WO1999047667A1 - Gene humain de sous-unite gamma sec61: cbdame07 - Google Patents

Gene humain de sous-unite gamma sec61: cbdame07 Download PDF

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Publication number
WO1999047667A1
WO1999047667A1 PCT/CN1998/000046 CN9800046W WO9947667A1 WO 1999047667 A1 WO1999047667 A1 WO 1999047667A1 CN 9800046 W CN9800046 W CN 9800046W WO 9947667 A1 WO9947667 A1 WO 9947667A1
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Prior art keywords
polypeptide
cbdame07
seq
polynucleotide
nucleotide sequence
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PCT/CN1998/000046
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English (en)
Inventor
Jisheng Wu
Mao Mao
Gang Fu
Yu Shen
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Shanghai Second Medical University
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Priority to PCT/CN1998/000046 priority Critical patent/WO1999047667A1/fr
Publication of WO1999047667A1 publication Critical patent/WO1999047667A1/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
    • 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 protein transporter sec61 family, hereinafter referred to as CBDAME07 . The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
  • the protein transporter sec61 family is highly conserved in organisms including C. elegans, yeast, rice to mammals. This indicates that the protein transporter sec ⁇ l family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further members of the protein transporter sec61 family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer, AIDS, metabolic disorders, and insulin dependent diabetes mellitus (IDDM).
  • IDDM insulin dependent diabetes mellitus
  • the invention relates to CBDAME07 polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such CBDAME07 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 CBDAME07 imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBDAME07 activity or levels.
  • CBDAME07 refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NOJ or an allelic variant thereof.
  • CBDAME07 activity or CBDAME07 polypeptide activity refers to the metabolic or physiologic function of said CBDAME07 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said CBDAME07
  • CBDAME07 gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or alle c vanants thereof and/or their complements
  • Antibodies as used herem includes polyclonal and monoclonal antibodies, chimenc, smgle chain, and humamzed antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulin expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated ' but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated”, as the term is employed herein
  • Polynucleotide generally refers to any polynbonucleotide 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 smgle- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsing DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions comprising RNA or DNA or both RNA and DNA The term polynucleotide also mcludes DNAs or RNAs contammg one or more modified bases and DNAs
  • 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
  • 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
  • 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 vanant, or it may be a vanant that is not known to occur naturally
  • Non-naturally occurring vanants 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 companng 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, Biocomputmg Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Gnffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Pnmer, Gn
  • polypeptide sequence companson examples include the following 1) Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4
  • Preferred polynucleotide embodiments further mclude an isolated polynucleotide comprising a polynucleotide havmg 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 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, mterspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides
  • 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%
  • any non-integer product of xn and y is rounded down to the nearest integer 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 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 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 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 NO:2 by the numerical percent of the respective percent identity and subtracting that product from said total number of amino acids in SEQ ID NO:2, or:
  • na is the number of amino acid alterations
  • xa 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 xa and y is rounded down to the nearest integer prior to subtracting it from xa.
  • the present invention relates to CBDAME07 polypeptides (or CBDAME07 proteins).
  • the CBDAME07 polypeptides include the polypeptide of SEQ ID NOJ; 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 NOJ 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.
  • CBDAME07 polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NOJ 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 Preferably CBDAME07 polypeptide exhibit at least one biological activity of CBDAME07
  • the CBDAME07 polypeptides may be m the form of the "mature" protem or may be a part of a larger protem such as a fusion protem It is often advantageous to mclude an additional ammo acid sequence which contams 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 CBDAME07 polypeptides
  • fragments may be "free-standmg,” or compnsed 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 ammo acid number 1-20, 21-40, 41-60, 61- 80, 81-100, and 101 to the end of CBDAME07 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 havmg the ammo acid sequence of CBDAME07 polypeptides, except for deletion of a continuous senes of residues that mcludes the amino 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 compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigemc mdex regions
  • Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate
  • the CBDAME07 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.
  • CBDAME07 polynucleotides include isolated polynucleotides which encode the CBDAME07 polypeptides and fragments, and polynucleotides closely related thereto. More specifically, CBDAME07 polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a CBDAME07 polypeptide of SEQ ID NO: 2, and polynucleotide having the particular sequence of SEQ ID NO: 1.
  • CBDAME07 polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBDAME07 polypeptide of SEQ ID NOJ, and a polynucleotide comprising a nucleotide sequence that is at least 80% 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.
