Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• This invention relates to newly identified splice- variant polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the cell adhesion molecule family, hereinafter referred to as NrCAMvar. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
• NrCAM NgCAM-related cell adhesion molecule
• This cell surface glycoprotein is a member of the immunoglobulin (Ig) superfamily, and is very similar in structure to chick NgCAM, human and mouse LI and chick neurofascin. Each consists of six Ig domains, five fibronectin type Ill-like repeats, a transmembrane domain and an intracellular region.
• NrCAM chick and rat NrCAM
• a role for NrCAM in the in vivo guidance of chick commissural neurones has been identified and distinguished from that of NgCAM (Stoeckli and Landmesser, 1995).
• Chick NrCAM in floor plate cells together with axonin-1 on commissural growth cones is essential for accurate pathfinding at the midline whereas NgCAM is required for fasciculation of the commissural neurites.
• NrCAM can also bind at the cell surface with FI 1 , another member of the Ig superfamily (Morales et al., 1993). Recently a highly conserved human homologue to chick NrCAM was described
• AE19 encodes a 19-amino-acid section between Ig ⁇ and Iglll while AE10 is a 10 amino-acid section between IgVI and FNIII-1 (Grumet et al., 1991).
• NrCAM probes Lane et al.
• RNA band of ⁇ 7.0kb in multiple brain tissues including amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus, and thalamus.
• chick the same size of RNA was found in brain tissue but not in embryo heart, gizzard or liver on Northern blots.
• the invention relates to NrCAMvar polypeptides and recombinant materials and methods for their productioa
• Another aspect of the invention relates to methods for using such NrCAMvar polypeptides and polynucleotides. Such uses include the treatment of diabetes, obesity and cancer, among others.
• the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with NrCAMvar imbalance with the identified compounds, including diabetes, obesity and cancer.
• Figure 1 shows the nucleotide and deduced amino acid sequence of a human NrCAMvar; SEQ ID NOS: 1 and 2, respectively.
• Figure 2 shows a comparison of the sequences of human NrCAMvar of the present invention and human and chick NrCAM cDNAs.
• NeCAMvar refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allelic variant thereof.
• NrCAMvar activity or NrCAMvar polypeptide activity refers to the metabolic or physiological function of said NrCAMvar including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said NrCAMvar.
• NeCAMvar gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or allelic variants thereof and/or their complements.
• Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
• Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
• a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
• Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
• polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
• Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
• Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
• Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
• Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching.
• Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
• Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
• a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
• a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
• a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
• a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
• a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
• Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques.
• identity is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48: 1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs.
• Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al, Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al, J Molec Biol (1990) 215:403).
• NrCAMvar a new splice variant of NrCAM (termed NrCAMvar) which comprises the AE10K2 sequence, which is absent in the published human NrCAM sequence (Lane et al, 1996) but which is present in the chick sequence.
• NrCAMvar does not have the AE10K1 sequence which is present in the Lane et al human sequence ( Figure 2).
• NrCAMvar is expressed at high levels in the brain, pancreas and adrenal cortex and at lower levels in placenta, adrenal medulla, thyroid and testis.
• the published human NrCAM however, appears not to be expressed in the pancreas.
• the present invention relates to novel NrCAMvar polypeptides.
• the NrCAMvar polypeptides include the polypeptide of SEQ ID NO:2; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2.
• Preferably NrCAMvar polypeptide exhibit at least one biological activity of NrCAMvar.
• NrCAMvar polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
• a fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned NrCAMvar polypeptides.
• fragments may be "free-standing,” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous regioa
• Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of NrCAMvar polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
• fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha- helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydropnobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
• Biologically active fragments are those that mediate NrCAMvar activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity.
• variants of the defined sequence and fragments also form part of the present inventioa Preferred variants are those that vary from the referents by conservative amino acid substitutions - i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and Ue; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
• NrCAMvar polypeptides of the invention can be prepared in any suitable manner.
• Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art
• NrCAMvar polynucleotides include isolated polynucleotides which encode the NrCAMvar polypeptides and fragments, and polynucleotides closely related thereto. More specifically, NrCAMvar polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 encoding a NrCAMvar polypeptide of SEQ LD NO: 2, and polynucleotides having the particular sequence of SEQ LD NO: 1.
• NrCAMvar polynucleotides are also included under NrCAMvar polynucleotides.
• a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO:l to hybridize under conditions useable for amplification or for use as a probe or marker.
• the invention also provides polynucleotides which are complementary to such NrCAMvar polynucleotides.
• NrCAMvar of the invention is structurally related to other proteins of the cell adhesion molecules, as shown by the results of sequencing the cDNA encoding human NrCAMvar.
• the cDNA sequence contains an open reading frame encoding a polypeptide of 1304 amino acids.
• Amino acid of sequence of Figure 1 (SEQ ID NO:2) has about >99% identity (using BlastP) in 1299 amino acid residues with Human NrCAM (Lane, RP et al, Genomics 35 (3), 456-465 (1996)).
