WO1999021885A1 - Gene transporteur 7 abc humain (habc7) - Google Patents

Gene transporteur 7 abc humain (habc7) Download PDF

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Publication number
WO1999021885A1
WO1999021885A1 PCT/CN1997/000120 CN9700120W WO9921885A1 WO 1999021885 A1 WO1999021885 A1 WO 1999021885A1 CN 9700120 W CN9700120 W CN 9700120W WO 9921885 A1 WO9921885 A1 WO 9921885A1
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polypeptide
habc7
seq
nucleotide sequence
polynucleotide
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PCT/CN1997/000120
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English (en)
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Qing-Hua Zhang
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Shanghai Second Medical University
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Priority to PCT/CN1997/000120 priority Critical patent/WO1999021885A1/fr
Publication of WO1999021885A1 publication Critical patent/WO1999021885A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified 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 ABC transporter gene family, hereinafter referred to as HABC7. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
  • the murine ABC7 transporter protein is a typical half-transporter, having six transmembrane domains in the N-terminal region and an ATP binding cassette (ABC motif) in the C-terminal region. It functions as a membrane pump required for other half-transporters such as TAP1 and TAP2 to form either homodimers or heterodimers.
  • ABC transporter gene family has an established, proven history as therapeutic targets.
  • Clearly there is a need for identification and characteri ⁇ ation of further members of the ABC transporter gene family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM.
  • the invention relates to HABC7 polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such HABC7 polypeptides and polynucleotides. Such uses include the treatment of cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with HABC7 imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate HABC7 activity or levels.
  • HABC7 refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO 2 or an allelic variant thereof
  • HABC7 activity or HABC7 polypeptide activity refers to the metabolic or physiologic function of said HABC7 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and immunogenic activities of said HABC7
  • HABC7 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, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, mcluding the products of an Fab or other lmmunoglobuhn 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 For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexistmg materials of its natural state is “isolated", as the term is employed herein
  • Polynucleotide generally refers to any polyribonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation smgle- 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 comp ⁇ sing RNA or DNA or both RNA and DNA
  • polynucleotide also mcludes 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
  • Polypeptide refers to any peptide or protein comp ⁇ sing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres "Polypeptide” refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins Polypeptides may contain ammo 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.
  • 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 mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • 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., S M 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., SUM 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, JMolec Biol (1990) 215:403).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the present invention relates to HABC7 polypeptides (or HABC7 proteins).
  • the HABC7 polypeptides include the polypeptide of SEQ ID NO:2; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the amino acid sequence which have at least 84% identity to that of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly preferred.
  • HABC7 polypeptides having the amino acid sequence which have at least 84% identity to the polypeptide having the amino acid sequence of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO:2. Furthermore, those with at least 97-99% are highly preferred.
  • HABC7 polypeptide exhibit at least one biological activity of HABC7.
  • the HABC7 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. Fragments of the HABC7 polypeptides are also included in the invention. 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 HABC7 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 region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of HABC7 polypeptide.
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of HABC7 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, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Other preferred fragments are biologically active fragments.
  • Biologically active fragments are those that mediate HABC7 activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
  • 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 He; 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. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • the HABC7 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.
  • Polynucleotides of the Invention are well understood in the art.
  • HABC7 polynucleotides include isolated polynucleotides which encode the HABC7 polypeptides and fragments, and polynucleotides closely related thereto. More specifically, HABC7 polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a HABC7 polypeptide of SEQ ID NO: 2, and polynucleotide having the particular sequence of SEQ ID NO:l.
  • HABC7 polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 82% identity over its entire length to a nucleotide sequence encoding the HABC7 polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 82% identical to of SEQ ID NO: 1 over its entire length.
  • polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred.
  • those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred.
  • HABC7 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 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 HABC7 polynucleotides.
  • HABC7 of the invention is structurally related to other proteins of the ABC transporter gene family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1) encoding human HABC7.
  • the cDNA sequence of SEQ ID NO:l contains an open reading frame (nucleotide number 9 to 2264) encoding a polypeptide of 752 amino acids of SEQ ID NO:2.
  • Table 2 (SEQ ID NO:2) has about 84% identity (using FASTA) in 752 amino acid residues with mouse ABC transporter-7 (S.Savary et al.,Genomics,41:275-278, 1997).
  • the nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 81.4% identity (using FASTA) in 2384 nucleotide residues with mouse ABC transporter-7 (S.Savary et al.,Genomics,41:275-278,1997).
  • HABC7 polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.
  • a nucleotide sequence of a human HABC7 (SEQ ID NO: 1).
  • One polynucleotide of the present invention encoding HABC7 may be obtained using standard cloning and screening, from a cDNA Hbrary derived from mRNA in cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651-1656; Adams, M.D. et al, Nature, (1992) 355:632-634; 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.
  • nucleotide sequence encoding HABC7 polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 9 to 2264 of SEQ ID NO:l), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • polynucleotides of the invention are used for the recombinant production of
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding 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 NatlAcadSci 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 mRNA.
  • polynucleotides encoding HABC7 variants comprise the amino acid sequence HABC7 polypeptide of Table 2 (SEQ ID 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 combination.
  • 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 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences.
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding HABC7 polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the HABC7 gene.
  • hybridization techniques are known to those of skill in the art.
  • these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% 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. Particularly preferred probes will range between 30 and 50 nucleotides.
