Immunol. 147:60 (1991).
  • Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.
  • bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • Fc ⁇ R Fc receptors for IgG
  • Fc ⁇ R Fc ⁇ RI
  • CD32 Fc ⁇ RII
  • Fc ⁇ RIII CD 16
  • Heteroconjugate antibodies are also within the scope ofthe present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • suitable reagents for this pu ⁇ ose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. Effector Function Engineering
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. hnmunol., 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design, 3: 219-230 (1989).
  • Immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeraginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 1, 131 h ⁇ , 90 Y, and 186 Re.
  • Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-
  • a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibody in another embodiment, can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such streptavidin
  • a "ligand” e.g., avidin
  • 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 NOVX protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX protein possessing such a domain.
  • antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • Anti-NOVX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an NONX protein (e.g., for use in measuring levels ofthe NONX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies for ⁇ ONX 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- ⁇ ONX antibody (e.g., monoclonal antibody) can be used to isolate an ⁇ ONX polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti- ⁇ ONX antibody can facilitate the purification of natural ⁇ ONX polypeptide from cells and of recombinantly-produced ⁇ ONX polypeptide expressed in host cells.
  • an anti- ⁇ ONX antibody can be used to detect ⁇ ONX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe ⁇ ONX protein.
  • Anti- ⁇ ONX 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;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I,
  • vectors preferably expression vectors, containing a nucleic acid encoding an ⁇ OVX 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.
  • vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Plasmid which refers to a circular double stranded D ⁇ A loop into which additional D ⁇ A segments can be ligated.
  • a viral vector Another type of vector is a viral vector, wherein additional D ⁇ A segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • 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 retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe 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 ofthe 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 ofthe 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 ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors ofthe 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., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
  • the recombinant expression vectors ofthe invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells.
  • NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, 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 ofthe recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: (i) to increase expression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech e; Smith and Johnson, 1988.
  • pMAL New England Biolabs, Beverly, Mass.
  • pRIT5 Pharmacia, Piscataway, N.J.
  • GST glutathione S-transferase
  • maltose E binding protein or protein A, respectively, to the target recombinant protein.
  • suitable inducible non-fusion E. coli expression vectors include pTrc
  • 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, e.g., 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 ofthe 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 (see, e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
  • the NOVX expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Co ⁇ oration, San Diego, Calif.), and picZ (InVitrogen Co ⁇ , San Diego, Calif).
  • NOVX 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 ofthe invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al, 1987. EMBO J. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of 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.
  • the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 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 ofthe 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 ofthe DNA molecule) of an RNA molecule that is antisense to NOVX mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression ofthe 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.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • NOVX 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 NOVX 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 inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein.
  • the invention further provides methods for producing NOVX protein using the host cells ofthe invention, h one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced, another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
  • the host cells ofthe invention can also be used to produce non-human transgenic animals.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered.
  • Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein 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 ofthe cells ofthe 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 ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.
  • a transgenic animal ofthe invention can be created by introducing NOVX-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 NOVX cDNA sequences SEQ ID NOS: 1 or 3 can be introduced as a transgene into the genome of a non-human animal.
  • a non-human homologue ofthe human NOVX gene such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene.
  • a tissue-specific regulatory sequence(s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells.
  • a transgenic founder animal can be identified based upon the presence ofthe NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene.
  • the NOVX gene can be a human gene (e.g., the cDNA of SEQ ID NOS: 1 or 3), but more preferably, is a non-human homologue of a human NOVX gene.
  • a mouse homologue of human NOVX gene of SEQ ID NOS: 1 or 3 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome, hi one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX 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 NOVX 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 NOVX protein).
  • the altered portion ofthe NOVX gene is flanked at its 5'- and 3 '-termini by additional nucleic acid ofthe NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell.
  • flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5'- and 3'-termini
  • the vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., U, et al, 1992. Cell 9: 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 ofthe animal contain the homologously-recombined DNA by germline transmission ofthe transgene.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system See, e.g., Lakso, et al, 1992.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355. Ifa cre/loxP recombinase system is used to regulate expression ofthe transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones ofthe 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 ofthe 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 ofthe animal from which the cell (e.g., the somatic cell) is isolated.
  • compositions suitable for administration can be inco ⁇ orated 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 abso ⁇ tion 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 inco ⁇ orated herein by reference.