• Nucleotide sequence of Figure 1 (SEQ LD NO:l) has about >99% identity (using BlastN) in 3897 nucleotide residues with Human NrCAM (Genomics 35 (3), 456-465 (1996)).
• Figure 2 shows the splice variant AE10K.
• One polynucleotide of the present invention encoding NrCAMvar may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human adrenal using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651-1656; Adams, M.D. et al, Nature, (1992) J55:632-634; Adams, M.D., et al, Nature (1995) 377 Supp:3-174).
• Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
• the nucleotide sequence encoding NrCAMvar polypeptide of SEQ ID NO:2 may be identical over its entire length to the coding sequence set forth in Figure 1 (SEQ ID NO: 1), or may be a degenerate form of this nucleotide sequence encoding the polypeptide of SEQ ID NO:2, or may be highly identical to a nucleotide sequence that encodes the polypeptide of SEQ ID NO: 2.
• the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the cod g sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
• a marker sequence which facilitates purification of the fused polypeptide can be encoded.
• the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
• the polynucleotide may also contain non-coding 5 ' and 3 ' sequences, such as transcribed, non- translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRN A
• NrCAMvar variants comprise the amino acid sequence NrCAMvar polypeptide of Figure 1 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combinatioa
• the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
• the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
• stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
• Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 , may be used as hybridization probes for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding NrCAMvar polypeptide and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the NrCAMvar gene.
• hybridization techniques are known to those of skill in the art.
• these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical to that of the referent
• the probes generally will comprise at least 15 nucleotides.
• such probes will have at least 30 nucleotides and may have at least 50 nucleotides.
• a polynucleotide encoding NrCAMvar comprises the steps of screening an appropriate library under stingent hybridization conditions with a labeled probe having the SEQ ED NO: 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
• hybridization techniques are well known to those of skill in the art.
• Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 miCTOgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C.
• the polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
• Vectors, Host Cells, Expression also relates to vectors which comprise a polynucleotide or polynucleotides of the present inventioa and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques.
• RNAs derived from the DNA constructs of the present inventioa can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present inventioa
• Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et aL, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
• bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
• fungal cells such as yeast cells and Aspergillus cells
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
• plant cells include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
• fungal cells such as yeast cells and Aspergillus cells
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
• plant cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanom
• chromosomal, episomal and virus-derived systems e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and rettoviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
• viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and rettoviruses
• vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and
• the expression systems may contain control regions that regulate as well as engender expressioa
• any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used
• the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL ⁇ supra).
• appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
• NrCAMvar polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If NrCAMvar polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
• NrCAMvar polypeptides can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purificatioa Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purificatioa
• This invention also relates to the use of NrCAMvar polynucleotides for use as diagnostic reagents. Detection of a mutated form of NrCAMvar gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of NrCAMvar. Individuals carrying mutations in the NrCAMvar gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
• RNA or cDNA may also be used in similar fashioa Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
• Point mutations can be identified by hybridizing amplified DNA to labeled NrCAMvar nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA se ⁇ encing. See, e.g., Myers et al, Science (1985) 230:1242.
• nuclease protection assays such as RNase and S 1 protection or the chemical cleavage method See Cotton et al. , Proc Natl Acad Sci USA (1985) 85: 4397-4401.
• an array of oligonucleotides probes comprising NrCAMvar nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
• Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
• the diagnostic assays offer a process for diagnosing or dete ⁇ riining a susceptibility to Diabetes, obesity and cancer through detection of mutation in the NrCAMvar gene by the methods described
• Diabetes, obesity and cancer can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of NrCAMvar polypeptide or NrCAMvar mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as an NrCAMvar polypeptide, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
• the nucleotide sequences of the present invention are also valuable for chromosome identificationioa
• the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
• the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns H ⁇ kins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
• the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
• polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies in ⁇ mur ⁇ )specific for the NrCAMvar polypeptides.
• immunospecific means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
• Antibodies generated against the NrCAMvar polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols.
• any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
• the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
• Antibodies against NrCAMvar polypeptides may also be employed to treat Diabetes, obesity and cancer, among others.
• Vaccines Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with NrCAMvar polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from Diabetes, obesity and cancer, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering NrCAMvar polypeptide via a vector directing expression of NrCAMvar polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
• composition which, when introduced into a mammalian host, induces an immunological response in that mammal to a NrCAMvar polypeptide wherein the composition comprises a NrCAMvar polypeptide or NrCAMvar gene.
• the vaccine formulation may further comprise a suitable carrier. Since NrCAMvar polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
• the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
• NrCAMvar polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the NiC AMvar polypeptide of the present inventioa
• polypeptides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
• agonists or antagonists may be natural substrates, ligands, receptors, etc., as the case may be, of the polypeptide of the present invention; or may be structural or functional mimetics of the polypeptide of the present inventioa See Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991).