  • CBDAME07 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO:l to hybridize under conditions useable for amplification or for use as a probe or marker.
  • the invention also provides polynucleotides which are complementary to such CBDAME07 polynucleotides.
  • the CBDAME07 of the invention is structurally related to other proteins of the protein transporter sec61 family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1) encoding human CBDAME07.
  • the cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 91 to 294) encoding a polypeptide of 68 amino acids of SEQ ID NOJ.
  • the amino acid sequence of Table 2 (SEQ ID NOJ) has about 99% identity (using FASTA) in 68 amino acid residues with canine and murine Sec61 gamma subunit (E. Hartmann, et al. Nature. 1994, 367: 654- 657).
  • the nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 70.1% identity (using FASTA) in 482 nucleotide residues with canine and murine Sec61 gamma subunit (E. Hartmann, et al. Nature 1994, 367: 654-657).
  • CBDAME07 polypeptides and polynucleotides of the present invention are expected to have, inter aha, 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 CBDAME07 (SEQ ID NO: 1).
  • One polynucleotide of the present invention encoding CBDAME07 may be obtained using standard cloning and screening, from a cDNA library 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) 55:632-634; 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 usmg well known and commercially available techniques
  • the nucleotide sequence encodmg CBDAME07 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encodmg sequence contained m Table 1 (nucleotide number 91 to 294 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 mclude the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment m readmg frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem 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-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc NatlAcad Set USA (1989) 86 821- 824, or is an HA tag
  • the polynucleotide may also contain non-coding 5 '
  • compositions encodmg CBDAME07 vanants compnse the ammo acid sequence CBDAME07 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 invention further relates to polynucleotides that hybndize to the herem above- descnbed sequences
  • the present invention especially relates to polynucleotides which hybndize under stringent conditions to the herem above-descnbed polynucleotides
  • stringent 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 invention 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 CBDAME07 polypeptide and to isolate cDNA and genomic clones of other genes (mcludmg genes encodmg homologs and orthologs from species other than human) that have a high sequence similanty to the CBDAME07 gene
  • Such hybndization 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 compnse at least 15 nucleotides Preferably, such probes will have at
  • 10 least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
  • CBDAME07 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.
  • CBDAME07 polypeptides are polypeptide 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 a ⁇ ., 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.
  • appropnate hosts mclude bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs 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, and plant cells
  • chromosomal, episomal and virus-denved systems e g , vectors denved from bactenal plas ⁇ uds, 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, adenovirus
  • appropnate secretion signals may be incorporated into the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • CBDAME07 polypeptide is to be expressed for use in screemng assays, generally, it is preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use m the screening assay If CBDAME07 polypeptide is secreted mto the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered
  • CBDAME07 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techmques for refoldmg proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication
  • This invention also relates to the use of CBDAME07 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBDAME07 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 CBDAME07 Individuals carrying mutations m the CBDAME07 gene may be detected at the DNA level by a vanety of techmques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, 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 techmques pnor to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions can be detected by a change m size of the amplified product in companson to the normal genotype
  • Pomt mutations can be identified by hybndizing amplified DNA to labeled CBDAME07 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 electiophoretic 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
  • cancer, AIDS, metabolic disorders, and IDDM can be diagnosed by methods comprising determining from a sample denved from a subject an abnormally decreased or increased level of CBDAME07 polypeptide or CBDAME07 mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybndization methods Assay techmques that can be used to determme levels of a protem, such as an CBDAME07 polypeptide, m a sample denved from a host are well-
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present mvention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly cancer, AIDS, metabolic disorders, and IDDM , which compnses
  • CBDAME07 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,
  • CBDAME07 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 accordmg to the present mvention is an important first step m 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
  • genetic map data are found, for example, m V McKusick, Mendehan Inhentance m Man (available on lme through Johns Hopkins Umversity Welch Medical Library)
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (comhentance 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 m some or all of the affected individuals but not m 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 expressing them can also be used as lmmunogens to produce antibodies lmmunospecific for the CBDAME07
  • Antibodies generated against the CBDAME07 polypeptides can be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tnoma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 472) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)
  • 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 CBDAME07 polypeptides may also be employed to treat cancer, AIDS, metabolic disorders, and IDDM , among others