  • Preferred examples of 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 inco ⁇ orated into the compositions.
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., 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 (EDTA); 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 EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid 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 ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion ofthe injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., an NOVX protein or anti-NOVX 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 NOVX protein or anti-NOVX antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above, hi the case of sterile powders for the preparation of sterile injectable solutions, 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 pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057).
  • the pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells 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 ofthe invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below.
  • the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias.
  • the anti-NOVX antibodies ofthe invention can be used to detect and isolate NOVX proteins and modulate NOVX activity.
  • the invention can be used in methods to influence appetite, abso ⁇ tion of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
  • the invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra. 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 NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOV
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity ofthe membrane-bound form of an NOVX protein or polypeptide or biologically-active portion thereof.
  • the test compounds of the invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 ' . Anticancer Drug Design 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 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 ofthe assays ofthe invention.
  • Biotechniques 13: 412-421 or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl. Acad. Sci. U.S.A.
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to an NOVX protein determined.
  • the cell for example, can of mammalian origin or a yeast cell.
  • Determining the ability ofthe test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the NOVX 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 NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX protein 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 NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe NOVX protein or bio logically- active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX or a biologically- active portion thereof can be accomplished, for example, by determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule.
  • a "target molecule” is a molecule with which an NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX 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 NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide ofthe invention.
  • an NOVX 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 NOVX.
  • Determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity ofthe target molecule. For example, the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e.
  • a reporter gene comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • a cellular response for example, cell survival, cellular differentiation, or cell proliferation.
  • an assay ofthe invention is a cell-free assay comprising contacting an NOVX protein or biologically- active portion thereof with a test compound and determining the ability ofthe test compound to bind to the NOVX protein or biologically- active portion thereof. Binding ofthe test compound to the NOVX protein can be determined either directly or indirectly as described above.
  • the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to modulate (e.g.
  • Determining the ability ofthe test compound to modulate the activity of NOVX can be accomplished, for example, by dete ⁇ nining the ability ofthe NOVX protein to bind to an NOVX target molecule by one ofthe methods described above for determining direct binding.
  • determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability ofthe NOVX protein further modulate an NOVX target molecule.
  • the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described, supra.
  • the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe NOVX protein to preferentially bind to or modulate the activity of an NOVX target molecule.
  • the cell-free assays ofthe invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein.
  • 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-l 14, 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-l-propane sulfonate (CHAPSO).
  • binding of a test compound to NOVX protein, or interaction of NOVX protein 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, h one embodiment, a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • GST-NO VX 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 NOVX 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, supra.
  • glutathione sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized microtiter plates glutathione derivatized microtiter plates
  • the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays ofthe invention.
  • either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within 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 NOVX protein or target molecules can be derivatized to the wells ofthe plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
  • modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence o the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression.
  • the candidate compound when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression.
  • the level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
  • the NONX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al, 1993. Cell 72: 223-232; Madura, et al, 1993. J. Biol. Chem.
  • NOVX-binding proteins proteins that bind to or interact with NOVX
  • NOVX-binding proteins proteins that bind to or interact with NOVX
  • NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements ofthe NOVX 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 NOVX 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 ofthe known transcription factor. If the "bait” and the "prey” proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity.
  • reporter gene e.g., LacZ
  • expression ofthe 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 NOVX.
  • the invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents.
  • these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample.
  • this sequence can be used to map the location ofthe gene on a chromosome.
  • This process is called chromosome mapping.
  • portions or fragments ofthe NOVX sequences, SEQ ID NOS: 1 or 3, or fragments or derivatives thereof, can be used to map the location ofthe NOVX genes, respectively, on a chromosome.
  • the mapping ofthe NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease. Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
  • 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 NOVX 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 camiot 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.
  • mammals e.g., human and mouse cells.
  • 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 NOVX sequences to design oligonucleotide primers, sub- localization can be achieved with panels of fragments from specific chromosomes.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase cliromosomal 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 cliromosomal 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 ofthe genes actually are preferred for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping. Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data.
  • a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe 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 D ⁇ A sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymo ⁇ hisms.
  • the ⁇ OVX sequences ofthe invention can also be used to identify individuals from minute biological samples.
