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WO2001051632A9 - Nouveaux polypeptides et acides nucleiques codant pour ceux-ci - Google Patents

Nouveaux polypeptides et acides nucleiques codant pour ceux-ci

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Publication number
WO2001051632A9
WO2001051632A9 PCT/US2001/001513 US0101513W WO0151632A9 WO 2001051632 A9 WO2001051632 A9 WO 2001051632A9 US 0101513 W US0101513 W US 0101513W WO 0151632 A9 WO0151632 A9 WO 0151632A9
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WO
WIPO (PCT)
Prior art keywords
ofthe
polypeptide
nucleic acid
seq
novx
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PCT/US2001/001513
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English (en)
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WO2001051632A3 (fr
WO2001051632A2 (fr
Inventor
Muralidhar Padigaru
Sudhirdas K Prayaga
Raymond J Taupier Jr
Vishnu Mishra
Velizar T Tchernev
Kimberley A Spytek
Li Li
Original Assignee
Curagen Corp
Muralidhar Padigaru
Sudhirdas K Prayaga
Raymond J Taupier Jr
Vishnu Mishra
Velizar T Tchernev
Kimberley A Spytek
Li Li
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Application filed by Curagen Corp, Muralidhar Padigaru, Sudhirdas K Prayaga, Raymond J Taupier Jr, Vishnu Mishra, Velizar T Tchernev, Kimberley A Spytek, Li Li filed Critical Curagen Corp
Priority to AU2001227925A priority Critical patent/AU2001227925A1/en
Publication of WO2001051632A2 publication Critical patent/WO2001051632A2/fr
Publication of WO2001051632A3 publication Critical patent/WO2001051632A3/fr
Publication of WO2001051632A9 publication Critical patent/WO2001051632A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention generally relates to nucleic acids and polypeptides encoded therefrom.
  • odor detection is a universal tool used for social interaction, predation, and reproduction.
  • Chemosensitivity in vertebrates is modulated by bipolar sensory neurons located in the olfactory epithelium, which extend a single, highly arborized dendrite into the mucosa while projecting axons to relay neurons within the olfactory bulb.
  • the many ciliae on the neurons bear odorant (or olfactory) receptors (ORs), which cause depolarization and formation of action potentials upon contact with specific odorants.
  • ORs may also function as axonal guidance molecules, a necessary function as the sensory neurons are normally renewed continuously through adulthood by underlying populations of basal cells.
  • Odorant receptors are believed to be encoded by an extremely large subfamily of G protein-coupled receptors. These receptors share a 7-transmembrane domain structure with many neurotransmitter and hormone receptors and are likely to underlie the recognition and G-protein- mediated transduction of odorant signals and possibly other chemosensing responses as well.
  • the genes encoding these receptors are devoid of introns within their coding regions.
  • Schurmans and co-workers cloned a member of this family of genes, OLFR1, from a genomic library by cross-hybridization with a gene fragment obtained by PCR. See Schurmans et al, Cyto genet. Cell Genet.. 1993, 63(3):200. By isotopic in situ hybridization, they mapped the gene to 17pl3- pl2 with a peak at band 17pl3. A minor peak was detected on chromosome 3, with a maximum in the region 3ql3-q21. After Mspl digestion, a restriction fragment length polymorphism (RFLP) was demonstrated.
  • RFLP restriction fragment length polymorphism
  • the OLFR genes in the cluster belong to 4 different gene subfamilies, displaying as much sequence variability as any randomly selected group of OLFRs. This suggested that the cluster may be one of several copies of an ancestral OLFR gene repertoire whose existence may have predated the divergence of mammals. Localization to 17pl3.3 was performed by fluorescence in situ hybridization as well as by somatic cell hybrid mapping.
  • OR genes cloned in different species were from disparate locations in the respective genomes.
  • the human OR genes on the other hand, lack introns and may be segregated into four different gene subfamilies, displaying great sequence variability. These genes are primarily expressed in olfactory epithelium, but may be found in other chemoresponsive cells and tissues as well.
  • OL2 polymerase chain reaction
  • PCR analysis reveals that the transcript is present mainly in the rat spleen and in a mouse insulin- secreting cell line (MIN6). No correlation was found between the tissue distribution of OL2 and that ofthe olfaction-related GTP-binding protein Golf alpha subunit.
  • Olfactory loss may be induced by trauma or by neoplastic growths in the olfactory neuroepithelium. There is currently no treatment available that effectively restores olfaction in the case of sensorineural olfactory losses. See Harrison's Principles of Internal Medicine, 14 th Ed., Fauci, AS et al. (eds.), McGraw-Hill, New York, 1998, 173. There thus remains a need for effective treatment to restore olfaction in pathologies related to neural olfactory loss.
  • the invention is based, in part, upon the discovery of novel polynucleotide sequences encoding novel polypeptides.
  • the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can include, e.g. , a nucleic acid sequence encoding a polypeptide at least 85% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
  • Also included in the invention is a vector containing one or more ofthe nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
  • the invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
  • the invention includes a pharmaceutical composition that includes a NOVX nucleic acid and a pharmaceutically acceptable carrier or diluent.
  • the invention includes a substantially purified NOVX polypeptide, e.g., any ofthe NOVX polypeptides encoded by an NONX nucleic acid, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition that includes an ⁇ ONX polypeptide and a pharmaceutically acceptable carrier or diluent.
  • the invention provides an antibody that binds specifically to an
  • the antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition including ⁇ ONX antibody and a pharmaceutically acceptable carrier or diluent.
  • the invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any ofthe nucleic acid molecules described above.
  • the invention also includes kits comprising any ofthe pharmaceutical compositions described above.
  • the invention further provides a method for producing an NONX polypeptide by providing a cell containing an ⁇ ONX nucleic acid, e.g., a vector that includes an ⁇ OVX nucleic acid, and culturing the cell under conditions sufficient to express the ⁇ ONX polypeptide encoded by the nucleic acid.
  • the expressed ⁇ ONX polypeptide is then recovered from the cell.
  • the cell produces little or no endogenous ⁇ ONX polypeptide.
  • the cell can be, e.g., a prokaryotic cell or eukaryotic cell.
  • the invention is also directed to methods of identifying an ⁇ ONX polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present.
  • the invention further provides methods of identifying a compound that modulates the activity of an ⁇ ONX polypeptide by contacting an ⁇ ONX polypeptide with a compound and determining whether the ⁇ ONX polypeptide activity is modified.
  • the invention is also directed to compounds that modulate ⁇ ONX polypeptide activity identified by contacting an ⁇ OVX polypeptide with the compound and determining whether the compound modifies activity ofthe ⁇ ONX polypeptide, binds to the ⁇ OVX polypeptide, or binds to a nucleic acid molecule encoding an ⁇ OVX polypeptide.
  • the invention provides a method of determining the presence of or predisposition of an ⁇ OVX-associated disorder in a subject.
  • the method includes providing a sample from the subject and measuring the amount of ⁇ OVX polypeptide in the subject sample.
  • the amount of ⁇ OVX polypeptide in the subject sample is then compared to the amount of ⁇ OVX polypeptide in a control sample.
  • An alteration in the amount of ⁇ OVX polypeptide in the subject protein sample relative to the amount of ⁇ OVX polypeptide in the control protein sample indicates the subject has a tissue proliferation-associated condition.
  • a control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having a tissue proliferation-associated condition.
  • the control sample may be taken from the subject at a time when the subject is not suspected of having a tissue proliferation-associated disorder.
  • the ⁇ OVX is detected using an ⁇ OVX antibody.
  • the invention provides a method of determining the presence of or predisposition of an ⁇ OVX-associated disorder in a subject.
  • the method includes providing a nucleic acid sample, e.g., R ⁇ A or D ⁇ A, or both, from the subject and measuring the amount of the ⁇ OVX nucleic acid in the subject nucleic acid sample.
  • the amount of ⁇ OVX nucleic acid sample in the subject nucleic acid is then compared to the amount of an NOVX nucleic acid in a control sample.
  • An alteration in the amount of NOVX nucleic acid in the sample relative to the amount of NOVX in the control sample indicates the subject has a NOVX-associated disorder.
  • the invention provides a method of treating or preventing or delaying an NOVX-associated disorder.
  • the method includes administering to a subject in which such treatment or prevention or delay is desired an NOVX nucleic acid, an NOVX polypeptide, or an NOVX antibody in an amount sufficient to treat, prevent, or delay a NOVX- associated disorder in the subject.
  • Olfactory receptors are the largest family of G-protein-coupled receptors (GPCRs) and belong to the first family (Class A) of GPCRs, along with catecholamine receptors and opsins.
  • GPCRs G-protein-coupled receptors
  • Class A the first family of GPCRs
  • the OR family contains over 1,000 members that traverse the phylogenetic spectrum from C. elegans to mammals. ORs most likely emerged from prototypic GPCRs several times independently, extending the structural diversity necessary both within and between species in order to differentiate the multitude of ligands. Individual olfactory sensory neurons are predicted to express a single, or at most a few, ORs.
  • ORs are believed to contain seven ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus.
  • the pocket of OR ligand binding is expected to be between the second and sixth transmembrane domains ofthe proteins.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%, and genes greater than 80% identical to one another at the amino acid level are considered to belong to the same subfamily.
