WO1999046292A1 - Gene humain p18 (cbdara04) - Google Patents

Gene humain p18 (cbdara04) Download PDF

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Publication number
WO1999046292A1
WO1999046292A1 PCT/CN1998/000034 CN9800034W WO9946292A1 WO 1999046292 A1 WO1999046292 A1 WO 1999046292A1 CN 9800034 W CN9800034 W CN 9800034W WO 9946292 A1 WO9946292 A1 WO 9946292A1
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Prior art keywords
polypeptide
cbdara04
seq
polynucleotide
nucleotide sequence
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PCT/CN1998/000034
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English (en)
Inventor
Juan Zhou
Qinghua Zhang
Jian Gu
Jisheng Wu
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Shanghai Second Medical University
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Priority to PCT/CN1998/000034 priority Critical patent/WO1999046292A1/fr
Publication of WO1999046292A1 publication Critical patent/WO1999046292A1/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
    • 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 eukaryotic transcription elongation factor family, hereinafter referred to as CBDARA04. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
  • Protein synthesis consists of three stages: initiation, elongation, and termination. Initiation involves the reactions which precede formation of the peptide bond between the first two amino acids of the protein. The process requires the ribosome to bind to the mRNA, forming an initiation complex with the first aminoacyl-tRNA. In addition to the critical 3 OS ribosomal subunit, a number of proteins known as initiation factors are required for this step of protein synthesis. Elongation consists of all of the reactions from synthesis of the first peptide bond to addition of the last amino acid. Amino acids are added to the polypeptide chain one at a time; the addition of an amino acid is the most rapid step in protein synthesis.
  • Elongation factors are proteins which associate with ribosomes cyclically, during the addition of each amino acid to the polypeptide chain. Members of the elongation factor family are evolutionarily conserved. This indicates that the eukaryotic transcription elongation factor family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further members of the eukaryotic transcription elongation factor family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer and AIDS.
  • the invention relates to CBDARA04 polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such CBDARA04 polypeptides and polynucleotides. Such uses include the treatment of cancer and AIDS, 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 CBDARA04 imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBDARA04 activity or levels. DESCRIPTION OF THE INVENTION
  • CBDARA04 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
  • CBDARA04 activity or CBDARA04 polypeptide activity refers to the metabolic or physiologic function of said CBDARA04 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogemc activities of said CBDARA04
  • CBDARA04 gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allehc 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 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 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 triple-stranded regions comprising RNA or DNA or both RNA and DNA
  • 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 ⁇ tylated
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, 1 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 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 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 ammo acid side-chains and the ammo 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
  • 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 m nucleotide sequence from another, reference polynucleotide
  • 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, m 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 allehc variant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring variants of polynucleotides and polypeptides may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally
  • 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.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al. , Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S F et al. , J Molec. Biol.
  • the BLAST X program is publicly available from NCBI and other sources ⁇ BLAST Manual , Altschul, S., et al , NCBI NLM NIH 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) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4
  • a program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI.
  • the aforementioned parameters are the default parameters for polynucleotide comparisons.
  • Preferred polynucleotide embodiments further include an isolated polynucleotide comprising 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.J, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: l
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO: l
  • 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%
  • any non- integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NOJ 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 include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NOJ, 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 amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino 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 in SEQ ID NOJ by the numerical percent of the respective percent identity and subtracting that product from said total number of amino acids in SEQ ID NOJ, or:
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NOJ
  • 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 x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • the present invention relates to CBDARA04 polypeptides (or CBDARA04 proteins).
  • the CBDARA04 polypeptides include the polypeptide of SEQ ID NOJ, as well as
  • CBDARA04 polypeptides comprising the amino acid sequence of SEQ ID NO 2
  • 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
  • those with at least 97-99% are highly preferred
  • those with at least 97-99% are highly preferred
  • CBDARA04 polypeptide exhibit at least one biological activity of CBDARA04
  • the CBDARA04 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,
  • Preferred fragments m include, for example, truncation polypeptides having the ammo acid sequence of CBDARA04 polypeptides, except for deletion of a continuous se ⁇ es of residues that mcludes the ammo terminus, or a continuous se ⁇ es of residues that mcludes the carboxyl terminus or deletion of two continuous se ⁇ es of residues, one including the ammo terminus and one including the carboxyl terminus
  • fragments characte ⁇ zed by structural or functional attributes such as fragments that comp ⁇ se alpha-helix and alpha-helix formmg regions, beta-sheet and beta-sheet- formmg 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 mdex regions
  • Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate CBD
  • the CBDARA04 polypeptides of the mvention 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
  • CBDARA04 polynucleotides mclude isolated polynucleotides which encode the CBDARA04 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBDARA04 polynucleotide of the mvention mclude a polynucleotde compnsmg the nucleotide sequence contamed m SEQ ID NO 1 encoding a CBDARA04 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBDARA04 polynucleotides further mclude a polynucleotide compnsmg a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBDARA04 polypeptide of SEQ ID NO 2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to of
  • the CBDARA04 of the mvention is structurally related to other proteins of the eukaryotic transc ⁇ ption elongation factor family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human CBDARA04
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 29 to 550) encoding a polypeptide of 174 ammo acids of SEQ ID NO 2
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 91 % identity (usmg FASTA) m 174 ammo acid residues with the rat P18 gene (S Quevillonet al , FEBS Lett 395 63-67,1997)
  • the nucleotide sequence of Table 1 (SEQ ID NO 1) has about 77 5% identity (usmg FASTA) m 755 nucleotide residues with the rat PI 8 gene (S Quevillonet al , FEBS Lett 3
  • a nucleotide sequence of a human CBDARA04 (SEQ ID NO 1)
  • One polynucleotide of the present mvention encoding CBDARA04 may be obtained usmg standard cloning and screening, from a cDNA library denved from mRNA m 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)
  • EST expressed sequence tag
  • 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 CBDARA04 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 29 to 550 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
  • the polynucleotide may include the coding sequence for the mature
  • 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 punfication of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidme peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed m 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 transcnbed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that
  • the present mvention further relates to polynucleotides that hybndize to the herem above- descnbed sequences
  • the present mvention especially relates to polynucleotides which hybndize under strmgent conditions to the herem above-descnbed polynucleotides
  • strmgent conditions means hyb ⁇ dization 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 contamed 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 encoding CBDARA04 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 sinula ⁇ ty to the CBDARA04 gene
  • Such hybndization techniques are known to those of skill m the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent
  • the 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 preferred probes will range between 30 and 50 nucleotides
  • CBDARA04 polynucleotides of the present mvention further mclude a nucleotide sequence compnsmg a nucleotide sequence that hybndize under strmgent condition to a
  • CBDARA04 polypeptides are polypeptide compnsmg ammo acid sequence encoded by nucleotide sequence obtained by the above hybndization condition
  • hybndization techniques are well known to those of skill in the art Strmgent hybndization conditions are as defined above or, alternatively, conditions under overnight mcubation at 42°C m a solution compnsmg 50% formamide, 5xSSC (150mM NaCl, 15mM tnsodium 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 lx SSC at about 65°C
  • polynucleotides and polypeptides of the present mvention may be employed as research reagents and mate ⁇ als for discovery of treatments and diagnostics to animal and human disease
  • the present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engineered 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 descnbed m 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 pid-mediated transfection, electroporation, transduction, scrape loading, ballistic mtroduction or infection
  • approp ⁇ ate hosts mclude bactenal cells, such as streptococci, staphylococci, E colt, Streptomyces and Bacillus subtihs cells, fiingal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • chromosomal, episomal and virus-de ⁇ ved systems e g , vectors denved 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, a
  • 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 approp ⁇ ate nucleotide sequence may be inserted mto an expression system by any of a vanety of well-known and routme techniques, such as, for example, those set forth in Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL ⁇ supra)
  • appropnate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • CBDARA04 polypeptide is to be expressed for use m screenmg assays, generally, it is preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested pnor to use in the screenmg assay If CBDARA04 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide, if produced mtracellularly, the cells must first be lysed before the polypeptide is recovered CBDARA04 polypeptides can be recovered and punfied 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 lectm chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured
  • This mvention also relates to the use of CBDARA04 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBD ARA04 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 CBDARA04 Individuals carrying mutations m the CBDARA04 gene may be detected at the DNA level by a vanety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, unne, saliva, tissue biopsy or autopsy matenal
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification techniques pnor to analysis RNA or
  • 13 cDNA may also be used m similar fashion Deletions and insertions can be detected by a change m size of the amplified product m compa ⁇ son to the normal genotype
  • Pomt mutations can be identified by hybndizmg amplified DNA to labeled CBDARA04 nucleotide sequences
  • Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments m gels, with or without denaturing agents, or by direct DNA sequencmg 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
  • cancer and AIDS can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of CBDARA04 polypeptide or CBDARA04 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 CBDARA04 polypeptide, in a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly cancer and AIDS, which comprises (a) a CBDARA04 polynucleotide, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof, (b) a nucleotide sequence complementary to that of (a),
  • CBDARA04 polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • 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 in correlating those sequences with gene associated disease
