WO1999047664A1 - Gene humain homologue du proteolipide mitochondrial mp68: cbnaac01 - Google Patents

Gene humain homologue du proteolipide mitochondrial mp68: cbnaac01 Download PDF

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Publication number
WO1999047664A1
WO1999047664A1 PCT/CN1998/000043 CN9800043W WO9947664A1 WO 1999047664 A1 WO1999047664 A1 WO 1999047664A1 CN 9800043 W CN9800043 W CN 9800043W WO 9947664 A1 WO9947664 A1 WO 9947664A1
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Prior art keywords
polypeptide
cbnaacol
seq
polynucleotide
nucleotide sequence
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PCT/CN1998/000043
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English (en)
Inventor
Qinghua Zhang
Yu Shen
Mao Mao
Yaping Yu
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Shanghai Second Medical University
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Priority to PCT/CN1998/000043 priority Critical patent/WO1999047664A1/fr
Publication of WO1999047664A1 publication Critical patent/WO1999047664A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to newly identified polynucleotides. polypeptides encoded b ⁇ them and to the use of such polynucleotides and polypeptides. and to their production More particularly, the polynucleotides and polypeptides of the present invention relate to the famih , hereinafter referred to as CBNAACOl. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides
  • the mitochondrion is a key subcellular organelle involved in energy metabolism of the cell
  • the MP68 gene product is a mitochondria] membrane component This indicates that the MP68 family has an established, proven history as therapeutic targets Clearly there is a need for identification and characten.zat ⁇ on of further members of the family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer, AIDS, metabolic disorders, and hepatitis.
  • the invention relates to CBNAACOl polypeptides and recombinant matc ⁇ als and methods for their production ./Vnother aspect of the invention relates to methods for using such CBNAACOl polypeptides and polynucleotides. Such uses include the treatment of cancer, .AIDS, metabolic disorders, and hepatitis, among others.
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBN ⁇ ⁇ ACOl imbalance with the identified compounds.
  • Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBN.AAC01 activity or levels.
  • CBNAACOl refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allelic variant thereof.
  • CBNAACOl activity or CBNAACOl polypeptide activity or "biological activity of the CBNAACOl or CBNAACOl polypeptide” refers to the metabolic or physiologic function of said CBNAACOl including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said CBNAACOl
  • CBNAACOl 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 lmmunoglobulin expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated ' composition or substance occurs in nature, it has been changed or removed from its original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated/' but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the teim is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, t ⁇ tylated
  • Polypeptide refers to any peptide or protem comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, 1 e , peptide isosteres
  • Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins
  • Polypeptides may contain ammo acids other than the 20 gene-encoded amino acids
  • Polypeptides include amino acid sequences modified either by
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, branched and branched cvchc polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include acetylation.
  • acylation ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a pid or pid derivative, covalent attachment of phosphotidy nositol, cross-linking, cychzation.
  • disulfide bond formation demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation.
  • GPI anchor formation hydroxylation.
  • 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 naturalh occurring such as an allclic 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 bv mutagenesis techniques or b ⁇ direct synthesis
  • Identity ' as l nown in the art 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 b ⁇ the match between strings of such sequences "Identity” and “similarity” can be readily calculated by .known methods, including but not limited to those described in (Computational Molecular Biolog ⁇ , Lesk.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested
  • Methods to determine identity and similarity are codified in publicly available computer programs
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S F et al , J Molec Biol 215 403-410 (1990)
  • 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 Wate ⁇ nan algorithm may also be used to dete ⁇ nine identity
  • Preferred parameters for polypeptide sequence comparison include the following 1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4
  • 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 insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 by the numerical percent of
  • nn is the number of nucleotide alterations
  • xn is the total number of nucleotides in SEQ ID NO 1
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Preferred polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said reference sequence may be identical to the sequence of SEQ ID NO 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the numerical percent of the respective percent identity and subtracting that product from said total number of ammo acids in SEQ ID NO 2, or
  • na is the number of ammo 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 prior to subtracting it from xa
  • the present invention relates to CBNAACOl polypeptides (or CBNAACOl proteins)
  • the CBNAACOl 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 CBNAACOl polypeptides are polypeptides having the ammo acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% arc highly preferred Preferably CBNAACOl polypeptide exhibit at least one biological
  • the CBNAACOl polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned CBNAACOl polypeptides
  • fragments may be "free-standing," or compnsed within a larger polypeptide of which they farm a part or region, most preferably as a single continuous region
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61- 80, 81-100, and 101 to the end of CBNAACOl polypeptide
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of CBNAACOl polypeptides, except for deletion of a continuous senes of residues that includes the amino terminus, or a continuous senes of residues that includes the carboxyl terminus or deletion of two continuous senes of residues, one including the .amino terminus and one including the carboxyl terminus .