  • ORs Since the first ORs were cloned in 1991, outstanding progress has been made into their mechanisms of action and potential dysregulation during disease and disorder. It is understood that some human diseases result from rare mutations within GPCRs. Drug discovery avenues could be used to produce highly specific compounds on the basis of minute structural differences of OR subtypes, which are now being appreciated with in vivo manipulation of OR levels in transgenic and knock-out animals. Furthermore, due to the intracellular homogeneity and ligand specificity of ORs, renewal of specific odorant-sensing neurons lost in disease or disorder is possible by the introduction of individual ORs into basal cells. Additionally, new therapeutic strategies may be elucidated by further study of so-called orphan receptors, whose ligand(s) remain to be discovered.
  • OR proteins bind odorant ligands and transmit a G-protein-mediated intracellular signal, resulting in generation of an action potential.
  • the accumulation of DNA sequences of hundreds of OR genes provides an opportunity to predict features related to their structure, function and evolutionary diversification. See Pilpel Y, etal., Essays Biochem 1998;33:93-104.
  • the OR repertoire has evolved a variable ligand-binding site that ascertains recognition of multiple odorants, coupled to constant regions that mediate the cAMP-mediated signal transduction.
  • the cellular second messenger underlies the responses to diverse odorants through the direct gating of olfactory-specific cation channels.
  • ORs odorant receptors
  • ORs are expressed by nasal olfactory sensory neurons, and each neuron expresses only 1 allele of a single OR gene.
  • different sets of ORs are expressed in distinct spatial zones. Neurons that express the same OR gene are located in the same zone; however, in that zone they are randomly interspersed with neurons expressing other ORs.
  • the cell chooses an OR gene for expression, it may be restricted to a specific zonal gene set, but it may select from that set by a stochastic mechanism.
  • Proposed models of OR gene choice fall into 2 classes: locus-dependent and locus-independent.
  • Locus-dependent models posit that OR genes are clustered in the genome, perhaps with members of different zonal gene sets clustered at distinct loci. In contrast, locus-independent models do not require that OR genes be clustered.
  • OR genes have been mapped to 11 different regions on 7 chromosomes. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Studies have shown that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones.
  • Issel-Tarver and Rine characterized 4 members ofthe canine olfactory receptor gene family.
  • the 4 subfamilies comprised genes expressed exclusively in olfactory epithelium.
  • Analysis of large DNA fragments using Southern blots of pulsed field gels indicated that subfamily members were clustered together, and that two ofthe subfamilies were closely linked in the dog genome.
  • Analysis ofthe four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.
  • Issel-Tarver and Rine performed a comparative study of four subfamilies of olfactory receptor genes first identified in the dog to assess changes in the gene family during mammalian evolution, and to begin linking the dog genetic map to that of humans.
  • These four families were designated by them OLFl, OLF2, OLF3, and OLF4 in the canine genome.
  • the subfamilies represented by these four genes range in size from 2 to 20 genes. They are all expressed in canine olfactory epithelium but were not detectably expressed in canine lung, liver, ovary, spleen, testis, or tongue.
  • the OLFl and OLF2 subfamilies are tightly linked in the dog genome and also in the human genome.
  • the smallest family is represented by the canine OLFl gene.
  • dog gene probes individually to hybridize to Southern blots of genomic DNA from 24 somatic cell hybrid lines. They showed that the human homologous OLFl subfamily maps to human chromosome 11. The human gene with the strongest similarity to the canine OLF2 gene also mapped to chromosome 11. Both members ofthe human subfamily that hybridized to canine OLF3 were located on chromosome 7. It was difficult to determine to which chromosome or chromosomes the human genes that hybridized to the canine OLF4 probe mapped. This subfamily is large in mouse and hamster as well as human, so the rodent background largely obscured the human cross-hybridizing bands.
  • Issel-Tarver and Rine demonstrated that in the human OLFl and OLF2 homologs are likewise closely linked.
  • Issel-Tarver and Rine found that the human OLF3 homolog maps to 7q35.
  • a chromosome 19-specific cosmid library was screened by hybridization with the canine OLF4 gene probe, and clones that hybridized strongly to the probe even at high stringency were localized to 19p 13.1 and 19pl3.2. These clones accounted, however, for a small fraction ofthe homologous human bands.
  • Rouquier et al. demonstrated that members ofthe olfactory receptor gene family are distributed on all but a few human chromosomes. Through fluorescence in situ hybridization analysis, they showed that OR sequences reside at more than 25 locations in the human genome. Their distribution was biased for terminal bands of chromosome arms. Flow-sorted chromosomes were used to isolate 87 OR sequences derived from 16 chromosomes. Their sequence relationships indicated the inter- and intrachromosomal duplications responsible for OR family expansion. Rouquier et al. (1998) determined that the human genome has accumulated a striking number of dysfunctional copies: 72% of these sequences were found to be pseudogenes. ORF-containing sequences predominate on chromosomes 7, 16, and 17.
  • telomere a subtelomeric DNA duplication that provided insight into the variability, complexity, and evolutionary history of that unusual region ofthe human genome, the telomere.
  • ORs are likely G protein-coupled receptors, which characteristically are 7-transmembrane proteins.
  • ORs are likely members of a multigene family of considerable size, because an immense number of chemicals with vastly different structures can be detected and discriminated by the vertebrate olfactory system.
  • ORs are likely expressed selectively in olfactory sensory neurons. Ben-Arie et al. (1994) focused attention on a cluster of human OR genes on 17p, to which the first human OR gene, OR1D2, had been mapped by Schurmans et al. (1993). According to Mombaerts (1999), the sequences of more than 150 human OR clones had been reported.
  • the human OR genes differ markedly from their counterparts in other species by their high frequency of pseudogenes, except the testicular OR genes. Research showed that individual olfactory sensory neurons express a small subset ofthe OR repertoire. In rat and mouse, axons of neurons expressing the same OR converge onto defined glomeruli in the olfactory bulb.
  • the present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their polypeptides. The sequences are collectively referred to as “NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any ofthe novel sequences disclosed herein. Table 1 provides a summary ofthe NOVX nucleic acids and their encoded polypeptides. Example 1 provides a description of how the novel nucleic acids were identified.
  • OR GPCR is an odorant receptor ofthe G-protein coupled-receptor family.
  • NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
  • the various NOVX 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, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members ofthe family to which the NOVX polypeptides belong.
  • NOVl-10 are homologous to members ofthe odorant receptor (OR) family ofthe human G-protein coupled receptor (GPCR) superfamily of proteins, as shown in Table 56.
  • OR odorant receptor
  • GPCR human G-protein coupled receptor
  • the NOVl-10 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in disorders of olfactory loss, e.g., trauma, HIV illness, neoplastic growth and neurological disorders e.g. Parkinson's disease and Alzheimer's disease.
  • the NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function.
  • 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 motility, cell proliferation and angiogenesis. Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein. NOV1
  • a NO VI sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV1 nucleic acid and its encoded polypeptide includes the sequences shown in Table 2.
  • the disclosed nucleic acid (SEQ ID NO:l) is 1,071 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 42-44 and ends with a TAA stop codon at nucleotides 1,053-1,055.
  • the representative ORF encodes a 337 amino acid polypeptide (SEQ ID NO:2). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 1.
  • the NOVl nucleic acid sequence has homology with several fragments ofthe human olfactory receptor 17-93 (OLFR) (GenBank Accession No.: HSU76377), as shown in Table 3. Also, the NOVl polypeptide has homology (approximately 61% identity, 74% similarity) to human olfactory receptor, family 1, subfamily F, member 8 (OLFR) (GenBank Accession No.: XP007973), as is shown in Table 4. Furthermore, the NOVl polypeptide has homology (approximately 61% identity, 75% similarity) to a human olfactory protein (OLFR)(EMBL Accession No.: 043749), as is shown in Table 5.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence augment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOVl is predicted to have a seven transmembrane region and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288), as is shown in Table 6.
