WO1999062947A1 - Gene homologue ci-pdsw de la sous-unite de l'ubiquinone oxyreductase humaine (cblaic08) - Google Patents

Gene homologue ci-pdsw de la sous-unite de l'ubiquinone oxyreductase humaine (cblaic08) Download PDF

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Publication number
WO1999062947A1
WO1999062947A1 PCT/CN1998/000084 CN9800084W WO9962947A1 WO 1999062947 A1 WO1999062947 A1 WO 1999062947A1 CN 9800084 W CN9800084 W CN 9800084W WO 9962947 A1 WO9962947 A1 WO 9962947A1
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Prior art keywords
polypeptide
identity
seq
ofthe
amino acid
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PCT/CN1998/000084
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English (en)
Inventor
Yu Shen
Min Ye
Jisheng Wu
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Shanghai Second Medical University
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Priority to PCT/CN1998/000084 priority Critical patent/WO1999062947A1/fr
Publication of WO1999062947A1 publication Critical patent/WO1999062947A1/fr

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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)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists, antagonists and/or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
  • the present invention relates to CBLAIC08, in particular CBLAIC08 polypeptides and CBLAIC08 polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of AIDS, cancer, autoimmune disease, hepatitis, and diabetes, hereinafter referred to as "the Diseases", amongst others.
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with CBLAIC08 imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBLAIC08 activity or levels.
  • the present invention relates to CBLAIC08 polypeptides.
  • Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2
  • Such polypeptides mclude those compnsmg the ammo acid of SEQ ID NO 2
  • peptides of the present mvention mclude isolated polypeptides in which the ammo acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2
  • polypeptides include the polypeptide of SEQ ID NO 2
  • peptides of the present mvention mclude isolated polypeptides encoded by a polynucleotide compnsmg the sequence contained m SEQ ID NO 1
  • Polypeptides of the present mvention are believed to be members of the ubiqumone oxidoreductase family of polypeptides They are therefore of interest because ubiqumone oxidoreductase is a mitochondria protein complex encoded by nuclear genome, it is composed of many subumts, and it participates m metabolism These properties are hereinafter refened to as "CBLAIC08 activity” or “CBLAIC08 polypeptide activity” or “biological activity of CBLAIC08” Also included amongst these activities are antigenic and immunogenic activities of said CBLAIC08 polypepudes, m particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO 2 Preferably, a polypeptide of the present mvention exhibits at least one biological activity of CBLAIC08
  • the polypeptides of the present mvention 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
  • polynucleotides of the present mvention mclude isolated polynucleotides compnsing a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO 1 over the entire length of SEQ ID NO 1
  • polynucleotides which have at least 97% identity are highly prefened, whilst those with at least 98-99% identiy are more highly preferred, and those with at least 99% identity are most highly prefened
  • Such polynucleotides mclude a polynucleotide compnsmg the polynucleotide of SEQ LD NO 1 as well as the polynucleotide of SEQ ID NO 1
  • the invention also provides polynucleotides which are complementary to all the above descnbed polynucleotides
  • the nucleotide sequence of SEQ ID NO 1 shows homology with X63224,bovme ubiqumone oxidoreductase complex subumt CI-PDSW (J E Walker, et al J Mol Biol 1992,226 1051-1072 )
  • the nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and compnses a polypeptide encodmg sequence (nucleotide 63 to 578) encodmg a polypeptide of 172 ammo acids, the polypeptide of SEQ LD NO 2
  • the nucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained m SEQ ID NO 1 or it may be a sequence other than the one contained m SEQ ID NO 1, which, as a result of
  • Prefened polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, prefened polypeptides and polynucleotides of the present invention have at least one CBLAIC08 activity.
  • Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA hbrary derived from mRNA in cells of human umbilical 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
  • 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 polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gmtz et al., ProcNatlAcadSci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polypeptide variants which comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination.
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to SEQ ID NO: 1.
  • PCR nucleic acid amplification
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent
  • the probes or p ⁇ mers will generally compnse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides Particularly prefened probes will have between 30 and 50 nucleotides
  • a polynucleotide encoding a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than human, may be obtamed by a process which comp ⁇ ses the steps of screenmg an appropnate library under st ⁇ ngent hybndization conditions with a labeled probe havmg the sequence of SEQ LD NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence
  • Such hybndization techniques are well known
  • an isolated cDNA sequence will be mcomplete, m that the region coding for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transc ⁇ ptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remam attached to the template dunng the polymerisation reaction), failing to complete a DNA copy of the mRNA template dunng 1st strand cDNA synthesis
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems 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. For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Prefened such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
  • Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen ofthe endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • a polypeptide ofthe present invention is to be expressed for use in screening assays, it is generally prefened that the polypeptide be produced at the surface ofthe cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides ofthe present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphoceUulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. This invention also relates to the use of polynucleotides ofthe present invention as diagnostic reagents.
  • Detection of a mutated form ofthe gene characterised by the polynucleotide of SEQ ID NO: 1 which is 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 ofthe gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acids for diagnosis may be obtained from a subject's ceUs, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled CBLAIC08 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
  • DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (ee, 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 etal., ProcNatlAcadSci USA (1985) 85: 4397-4401).
  • an anay of oligonucleotides probes comprising CBLAIC08 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the CBLAIC08 gene by the methods described.
  • diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any ofthe methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • the present invention relates to a diagonostic kit which comprises:
  • a polynucleotide ofthe present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ;
  • polypeptide ofthe present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof;
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or suspectability to a disease, particularly AIDS, cancer, autoimmune disease, hepatitis, and diabetes, amongst others.
  • the nucleotide sequences ofthe present invention are also valuable for chromosome identification.
  • the sequence is specificaUy targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in conelating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be conelated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • 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 ofthe affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent ofthe disease.
  • polypeptides ofthe invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for polypeptides ofthe present invention.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides ofthe invention than their affinity for other related polypeptides in the prior art.
  • Antibodies generated against polypeptides ofthe present invention may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G.
  • No. 4,946,778 can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • Antibodies against polypeptides ofthe present invention may also be employed to treat the Diseases, amongst others.
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide ofthe present invention, or a fragment thereof, and various portions ofthe constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part ofthe heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
