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WO1999047666A1 - Gene humain homologue de ci-mnll: cbdfmd04 - Google Patents

Gene humain homologue de ci-mnll: cbdfmd04 Download PDF

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Publication number
WO1999047666A1
WO1999047666A1 PCT/CN1998/000045 CN9800045W WO9947666A1 WO 1999047666 A1 WO1999047666 A1 WO 1999047666A1 CN 9800045 W CN9800045 W CN 9800045W WO 9947666 A1 WO9947666 A1 WO 9947666A1
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Prior art keywords
polypeptide
cbfbmd04
seq
polynucleotide
nucleotide sequence
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Application number
PCT/CN1998/000045
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English (en)
Inventor
Mao Mao
Gang Fu
Juan Zhou
Kaili He
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Shanghai Second Medical University
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Publication date
Application filed by Shanghai Second Medical University filed Critical Shanghai Second Medical University
Priority to PCT/CN1998/000045 priority Critical patent/WO1999047666A1/fr
Publication of WO1999047666A1 publication Critical patent/WO1999047666A1/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)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present mvention relate to the NADH dehydrogenase complex family, hereinafter referred to as CBFBMD04 The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides
  • NADH dehydrogenase is a mitochondnal complex composed of many subunits and is associated with metabolism
  • the CI-MNLL protein is a subunit of NADH dehydrogenase This indicates that the NADH dehydrogenase complex family has an established, proven history as therapeutic targets Clearly there is a need for identification and characterization of further members of the NADH dehydrogenase complex 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 melhtus (IDDM)
  • IDDM insulin dependent diabetes melhtus
  • the invention relates to CBFBMD04 polypeptides and recombinant matenals and methods for their production
  • Another aspect of the invention relates to methods for using such CBFBMD04 polypeptides and polynucleotides
  • Such uses include the treatment of cancer, AIDS, metabolic disorders, and IDDM, among others
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBFBMD04 imbalance with the identified compounds
  • CBFBMD04 refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO 2 or an allehc variant thereof
  • CBFBMD04 activity or CBFBMD04 polypeptide activity refers to the metabolic or physiologic function of said CBFBMD04 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said CBFBMD04
  • CBFBMD04 gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allehc variants thereof and/or their complements
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation smgle- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg 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 triple-stranded regions comprising RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for
  • 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, o gopeptides or ohgomers, and to
  • Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids
  • Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present m the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branchmg Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include ace
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide 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 m 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
  • a variant and reference polypeptide are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described m (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, G ⁇ ffin,
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990)
  • the well known Smith Waterman algorithm may also be used to determine identity
  • Preferred parameters for polypeptide sequence comparison include the following l) Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)
  • Preferred parameters for polynucleotide compa ⁇ son include the following
  • Preferred polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polynucleotide reference sequence of SEQ ID NO 1, wherein said reference sequence may be identical to the sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and trans version, or msertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 by the
  • nn is the number of nucleotide alterations
  • xn is the total number of nucleotides
  • SEQ ID NO 1 and y is 0 50 for 50%. 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and wherein any non-integer product of xn and y is rounded down to the nearest mteger prior to subtracting it from ⁇ n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense
  • 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 ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the nume ⁇ cal percent of the respective percent identity and subtracting that product from said total number of am
  • na is the number of amino acid alterations
  • xa is the total number of amino acids in SEQ ID NO 2
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and wherein any non-integer product of xa and y is rounded down to the nearest integer p ⁇ or to subtracting it from xa
  • the present invention relates to CBFBMD04 polypeptides (or CBFBMD04 proteins)
  • the CBFBMD04 polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comprising the amino acid sequence of SEQ ID NO 2, and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99%> are highly preferred Also included within CBFBMD04 polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO 2 over
  • CBFBMD04 polypeptide exhibit at least one biological activity of CBFBMD04
  • the CBFBMD04 polypeptides may be in the form of the "mature" protein or may be a part of a larger protem such as a fusion protein It is often advantageous to mclude an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidme residues, or an additional sequence for stability during recombinant production