  • GPCR dopamine
  • NOVl 1034 GGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCAT 1093
  • OLFR 41260 GAGGTCAGTTGTTCGAGACCAACCTGGTCAAC 41291 (SEQ ID NO. 37)
  • NOVl 1032 CCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATC 1091
  • OLFR 1 CTGGGCTCGGTGGCTCACACGTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATC 60 NOVl: 1092 A--TGAGGTCAGGAGATCGAGACCATCCTGGCTAAC 1125 (SEQ ID NO. 41)
  • OLFR 61 ACATGAGGTCAGGAGTTCGAGACCAGCCTGGTCAAC 96 (SEQ ID NO. 47)
  • OLFR 4688 GTTAGCCAGGATGGTCTCAATCTCCTGACCTCGTGATCCGCCTGCCTTGGCCTCCCAAAG 4747 NOVl: 1065 TGCTGGGATTACAGGCGTGAGCCACCGCGCCCGG 1032 (SEQ ID NO. 48) OLFR: 4748 TGCTGGGATTACAGGCATGAGCCACTGCGCCCGG 4781 (SEQ ID NO. 52)
  • NOVl 1 MSGTNQSSVSEFLLLGLSRQPQQQHLLFVFFLSMYLATVLGNLLIILSVSIDSCLHTPMY 60 * * ** 4 .. .******* * *** ** _
  • OLFR 1 MEGKNQTNISEFLLLGFSS QQQQVLLFALFLCLYLTGLFGNLLILLAIGSDHCLHTPMY 60
  • OLFR 121 DRYVAICHPLHYSTIMALRLCASLVAAPWVIAILNPLLHTLi AHLHFCSDNVIHHFFCD 180
  • NOVl 181 VTPLLKLSCSDTHLNEVIILSEGALVMITPFLCILASYMHITCTVLKVPSTKGRWKAFST 240 . ** ****** **++ 4.*+ *4- 4. * 4.*** **4- * * ⁇ .**** +*+ *****
  • OLFR 181 INSLLPLSCSDTSLNQLSVLATVGLIFWPSVCILVSYILIVSAVMKVPSAQGKLKAFST 240 NOVl: 241 CGSHLAVVLLFYSTIIAVYFNPLSSHSAEKDT ATVLYTVVTP LNPFIYSLRNRYLKGA 300
  • NOVl 1 ⁇ GKNQTNISEFLLLGFSS QQQQVLLFALFLCLYLTGLFGNLLILLAIGSDHCLHTPMY 60 * * **++ 4 -******* * *** *** ** +** + *****+*-
  • OLFR 61 FFLSNLSFVDICFSFTTVPK LANHILETQTISFCGCLTQMYFVFMFVD DNFLLAVMAY 120 NOVl: 121 DRYVAICHPLHYSTIIVLALRLCASLVAAP VIAILNPLLHTLJMMAHLHFCSDNVIHHFFCD 180
  • OLFR 121 DHFVAVCHPLHYTAKMTHQLCALLVAGLWWANLNVLLHTLLMAPLSFCADNAITHFFCD 180
  • OLFR 181 VTPLLKLSCSDTHLNEVIILSEGALVMITPFLCILASYi HITCTVLKVPSTKGRWKAFST 240
  • NOVl 241 CGSHLALVILFYGAITGVYMSPLSNHSTEKDSAASVIFMVVAPVLNPFIYSLRNNELKG 299
  • NOVl 108 ANro FLLT MAYDRY/AICHPLHYS IM- LRLCASLVAAPWVIAILNP LH MAHL 166
  • NOVl can be used to detect nasal epithelial neuronal tissue.
  • NOVl Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing. NOV2
  • a NOV2 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV2 nucleic acid and its encoded polypeptide includes the sequences shown in Table 7.
  • the disclosed nucleic acid (SEQ ID NO:3) is 1,040 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 82-84 and ends with a TGA stop codon at nucleotides 1,012-1,014.
  • the representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:4). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 3.
  • NOV2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • a NOV2 nucleic acid was identified on human chromosome 6.
  • the NOV2 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 6 (CHR6) (GenBank Accession No.: AL135904), as shown in Table 8. Additionally, the NOV2 polypeptide has a high degree of homology (approximately 95% identity) to a human olfactory receptor (OLFR) (GenBank Accession No.: AL135904), as shown in Table 9. Furthermore, the NOV2 polypeptide has a high degree of homology (approximately 91% identity) to a human olfactory protein (OLFR) (EMBL Accession No.: AC005587), as shown in Table 10.
  • OR proteins have seven transmembrane -helices separated by three extracellular and three cytoplasmic loops, along with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence augment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV2 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 11.
  • GPCR dopamine
  • N0V2 241 ctgcacgcccccatgtacttcttcctctcacacctggcggtcgtcgacatcgcctacgcc 300 CHR6 : 22339 ctgcacgcccccatgtacttcttcctctcacacctggcggtcgtcgacatcgcctacgcc 22280
  • N0V2 301 tgcaacacggtgccccggatgctggtgaacctcctgcatccagccaagcccatctccttt 360
  • N0V2 361 gcgggccgcatgatgcagacctttctgttttccacttttgctgtcacagaatgtctcctc 420
  • N0V2 421 ctggtggtgatgtcctatgatctgtacgtggccatctgccaccccctcgatatttggcc 480
  • N0V2 481 atcatgacctggagagtctgcatcaccctcgcggtgacttcctggaccactggagtcctt 540
  • CHR6 22099 atcatgacctggagagtctgcatcaccctcgcggtgacttcctggaccactggagtcctt 22040
  • N0V2 541 ttatccttgattcatcttgtgttacttctacctttacccttctgtaggccccagaaaatt 600
  • N0V2 601 tatcacniiiinnnngtgaaatcttggctgttctcaaacttgcctgtgcagatacccacatc 660
  • N0V2 661 aatgagaacatggtcttggccggagcaatttctgggctggtgggacccttgtccacaatt 720
  • N0V2 721 gtagtttcatatatgtgcatcctctgtgctatccttcagatccaatcaagggaagttcag 780
  • NOV2 901 ctgctgtttcacag ⁇ ctctttaatcccatgctcaatccccttatctgtagtcttaggaac 960 5 CHR6 : 21679 ctgctgtttcacagcctctttaatcccatgctcaatccccttatctgtagtcttaggaac 21620
  • N0V2 961 tcagaagtgaagaatactttgaagagagtgctgggagtagaaagggctttatgaaagga 1020 0 CHR6 : 21619 tcagaagtgaagaatactttgaagagagtgctgggagtagaaagggctttatgaaagga 21560 NOV2: 1021 ttatggcattgtgactgaca 1040 (SEQ ID NO. 3)
  • OLFR 73 CLLLWMSYDLYVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRP 132 5 NOV2 : 171 QKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSY CILCAILQIQSR 230
  • OLFR 133 QKIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSR 192 NOV2 : 231 EVQRKAFRTCFSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICS 290 (_) *** * * * ****** * **** **** *********** ** * * * ***** *
  • OLFR 193 EVQRKAFRTCFSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICS 252 NOV2: 291 LRNSEVKNTLKRVLGVERAL 310 (SEQ ID NO. 35)
  • NOV2 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFXXXXXXXXXXXXXXXXXXDSRLHAPMYF 60 0******************************************
  • OLFR 1 MGDNITSIR ⁇ FLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOV2: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRiyiMQTFLFSTFAVTECLLLVVMSYD 120*******************************************
  • OLFR 121 LYVAICHPLRYLAI T RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
  • NOV2 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIVVSYMCILCAILQIQSREVQRKAFRTC 240************************************************
  • OLFR 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOV2: 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
  • the OR family ofthe GPCR superfamily is involved in the initial steps ofthe olfactory signal transduction cascade. Therefore, the NOV2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV2 can be used to provide new diagnostic and/or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are also useful in the treatment of a variety of diseases and pathologies, including but not limited to, those involving neurogenesis, cancer, and wound healing.
  • NOV3 is also useful in the treatment of a variety of diseases and pathologies, including but not limited to, those involving neurogenesis, cancer, and wound healing.
  • a NOV3 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV3 nucleic acid and its encoded polypeptide includes the sequences shown in Table 12.
  • the disclosed nucleic acid (SEQ ID NO:5) is 1,090 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAA stop codon at nucleotides 1,061-1,063.
  • the representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 6). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 5.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • a NOV3 nucleic acid was identified on human chromosome 1.
  • the NOV3 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 1 (CHR1) (GenBank Accession No.:AL121986), as is shown in Table 13.
  • NOV3 polypeptide has homology (approximately 50% identity, 70% similarity) to a human olfactory receptor (OLFR) (GenBank Accession No.: F20722), as is shown in Table 14.
  • OLFR human olfactory receptor
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus.
  • NOV3 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 15.
  • GPCR dopamine
  • NOV3 1 aagaagttcttcagatgcgaggtttcaacaaaccactgtggttacacagttcatcctgg 60
  • N0V3 121 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 180
  • NOV3 241 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccctca 300 M l
  • NOV3 481 ttgctttggtggccaccaacctcatttgtgacatgcgtttttgtggccccaacagggtta 540
  • N0V3 601 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 660
  • NOV3 661 tcatatcctatggcttcatagtcaacaccatcctgaagatcccctcagctgagggcaaga 720 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
  • NOV3 781 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 840
  • N0V3 901 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 960
  • N0V3 961 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 1020
  • NOV3 1021 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 1080
  • N0V3 1081 tattttctcc 1090 (SEQ ID NO. 5) CHRl: 144815 tattttctcc 144806 (SEQ ID NO. 42)
  • NOV3 1 MRGFNKTTWTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTIMAVIRFS TLHTPM 59
  • OLFR 60 YLFLCVLSVSEILYTVAIIPRMLADLLSTQRSIAFLACASQMFFSFSFGFTHSFLLTVMG 119
  • OLFR 120 YDRYVAICHPLRYNVLMSPRGCACLVGCS AGGSVMGMWTSAIFQLTFCGSHEIQHFLC 179 NOV3: 180 DMAPVIKLAC-TDTHVKELALFSLSILVI VPFLLILISYGFIVNTILKIPSAEGK-KAF 239
  • OLFR 180 HVPPLLKLACGNNVPAVALGVGLVCIMALLGCFLLILLSYAFIVADILKIPSA ⁇ GRNKAF 239 NOV3: 240 VTCASHLTWFVHYGCASIIYLRPKSKSASDKDQLVAVTYTWTPLLNPLVYSLRNKEVK 299 ****** ** **** **_***.j_** . . * * ⁇ .* ** *-j-** *_j.* ⁇ .. ⁇ .****** ⁇ .*
  • OLFR 240 STCASHLIWIVHYGFASVIYLKPKGPHSQEGDTLMATTYAVLTPFLSPIIFSLRNKELK 299 NOV3: 300 TALKR 304 (SEQ ID NO. 43)
  • N0V3 222 IVNTILKI 229 (SEQ ID NO . 45 )
  • GPCR 162 IYIVLRRR 169 (SEQ ID NO . 46 )
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, in one embodiment, the NOV3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. ' Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV3 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV4 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • the NOV3 nucleic acid sequence (SEQ ID NO.: 5) was further analyzed by exon linking and the resulting sequence was identified as NOV4.
