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WO1999047665A1 - GENE HUMAIN HOMOLOGUE DE CI-B14.5a: CBUACD09 - Google Patents

GENE HUMAIN HOMOLOGUE DE CI-B14.5a: CBUACD09 Download PDF

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Publication number
WO1999047665A1
WO1999047665A1 PCT/CN1998/000044 CN9800044W WO9947665A1 WO 1999047665 A1 WO1999047665 A1 WO 1999047665A1 CN 9800044 W CN9800044 W CN 9800044W WO 9947665 A1 WO9947665 A1 WO 9947665A1
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Prior art keywords
polypeptide
cbuacd09
seq
polynucleotide
nucleotide sequence
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PCT/CN1998/000044
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English (en)
Inventor
Jisheng Wu
Juan Zhou
Yaxin Wang
Baiwei Gu
Shuhua Xu
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Shanghai Second Medical University
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Shanghai Second Medical University
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Priority to PCT/CN1998/000044 priority Critical patent/WO1999047665A1/fr
Publication of WO1999047665A1 publication Critical patent/WO1999047665A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

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 NADH dehydrogenase complex family, hereinafter referred to as CBUACD09. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
  • the NADH dehydrogenase is a mitochondrial complex composed of many subunits, and is associated with metabolism.
  • the CI-B14.5a protein is a subunit of NADH dehydrogenase. This indicates that the NADH dehydrogenase complex family has an established, proven history as therapeutic targets.
  • IDDM insulin dependent diabetes mellitus
  • the invention relates to CBUACD09 polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such CBUACD09 polypeptides and polynucleotides. Such uses include the treatment of cancer, AIDS, metabolic 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 CBUACD09 imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBUACD09 activity or levels.
  • CBUACD09 refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO 2 or an alle c variant thereof
  • CBUACD09 activity or CBUACD09 polypeptide activity or “biological activity of the CBUACD09 or CBUACD09 polypeptide” refers to the metabolic or physiologic function of said CBUACD09 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigemc and lmmunogenic activities of said CBUACD09
  • CBUACD09 gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or alle c 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, 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 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 coexisting materials of its natural state is “isolated", as the term is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, 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 t ⁇ ple-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, t ⁇ t
  • Polypeptide refers to any peptide or protein comprising 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 o gomers, and to
  • Polypeptides may contain amino acids other than the 20 gene-encoded ammo 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 m a polypeptide, including the peptide backbone, the ammo 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 ubiquitmation, 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
  • 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 ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations m 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
  • a variant and reference polypeptide are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code
  • a vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a va ⁇ ant 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 relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin,
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NM Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990)
  • the well known Smith Waterman algorithm may also be used to determine identity
  • Preferred parameters for polypeptide sequence comparison include the following 1) Algorithm Needleman and Wunsch. J Mol Biol 48 443-453 (1970)
  • Preferred parameters for polynucleotide comparison include the following
  • Preferred polynucleotide embodiments further include an isolated polynucleotide comp ⁇ sing a polynucleotide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polynucleotide reference sequence of SEQ ID NO 1 , wherem said reference sequence may be identical to the sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or msertion, and wherem said alterations may occur at the 5 ' or 3' termmal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number
  • nn is the number of nucleotide alterations
  • xn is the total number of nucleotides in SEQ ID NO 1
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and wherein any non-integer product of xn and y is rounded down to the nearest mteger prior to subtracting it from xn
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Preferred polypeptide embodiments further mclude an isolated polypeptide comprising a polypeptide havmg at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said reference sequence may be identical to the sequence of SEQ ID NO 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids m SEQ ID NO 2 by the nume ⁇ cal percent of the respective percent identity and subtracting that product from said total number of amino
  • na is the number of amino acid alterations
  • xa is the total number of amino acids in SEQ ID NO 2
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and wherem any non-mteger product of xa and y is rounded down to the nearest integer p ⁇ or to subtracting it from xa
  • the present invention relates to CBUACD09 polypeptides (or CBUACD09 proteins)
  • the CBUACD09 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 80% 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 prefe ⁇ ed Also included within CBUACD09 polypeptides are polypeptides having the amino acid sequence which have at least 80% 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
  • CBUACD09 polypeptide exhibit at least one biological activity of CBUACD09
  • the CBUACD09 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 ammo acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned CBUACD09 polypeptides
  • fragments may be "free-standing,” or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region
  • Representative examples of polypeptide fragments of the invention, m clude, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61- 80, 81-100, and 101 to the end of CBUACD09 polypeptide
  • “about” m cludes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 ammo acid at either extreme or at both extremes
  • Preferred fragments m include, for example, truncation polypeptides having the ammo acid sequence of CBUACD09 polypeptides, except for deletion of a continuous se ⁇ es of residues that mcludes the ammo terminus, or a continuous senes of residues that mcludes the carboxyl terminus or deletion of two continuous senes of residues, one mcludmg the ammo terminus and one mcludmg the carboxyl terminus
  • fragments characterized by structural or functional attributes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- forming regions, turn and turn-forming regions, coil and co ⁇ l-fo ⁇ rung regions, hydroph ⁇ ic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic mdex regions