  • Vaccines Another aspect of the mvention relates to a method for mducmg an lmmunological response in a mammal which compnses moculatmg the mammal with CBDAME07 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 mducmg lmmunological response m a mammal which comprises, dehvermg CBDAME07 polypeptide via a vector directmg expression of CBDAME07 polynucleotide in vivo m order to mduce such an lmmunological response to produce antibody to protect said animal from diseases
  • composition which, when introduced into a mammalian host, induces an lmmunological response in that mammal to a CBDAME07 polypeptide wherem the composition comprises a CBDAME07 polypeptide or CBDAME07 gene
  • the vaccine formulation may further compnse a suitable carrier Since CBDAME07 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, mtradermal etc injection)
  • parenterally including subcutaneous, intramuscular, intravenous, mtradermal etc injection
  • Formulations suitable for parenteral administration mclude 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 CBDAME07 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 CBDAME07 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.
  • These 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 et al. , Current Protocols in Immunology l(2):Chapter 5 (1991).
  • CBDAME07 polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate CBDAME07 polypeptide on the one hand and which can inhibit the function of CBDAME07 polypeptide on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer, AIDS, metabolic disorders, and IDDM .
  • Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as cancer, AIDS, metabolic disorders, and IDDM .
  • such screening procedures may involve using appropriate cells which express the CBDAME07 polypeptide or respond to CBDAME07 polypeptide of the present invention.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • Cells which express the CBDAME07 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBDAME07 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 CBDAME07 activity.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the CBDAME07 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or m 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 CBDAME07 polypeptide, using detection systems appropnate to the cells beanng the CBDAME07 polypeptide Inhibitors of activation are generally assayed m the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed
  • the assays may simply compnse the steps of mixing a candidate compound with a solution contammg a CBDAME07 polypeptide to form a mixture, measurmg CBDAME07 activity m the mixture, and comparing the CBDAME07 activity of the mixture to a standard
  • the CBDAME07 cDNA, protein and antibodies to the protem may also be used to configure assays for detectmg the effect of added compounds on the production of CBDAME07 mRNA and protein m cells
  • an ELISA may be constructed for measunng secreted or cell associated levels of CBDAME07 protem 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 CBDAME07 (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues
  • the CBDAME07 protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor bindmg techmques known in the art These mclude, but are not limited to, hgand bindmg and crosshnking assays in which the CBDAME07 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or punfication, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods mclude biophysical techmques 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 CBDAME07 which compete with the binding of CBDAME07 to its receptors, if any Standard methods for conducting screemng assays are well understood in the art Examples of potential CBDAME
  • the present invention relates to a screening kit for identifying agonists, antagonists, Hgands, receptors, substrates, enzymes, etc. for CBDAME07 polypeptides; or compounds which decrease or enhance the production of CBDAME07 polypeptides, which comprises: (a) a CBDAME07 polypeptide, preferably that of SEQ ID NOJ;
  • kits may comprise a substantial component.
  • This invention provides methods of treating abnormal conditions such as, cancer, AIDS, metabolic disorders, and IDDM , related to both an excess of and insufficient amounts of CBDAME07 polypeptide activity.
  • CBDAME07 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 CBDAME07 polypeptide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of CBDAME07 polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous CBDAME07 polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the CBDAME07 polypeptide.
  • expression of the gene encoding endogenous CBDAME07 polypeptide can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
  • oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al, Nucleic Acids Res (1979) 3:173; Cooney et al, Science (1988) 241:456; Dervan ef ⁇ /, Science (1991) 251: 1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • One approach comprises administering to a subject a
  • a compound which activates CBDAME07 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 CBDAME07 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.
  • producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
  • gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).
  • Another approach is to administer a therapeutic amount of CBDAME07 polypeptides in combination with a suitable pharmaceutical carrier.
  • Peptides such as the soluble form of CBDAME07 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 J - 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 RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • ADDRESSEE RATNER & PRESTIA
  • STREET P.O. BOX 980
  • NAME PRESTIA, PAUL F

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBDAME07 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 CBDAM07 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/CN1998/000046 1998-03-18 1998-03-18 Gene humain de sous-unite gamma sec61: cbdame07 WO1999047667A1 (fr)

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PCT/CN1998/000046 WO1999047667A1 (fr) 1998-03-18 1998-03-18 Gene humain de sous-unite gamma sec61: cbdame07

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7179890B2 (en) 1998-12-22 2007-02-20 Janssen Pharmaceutica.N.V. Vascular endothelial growth factor-x

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENBANK, gb/U11027/MMU11027, 30 Jun. 1994, YOTOV W.V. and St-ARNAUD R., "Nucleotide Sequence of Mouse Sec61-gamma cDNA". *
J. CELL BIOL., 134(3), (1996), IWASAKI K. et al., "Emo-1, a Caenorhabditis Elegans Sec61p gamma Homologue, is Required for Oocyte Development and Ovulation", pages 699-714. *
NATURE, 367(6464), (1994), HARTMANN E. et al., "Evolutionary Conservation of Components of the Protein", pages 654-657. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7179890B2 (en) 1998-12-22 2007-02-20 Janssen Pharmaceutica.N.V. Vascular endothelial growth factor-x

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