  • a NOV4 nucleic acid and its encoded polypeptide includes the sequences shown in Table 16.
  • the disclosed nucleic acid (SEQ ID NO:7) is 1,090 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 15-17 and ends with a TAA stop codon at nucleotides 1,061-1,063.
  • the representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 8). Putative untranslated regions upstream and downstream ofthe coding sequence are underlined in SEQ ID NO: 7. TABLE 16.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV4 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • a NOV4 nucleic acid was identified on human chromosome 1.
  • the NOV4 nucleic acid sequence has a high degree of homology (99% identity) with a human genomic clone corresponding to chromosome 1 (CHRl) (GenBank Accession No.:AL121986), as is shown in Table 17.
  • the NOV4 nucleic acid sequence also has a high degree of homology with the NOV3 sequence (99% identity), as is shown in Table 18.
  • the NOV3 polypeptide has homology (approximately 53% identity, 71% similarity) to the human olfactory receptor 10J1 (OLFR) (GenBank Accession No.: P30954), as is shown in Table 19.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV4 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 20.
  • GPCR dopamine
  • NOV4 1 aagaagttcttcagatgcgaggtttcaacaaaccactgtggttacacagttcatcctgg 60
  • NOV4 61 tgggtttctccagcctgggggagct ⁇ cagctgctactttttgtcatctttcttctcctat 120
  • NOV4 121 acttgacaatcctggtggccaatgtgaccatcatggccgttattcgcttcagctggactc 180
  • NOV4 241 ttgtcatcatccctcagctgctggtccacctgctctcagacaccaagaccatctccctca 300
  • N0V4 541 accactatttctgtgacatggcacctgttatcaagttagcctgcactgacacccatgtga 600
  • N0V4 601 aagagctggctttatttagcctcagcatcctggtaattatggtgccttttctgttaattc 660
  • N0V4 661 tcatatcctatggcttcatagtcaacaccatcctgaagatcccctcagctgagggcaaga 720
  • N0V4 721 aggcctttgtcacctgtgcctcacatctcactgtggtctttgtccactatgactgtgcct 780 i I i 111111 M 11 ! 11 M 11 M ; ! I i ' 111 M 111111 i 111 i 11 ; i 11 IIIHI
  • N0V4 781 ctatcatctatctgcggcccaagtccaagtctgcctcagacaaggatcagttggtggcag 840
  • N0V4 841 tgacctacgcagtggttactcccttacttaatcctcttgtctacagtctgaggaacaag 900
  • N0V4 901 aggtaaaaactgcattgaaaagagttcttggaatgcctgtggcaaccaagatgagctaac 960
  • N0V4 961 aaaaaataataataaaattaactaggatagtcacagaagaaatcaaaggcataaaatttt 1020 llll II IIIMIII II II lllll III lllll lllll II I
  • N0V4 1021 ctgacctttaatgcatgtctcagacagtgtttccaaggattaagactactcttgcctttt 1080
  • N0V4 1081 tattttctcc 1090 (SEQ ID NO. 4)
  • NOV3 121 ACTTGACAATCCTGGTGGCCAATGTGACCATCATGGCCGTTATTCGCTTCAGCTGGACTC 180 NOV4: 181 TCCACACTCCCATGTATGGCTTTCTATTCATCCTTTCATTTTCTGAGTCCTGCTACACTT 240
  • N0V3 241 TTGTCATCATCCCTCAGCTGCTGGTCCACCTGCTCTCAGACACCAAGACCATCTCCTTCA 300
  • NOV4 301 TGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCACCAACTGCCTCCTCA 360
  • N0V3 301 TGGCCTGTGCCACCCAGCTGTTCTTTTTCCTTGGCTTTGCTTGCACCAACTGCCTCCTCA 360
  • N0V4 361 TTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCTCTGAGGTACACACTCA 420
  • N0V3 361 TTGCTGTGATGGGATATGATCGCTATGTAGCAATTTGTCACCCTCTGAGGTACACACTCA 420
  • N0V4 421 TCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCAGGGGCCACAGGTTTCTTTA 480 1 1 1 1 1 1 1 1 I I 1 1 1 1 1 1 1 1 1
  • N0V3 421 TCATAAACAAAAGGCTGGGGTTGGAGTTGATTTCTCTCTCAGGAGCCACAGGTTTCTTTA 480
  • N0V4 481 TTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTTTTTGTGGCCCCAACAGGGTTA 540
  • N0V3 481 TTGCTTTGGTGGCCACCAACCTCATTTGTGACATGCGTTTTTGTGGCCCCAACAGGGTTA 540
  • N0V4 541 ACCACTATTTCTGTGACATGGCACCTGTTATCAAGTTAGCCTGCACTGACACCCATGTGA 600
  • N0V3 541 ACCACTATTTCTGTGACATGGCACCTGTTATCAAGTTAGCCTGCACTGACACCCATGTGA 600
  • N0V4 601 AAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGTAATTATGGTGCCTTTTCTGTTAATTC 660
  • N0V3 601 AAGAGCTGGCTTTATTTAGCCTCAGCATCCTGGTAATTATGGTGCCTTTTCTGTTAATTC 660
  • N0V4 661 TCATATCCTATGGCTTCATAGTCAACACCATCCTGAAGATCCCCTCAGCTGAGGGCAAGA 720 11 II 111111111111 II 1111111111111111111
  • N0V3 661 TCATATCCTATGGCTTCATAGTTAACACCATCCTGAAGATCCCCTCAGCTGAGGGCAAGA 720 N0V4 : 721 AGGCCTTTGTCACCTGTGCCTCACATCTCACTGTGGTCTTTGTCCACTATGACTGTGCCT 780 M I N I M
  • N0V3 721 AGGCCTTTGTCACCTGTGCCTCACATCTCACTGTGGTCTTTGTCCACTATGGCTGTGCCT 780
  • N0V4 781 CTATCATCTATCTGCGGCCCAAGTCCAAGTCTGCCTCAGACAAGGATCAGTTGGTGGCAG 840
  • N0V3 781 CTATCATCTATCTGCGGCCCAAGTCCAAGTCTGCCTCAGACAAGGATCAGTTGGTGGCAG 840
  • N0V4 841 TGACCTACGCAGTGGTTACTCCCTTACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAG 900
  • N0V3 841 TGACCTACACAGTGGTTACTCCCTTACTTAATCCTCTTGTCTACAGTCTGAGGAACAAAG 900
  • N0V4 901 AGGTAAAAACTGCATTGAAAAGAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAAC 960
  • N0V3 901 AGGTAAAAACTGCATTGAAAAGAGTTCTTGGAATGCCTGTGGCAACCAAGATGAGCTAAC 960 NOV4 : 961 AAAAAATAATAATAAAATTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTT 1020 lllll 11111111 II 111111111 M 111 ! I )! 1111111 N 1111111 lllll
  • NOV3 961 AAAAAATAATAATAAAATTAACTAGGATAGTCACAGAAGAAATCAAAGGCATAAAATTTT 1020
  • NOV4 1021 CTGACCTTTAATGCATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTT 1080
  • NOV3 1021 CTGACCTTTAATGCATGTCTCAGACAGTGTTTCCAAGGATTAAGACTACTCTTGCCTTTTTT 1080
  • NOV4 1081 TATTTTCTCC 1090 (SEQ ID NO. 7)
  • NOV3 1081 TATTTTCTCC 1090 (SEQ ID NO. 5)
  • NOV4 18 TLITDFVFQGFSSFHEQQITLFGVFLALYILTLAGNIIIVTIIRIDLHLHTPMYFFLSML 77 *4-4.* * - **** * * * _ ** 4.** ** * * *4- * _ 4** ****** ** 4,* OLFR: 8 TWTQFILVGFSSLGELQLLLFVIFLLLYLTILVANVTI AVIRFSWTLHTPMYGFLFIL 67
  • NOV4 78 STSETVYTLVILPRMLSSLVGMSQP SLAGCATQMFFFVTFGITNCFLLTAMGYDRYVAI 137 * * *_ ** ** -*_ j ._ * *+ 4.4. 4.* * **** *** . * *** * -*******
  • OLFR 68 SFSESCYTFVIIPQLLVHLLSDTKTISLMACATQLFFFLGFACTNCLLIAVMGYDRYVAI 127
  • NOV4 138 CNPLRYMVIJNKRLRIQLVLGACSIGLIVAITQVTSVFRLPFCA-RKVPHFFCDIRPVMK 196 *4**** 4_* **** 44*4. 4 4 * 4*+ 4 4- ** 4-* *4-***4- **4*
  • OLFR 128 CHPLRYTLIINKRLGLELISLSGATGFFIALVATNLICDMRFCGPNRVNHYFCDMAPVIK 187 NOV4: 197 LSCIDTTVNEXXXXXXXXXXXPMGLVFISYVLIISTILKIASVEGRKKAFATCASHLT 256
  • OLFR 188 LACTDTHVKELALFSLSILVIMVPFLLILISYGFIVNTILKIPSAEG-KKAFVTCASHLT 246
  • N0V4 103 FFFLGFACTNCLLIAVMGYDRYVAICHPLRYTLIIN-KRLGLELISLSGATGFFIALVAT 161
  • NOV4 162 NLICDMRFCGPNRVNHYFCDMAPVIKLACTDTHVKELALFSLSILVIMVPFLLILISYGF 221 GPCR: 122 FGLNNTDQNEC IIANPAFWYSSIVSFYVPFIVTLLVYIK 161
  • NOV4 222 IVNTILKI 229 (SEQ ID NO. 51)
  • GPCR 162 IYIVLRRR 169 (SEQ ID NO. 46)
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV4 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV4 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV4 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in treating and/or diagnosing a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV5 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV5 nucleic acid and its encoded polypeptide includes the sequences shown in Table 21.