  • Other preferred fragments are biologically active fragments Biologically active fragment
  • the CBUACD09 polypeptides of the invention can be prepared m any suitable manner Such polypeptides mclude 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 m the art
  • CBUACD09 polynucleotides mclude isolated polynucleotides which encode the CBUACD09 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBUACD09 polynucleotide of the invention mclude a polynucleotide compnsing the nucleotide sequence contained m SEQ ED NO 1 encodmg a CBUACD09 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBUACD09 polynucleotides further mclude a polynucleotide compnsing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encodmg the CBUACD09 polypeptide of SEQ ID NO 2, and a polynucleotide comprising a nucle
  • CBUACD09 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 CBUACD09 polynucleotides
  • CBUACD09 of the invention is structurally related to other proteins of the NADH dehydrogenase complex family, as shown by the results of sequencmg the cDNA of Table 1 (SEQ ID NO 1) encodmg human CBUACD09
  • the cDNA sequence of SEQ ED NO 1 contains an open reading frame (nucleotide number 12 to 365) encodmg a polypeptide of 118 ammo acids of SEQ ID NO 2
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 79 7% identity (using FASTA) m 118 ammo acid residues
  • a nucleotide sequence of a human CBUACD09 (SEQ ID NO 1)
  • One polynucleotide of the present mvention encodmg CBUACD09 may be obtamed usmg standard clonmg and screemng, from a cDNA library de ⁇ ved from mRNA m cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M O , 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 mvention 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 CBUACD09 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encodmg sequence contained m Table 1 (nucleotide number 12 to 365 of SEQ ID NO 1), 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 When the 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 m 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 pu ⁇ fication of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and desc ⁇ bed m Gentz et al , Proc Natl Acad Set USA ( 1989) 86 821 - 824, or is an HA tag
  • the polynucleotide may also contain non-coding 5 ' and 3 ' sequences, such as transc ⁇ bed, non-
  • polynucleotides encodmg CBUACD09 va ⁇ ants compnse the ammo acid sequence CBUACD09 polypeptide of Table 2 (SEQ ID NO 2) m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination
  • the present mvention further relates to polynucleotides that hyb ⁇ dize to the herem above- desc ⁇ bed sequences
  • the present mvention especially relates to polynucleotides which hyb ⁇ dize under st ⁇ ngent conditions to the herem above-desenbed polynucleotides
  • st ⁇ ngent conditions means hybndization 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 mvention which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ID NO 1 or a fragment thereof, may be used as hybndization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encodmg CBUACD09 polypeptide and to isolate cDNA and genomic clones of other
  • probes generally will compnse at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly prefe ⁇ ed probes will range between 30 and 50 nucleotides
  • CBUACD09 polynucleotides of the present mvention further mclude a nucleotide sequence compnsing a nucleotide sequence that hybndize under st ⁇ ngent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof. Also mcluded with CBUACD09 polypeptides are polypeptide compnsing ammo acid sequence encoded by nucleotide sequence obtamed by the above
  • the present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engmeered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the present mvention
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides mto host cells can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sa brook 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,
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtths cells, fiingal cells, such as yeast cells and Aspergillus cells, insect cells such as DrosopMa S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • bacte ⁇ al cells such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtths cells
  • fiingal cells such as yeast cells and Aspergillus cells
  • insect cells such as DrosopMa S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • a great vanety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors de ⁇ ved from bactenal plasmids, from bactenophage, 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 denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used The approp ⁇ ate nucleotide sequence may
  • approp ⁇ ate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • the cells may be harvested pnor to use in the screemng assay If CBUACD09 polypeptide is secreted mto 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 CBUACD09 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, aruon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affimty chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techniques for
  • This mvention also relates to the use of CBUACD09 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBUACD09 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 CBUACD09 Individuals carrying mutations m the CBUACD09 gene may be detected at the DNA level by a vanety of techniques
  • Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, sahva, tissue biopsy or autopsy mate ⁇ al
  • the genomic DNA may be used directly for detection or may be amplified enzymatica-ly by usmg PCR or other amplification techniques p ⁇ or to analysis RNA or cDNA may also be used m similar fashion
  • Deletions and insertions can be detected by a change m size of the amplified product m companson to the normal genotype
  • Pomt mutations can be identified by hybndizing amplified DNA to labeled CBUACD09 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in meltmg temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing See, e g , Myers et al
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to cancer, AIDS, metabolic disorders, and IDDM through detection of mutation m the CBUACD09 gene by the methods desc ⁇ bed
  • cancer can be diagnosed by methods comprising determining from a sample denved from a subject an abnormally decreased or increased level of CBUACD09 polypeptide or CBUACD09 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
  • the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly cancer, AIDS, metabolic disorders, and IDDM, which comprises
  • a CBUACD09 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,
  • any such kit, (a), (b), (c) or (d) may comprise a substantial component
  • the nucleotide sequences of the present mvention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hybndize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step m co ⁇ elatmg those sequences with gene associated disease
  • the physical position of the sequence on the chromosome can be co ⁇ elated with genetic map data
  • genetic map data are found, for example, m V McKusick, Mendehan Inhentance m Man (available on lme 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 (coinhentance 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 mvention or their fragments or analogs thereof, or cells expressmg them can also be used as -mmunogens to produce antibodies immunospecific for the CBUACD09 polypeptides