  • a further aspect ofthe invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914.
  • Another aspect ofthe invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with a polypeptide ofthe present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others.
  • Yet another aspect ofthe invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide ofthe present invention via a vector directing expression ofthe polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • a further aspect ofthe invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide ofthe present invention wherein the composition comprises a polypeptide or polynucleotide ofthe present invention.
  • the vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood ofthe 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 ofthe sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity ofthe vaccine and can be readily determined by routine experimentation.
  • Polypeptides ofthe present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function ofthe polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function ofthe polypeptide.
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.
  • Compounds may be identified from a variety of sources, for example, ceUs, cell-free preparations, chemical libraries, and natural product mixtures.
  • Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, ofthe polypeptide; or may be structural or functional mimetics thereof (see Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve competition with a labeled competitor.
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition ofthe polypeptide, using detection systems appropriate to the cells bearing the 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 ofthe candidate compound is observed.
  • Constitutively active polpypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation ofthe polypeptide. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide ofthe present invention, to form a mixture, measuring CBLAIC08 activity in the mixture, and comparing the CBLAIC08 activity ofthe mixture to a standard. Fusion proteins, such as those made from Fc portion and CBLAIC08 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide ofthe present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995)).
  • polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells.
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source ofthe putative receptor ⁇
  • a radioactive isotope for instance, ⁇ 1
  • chemically modified for instance, biotinylated
  • polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc., as the case may be, ofthe polypeptide, e.g., a fragment ofthe hgands, substrates, receptors, enzymes, etc.; or smaU molecules which bind to the polypeptide ofthe present invention but do not elicit a response, so that the activity ofthe polypeptide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides ofthe present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:
  • polypeptide of the present invention (c) a cell membrane expressing a polypeptide ofthe present invention; or (d) antibody to a polypeptide ofthe present invention; which polypeptide is preferably that of SEQ ID NO:2.
  • kits may comprise a substantial component.
  • polypeptide ofthe present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor ofthe polypeptide, by:
  • the present invention provides methods of treating abnormal conditions such as, for instance, AIDS, cancer, autoimmune disease, hepatitis, and diabetes, related to either an excess of, or an under-expression of, CBLAIC08 polypeptide activity.
  • One approach comprises administering to a subject in need thereof an inhibitor compound (antagonist) as hereinabove described, optionaUy in combination with a pharmaceuticaUy acceptable carrier, in an amount effective to inhibit the function ofthe polypeptide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby aUeviating the abnormal condition.
  • soluble forms ofthe polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide may be administered. Typical examples of such competitors include fragments ofthe CBLAIC08 polypeptide.
  • expression ofthe gene encoding endogenous CBLAIC08 polypeptide can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al, Nucleic Acids Res (1979) 3:173; Cooney et al, Science (1988) 241:456; Dervan et ⁇ /., Science (1991) 251:1360). These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo.
  • a therapeuticaUy effective amount of a compound which activates a polypeptide ofthe present invention i.e., an agonist as described above
  • a pharmaceutically acceptable carrier i.e., a pharmaceutically acceptable carrier
  • gene therapy may be employed to effect the endogenous production of CBLAIC08 by the relevant cells in the subject.
  • a polynucleotide ofthe 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 ofthe present invention such that the packaging ceU now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression ofthe polypeptide in vivo.
  • Another approach is to administer a therapeutic amount of a polypeptide ofthe present invention in combination with a suitable pharmaceutical carrier.
  • the present invention provides for pharmaceutical compositions comprising a therapeuticaUy effective amount of a polypeptide, such as the soluble form of a polypeptide ofthe present invention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceuticaUy acceptable carrier or excipient.
  • a polypeptide such as the soluble form of a polypeptide ofthe present invention, agonist/antagonist peptide or small molecule compound
  • a pharmaceuticaUy acceptable carrier or excipient include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers fiUed with one or more ofthe ingredients ofthe aforementioned compositions ofthe invention.
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route.
  • Prefened forms of systemic administration 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 or other compounds ofthe present invention, the route of administration, the nature ofthe formulation, the nature ofthe subject's condition, and the judgment ofthe attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view ofthe 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 weU understood in the art.
  • Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often refened 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.
  • Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology. This is most easily facilitated by storing the sequence in a computer readable medium and then using the stored data to search a sequence database using weU known searching tools, such as GCC. Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO:l and/or a polypeptide sequence encoded thereby.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides .
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational 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 may 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 to 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 post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, PROTEINS - STRUC
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence ofthe 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.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, 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 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, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, 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.
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1, wherein said polynucleotide sequence may be identical to the reference 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 where
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NOT
  • 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%
  • • is the symbol for the multiplication operator, and wherein 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 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.
  • a polynucleotide sequence ofthe present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions ofthe reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or:
  • n n is the number of amino acid alterations
  • x n is the total number of amino acids in SEQ ID NO:2
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • 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 polypeptide sequence may be identical to the reference 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 ofthe 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 integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:
  • n a is the number of amino acid alterations
  • x a 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%
  • • is the symbol for the multiplication operator, 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 .
  • a polypeptide sequence ofthe present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such 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 ofthe 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.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or:
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, 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 .
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262].