  • the disclosed nucleic acid (SEQ ID NO: 9) is 822 nucleotides in length and contains an open reading frame (ORF) that begins at nucleotide 6 and ends with a TGA stop codon at nucleotides 800-802.
  • ORF open reading frame
  • C indicates 'G' to 'C substitutions in the sequence to correct stop codons.
  • a representative ORF encodes a 265 amino acid polypeptide (SEQ ID NO: 10).
  • a putative untranslated region downstream ofthe coding sequence is underlined in SEQ ID NO: 9.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV5 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV5 nucleic acid sequence has a high degree of homology (94% identity) with a human genomic clone cotaining an OR pseudogene (OLFR) (GenBank Accession No.:AF065864), as is shown in Table 22.
  • the NOV5 polypeptide has homology (approximately 67% identity, 79% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:043789), as is shown in Table 23.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV5 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 24.
  • GPCR dopamine
  • OLFR 196 GCCACGGTTCCTAAGATGATTGTGGACATGCAGTCTCATACCAGAGTCATCTCTTATGAG 255
  • OLFR 256 GGCTGCCTGACACGGATATCTTTCTTGGTCCTTTTTGCATGTATAGAAGACATGCTCCTG 315
  • OLFR 316 ACTGTGATGGCCTATGACTGCTTTGTAGCCATCTGTCGCCCTCTGCACTACCCAGTCATC 375
  • NOV5 241 GTGAATCCTCACCTCTGTGTCTTCTTCGTCTTGGTGTCCTTTTTCCTTAGCCCGTTGGAT 300
  • OLFR 376 GTGAATCCTCACCTCTGTGTCTTCTTCCTTTTGGTATACTTTTTCCTTAGCTTGTTGGAT 435
  • NOV5 301 TCCCAGCTGCACAGTTGGATTGTGTTACTATTCACCATCATCAAGAATGTGGAAATCACT 360
  • OLFR 436 TCCCAGCTGCACAGTTGGATTGTGTTACAATTCACCATCATCAAGAATGTGGAAATCTCT 495
  • NOV5 421 AACATATTCATATATTTCGATAGTACTATGTTTGGTTTTCTTCCCATTTCAGGGATCCTT 480
  • NOV5 481 TTGTCTTACTATAAAATTGTCCCCTCCATTCTAAGGATGTCATCGTCAGATGGGAAGTAT 540
  • NOV5 721 GATATACAAAGTGTCCTGCGGAGGCTGTGCAGCAGAACAGTCGAATCTCATGATATGTTC 780
  • OLFR 916 CATCCTTT 923 (SEQ ID NO. 54)
  • OLFR 1 PiMYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSV 60 NOV5: 187 MAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHS IVLLFTIIKNVEITNF 366 **** ***** **** 4.* ⁇ .** ** * _ j _*4-***4-* ****** ⁇ _ *_ j _* * *4>*4.* .**
  • OLFR 61 MAYDRFVAICHPLHYRIIMNPRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNF 120
  • NOV5 367 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 546 *4-*****4-* ***4_ ** 4. *** 4.******* ****** *** - 4.******
  • OLFR 121 FCDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKA 180
  • NOV5 547 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASV YAWTPMLNLFIYSLGKRDI 726 ******_ ⁇ .***** * ***4 > **_ ⁇ _*** * ** 4.*********** ***** 4.**
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • the NOV5 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV5 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV5 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV6 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV6 nucleic acid and its encoded polypeptide includes the sequences shown in Table 25.
  • the disclosed nucleic acid (SEQ ID NO:l 1) is 930 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 22-24 and ends with a TAA stop codon at nucleotides 907-909.
  • ORF open reading frame
  • C indicates 'G' to 'C substitutions in the sequence to correct stop codons.
  • the representative ORF encodes a 294 amino acid polypeptide (SEQ ID NO: 12). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 11.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • the NOV6 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV6 nucleic acid sequence has a high degree of homology (94% identity) with a human genomic clone cotaining an OR pseudogene (OLFR) (GenBank Accession No.:AF065864), as is shown in Table 26.
  • the NOV6 polypeptide has homology (approximately 67% identity, 79% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:043789), as is shown in Table 27.
  • NOV6 polypeptide also has a high degree of homology (99% identity) with the NOV5 polypeptide.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • NOV5 and NOV6 belong to the same OR subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus.
  • NOV6 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 29.
  • GPCR dopamine
  • NOV6 10 ctgtccctgtccatgtatatggtcacggtgctgaggaacctgctcagcatcctggctgtc 69
  • NOV6 70 agctctgactccccgctccacacccccatgtgcttcttcctctccaaactgtgctcagct 129 OLFR: 118 agctctgacccccacctccacacccccatgtgcttcttcctctccaacctgtgctgggct 177 NOV6 : 130 gacatcggtttcaccttggccatggttcccaagatgattgtgaacatgcagtcgcatagc 189
  • NOV6 190 agagtcatctcttatgagggctgcctgacacggatgtctttctttgtcctttttgcatgt 249 OLFR: 238 agagtcatctcttatgagggctgcctgacacggatatctttcttggtcctttttgcatgt 297
  • N0V6 250 atggaagacatgctcctgactgtgatggcctatgactgctttgtagccat ⁇ tgtcgccct 309
  • N0V6 310 ctgcactacccagtcatcgtgaatcctcacctctgtgtcttcttcgtcttggtgtccttt 369
  • NOV6 370 ttccttagcccgttggattcccagctgcacagttggattgtgttactattcaccatcatc 429 OLFR: 418 ttccttagcttgttggattcccagctgcacagttggattgtgttacaattcaccatcatc 477
  • NOV6 430 aagaatgtggaaatcactaattttgtctgtgaaccctctcaacttctcaaccttgcttgt 489
  • NOV6 550 cccatttcagggatccttttgtcttactataaaattgtccct ⁇ cattctaaggatgtca 609
  • NOV6 610 tcgtcagatgggaagtataaaggcttctcacctgtggct ⁇ ttacctggcagttgtttgc 669
  • NOV6 670 tcatttgatggaacaggcattggcatgtacctgacttcagctgtgtcaccaccccccagg 729 OLFR: 718 tgattttatggaacaggcattggcgtgtacctgacttcagctgtgtcaccaccccccagg 777
  • NOV6 7 P CFFLSKLCSADIGFTI-AMVPK IVNMQSHSRVISYEGCLTRMSFFVLFACMEDMLLTV 186 ** **** * ****** ******4** +********** +********4**** + *
  • NOV6 187 MAYDCFVAICRPLHYPVIWPHLCVFFVLVSFFLSPLDSQLHSWIVLLFTIIKJ EITNF 366 **** ***** **** 4*4** ** * 4*4***4* ****** + * * * * * + *4* + **
  • OLFR 61 MAYDRFVAICHPLHYRIIM PRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNF 120
  • NOV6 367 VCEPSQ LNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 546 *4*****4* *** + ** *** 4******* ****** ***4 + ******
  • OLFR 121 FCDPSQLLHLRCSDTFINEMVIYFMGAIFGCLPISGILFSYYKIVSPILRVPTSDGKYKA 180
  • NOV6 547 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASVMYAWTPMLNLFIYSLGKRDI 726
  • NOV6 85 MAYDCFVAICRPLHYPVIVNPHLCXXXXXXXXXXXXXQLHS IVLLFTIIKNVEITNF 144*****************************************************
  • NOV5 61 MAYDCFVAICRPLHYPVIVNPHLCVFFVLVSFFLSPLDSQLHS IVLLFTIIKNVEITNF 120
  • NOV6 145 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 204
  • NOV5 121 VCEPSQLLNLACSDSVINNIFIYFDSTMFGFLPISGILLSYYKIVPSILRMSSSDGKYKG 180
  • NOV6 205 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNG-VASV YAWTPMLNLFILSLGKRDI 263
  • NOV5 181 FSTCGSYLAWCSFDGTGIGMYLTSAVSPPPRNGWASVMYAWTPMLNLFIYSLGKRDI 240
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV6 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV6 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV6 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV7 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV7 nucleic acid and its encoded polypeptide includes the sequences shown in Table 30.
  • the disclosed nucleic acid (SEQ ID NO: 13) is 930 nucleotides in length and contains an open reading frame (ORF) that begins with an ACG initiation codon at nucleotides 10-12 and ends with a TGA stop codon at nucleotides 882-884.