  • immunospecific means that the antibodies have substantiall greater affimty for the polypeptides of the mvention than their affimty for other related polypeptides m the p ⁇ or art
  • Antibodies generated against the CBUACD09 polypeptides can be obtamed by administering the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routme protocols
  • any technique which provides antibodies produced by continuous cell lme cultures can be used Examples include the hyb ⁇ doma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tnoma technique, the human B-cell hyb ⁇ doma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985) Techniques for the production of single chain antibodies (U S Patent No 4,946,778) can also be adapted to produce sm
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to punfy the polypeptides by affimty chromatography
  • Antibodies against CBUACD09 polypeptides may also be employed to treat cancer, AIDS, metabolic disorders, and IDDM, among others
  • Another aspect of the invention relates to a method for inducmg an immunological response in a mammal which comprises inoculating the mammal with CBUACD09 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer, AIDS, metabolic disorders, and IDDM, among others
  • Yet another aspect of the invention relates to a method of inducing immunological response m a mammal which compnses, delivering CBUACD09 polypeptide via a vector directing expression of CBUACD09 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 CBUACD09 polypeptide wherein the composition comprises a CBUACD09
  • the vaccine formulation may further compnse a suitable carrier Since CBUACD09 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 mclude aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation lnstonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may mclude suspending agents or thickening agents
  • the formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid earner immediately pnor to use
  • the vaccme formulation may also mclude adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-
  • the CBUACD09 polypeptide of the present mvention may be employed m a screemng process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBUACD09 polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
  • These agonists or antagonists may be natural or modified substrates, gands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Cohgan et al , Current Protocols m Immunology 1(2) Chapter 5 (1991)
  • CBUACD09 polypeptides are responsible for many biological functions, mcludmg many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBUACD
  • CBUACD09 polypeptide or respond to CBUACD09 polypeptide of the present mvention Such cells mclude cells from mammals, yeast, DrosopMa or E colt Cells which express the CBUACD09 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBUACD09 polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a
  • the assays may simply test binding of a candidate compound wherem adherence to the cells beanng the CBUACD09 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 CBUACD09 polypeptide, using detection systems approp ⁇ ate to the cells bearmg the CBUACD09 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 mixmg a candidate compound with a solution containing a CBUACD09 polypeptide to form a mixture, measuring CBUACD09 activity in the mixture, and comparing the CBUACD09 activity of the mixture to a standard
  • the CBUACD09 cDNA, protein and antibodies to the protem may also be used to configure assays for detecting the effect of added compounds on the production of CBUACD09 mRNA and protein in cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of CBUACD09 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 CBUACD09 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBUACD09 protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These mclude, but are not limited to, hgand binding and crosslinking assays in which the CBUACD09 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 In addition to being used for purification and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBUACD09 which compete with the binding of CBUACD09 to its receptors, if any Standard methods for conductmg screening assays are well understood in the art Examples of potential CBUACD09 polypeptide antagonists
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for CBUACD09 polypeptides, or compounds which decrease or enhance the production of CBUACD09 polypeptides, which compnses
  • This mvention provides methods of treating abnormal conditions such as, cancer, AIDS, metabolic disorders, and IDDM, related to both an excess of and insufficient amounts of CBUACD09 polypeptide activity
  • CBUACD09 polypeptide If the activity of CBUACD09 polypeptide is m excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove desc ⁇ bed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBUACD09 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. In another approach, soluble forms of CBUACD09 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc in competition with endogenous CBUACD09 polypeptide may be admmistered Typical embodiments of such competitors comprise fragments of the CBUACD09 polypeptide
  • expression of the gene encoding endogenous CBUACD09 polypeptide can be inhibited usmg 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 m Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.
  • oligonucleotides which form triple helices with the gene can be supplied See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360 These o gomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • a polynucleotide of the mvention may be engmeered for expression m a replication defective retroviral vector, as discussed above
  • the retroviral expression construct may then be isolated and introduced mto a packagmg cell transduced with a retroviral plasmid vector containmg RNA encodmg a polypeptide of the present mvention such that the packagmg cell now produces infectious viral particles containmg the gene of interest
  • These producer cells may be admmistered
  • Peptides such as the soluble form of CBUACD09 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner
  • a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient
  • earners mclude 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 mvention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • Polypeptides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • Prefe ⁇ ed forms of systemic administration of the pharmaceutical compositions mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal admimstration usmg penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be used.
  • 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 m the range of 0 1 - 100 ⁇ g/kg of subject Wide vanations m the needed dosage, however, are to be expected m view of the vanety of compounds available and the differing efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by mtravenous injection Vanations m these dosage levels can be adjusted usmg standard empincal routmes for optimization, as is well understood m the art
  • Polypeptides used in treatment can also be generated endogenously m the subject, in treatment modalities often refe ⁇ ed to as "gene therapy" as desc ⁇ bed above
  • cells from a subject may be engmeered 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 mto the subject