  • HABC7 polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ID NO: 1 or a fragment thereof.
  • HABC7 polypeptides comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition.
  • 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 microgram/ml denatured, sheared salmon sperm DNA followed by washing the filters in 0. Ix SSC at about 65°C.
  • 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.
  • the present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • 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. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • 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, HEK 293 and Bowes melanoma cells
  • plant cells A great variety of expression systems can be used.
  • Such systems include, among others, 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 retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression systems may contain control regions that regulate as well as engender expression.
  • 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).
  • secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide be produced at the surface of the cell.
  • the cells may be harvested prior to use in the screening assay. If HABC7 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.
  • HABC7 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, afi_nity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • This invention also relates to the use of HABC7 polynucleotides for use as diagnostic reagents. Detection of a mutated form of HABC7 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 HABC7. Individuals carrying mutations in the HABC7 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 fashion. 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 HABC7 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 sequencing. See, e.g, Myers et al. , Science (1985) 230: 1242. Sequence changes at specific locations may also be revealed by 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 HABC7 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 determining a susceptibility to cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM through detection of mutation in the HABC7 gene by the methods described.
  • cancer can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of HABC7 polypeptide or HABC7 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 HABC7 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 present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM, which comprises:
  • HABC7 polynucleotide preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ;
  • HABC7 polypeptide preferably the polypeptide of SEQ ID NO: 2, or a fragment thereof; or (d) an antibody to a HABC7 polypeptide, preferably to the polypeptide of SEQ ID NO: 2.
  • kits may comprise a substantial component.
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • 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 Hopkins 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).
  • 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.
  • the HABC7 gene has been localized to the X chromosome on q21.3.
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the HABC7 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 HABC7 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 HABC7 polypeptides may also be employed to treat cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM, among others.
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with HABC7 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM, among others.
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering HABC7 polypeptide via a vector directing expression of HABC7 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 HABC7 polypeptide wherein the composition comprises a HABC7 polypeptide or HABC7 gene.
  • the vaccine formulation may further comprise a suitable carrier. Since HABC7 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.
  • the HABC7 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 HABC7 polypeptide of the present invention.
  • 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 or modified substrates, ligands, receptors, enzymes, 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 invention. See Coligan etal, Current Protocols in Immunology l(2):Chapter 5 (1991).
  • HABC7 polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate HABC7 polypeptide on the one hand and which can inhibit the function of HABC7 polypeptide on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM.
  • Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM.
  • such screening procedures may involve using appropriate cells which express the HABC7 polypeptide or respond to HABC7 polypeptide of the present invention.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • Cells which express the HABC7 polypeptide (or cell membrane containing the expressed polypeptide) or respond to HABC7 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 HABC7 activity.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the HABC7 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 HABC7 polypeptide, using detection systems appropriate to the cells bearing the HABC7 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.
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing a HABC7 polypeptide to form a mixture, measuring HABC7 activity in the mixture, and comparing the HABC7 activity of the mixture to a standard.
  • the HABC7 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of HABC7 mRNA and protein in cells.
  • an ELISA may be constructed for measuring secreted or cell associated levels of HABC7 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of HABC7 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the HABC7 protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the HABC7 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
  • a radioactive isotope eg 1251
  • chemically modified eg biotinylated
  • these binding assays can be used to identify agonists and antagonists of HABC7 which compete with the binding of HABC7 to its receptors, if any. Standard methods for conducting screening assays are well understood in the art.
  • HABC7 polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc, as the case may be, of the HABC7 polypeptide, e.g, a fragment of the ligands, substrates, receptors, enzymes, etc.; 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.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for HABC7 polypeptides; or compounds which decrease or enhance the production of HABC7 polypeptides, which comprises:
  • kits may comprise a substantial component.
  • This invention provides methods of treating abnormal conditions such as, cancer, autoimmune disease, Addison's disease, microsomal disorders, and IDDM, related to both an excess of and insufficient amounts of HABC7 polypeptide activity.
  • HABC7 polypeptide is in excess.
  • 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 the function of the HABC7 polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of HABC7 polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous HABC7 polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the HABC7 polypeptide.
  • expression of the gene encoding endogenous HABC7 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.
  • oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • HABC7 For treating abnormal conditions related to an under-expression of HABC7 and its activity, several approaches are also available.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound which activates HABC7 polypeptide, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition.
  • gene therapy may be employed to effect the endogenous production of HABC7 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.
  • Another approach is to administer a therapeutic amount of HABC7 polypeptides in combination with a suitable pharmaceutical carrier.
  • Peptides such as the soluble form of HABC7 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 administration, 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 invention.
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
  • 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 fusidic acids or other detergents.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible.
  • Administration of these compounds 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 compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, 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 R A 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.