  • a fragment is a polypeptide havmg an ammo acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned CBFBMD04 polypeptides
  • fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region
  • Representative examples of polypeptide fragments of the invention, m clude, for example, fragments from about ammo acid number 1-20, 21-40, 41-60, 61- 80, 81 - 100, and 101 to the end of CBFBMD04 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 CBFBMD04 polypeptides, except for deletion of a continuous senes of residues that mcludes the amino termmus, or a contmuous senes of residues that mcludes the carboxyl termmus or deletion of two continuous senes of residues, one including the ammo termmus and one including 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, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic index regions
  • Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate CBFBMD04 activity, including those with a
  • the CBFBMD04 polypeptides of the invention can be prepared m any suitable manner Such polypeptides mclude isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood m the art
  • CBFBMD04 polynucleotides mclude isolated polynucleotides which encode the CBFBMD04 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBFBMD04 polynucleotide of the mvention mclude a polynucleotide compnsing the nucleotide sequence contained in SEQ ID NO 1 encoding a CBFBMD04 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBFBMD04 polynucleotides further mclude a polynucleotide compnsing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBFBMD04 polypeptide of SEQ ID NO 2, and a polynucleot
  • CBFBMD04 of the mvention is structurally related to other proteins of the NADH dehydrogenase complex family, as shown by the results of sequencmg the cDNA of Table 1 (SEQ ID NO 1) encoding human CBFBMD04
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 163 to 336) encoding a polypeptide of 58 ammo acids of SEQ ID NO 2
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 81% identity (usmg FASTA) m 58 ammo acid residues with rat CI-MNLL mRNA for ubiqumone oxidoreductase (J E Walker, et al J Mol Biol 1992, 226 1051-1072)
  • the nucleotide sequence of Table 1 (SEQ ID NO 1) has about 55% identity (usmg FASTA) m 437 nucleotide residues with rat CI-MN
  • One polynucleotide of the present mvention encoding CBFBMD04 may be obtained usmg standard clonmg and screenmg, from a cDNA library denved from mRNA m cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M D , et al Science (1991) 252 1651- 1656, Adams, M D et al , Nature, (1992) 355 632-634, Adams, M O , et al , Nature (1995) 377 Supp 3-174)
  • EST expressed sequence tag
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques
  • the nucleotide sequence encoding CBFBMD04 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 163 to 336 of SEQ ID NO 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2
  • the polynucleotide may include the codmg sequence for the mature polypeptide or a fragment thereof, by itself, the codmg sequence for the mature polypeptide or fragment m readmg frame with other codmg sequences, such as those encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protem 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 m the pQE vector (Qiagen, Inc ) and desc ⁇ bed in Gentz et al , Proc Natl Acad Set USA ( 1989) 86 821 - 824, or is an HA tag
  • the polynucleotide may include the codmg sequence for the mature polypeptide or a fragment thereof, by itself, the codmg sequence for the mature polypeptide or fragment m readmg frame with other
  • polynucleotides encodmg CBFBMD04 vanants comp ⁇ se the ammo acid sequence CBFBMD04 polypeptide of Table 2 (SEQ ID NO 2) m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination
  • the present mvention further relates to polynucleotides that hyb ⁇ dize to the herein above- desenbed sequences
  • the present mvention especially relates to polynucleotides which hyb ⁇ dize under stringent conditions to the herein above-desenbed polynucleotides
  • stringent conditions means hyb ⁇ dization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences
  • Polynucleotides of the mvention which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ID NO 1 or a fragment thereof, may be used as hyb ⁇ dization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encodmg CBFBMD04 polypeptide and to isolate cDN A and genomic clones of other genes (mcludmg genes encodmg homologs and orthologs from species other than human) that have a high sequence simila ⁇ ty to the CBFBMD04 gene
  • Such hybndrzation 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 comp ⁇ se at least 15 nucleotides Preferably, such probes will have at
  • CBFBMD04 polynucleotides of the present mvention further mclude a nucleotide sequence compnsing a nucleotide sequence that hybndize under stringent condition to a nucleotide sequence havmg SEQ ID NO 1 or a fragment thereof. Also mcluded with CBFBMD04 polypeptides are polypeptide comp ⁇ smg ammo acid sequence encoded by nucleotide sequence obtamed by the
  • the present mvention also relates to vectors which comp ⁇ se a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engineered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the present mvention
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides mto host cells can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al ,MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N Y (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microi ⁇ jection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
  • approp ⁇ ate hosts mclude bacte ⁇ al cells, such as streptococci, staphylococci, E colt, Streptomyces and Bacillus subtilis cells, fungal cells, such as yeast cells and Aspergtllus 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-de ⁇ ved systems e g , vectors de ⁇ ved from bactenal plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovmises, papova viruses, such as SV40, vaccinia viruses,