  • ORF open reading frame
  • C indicates 'G' to 'C substitutions in the sequence to correct stop codons.
  • the representative ORF encodes a 309 amino acid polypeptide (SEQ ID NO: 12). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 13
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • the NOV7 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV7 nucleic acid sequence has a high degree of homology (94% identity) with the human genomic clone pDJ392al7 from chromosome 11 (CHRl 1) (GenBank Accession No.:AC000385), as is shown in Table 31.
  • the NOV7 polypeptide has homology (approximately 68% identity, 78% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No..-043789), as is shown in Table 32.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV7 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 33.
  • GPCR dopamine
  • NOV7 1 cacagagccacggaatctcacaggtgtctcagaattcctcctgggactctcagagga 60
  • NOV7 241 tcccaagatgattgtggacatgcagtggtatagcagagtcatctctcatgcgggctgcct 300
  • N0V7 661 ctataaaattgtcccctccattctaaggatgtcatcgtcagatgggaagtataaaacttt 720
  • N0V7 781 gtacctggcttcagctatgtcaccaacccccaggaatggtgtggtggtgtcagtgatgta 840 MMMMIMMMMMM 111111 ; 1111
  • N0V7 841 agctgtggtcacccccatgctgaaccttttcatctacagcctgagaaacagggacataca 900
  • N0V7 179 PTYFFLSILCWADIGFTSATVPKMIVDMQWYSRVISHAGCLTQMSFLVLFACIEGMLLTV 358 * ***** ***********4*****4****** OLFR: 1 PMYFFLSNLSLADIGFTSTTVPKMIVDMQTHSRVISYEGCLTQMSFFVLFACMDDMLLSV 60 NOV7: 359 MAYDCFVGIYRPLHYPVIVNPHLCVFFVLVSFFLSLLDSQLHSWIVLQFTIIKNVEISNF 538 **** ** * * * * * ** _ j _* ** ** * 4*4***4********4.
  • the OR family ofthe GPCR superfamily is a group of related proteins that are specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV7 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV7 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV7 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV8 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV8 nucleic acid and its encoded polypeptide includes the sequences shown in Table 34.
  • the disclosed nucleic acid (SEQ ID NO: 15) is 994 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 27-29 and ends with a TGA stop codon at nucleotides 969-971.
  • the representative ORF encodes a 314 amino acid polypeptide (SEQ ID NO: 16). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 15.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade.
  • NOV8 nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV8 polypeptide has homology (approximately 44% identity, 65% similarity) to the human olfactory receptor family 2 subfamily F, member 1 (OLFR) (EMBL Accession No.:NP 036501), as is shown in Table 35.
  • the NOV8 polypeptide also has homology (44% identity, 65% similarity) to the rat olfactor receptor-like protein OLF3 (SwissProt Accession No.: Q13607), as is shown in Table 36.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV8 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 37.
  • GPCR dopamine
  • N0V8 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60 ** **4- *4***4-** * 4.* ** * ** .*4- 4*** 4.4.4- ***4*********
  • OLFR 1 MGTDNQT VSEFILLGLSSDWDTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
  • NOV8 61 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 120 * * 4 - *** * 4 - 4 .*-_ 4 . ** * 4 . ***.** _ ⁇ .* * *** 4 -* 4 _*
  • OLFR 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGIEFVLLAVMAY 120
  • OLFR 121 DRYVAVCDALRYSAIMHGGLCARLAITS VSGFISSPVQTAITFQLPMCRNKFIDHISCE 180
  • OLFR 181 LLAWRLACVDTSSNEVTIMVSSIVLLMTPLCLVLLSYIQIISTILKIQSREGRKKAFHT 240 NOV8: 241 CSSHITVVGLFYGAAVFMYMVPCAYHSPQQDNVVSLFYSLVTPTLNPLIYSLRNPEV MA 300
  • NOV8 27 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 206 ** ** _ * _*** ⁇ .** * _* ** * **4-* - ⁇ .*** +4-4- ***4-********* OLFR: 1 MGTDNQT VS ⁇ FILLGLSSD DTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
  • NOV8 207 FLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQIFFLTLMGVAEGVLLVLMSY 386 * *4_ *** *4. 4.*4.4- 4. ** * 4. ***4-** 4.* * *** -* -*
  • OLFR 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGI ⁇ FVLLAVMAY 120
  • NOV8 41 GNTVLLFLIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQ 100 GPCR: 1 GNVLVCMAVSREKALQTTTIWLIVSIAVADLLVATLVMP VVYLEVVGE KFSRIHCDIF 60
  • the OR family ofthe GPCR superfamily is a group of related proteins located at the ciliated surface of olfactory sensory neurons in the nasal epithelium.
  • the OR family is involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV8 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV8 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV8 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOV9 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV9 nucleic acid and its encoded polypeptide includes the sequences shown in Table 38.
  • the NOV8 nucleic acid sequence (SEQ ID NO.: 15) was further analyzed by exon linking, and the resulting sequence was identified as NOV9.
  • the disclosed nucleic acid (SEQ ID NO: 17) is 994 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 28-30 and ends with a TAG stop codon at nucleotides 979-981.
  • the representative ORF encodes a 317 amino acid polypeptide (SEQ ID NO: 18). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 17.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV9 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV9 polypeptide has homology (approximately 44% identity, 65% similarity) to the human olfactory receptor family 2 subfamily F, member 1 (OLFR) (EMBL Accession No.:NP 036501), as is shown in Table 39.
  • the NOV9 polypeptide also has a high degree of homology (99% identity) to the NOV8 polypeptide as shown in Table 40.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. Thus NOVS andNOV9 belong to the same subfamily of ORs.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino-terminus and a cytoplasmic carboxy- terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOV9 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 41.
  • GPCR dopamine
  • OLFR 1 MGTDNQTWVSEFILLGLSSD DTRVSLFVLFLVMYWTVLGNCLIVLLIRLDSRLHTPMY 60
  • OLFR 61 FFLTNLSLVDVSYATSWPQLLAHFLAEHKAIPFQSCAAQLFFSLALGGIEFVLLAVMAY 120
  • OLFR 121 DRYVAVCDALRYSAIMHGGLCARLAITS VSGFISSPVQTAITFQLPMCRNKFIDHISCE 180
  • OLFR 181 LLAWRLACVDTSSNEVTIMVSSIVLLMTPLCLVLLSYIQIISTILKIQSREGRKKAFHT 240
  • NOV8 1 MGDVNQSVASDFILVGLFSHSGSRQLLFSLVAVMFVIGLLGNTVLLFLIRVDSRLHTPMY 60
  • N0V9 181 VPALLKLSCADTCAY ⁇ MALSTSGVLILMLPLSLIATSYGHVLQAVLSMRSE ⁇ ARHKAVTT 240*******************************************
  • NOV9 41 GNTVLLFLIRVDSRLHTPMYFLLSQLSLFDIGCPMVTIPKMASDFLRGEGATSYGGGAAQ 100 GPCR: 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMP WYL ⁇ WGE KFSRIHCDIF 60 NOV9: 101 IFFLTLMGVAEGVLLVLMSYDRYVAVCQPLQYPVLM-RRQVCLLMMGSSWWGVLNASIQ 159 GPCR: 61 VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM 120
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV9 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOV9 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV9 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing. NOV10
  • a NOV10 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOV10 nucleic acid and its encoded polypeptide includes the sequences shown in Table 42.
  • the disclosed nucleic acid (SEQ ID NO: 19) is 1,077 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 31-33 and ends with a TAG stop codon at nucleotides 1,030- 1,032.
  • the representative ORF encodes a 318 amino acid polypeptide (SEQ ID NO:20). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 19. Exon linking was used to confirm the sequence.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV10 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV10 polypeptide has homology (approximately 55% identity, 72% similarity) to the olfactory receptor MOR83 (OLFR) (EMBL Accession No. :BAA86125), as is shown in Table 43.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus.
  • NOV10 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 44.
  • GPCR dopamine
  • NOVIO 79 MNPANHSQVAGFVLLGLSQV ELRFVFFTVFSAVYFMTWGNLLIWIVTSDPHLHTTMY 258
  • OLFR 1 MGALNQTRVTEFIFLGLTDNWVLEILFFVPFTVTYMLTLLGNFLIWTIVFTPRLHNPMY 60 NOVIO: 259 FLLGNLSFLDFCYSSITAPRMLVDLLSGNPTISFGGCLTQLFFFHFIGGIKIFLLTVMAY 438
  • OLFR 61 FFLSNLSFIDICHSSVTVPKMLEGLLLERKTISFDNCIAQLFFLHLFACSEIFLLTIMAY 120
  • OLFR 121 DRYVAICIPLHYSNVMNMKVCVQLVFALWLGGTIHSLVQTFLTIRLPYCGPNIIDSYFCD 180
  • GPCR 1 GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWWYLEWGEWKFSRIHCDIF 60
  • NOVIO 101 LFFFHFIGGIKIFLLTVMAYDRYIAISQPLHYTLIMNQ-TVCALLMAAS VGGFIHSIVQ 159
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOV10 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue. Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOV10 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins. Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOVl 1 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • OR human odorant receptor
  • GPCR G-protein coupled receptor
  • a NOVl 1 nucleic acid was discovered by exon linking analysis of NOV2 (SEQ ID NO.: 3).