Abstract

L'invention concerne des polypeptides et des polynucléotides HABC7 et des procédés de production de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides HABC7 dans la conception de protocoles pour le traitement du cancer, de maladies auto-immunes, de la maladie d'Addison, de troubles microsomaux et du diabète sucré insulino-dépendant, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1997/000120 1997-10-29 1997-10-29 Gene transporteur 7 abc humain (habc7) WO1999021885A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN1997/000120 WO1999021885A1 (fr) 1997-10-29 1997-10-29 Gene transporteur 7 abc humain (habc7)

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Application Number Priority Date Filing Date Title
PCT/CN1997/000120 WO1999021885A1 (fr) 1997-10-29 1997-10-29 Gene transporteur 7 abc humain (habc7)

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WO1999021885A1 true WO1999021885A1 (fr) 1999-05-06

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078953A2 (fr) * 1999-06-17 2000-12-28 Incyte Genomics, Inc. Proteines de transport humaines
WO2002002633A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Transporteurs et canaux ioniques
WO2010056855A1 (fr) * 2008-11-13 2010-05-20 Teva Pharmaceutical Industries Ltd. Préparation de rétapamuline par l'intermédiaire de son précurseur pleuromutiline-thiol

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HUM. MOL. GENET., 5(10), 1996, ALLIKMETS RANDO et al., pages 1649-1655. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078953A2 (fr) * 1999-06-17 2000-12-28 Incyte Genomics, Inc. Proteines de transport humaines
WO2000078953A3 (fr) * 1999-06-17 2002-01-17 Incyte Genomics Inc Proteines de transport humaines
WO2002002633A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Transporteurs et canaux ioniques
WO2002002633A3 (fr) * 2000-06-29 2002-07-18 Incyte Genomics Inc Transporteurs et canaux ioniques
WO2010056855A1 (fr) * 2008-11-13 2010-05-20 Teva Pharmaceutical Industries Ltd. Préparation de rétapamuline par l'intermédiaire de son précurseur pleuromutiline-thiol

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