  • approp ⁇ ate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • the cells may be harvested prior to use in the screening assay If CBFBMD04 polypeptide is secreted into the medium, the medium can be recovered in order to recover and punfy the polypeptide, if produced rntracellularly, the cells must first be lysed before the polypeptide is recovered
  • CBFBMD04 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 pu ⁇ fication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or pu ⁇ fication
  • This mvention also relates to the use of CBFBMD04 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBFBMD04 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 CBFBMD04 Individuals carrying mutations m the CBFBMD04 gene may be detected at the DNA level by a va ⁇ ety of techniques
  • Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy mate ⁇ al
  • the genomic DNA may be used directly for detection or may be amphfied enzymatically by usmg PCR or other amplification techniques p ⁇ or to analysis RNA or cDNA may also be used m similar fashion
  • Deletions and insertions can be detected by a change m size of the amphfied product m companson to the normal genotype
  • Point mutations can be identified by hyb ⁇ dizmg amphfied DNA to labeled CBFBMD04 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing See, e g , Myers
  • cancer, AIDS, metabolic disorders, and IDDM can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or mcreased level of CBFBMD04 polypeptide or CBFBMD04 mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods Assay techniques that can be used to determme levels of a protem, such as an CBFBMD04 polypeptide, m a sample de ⁇ ved from a host are well-known
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly cancer, ADDS, metabolic disorders, and IDDM, which comp ⁇ ses
  • a CBFBMD04 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1 , or a fragment thereof,
  • a CBFBMD04 polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • any such kit, (a), (b), (c) or (d) may comp ⁇ se a substantial component
  • the nucleotide sequences of the present mvention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hyb ⁇ dize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step m co ⁇ elating those sequences with gene associated disease
  • Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found, for example, m V McKusick, Mende an Inhe ⁇ tance m Man (available on lme through Johns Hopkins University Welch Medical Library)
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (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 in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease
  • polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as rmmunogens to produce antibodies lmmunospecific for the CBFBMD04
  • immunospecific means that the antibodies have substantiall greater affimty for the polypeptides of the mvention than their affinity for other related polypeptides m the p ⁇ or art
  • Antibodies generated against the CBFBMD04 polypeptides can be obtamed 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 lme cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256495-497), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hyb ⁇ doma 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 pu ⁇ fy the polypeptides by affinity chromatography
  • Antibodies against CBFBMD04 polypeptides may also be employed to treat cancer, AIDS, metabolic disorders, and IDDM, among others
  • Vaccines Another aspect of the invention relates to a method for inducing an lmmunological response m a mammal which comprises moculatmg the mammal with CBFBMD04 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 mvention relates to a method of inducmg lmmunological response in a mammal which comprises, delivering CBFBMD04 polypeptide via a vector directing expression of CBFBMD04 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 m that mammal to a CBFBMD04 polypeptide wherein the composition comp ⁇ ses a CBFBMD04 polypeptide or CBFBMD04 gene
  • the vaccine formulation may further comp ⁇ se a suitable carrier Since CBFBMD04 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc injection)
  • parenterally including subcutaneous, intramuscular, intravenous, intradermal etc injection
  • Formulations suitable for parenteral administration include aqueous and non-aqueous
  • sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation lnstonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspendmg 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 m a freeze-dried condition requiring only the addition of the stenle liquid earner immediately prior to use
  • the vaccine formulation may also mclude adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-m water systems and other systems known m the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expe ⁇ mentation
  • the CBFBMD04 polypeptide of the present mvention may be employed m a screenmg process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBFBMD04 polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical bra ⁇ es, and natural product mixtures
  • These agonists or antagonists may be natural or modified substrates, gands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Co gan etal , Current Protocols in Immunology 1(2) Chapter 5 (1991) CBFBMD04 polypeptides are responsible for many biological functions, mcludmg many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBFBMD04 polypeptide on the
  • such screening procedures may mvolve usmg appropnate cells which express the CBFBMD04 polypeptide or respond to CBFBMD04 polypeptide of the present mvention
  • Such cells mclude cells from mammals, yeast, Drosophila or E colt Cells which express the CBFBMD04 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBFBMD04 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 CBFBMD04 activity