  • genetic map data are found, for example, m V McKusick, Mendehan Inhentance m 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 (comhe ⁇ tance 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
  • the polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as immunogens to produce antibodies immunospecific for the CBDARA04 polypeptides
  • immunospecific means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides the pnor art Antibodies generated against the CBDARA04 polypeptides can be obtained by administering the polypeptides or epitope-beanng 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 mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today ( 1983) 4 72)
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to punfy the polypeptides by affinity chromatography
  • Antibodies against CBDARA04 polypeptides may also be employed to treat cancer and AIDS, 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 CBDARA04 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer and AIDS, among others
  • Yet another aspect of the mvention relates to a method of inducing immunological response in a mammal which compnses, delivering CBDARA04 polypeptide via a vector directing expression of CBDARA04 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases
  • an lmmunological/vaccine formulation composition which, when introduced into a mammalian host, induces an immunological response in that mammal to a CBDARA04 polypeptide wherein the composition comprises a CBDARA04 polypeptide or CBDARA04 gene
  • the vaccine formulation may further comprise a suitable carrier Since CBDARA04 polypeptide may be broken down in the stomach, it is preferably administered
  • the CBDARA04 polypeptide of the present mvention may be employed m a screenmg process for compounds which activate (agonists) or inhibit activation of (antagomsts, or otherwise called inhibitors) the CBDARA04 polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonist or antagomsts from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • agonists or antagomsts 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 Cokgan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991)
  • CBDARA04 polypeptides are responsible for many biological functions, including many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBDARA04 polypeptide on the one hand and which can inhibit the function of CBDARA04 polypepude on the other hand
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer and AIDS
  • Antagonists may be employed for a vanety of therapeutic and prophylactic purposes for such conditions as cancer and AIDS
  • such screening procedures may mvolve usmg appropnate cells which express the CBDARA04 polypeptide or respond to CBDARA04 polypeptide of the present mvention
  • Such cells mclude cells from mammals, yeast, Drosoph ⁇ a or E coli Cells which express the CBDARA04 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBDARA04 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 CBDARA04 activity
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the CBDARA04 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 CBDARA04 polypeptide, using detection systems appropriate to the cells bearing the CBDARA04 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 CBDARA04 polypeptide to form a mixture, measuring CBDARA04 activity in the mixture, and comparing the CBDARA04 activity of the mixture to a standard
  • the CBDARA04 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 CBDARA04 mRNA and protein in cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of CBDARA04 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 CBDARA04 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBDARA04 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, hgand binding and crosshnkmg assays in which the CBDARA04 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotmylated), 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 punfication and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBDARA04 which compete with the binding of CBDARA04 to its receptors, if any Standard methods for conducting screening assays are well understood in the art
  • Examples of potential CBDARA04 polypeptide antagonists mclude antibodies or, m some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the CBDARA04 polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • the present mvention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for CBDARA04 polypeptides, or compounds which decrease or enhance the production of CBDARA04 polypeptides, which comprises
  • This mvention provides methods of treating abnormal conditions such as, cancer and AIDS, related to both an excess of and insufficient amounts of CBDARA04 polypeptide activity
  • CBDARA04 polypeptide If the activity of CBDARA04 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 m an amount effective to inhibit the function of the CBDARA04 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 CBDARA04 polypeptides still capable of binding the gand, substrate, enzymes, receptors, etc in competition with endogenous CBDARA04 polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBDARA04 polypeptide
  • expression of the gene encoding endogenous CBDARA04 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 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)
  • oligonucleotides which form triple helices ith 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 oligomers can be administered per se or the relevant oligomers can be expressed in vivo
  • One approach compnses administering to a subject a therapeutically effective amount of a compound which activates CBDARA
  • Peptides such as the soluble form of CBDARA04 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m 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 admmistration, and is well within the skill of the art
  • the mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • systemic admmistration of the pharmaceutical compositions mclude injection, typically by intravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or mtrape ⁇ toneal
  • Alternative means for systemic admmistration include transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents
  • oral admmistration may also be possible
  • Admmistration 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 admmistration, 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 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 admmistration by mtravenous injection Vanations m these dosage levels can be adjusted usmg standard empincal routines for optimization, as is well understood the art Polypeptides used m treatment can also be generated endogenously m the subject, m treatment modalities often referred to as "gene therapy" as desenbed 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
  • TAATTCCCAC TAGAAGCTGT CCATGCCATA CAGAAGATCT ATTAAAAATG TTTTAAATGG 600