• NrCAMvar polypeptides are ubiquitous in the nrianrimalian host and are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate NrCAMvar polypeptide on the one hand and which can inhibit the function of NrCAMvar polypeptide on the other hand
• agonists are employed for therapeutic and prophylactic purposes for such conditions as Diabetes, obesity and cancer.
• Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as Diabetes, obesity and cancer.
• such screening procedures may involve using appropriate cells which express the NrCAMvar polypeptide or respond to NrCAMvar polypeptide of the present inventioa
• cells include cells from mammals, yeast, Drosophila or E. coli.
• Cells which express the NrCAMvar polypeptide (or cell membrane containing the expressed polypeptide) or respond to NrCAMvar polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
• the ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for NrCAMvar activity.
• the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the NrCAMvar polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the NrCAMvar polypeptide, using detection systems appropriate to the cells bearing the NrCAMvar polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Standard methods for conducting such screening assays are well understood in the art.
• NrCAMvar polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, etc., as the case may be, of the NrCAMvar polypeptide, e.g., a fragment of the ligands, substrates, receptors, or small molecules which bind to the polypetide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented
• This invention provides methods of treating an abnormal conditions related to both an excess of and insufficient amounts of NrCAMvar polypeptide activity, including diabetes, obesity and cancer. If the activity of NrCAMvar polypeptide is in excess, several approaches are available.
• One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the NrCAMvar polypeptide, or by inhibiting a second signal, and thereby alleviating the abnormal conditioa
• NrCAMvar polypeptides still capable of binding the ligand in competition with endogenous NrCAMvar polypeptide may be administered Typical embodiments of such competitors comprise fragments of the NrCAMvar polypeptide.
• NrCAMvar polypeptide expression of the gene encoding endogenous NrCAMvar polypeptide can be inhibited using expression blocking techniques.
• Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988).
• oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al, Nucleic Acids Res (1979) 3:173; Cooney et al, Science (1988) 241:456; Dervan et al, Science (1991) 251:1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
• NrCAMvar For treating abnormal conditions related to an under-expression of NrCAMvar and its activity, several approaches are also available.
• One approach comprises admmistering to a subject a therapeutically effective amount of a compound which activates NrCAMvar polypeptide, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal conditioa
• gene therapy may be employed to effect the endogenous production of NrCAMvar by the relevant cells in the subject.
• a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
• the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest
• These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
• gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).
• Peptides such as the soluble form of NrCAMvar polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
• suitable pharmaceutical carrier include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administratioa and is well within the skill of the art.
• the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the inventioa
• Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
• systemic adrninistration of the pharmaceutical compositions include injection, typically by intravenous injectioa Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used
• Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusi ic acids or other detergents.
• penetrants such as bile salts or fusi ic acids or other detergents.
• oral administration may also be possible.
• Administration of these conr ⁇ ounds may also be topical and/or localized in the form of salves, pastes, gels and the like.
• the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject Wide variations in the needed dosage, however, are to be expected in view of the variety of c»mpounds available and the differing efficiencies of various routes of administratioa For example, oral administration would be expected to require higher dosages than administration by intravenous injectioa Variations in these dosage levels can be adjusted using standard empirical routines for optimizatioa as is well understood in the art.
• Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
• cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
• HGS EST database was screened using the chick NrCAM sequence and three HGS EST clones (EST99669, EST237133, and EST373834) were obtained.
• EST99669 and EST237133 clones were from human adrenal cDNA library while EST373834 was from human striatum cDNA library.
• cDNA clones EST237133 and EST373834 contained several EcoRl or Eco RUXho I fragments, suggesting that inserts from several different genes may be present.
• EST99669 was found to contain a contiguous genomic sequence not homologous to any sequence of the chick gene. Examination of this sequence revealed a splice donor consensus sequence. This probably corresponds to an intron:exon boundary represented in cDNA due to incomplete mRNA processing. A donor and an acceptor were present in EST373834.
• AE12 and AE93 identified by Lane et al. (1996), AE19 was also found to be absent from some cDNA fragments obtained from human fetal brain cDNA.
• two novel alternatively spliced regions, encoding 10-amino-acid sections, were identified (see Figure 2). AE93 was absent in EST237133 which was from human adrenal cDNA library while both AE12 and AE93 were observed in PCR products from human adult brain cDNA.
• a mRNA band of ⁇ 7.0kb was observed for human brain, placenta, pancreas, adrenal medulla and cortex, thyroid, and testis tissues. The results also showed that this gene is highly expressed in brain, pancreas, and adrenal cortex tissues (the levels of mRNA on the blots used are controlled at Clontech and samples are tested for their integrity by hybridisation with an actin gene probe).
• Table 1 Data vectors obtained from testing Genebridge 4 Radiation Hydrids panel using primers sbpl2 and 13.
• NrCAMvar non-insulin-dependent diabetes mellitus

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