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBUACD09 et des procédés d'obtention de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides CBUACD09 dans la conception de protocoles pour le traitement de cancers, du SIDA, de troubles métaboliques et du diabète insulinodépendant, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000044 1998-03-18 1998-03-18 GENE HUMAIN HOMOLOGUE DE CI-B14.5a: CBUACD09 Ceased WO1999047665A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000044 WO1999047665A1 (fr) 1998-03-18 1998-03-18 GENE HUMAIN HOMOLOGUE DE CI-B14.5a: CBUACD09

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000044 WO1999047665A1 (fr) 1998-03-18 1998-03-18 GENE HUMAIN HOMOLOGUE DE CI-B14.5a: CBUACD09

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WO1999047665A1 true WO1999047665A1 (fr) 1999-09-23

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FEBS LETT., 313(1), (1992), WALKER J.E., "Complementary DNA Sequences of Two 14.5kDa Subunits of NADH:Ubiquinone Oxidoreductase from Bovine Heart Mitochondria. Completion of the Primary Structure of the Complex?", pages 80-84. *
GENBANK, dbj/D16847/MUSSCP1, 15 Jan. 1994, MARUYAMA K. et al., "Differential Cloning of a Gene Encoding SCP-1, a Transmembrane Protein Containing EGF-Like Repeats from Mouse Stromal Cell Lines PA6". *

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