  • the assays may simply test binding of a candidate compound wherem adherence to the cells bearing the CBFBMD04 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor Further, these assays may test whether the candidate compound results in a signal generated by activation of the CBFBMD04 polypeptide, using detection systems appropnate to the cells bearmg the CBFBMD04 polypeptide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the assays may simply comp ⁇ se the steps of mixing a candidate compound with a solution containing a CBFBMD04 polypeptide to form a mixture, measurmg CBFBMD04 activity m the mixture, and comparing the CBFBMD04 activity of the mixture to a standard
  • the CBFBMD04 cDNA, protem and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBFBMD04 mRNA and protein m cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of CBFBMD04 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of CBFBMD04 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBFBMD04 protem may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These mclude, but are not limited to, hgand binding and crosslinking assays in which the CBFBMD04 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy In addition to being used for punfication and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBFBMD04 which compete with the binding of CBFBMD04 to its receptors, if any Standard methods for conducting screening assays are well understood m the art Examples of potential CBFBMD04 polypeptide antagonists
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for CBFBMD04 polypeptides, or compounds which decrease or enhance the production of CBFBMD04 polypeptides, which comprises (a) a CBFBMD04 polypeptide, preferably that of SEQ ID NO 2,
  • a recombinant cell expressmg a CBFBMD04 polypeptide, preferably that of SEQ ID NO 2,
  • This mvention provides methods of treatmg abnormal conditions such as, cancer, AIDS, metabolic disorders, and IDDM, related to both an excess of and insufficient amounts of CBFBMD04 polypeptide activity
  • CBFBMD04 polypeptide If the activity of CBFBMD04 polypeptide is in excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove desc ⁇ bed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBFBMD04 polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of CBFBMD04 polypeptides still capable of bindmg the hgand, substrate, enzymes, receptors, etc in competition with endogenous CBFBMD04 polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBFBMD04 polypeptide In still another approach, expression of the gene encoding endogenous CBFBMD04 polypeptide can be inhibited usmg expression blocking techniques Known such techniques involve the use of antisense sequences, either
  • gene therapy may be employed to effect the endogenous production of CBFBMD04 by the relevant cells in the subject
  • a polynucleotide of the mvention may be engineered for expression in a replication defective retroviral vector, as discussed above
  • the retroviral expression construct may then be isolated and introduced mto a packaging cell transduced with a retroviral plasrmd vector containing RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containing the gene of interest
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo
  • Chapter 20 Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) m Human Molecular
  • Peptides such as the soluble form of CBFBMD04 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner
  • a suitable pharmaceutical earner Such formulations comp ⁇ se a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient
  • earners include but are not limited to, salme, buffered salme, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art
  • the mvention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • Prefened forms of systemic administration of the pharmaceutical compositions mclude injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg 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, m the form of salves, pastes, gels and the like
  • the dosage range required depends on the choice of peptide, the route of admimstration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are m the range of 0 1 - 100 ⁇ g/kg of subject Wide va ⁇ ations m the needed dosage, however, are to be expected in view of the va ⁇ ety of compounds available and the differmg efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Va ⁇ ations m these dosage levels can be adjusted usmg standard empincal routmes for optimization, as is well understood in the art
  • Polypeptides used m treatment can also be generated endogenously m the subject, m treatment modalities often refe ⁇ ed to as "gene therapy" as descnbed above
  • m treatment modalities often refe ⁇ ed to as "gene therapy" as descnbed 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 plasrmd vector The cells are then introduced mto the subject
  • ADDRESSEE RATNER & PRESTIA
  • STREET P.O. BOX 980
  • NAME PRESTIA, PAUL F
  • AGGGAATTGC AACCCAGTGA AGAAGTTACC TGGAAGTAAA GACTGGCTAG ATTATCGAAT 360

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBFBMD04 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 CBFBMD04 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/CN1998/000045 1998-03-18 1998-03-18 Gene humain homologue de ci-mnll: cbdfmd04 WO1999047666A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000045 WO1999047666A1 (fr) 1998-03-18 1998-03-18 Gene humain homologue de ci-mnll: cbdfmd04

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000045 WO1999047666A1 (fr) 1998-03-18 1998-03-18 Gene humain homologue de ci-mnll: cbdfmd04

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WO1999047666A1 true WO1999047666A1 (fr) 1999-09-23

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094537A3 (fr) * 2000-05-24 2004-04-08 Shanghai Biowindow Gene Dev Nouveau polypeptide, nadh-ubiquinone oxydoreductase humaine 21.89, et polynucleotide codant ce polypeptide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GENBANK, gb/AC003687/AC003687, 03 Feb. 1998, BIRREN B. et al., "Homo Sapiens Chromosome 17, Clone HRPC29G21". *
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. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094537A3 (fr) * 2000-05-24 2004-04-08 Shanghai Biowindow Gene Dev Nouveau polypeptide, nadh-ubiquinone oxydoreductase humaine 21.89, et polynucleotide codant ce polypeptide

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