  • a NOVl 1 nucleic acid and its encoded polypeptide includes the sequences shown in Table 45.
  • the disclosed nucleic acid (SEQ ID NO:21) is 1,012 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 54-56 and ends with a TGA stop codon at nucleotides 984-986.
  • the representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:22). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 1 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOVl 1 polypeptide has a high degree of homology (approximately 99% identity) to a human olfactory receptor (OLFR) (EMBL Accession No.:095047), as is shown in Table 46.
  • the NOVl 1 polypeptide also has a high degree of homology (approximately 99% identity) to NOV2, as is shown in Table 47.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences. 1999, 20:413. Therefore, NOVl 1 and NOV2 are two members ofthe same OR subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus.
  • NOVl 1 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 48.
  • GPCR dopamine
  • OLFR 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60 NOVll: 61 FLSHLAVVDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120*******************************************
  • OLFR 61 FLSHLAV ⁇ iAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLVVMSYD 120 NOVll: 121 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFC ⁇ I 180
  • OLFR 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240 NOVll : 241 FSHLCVIGLFYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
  • NOV2 1 MGDNITSIR ⁇ FLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 60
  • NOV2 61 FLSHLAWDIAYACNTVPRMLVNLLHPAK ISFAGRMMQTFLFSTFAVTECLLLWMSYD 120 NOVll: 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
  • NOVll 113 LLWMSYDLYVAICHPLRYLAIMT -RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQ 171
  • GPCR 74 NLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPMLFGLNNTDQNE- - 131
  • NOVll 172 KIYHFFCEILAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSRE 231
  • GPCR 132 - CIIANPAF WYSSIVSFYVPFIVTLLVYIKIYIVLRRKRV 173
  • the OR family of the GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 1 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOVl 1 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 1 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOVl 2 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins. .
  • a NOVl 2 nucleic acid was discovered by exon linking analysis of NOV2 (SEQ ID NO.: 3).
  • a NOV12 nucleic acid and its encoded polypeptide includes the sequences shown in Table 49.
  • the disclosed nucleic acid (SEQ ID NO:23) is 1,014 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 55-57 and ends with a TGA stop codon at nucleotides 985-987.
  • the representative ORF encodes a 310 amino acid polypeptide (SEQ ID NO:24). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 23.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV12 polypeptide has a high degree of homology (approximately 99% identity) to a human olfactory receptor (OLFR) (EMBL Accession No.:095047), as is shown in Table 50.
  • the NOVl 2 polypeptide also has a high degree of homology (approximately 99% identity) to NOV2, as is shown in Table 51.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%. OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413. Therefore, NOVl 2 andNOV2 are two members ofthe same OR subfamily.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOVl 2 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 52.
  • GPCR dopamine
  • OLFR 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF
  • NOV12 61 FLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYD 120
  • OLFR 121 LYVAICHPLRYLAIMTWRVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 180
  • OLFR 181 LAVLKLACADTHINENMVLAGAISGLVGPLSTIWSYMCILCAILQIQSREVQRKAFRTC 240
  • OLFR 241 FSHLCVIGLVYGTAIIMYVGPRYGNPKEQKKYLLLFHSLFNPMLNPLICSLRNSEVKNTL 300
  • NOV12 113 LLWMSYDLYVAICHPLRYLAIMTW-RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQ 171
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 2 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOVl 2 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 2 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • a NOVl 3 sequence according to the invention is a nucleic acid sequence encoding a polypeptide related to the human odorant receptor (OR) family ofthe G-protein coupled receptor (GPCR) superfamily of proteins.
  • a NOVl 3 nucleic acid and its encoded polypeptide includes the sequences shown in Table 53.
  • the disclosed nucleic acid (SEQ ID NO:25) is 908 nucleotides in length and contains an open reading frame (ORF) that begins with an ATG initiation codon at nucleotides 75-77 and ends with a TAA stop codon at nucleotides 901-903.
  • the representative ORF encodes a 270 amino acid polypeptide (SEQ ID NO:26). Putative untranslated regions up- and downstream ofthe coding sequence are underlined in SEQ ID NO: 25.
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium and are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • the NOV13 polypeptide has homology (approximately 73% identity, 83% similarity) to a human olfactory receptor (OLFR) (EMBL Accession No.:Q9UPJl), as is shown in Table 54.
  • Overall amino acid sequence identity within the mammalian OR family ranges from 45% to >80%.
  • OR genes that are 80% or more identical to each other at the amino acid level are considered by convention to belong to the same subfamily. See Dryer and Berghard, Trends in Pharmacological Sciences, 1999, 20:413.
  • OR proteins have seven transmembrane ⁇ -helices separated by three extracellular and three cytoplasmic loops, with an extracellular amino- terminus and a cytoplasmic carboxy-terminus. Multiple sequence aligment suggests that the ligand-binding domain ofthe ORs is between the second and sixth transmembrane domains.
  • NOVl 3 is predicted to have a seven transmembrane region, and is similar in that region to a representative GPCR, e.g. dopamine (GPCR) (GenBank Accession No.: P20288) as is shown in Table 55.
  • GPCR dopamine
  • NOV12 1 MYFFLSNLC ADIGFTLATVPKMIVDMGSHSRVISYEGCLTQMSFFVLFACIEDMLLTVM 60****** ****************** 4.*****************44**** 4 **
  • NOV12 61 AYDQFVAICHPLHYPVIMNPHLCVFLVLVSFFLSLLDSQLHS IVLQFTFFKNVEISNFF 120 ***+********** 4 -**** ** ** 4 -* 4 _***+********+ *+** * **+*+*****
  • OLFR 61 AYDRFVAICHPLHYRIIMNPRLCGFLILLSFFISLLDSQLHNLIMLQLTCFKDVDISNFF 120 NOV12: 121 CDPSQLLNLACSDGIINSIFIYLDSILFSFLPISGILLSYYKIVPSILRISSSDGKYKAF 180
  • NOV13 1 MYFFLSNLC ADIGFTLATVPKMIVDMGSHSRVISY ⁇ GCLTQMSFFVLFACIEDMLLTVM 60 GPCR: 19 TNYLIVSLAVADLLVATLVMPWWYLEWGE KFSRIHCDIFVTLDVMMCTASILNLCAI 78
  • the OR family ofthe GPCR superfamily is a group of related proteins specifically located at the ciliated surface of olfactory sensory neurons in the nasal epithelium that are involved in the initial steps ofthe olfactory signal transduction cascade. Accordingly, the NOVl 3 nucleic acid, polypeptide, antibodies and other compositions ofthe present invention can be used to detect nasal epithelial neuronal tissue.
  • NOVl 3 Based on its relatedness to the known members ofthe OR family ofthe GPCR superfamily, NOVl 3 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of OR family-like proteins.
  • Nucleic acids, polypeptides, antibodies, and other compositions ofthe present invention are useful in the treatment and/or diagnosis of a variety of diseases and pathologies, including by way of nonlimiting example, those involving neurogenesis, cancer and wound healing.
  • Table 56 shows a multiple sequence alignment of NOVl -13 polypeptides with the known human olfactory receptor 10J1 (GenBank Accession No.: P30954), indicating the homology between the present invention and known members of a protein family.
  • OR_10J1 is the known human olfactory receptor 10J1 (GenBank Accession No.: P30954).
  • nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in disorders ofthe neuro-olfactory system, such as those induced by trauma, surgery and/or neoplastic disorders.
  • a cDNA encoding the olfactory receptor protein may be useful in gene therapy for treating such disorders, and the olfactory receptor 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 disorders ofthe neuro-olfactory system.
  • novel nucleic acids encoding olfactory receptor protein, and the olfactory receptor protein ofthe invention, or fragments thereof, may further be useful in the treatment of adenocarcinoma; lymphoma; prostate cancer; uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, treatment of Albright hereditary ostoeodystrophy, development of powerful assay system for functional analysis of various human disorders which will help in understanding of pathology of the disease, and development of new drug targets for various disorders. They may also be used 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.
  • nucleic acids ofthe invention include those that encode a NOVX polypeptide or protein.
  • polypeptide and protein are interchangeable.
  • a NOVX nucleic acid encodes a mature NOVX polypeptide.
  • a "mature" form of a polypeptide or protein described herein relates to 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 open reading frame described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the 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 open reading frame, 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.
  • NOVX nucleic acids is the nucleic acid whose sequence is provided in SEQ
  • the invention includes mutant or variant nucleic acids of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a fragment thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, while still encoding a protein that maintains at least one of its NOVX-like activities and physiological functions (i.e., modulating angiogenesis, neuronal development).
  • the invention further includes the complement ofthe nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, including fragments, derivatives, analogs and homologs thereof.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • nucleic acid molecules that encode NOVX proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of NOVX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs ofthe DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • Probes refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source ofthe nucleic acid.
  • isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated NOVX nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a complement of any of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • NOVX nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et ⁇ l., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ h, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John 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 NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA 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 cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a complement thereof Oligonucleotides may be chemically synthesized and may be used as probes.
  • an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a portion of this nucleotide sequence.
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, 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, Von der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • nucleic acid molecule ofthe invention can comprise only a portion ofthe nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of NOVX.
  • 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.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs 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 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.
  • 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 a NOVX polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human NOVX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, as well as a polypeptide having NOVX activity. Biological activities ofthe NOVX proteins are described below.