  • an individual's genomic D ⁇ A is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • the sequences ofthe invention are useful as additional D ⁇ A markers for RFLP ("restriction fragment length polymo ⁇ hisms," described in U.S. Patent No. 5,272,057).
  • the sequences ofthe 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 NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini ofthe 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 ofthe invention can be used to obtain such identification sequences from individuals and from tissue.
  • the NOVX sequences ofthe invention uniquely represent portions ofthe 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 ofthe allelic variation is due to single nucleotide polymo ⁇ hisms (SNPs), which include restriction fragment length polymo ⁇ hisms (RFLPs).
  • SNPs single nucleotide polymo ⁇ hisms
  • RFLPs restriction fragment length polymo ⁇ hisms
  • each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses. Because greater numbers of polymo ⁇ hisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences 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. If predicted coding sequences, such as those in SEQ ID NOS: 1 or 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • the 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) pu ⁇ oses to thereby treat an individual prophylactically.
  • diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX 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 NOVX expression or activity.
  • the disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample.
  • Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
  • Another aspect ofthe invention provides methods for determining NOVX protein, nucleic acid expression or 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 ofthe individual (e.g. , the genotype ofthe individual examined to determine the ability ofthe individual to respond to a particular agent.)
  • Yet another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of NOVX 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 NOVX protein or nucleic acid (e.g. , mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample.
  • a compound or an agent capable of detecting NOVX protein or nucleic acid e.g. , mRNA, genomic DNA
  • An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS: 1 or 3, 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 NOVX mRNA or genomic DNA.
  • a full-length NOVX nucleic acid such as the nucleic acid of SEQ ID NOS: 1 or 3
  • 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 NOVX mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays ofthe invention are described herein.
  • An agent for detecting NOVX protein is an antibody capable of binding to NOVX 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 ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently- labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method ofthe invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX 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 NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of NOVX in a biological sample can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX 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 NOVX 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 NOVX 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 NOVX protein, nucleic acid expression or activity.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
  • the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX 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 NOVX expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder.
  • the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g. , wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).
  • the methods ofthe invention can also be used to detect genetic lesions in an NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression ofthe NOVX gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) aberrant modification of an NOVX gene, such as ofthe methylation pattern ofthe genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non- wild-type level of an NOVX protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein.
  • 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 ofthe lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al, 1988. Science 241 : 1077-1080; and Nakazawa, et al, 1994. Proc. Natl. Acad. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al, 1995. Nucl.
  • 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 ofthe sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification ofthe NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification ofthe NOVX gene (if present) occurs
  • detecting the presence or absence of an amplification product or detecting the size ofthe amplification product and comparing the length to a control sample.
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations
  • mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, e.g., U.S. Patent No. 5,493,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in NOVX 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. See, e.g., Cronin, et al, 1996. Human Mutation 1: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759.
  • genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al, supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This 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 NOVX gene and detect mutations by comparing the sequence ofthe sample NOVX with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al, 1995.
  • Biotechniques 19: 448 including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl. Biochem. Biotechnol. 38: 147-159).
  • Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al, 1985. Science 230: 1242.
  • mismatch cleavage starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent that cleaves single-stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • 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 ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol. 217: 286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX 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. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662.
  • a probe based on an NOVX sequence e.g., a wild-type NOVX sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in NOVX genes.
  • single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al, 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX 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 ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence, hi one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 1: 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).
  • 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. See, e.g., Rosenbaum and Reissner, 1981. 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. See, e.g., 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 ofthe molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238).
  • amplification may also be performed using Taq ligase for amplification. See, e.g., 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'-terminus ofthe 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing 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 NONX gene.
  • any cell type or tissue preferably peripheral blood leukocytes, in which ⁇ ONX 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 ⁇ ONX activity can be administered to individuals to treat (prophylactically or therapeutically) disorders
  • disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
  • the pharmacogenomics i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drag
  • the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NONX protein, expression of ⁇ ONX nucleic acid, or mutation content of ⁇ ONX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.
  • G6PD glucose-6-phosphate dehydrogenase
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drag action.
  • drug metabolizing enzymes e.g., ⁇ -acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 ⁇ -acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymo ⁇ hisms 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 polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drag 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 mo ⁇ hine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses.
  • the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of NONX protein, expression of ⁇ ONX nucleic acid, or mutation content of ⁇ ONX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype.
  • Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of ⁇ ONX can be applied not only in basic drag screening, but also in clinical trials.
  • agents e.g., drugs, compounds
  • the effectiveness of an agent determined by a screening assay as described herein to increase ⁇ ONX gene expression, protein levels, or upregulate ⁇ OVX activity can be monitored in clinical trails of subjects exhibiting decreased ⁇ OVX gene expression, protein levels, or downregulated ⁇ OVX activity.
  • the effectiveness of an agent determined by a screening assay to decrease ⁇ OVX gene expression, protein levels, or downregulate ⁇ OVX activity can be monitored in clinical trails of subjects exhibiting increased ⁇ OVX gene expression, protein levels, or upregulated ⁇ OVX activity.
  • the expression or activity of ⁇ OVX 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 ofthe immune responsiveness of a particular cell.
  • genes including ⁇ OVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates ⁇ OVX activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • ⁇ OVX activity e.g., identified in a screening assay as described herein
  • cells can be isolated and R ⁇ A prepared and analyzed for the levels of expression of ⁇ OVX and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of NOVX or other genes, h this manner, the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state maybe determined before, and at various points during, treatment ofthe individual with the agent.
  • the 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 drag candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of an NOVX protein, mRNA, or genomic DNA in the preadmmistration sample; (iii) obtaining one or more post- administration samples from the subject; (iv) detecting the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration ofthe agent to the subject accordingly.
  • an agent e
  • increased administration ofthe agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness ofthe agent.
  • decreased administration ofthe agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness ofthe agent.
  • the 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 NOVX expression or activity.
  • the disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn
  • 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; (iii) 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.
  • modulators i.e., inhibitors, agonists and antagonists, including additional peptide mimetic ofthe invention or antibodies specific to a peptide ofthe invention
  • modulators i.e., inhibitors, agonists and antagonists, including additional peptide mimetic ofthe invention or antibodies specific to a peptide ofthe invention
  • Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity.
  • Therapeutics that upregulate 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 ofthe 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 ofthe 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, and the like).
  • 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, and the like).
  • the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity.
  • Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an NOVX agonist or NOVX antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein. The prophylactic methods ofthe invention are further discussed in the following subsections.
  • the modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of NOVX protein activity associated with the cell.
  • An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of an NOVX protein, a peptide, an NOVX peptidomimetic, or other small molecule, hi one embodiment, the agent stimulates one or more NOVX protein activity.
  • stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell.
  • the agent inhibits one or more NOVX protein activity.
  • inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies.
  • the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an NOVX protein or nucleic acid molecule, hi one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering an NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • agents that modulates e.g., up-regulates or down-regulates
  • Stimulation of NOVX activity is desirable in stt ⁇ ations in which NOVX is abnormally downregulated and/or in which increased NOVX 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 ofthe 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.
  • suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
  • any ofthe animal model system known in the art may be used prior to administration to human subjects.
  • the NOVX nucleic acids and proteins ofthe invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
  • a cDNA encoding the NOVX protein ofthe invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof.
  • compositions ofthe invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias.
  • Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein ofthe invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • a further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties).
  • These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • Example 1 Quantitative expression analysis of clones in various cells and tissues The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on a Perkin- Elmer Biosystems ABI PRISM® 7700 Sequence Detection System.
  • Panel 1 containing cells and cell lines from normal and cancer sources
  • Panel 2 containing samples derived from tissues, in particular from surgical samples, from normal and cancer sources
  • Panel 3 containing samples derived from a wide variety of cancer sources
  • Panel 4 containing cells and cell lines from normal cells and cells related to inflammatory conditions
  • Panel CNSD.01 containing samples from normal and diseased brains.
  • the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, ⁇ -actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (PE Biosystems; Catalog No.
  • probes and primers selected were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200nM.
  • PCR conditions Normalized RNA from each tissue and each cell line was spotted in each well of a 96 well PCR plate (Perkin Elmer Biosystems). PCR cocktails including two probes (a probe specific for the target clone and another gene-specific probe multiplexed with the target probe) were set up using IX TaqManTM PCR Master Mix for the PE Biosystems 7700, with 5 mM MgCl2, dNTPs (dA, G, C, U at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq GoldTM (PE Biosystems), and 0.4 U/ ⁇ l RNase inhibitor, and 0.25 U/ ⁇ l reverse transcriptase.