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBDARA04 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 CBDARA04 dans la conception de protocoles pour le traitement de cancers et du SIDA, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000034 1998-03-12 1998-03-12 Gene humain p18 (cbdara04) WO1999046292A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000034 WO1999046292A1 (fr) 1998-03-12 1998-03-12 Gene humain p18 (cbdara04)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000034 WO1999046292A1 (fr) 1998-03-12 1998-03-12 Gene humain p18 (cbdara04)

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WO1999046292A1 true WO1999046292A1 (fr) 1999-09-16

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1617875A1 (fr) * 2004-04-27 2006-01-25 Seoul National University Industry Foundation Nouvelle utilisation de la proteine 3 multifonctionnelle interagissant avec l'aminoacyl-tarn synthase (aim3) comme suppresseur tumoral

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DDBJ: LOCUS AB011079, Accession AB011079, 21 Feb. 1998, MOTEGI H., NODA T., SHIBA K., "Cloning of cDNA for Human p18 from Human Testis". *
GENBANK: LOCUS CGU67146, Accession U67146, QUEVILLON S., MIRANDE M., "The p18 Component of the Multisynthetase COmplex Shares a Protein Motif with the beta and gamma Subunits of Eukaryotic Elongation Factor 1"; & FEBS LETT., 395(1), 14 oct. 1996, pages 63-67. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1617875A1 (fr) * 2004-04-27 2006-01-25 Seoul National University Industry Foundation Nouvelle utilisation de la proteine 3 multifonctionnelle interagissant avec l'aminoacyl-tarn synthase (aim3) comme suppresseur tumoral
EP1617875A4 (fr) * 2004-04-27 2007-10-10 Seoul Nat Univ Ind Foundation Nouvelle utilisation de la proteine 3 multifonctionnelle interagissant avec l'aminoacyl-tarn synthase (aim3) comme suppresseur tumoral
JP2007534748A (ja) * 2004-04-27 2007-11-29 ソウル ナショナル ユニバーシティー インダストリー ファウンデーション 腫瘍抑制因子として作用するａｉｍ３の新規用途
US7902165B2 (en) * 2004-04-27 2011-03-08 Seoul National University Industry Foundation Use of AIM3 acting as a tumor suppressor
JP4772782B2 (ja) * 2004-04-27 2011-09-14 ソウル ナショナル ユニバーシティー インダストリー ファウンデーション 腫瘍抑制因子として作用するａｉｍ３の新規用途

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