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Abstract

La présente invention concerne des polypeptides et des polynucléotides CBLAIC08 et des procédés permettant de produire ces polypeptides par des techniques de recombinaison. L'invention se rapporte également à des procédés d'utilisation de ces polypeptides et polynucléotides CBLAIC08 en thérapie et à des dosages diagnostiques de ces derniers.
PCT/CN1998/000084 1998-06-04 1998-06-04 Gene homologue ci-pdsw de la sous-unite de l'ubiquinone oxyreductase humaine (cblaic08) WO1999062947A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/CN1998/000084 WO1999062947A1 (fr) 1998-06-04 1998-06-04 Gene homologue ci-pdsw de la sous-unite de l'ubiquinone oxyreductase humaine (cblaic08)

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PCT/CN1998/000084 WO1999062947A1 (fr) 1998-06-04 1998-06-04 Gene homologue ci-pdsw de la sous-unite de l'ubiquinone oxyreductase humaine (cblaic08)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014128680A1 (fr) * 2013-02-25 2014-08-28 L'oreal Composition cosmétique de type gel

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENOMICS, 35(1), (1996), GU J.Z. et al., "The Human B22 Subunits of the NADH:Ubiquinone Oxidoreductase Maps to the Region of Chromosome 8 Involved in Branchio-oto-renal Syndrome", pages 6-10. *
J. MOL. BIOL., 226(4), (1992), WALKER J.E. et al., "Sequences of 20 Subunits of NADH:Ubiquinone Oxidoreductase from Bovine Heart Mitochondria, Application of a Novel Strategy for Sequencing Proteins Using the Polymerase Chain Reaction", pages 1051-1072. *
PEDIATRIC RESEARCH, 39(3), 1996, LANE R.H. et al., "Altered Hepatic Gene Expression of Enzymes Involved in Energy Metabolism in the Growth-Retarded Fetal Rat", pages 390-394. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014128680A1 (fr) * 2013-02-25 2014-08-28 L'oreal Composition cosmétique de type gel
FR3002449A1 (fr) * 2013-02-25 2014-08-29 Oreal Composition cosmetique de type gel
EP3473233A1 (fr) * 2013-02-25 2019-04-24 L'oreal Composition cosmétique de type gel

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