  • IX TaqManTM PCR Master Mix for the PE Biosystems 7700, with 5 mM MgCl2, dNTPs (dA, G, C, U at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq GoldTM (PE Biosystems), and 0.4 U/ ⁇ l RNase inhibitor,
  • Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cycles as follows: 95° C IO min, then 40 cycles of 95° C for 15 seconds, 60° C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.
  • the plates for Panel 2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI).
  • CHTN National Cancer Institute's Cooperative Human Tissue Network
  • NDRI National Disease Research Initiative
  • the tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins" obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted “NAT” in the results below.
  • the tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologists at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade.
  • RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen.
  • RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2: 1 to 2.5 : 1 28s: 18s) and the absence of low molecular weight RNAs that would be indicative of degradation products.
  • Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
  • PANEL 3D The plates of Panel 3D are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls.
  • the human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma ofthe tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines, hi addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures.
  • the cell lines in panel 3D and 1.3D are ofthe most common cell lines used in the scientific literature.
  • RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s: 18s) and the absence of low molecular weight RNAs that would be indicative of degradation products.
  • Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
  • Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4r) or cDNA (Panel 4d) isolated from various human cell lines or tissues related to inflammatory conditions.
  • RNA RNA from control normal tissues such as colon and lung (Stratagene ,La Jolla, CA) and thymus and kidney (Clontech) were employed.
  • Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, hie, Hayward, CA).
  • Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).
  • Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated.
  • cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1 %> serum.
  • Mononuclear cells were prepared from blood of employees at CuraGen Co ⁇ oration, using Ficoll.
  • LAK cells were prepared from these cells by culture in DMEM 5%> FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) and hiterleukin 2 for 4-6 days.
  • Cells were then either activated with 10-20 ng/ml PMA and 1-2 ⁇ g/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours.
  • mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 ⁇ g/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation.
  • FCS Hyclone
  • PHA phytohemagglutinin
  • PWM pokeweed mitogen
  • MLR mixed lymphocyte reaction
  • Monocytes were isolated from mononuclear cells using CD 14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5%> fetal calf serum (FCS) (Hyclone, Logan, UT), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days.
  • FCS fetal calf serum
  • Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10 '5 M (Gibco), 10 mM Hepes (Gibco) and 10%) AB Human Serum or MCSF at approximately 50 ng/ml.
  • Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 ⁇ g/ml for 6 and 12-14 hours.
  • LPS lipopolysaccharide
  • Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 ⁇ g/ml for 6 and 12-14 hours
  • CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions.
  • CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. Then CD45RO beads were used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes.
  • CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) and plated at 10 6 cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 ⁇ g/ml anti-CD28 (Pharmingen) and 3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation.
  • CD8 lymphocytes To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5%> FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days ofthe second expansion culture.
  • the isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
  • tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 10 6 cells/ml in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco). To activate the cells, we used PWM at 5 ⁇ g/ml or anti-CD40 (Pharmingen) at approximately 10 ⁇ g/ml and IL-4 at 5-10 ng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.
  • German Town, MD were cultured at 10 -10 cells/ml in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10 " 5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 ng/ml).
  • IL-12 (5 ng/ml) and anti-IL4 (1 Dg/ml) were used to direct to Thl
  • IL-4 (5 ng/ml) and anti-IFN gamma (1 Dg/ml) were used to direct to Th2
  • IL-10 at 5 ng/ml was used to direct to Trl.
  • Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml).
  • Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti- CD95L (1 Dg/ml) to prevent apoptosis.
  • the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles.
  • RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in hiterleukin 2.
  • leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5 xlO 5 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 xlO 5 cells/ml.
  • EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5 xlO 5 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 xlO 5 cells/ml.
  • DMEM or RPMI as recommended by the ATCC
  • RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 ⁇ g/ml for 6 and 14 hours.
  • Kefatinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco).
  • CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.
  • RNA was prepared by lysing approximately 10 7 cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Co ⁇ oration) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at -20 degrees C overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol.
  • Trizol Trizol
  • bromochloropropane Molecular Research Co ⁇ oration
  • the plates for Panel CNSD.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
  • the panel contains two brains from each ofthe following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy, Depression, and "Normal controls". Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex).
  • Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases.
  • Parkinson's disease is characterized by degeneration ofthe substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.
  • RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products.
  • Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
  • the labels employed to identify tissues in the CNS panel the following abbreviations are used:
  • PSP Progressive supranuclear palsy
  • NOV2 gene expression In addition to the high NOV2 gene expression seen in cerebellum, this gene is also more moderately expressed in other CNS tissues including amygdala, hippocampus, substantia nigra, thalamus, hypothalamus and spinal cord.
  • the NOV2 gene shows homology to BIG-2, an axon-associated cell adhesion molecule (AxCAM) (1).
  • AxCAMs are critical for the development and maintenance of neural networks within the brain. In the response to injury and/or neuronal death, gene expression during the process of compensatory synaptogenesis in many ways mirrors that seen during development.
  • the therapeutic expression ofthe NOV2 gene or its protein product may be beneficial in the treatment of CNS injury (stroke, head trauma, spinal cord injury) or neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, ALS, or any disease resulting in neuronal atrophy or death).
  • CNS injury stroke, head trauma, spinal cord injury
  • neurodegenerative diseases Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, ALS, or any disease resulting in neuronal atrophy or death.
  • this gene product may have a role in cell-cell communication in these tissues and thus be an antibody target for the treatment of diseases involving any or all of these tissues.
  • Panel 2.2 Summary: Ag3231 Expression ofthe NOV2 gene is highest in a sample derived from a kidney cancer (CT 32.1), although the overall levels of expression are low. In addition, there is significant expression detected in samples derived from two breast cancer metastases and normal stomach. Overall this pattern of expression, suggests that the NOV2 gene might be useful in distinguishing kidney, metastatic breast cancer and stomach from other tissues, h addition, therapeutic modulation ofthe function ofthe NOV2 gene product, through the use of antibodies or small molecule drags, might be of use in the treatment of metastatic breast cancer or kidney cancer.
  • Agl454/Agl465 Expression ofthe NOV2 gene is low/undetectable (CT values > 35) across all ofthe samples on this panel (data not shown).
  • NOV2 gene is expressed at low levels in normal thymus, lung, kidney and colon (CTs - 31-32). Interestingly, there is lowere expression in IBD colitis and Crohns disease samples as well as in lupus kidney, suggesting that this gene may play a role in these diseases. Thus, the NOV2 gene may be used to distinguish normal kidney from lupus kidney as well as normal colon from colon affected by IBD or Crohns disease, h addition, the NOV2 gene is expressed in an untreated eosinophil (EOL) cell line; however, EOL cells treated with PMA and ionomycin express this gene at much lower levels.
  • EOL eosinophil
  • the NOV2 gene encodes a protein that is related to BIG2, a neural adhesion molecule. Transcript expression is detected primarily in untreated tissues and is down regulated upon inflammation. Based on the the function of BIG2 as an adhesion and signaling molecule, the NOV2 protein may be important in the devlopment of normal organ structure and on the normal trafficking of eosinophils from the bone marrow into peripheral tissues. Therapies using the protein encoded by this transcript may therefore be important in reducing inflammation or in wound healing; similar therapies using other adhesion molecules which encourge neurite outgrowth have been proposed (2). Agl454/Agl465 Expression ofthe NOV2 gene is low/undetectable (CT values > 35) across all ofthe samples on this panel (data not shown).

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Abstract

L'invention concerne des séquences d'acide nucléique codant pour de nouveaux polypeptides. L'invention concerne également des polypeptides codés par ces séquences d'acide nucléique, et des anticorps, qui se lient de manière immunospécifique à ce polypeptide, ainsi que des dérivés, des variants, des mutants, ou des fragments du polypeptide, du polynucléotide ou de l'anticorps susmentionnés. L'invention concerne également des procédés thérapeutiques, de diagnostic et de recherche, pour le diagnostic, le traitement et la prévention de troubles impliquant un de ces nouveaux acides nucléiques et protéines humains.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1740167A2 (fr) * 2004-03-29 2007-01-10 The Feinstein Institute for Medical Research Traitement du diabete de type 1 a l'aide d'inhibiteurs du facteur d'inhibition de la migration des macrophages
EP1740167A4 (fr) * 2004-03-29 2008-12-10 The Feinstein Inst Medical Res Traitement du diabete de type 1 a l'aide d'inhibiteurs du facteur d'inhibition de la migration des macrophages

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