  • a homologous amino acid sequence does not encode the amino acid sequence of a human NOVX polypeptide.
  • the nucleotide sequence determined from the cloning ofthe human NOVX gene allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g., from other tissues, as well as NOVX homologues from other mammals.
  • the probe/primer typically comprises a 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 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; or an anti-sense strand nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; or of a naturally occurring mutant of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • Probes based on the human NOVX nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
  • polypeptide having a biologically active portion of NOVX refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically active portion of NOVX” can be prepared by isolating a portion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 that encodes a polypeptide having a NOVX biological activity (biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX.
  • a nucleic acid fragment encoding a biologically active portion of NOVX can optionally include an ATP-binding domain.
  • a nucleic acid fragment encoding a biologically active portion of NOVX includes one or more regions.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 due to the degeneracy ofthe genetic code.
  • These nucleic acids thus encode the same NOVX protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 e.g., the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of NOVX may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX gene may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a NOVX protein, preferably a mammalian NOVX protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe NOVX gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in NOVX that are the result of natural allelic variation and that do not alter the functional activity of NOVX are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding NOVX proteins from other species and thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • a soluble human NOVX cDNA can be isolated based on its homology to human membrane-bound NOVX.
  • a membrane-bound human NOVX cDNA can be isolated based on its homology to soluble human NOVX.
  • 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 NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length.
  • an isolated nucleic acid molecule ofthe invention hybridizes to the coding region.
  • 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 /. e., nucleic acids encoding NOVX 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 (T m ) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium.
  • 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 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 is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is 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 sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or fragments, analogs or derivatives thereof, under conditions of moderate stringency.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardf s solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, 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-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g. , as employed for cross-species hybridizations).
  • allelic variants ofthe NOVX sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, thereby leading to changes in the amino acid sequence ofthe encoded NOVX protein, without altering the functional ability ofthe NOVX protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of NOVX without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the NONX proteins ofthe present invention are predicted to be particularly unamenable to alteration.
  • nucleic acid molecules encoding ⁇ OVX proteins that contain changes in amino acid residues that are not essential for activity. Such ⁇ OVX proteins differ in amino acid sequence from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, 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 75% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8.
  • the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in NOVX is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • a mutant NOVX protein can be assayed for (1) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant NOVX protein and a NOVX receptor; (3) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind NOVX protein; or (5) the ability to specifically bind an anti-NOVX protein antibody.
  • 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 NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, 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 NOVX coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 are additionally provided.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding NOVX.
  • the term "coding region” refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human NOVX corresponds to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding NOVX.
  • 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). Given the coding strand sequences encoding NOVX disclosed herein (e.g., SEQ ID NO:
  • 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 NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis 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, N6-isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-me
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX 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 DNA 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 intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • 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 RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • NOVX Ribozymes and PNA moieties include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
  • 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 a mRNA, to which they have a complementary region.
  • ribozymes e. g. , hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)
  • a ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX DNA disclosed herein (i.e., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25).
  • a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in a NOVX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
  • NOVX mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418.
  • NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe NOVX (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe NOVX gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe NOVX e.g., the NOVX promoter and/or enhancers
  • the NOVX promoter and/or enhancers e.g., the NOVX promoter and/or enhancers
  • the nucleic acids of ⁇ OVX can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23).
  • the terms "peptide nucleic acids" or "P ⁇ As” refer to nucleic acid mimics, e.g., D ⁇ A mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs of NOVX can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g. , inducing transcription or translation arrest or inhibiting replication.
  • PNAs of NOVX can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
  • PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion ' would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
  • PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre etal, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre etal, 1987, Proc. Natl
  • oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • a NOVX polypeptide ofthe invention includes the NOVX-like protein whose sequence is provided in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 while still encoding a protein that maintains its NOVX-like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more ofthe residues may be so changed in the mutant or variant protein.
  • the NOVX polypeptide according to the invention is a mature polypeptide.
  • a NOVX -like variant that preserves NOVX-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 NOVX 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-NOVX antibodies.
  • native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • NOVX proteins are produced by recombinant DNA techniques.
  • a NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX 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 NOVX protein 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 NOVX protein having less than about 30% (by dry weight) of non-NOVX protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX protein, still more preferably less than about 10% of non-NOVX protein, and most preferably less than about 5% non-NOVX protein.
  • non-NOVX protein also referred to herein as a "contaminating protein”
  • NOVX protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX protein having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
  • Biologically active portions of a NOVX protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe NOVX protein, e.g., the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 that include fewer amino acids than the full length NOVX proteins, and exhibit at least one activity of a NOVX protein.
  • biologically active portions comprise a domain or motif with at least one activity ofthe NOVX protein.
  • a biologically active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a NOVX protein ofthe present invention may contain at least one ofthe above-identified domains conserved between the NOVX proteins, e.g. TSR modules. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native NOVX protein.
  • the NOVX protein has an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the NOVX protein is substantially homologous to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and retains the functional activity ofthe protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and retains the functional activity ofthe NOVX proteins of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either ofthe sequences being compared for optimal alignment between the sequences).
  • 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. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and 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 NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.
  • 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.
  • percentage of positive residues is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur 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 positive residues.
  • NOVX chimeric or fusion proteins As used herein, a NOVX "chimeric protein” or “fusion protein” comprises a NOVX polypeptide operatively linked to a non-NOVX polypeptide.
  • An "NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to NOVX
  • a non-NOVX polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism.
  • NOVX polypeptide can correspond to all or a portion of a NOVX protein.
  • a NOVX fusion protein comprises at least one biologically active portion of a NOVX protein.
  • a NOVX fusion protein comprises at least two biologically active portions of a NOVX protein.
  • the term "operatively linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame to each other.
  • the non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide.
  • a NOVX fusion protein comprises a NOVX polypeptide operably linked to the extracellular domain of a second protein.
  • fusion proteins can be further utilized in screening assays for compounds that modulate NOVX activity (such assays are described in detail below).
  • the fusion protein is a GST-NO VX fusion protein in which the NOVX sequences are fused to the C-terminus ofthe GST (i.e., glutathione S-transferase) sequences.
  • GST glutathione S-transferase
  • Such fusion proteins can facilitate the purification of recombinant NOVX.
  • the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences comprising one or more domains are fused to sequences derived from a member ofthe immunoglobulin protein family.
  • the NOVX-immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a NOVX ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo.
  • a contemplated NOVX ligand ofthe invention is the NOVX receptor.
  • the NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX cognate ligand. Inhibition ofthe NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival, as well as acute and chronic inflammatory disorders and hyperplastic wound healing, e.g. hypertrophic scars and keloids.
  • NOVX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand.
  • a NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
  • the present invention also pertains to variants ofthe NOVX proteins that function as either NOVX agonists (mimetics) or as NOVX antagonists.
  • Variants ofthe NOVX protein can be generated by mutagenesis, e.g., discrete point mutation or truncation ofthe NOVX protein.
  • An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein.
  • An antagonist ofthe NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX 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 ofthe NOVX proteins.
  • Variants ofthe NOVX protein that function as either NOVX agonists (mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, ofthe NOVX protein for NOVX protein agonist or antagonist activity.
  • a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
  • libraries of fragments ofthe NOVX protein coding sequence can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of a NOVX protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a 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 SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX protein.
  • Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of 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.
  • Recrusive 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 (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
  • 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 ab , and F (ab . )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 l5 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, such as an amino acid sequence shown in SEQ ID NO: 2, 4, 6 ,8 ,10, 12, 14, 16, 18, or 20, 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.
  • 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
  • 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 immunoaff ⁇ nity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • 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 purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown iv 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 ofthe 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-immunoglobulin 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.
  • 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.
  • 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 o the 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 In: 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. This approach is described, for example, in U.S. Patent Nos.
  • 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 incorporated, 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.
  • 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 ofthe 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 .
  • 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 of the 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.
  • 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. Additionally, 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') 2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • 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.
  • Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.
  • bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992).
  • 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.
  • the "diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
  • 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.
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al, J. Immunol. 152:5368 (1994).
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • 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. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • 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.
  • 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).
  • 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 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • 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 intemalization 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. Immunol., 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).
  • the invention also pertains to 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 aeruginosa), 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 mitogellin
  • restrictocin phenomycin, enomycin, and the tricothecenes.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, ]31 I, ,31 In, 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 l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl
  • 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
  • vectors preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • 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 purposes: (/) to increase expression of recombinant protein; (ii) to increase the solubility of the 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 Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, 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 ah, 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
  • 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 (Kaufman, et al, 1987. EMBOJ. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the recombinant mammalian expression vector is capable of directing expression 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 Gruss, 1990. Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule 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.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein.
  • 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 human, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

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Abstract

La présente invention concerne de nouveaux polynucléotides NOVX isolés et des polypeptides codés par les polynucléotides NOVX. L'invention concerne également des anticorps de liaison immunospécifique à un polypeptide NOVX ou à tout dérivé, variant, mutant ou fragment du polypeptide, du polynucléotide ou de l'anticorps NOVX. L'invention concerne en outre des méthodes dans lesquelles le polypeptide, le polynucléotide et l'anticorps NOVX sont utilisés pour la détection et le traitement d'une grande variété d'états pathologiques, ainsi que pour d'autres utilisations.
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