  • Also preferred are fragments charactenzed by ⁇ nictural or functional attnbutes 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, hydrophi c 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 CBNAA
  • vanants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination
  • the CBNAACO 1 polypeptides of the invention can be prepared in any suitable manner
  • Such polypeptides include isolated naturally occurring polypeptides, reco binantly produced polypeptides. synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood in the art
  • CBNAACOl polynucleotides include isolated polynucleotides which encode the CBNAACOl polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBNAACOl polynucleotide of the invention include a polynucleotide compnsing the nucleotide sequence contained in SEQ ID NO 1 encoding a CBNAACOl polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBNAACO 1 polynucleotides fuither include a polynucleotide compnsing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBNAACO 1 polypeptide of SEQ ID NO 2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to of
  • CBNAACOl polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO 1 to hybridize under conditions useable for amplification or for use as a probe or marker
  • the invention also provides polynucleotides which are complementary to such CBNAACOl polynucleotides
  • CBNAACOl of the invention is structurally related to other proteins of the MP68 family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human CBNAACOl
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 61 to 234) encoding a polypeptide of 58 amino acids of SEQ ID NO 2
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 80% identity (usmg FASTA) m 58 ammo acid residues with mouse 6 8 kd mitochondnal proteo pid MP68 (E
  • a nucleotide sequence of a human CBNAACO 1 (SEQ ID NO 1)
  • An ammo acid sequence of a human CBNAACOl (SEQ ID NO 2)
  • One polvnucleotide of the present mvention encoding CBNAACOl may be obtained using standard cloning and screening, from a cDNA library denved from mRNA in cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M O , et al Science (1991 ) 252 1651 - 1656. Adams, M D et al . Nature, (1992) 355 632-634, Adams, M D , et al . Nature (1995) 377 Supp 3-174)
  • Polynucleotides of the 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 CBNAACOl polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained m Table 1 (nucleotide number 61 to 234 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 in reading frame with other codmg sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions
  • a marker sequence which facilitates punfication of the fused polypeptide can be encoded
  • the marker sequence is a hexa-h ⁇ .st ⁇ d ⁇ ne peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed m Gentz et al , Proc Natl Acad Set USA ( 1989) 86 821 - 824, or is an .HA
  • polynucleotides encoding CBNAACOl vanants compnse the ammo acid sequence CBNAACOl polypeptide of Table 2 (SEQ ID NO 2) m wheh 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 hybndize to the herem above- desenbed sequences
  • the present mvention especially relates to polynucleotides which hybndize under stnngent conditions to the herein above-desenbed polynucleotides
  • stnngent 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 in SEQ ID NO 1 or a fragment thereof, may be used as hybndization probes for
  • probes generally wall comp ⁇ se at least 15 nucleotides
  • probes will have at least 30 nucleotides and may have at least 50 nucleotides
  • Particularly prefened probes will range between 30 and 50 nucleotides
  • CBNAACOl polynucleotides of the present invention further include a nucleotide sequence compnsing a nucleotide sequence that hybndize under stnngent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof
  • polypeptide compnsing ammo acid sequence encoded by nucleotide sequence obtained by the above hybndization condition Such hvbnd ⁇ .
  • the present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engmeered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such protems usmg RNAs denved from the DNA constructs of the present mvention
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides mto host cells can be effected by methods desenbed m many standard laboratory manuals, such as Davis
  • appropnate hosts include bactenal cells, such as streptococci, staphylococci, E colt, Streptomyces and Bacillus subtths cells, fungal cells, such as yeast cells and A ⁇ ergillus cells, insect cells such as Drosophtla S2 and Spodoptera Sf9 cells, animal cells such as CHO. COS, HeLa, C127, 3T3.
  • bactenal cells such as streptococci, staphylococci, E colt, Streptomyces and Bacillus subtths cells
  • fungal cells such as yeast cells and A ⁇ ergillus cells
  • insect cells such as Drosophtla S2 and Spodoptera Sf9 cells
  • animal cells such as CHO. COS, HeLa, C127, 3T3.
  • Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from msertion elements, from yeast chromosomal elements, from viruses such as baculoviruses.
  • papova vinises such as SV40, vaccinia viruses adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combmations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used
  • the appropnate nucleotide sequence may be inserted mto an expression system by any of a vanety of well-i nown and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL (supra)
  • appropnate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • the cells may be harvested pnor to use in the screening assay If CBNAACO 1 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
  • CBNAACOl polypeptides can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chr ⁇ matography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectm chromatography Most preferably, fiigh
  • This mvention also relates to the use of CBNAACO 1 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBNAACO 1 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 CBNAACO 1 Individuals caiTying mutations m the CBNAACO 1 gene may be detected at the DNA level by a vanety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy matenal
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification techniques
  • pnor to analysis RNA or cDNA may also be used similar fasliion Deletions and msertions can be detected by a change in size of the amplified product in companson to the normal genotype
  • Point mutations can be identified by hybndizmg amplified DNA to labeled CBNAACO 1 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m meltmg temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments m gels, with or without denaturing agents, or by direct DNA sequencmg See, e g , Myers et al
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to cancer, -AIDS, metabolic disorders, and hepatitis through detection of mutation m the CBNAACO 1 gene by the methods descnbed
  • cancer in addition, cancer, .AIDS, metabolic disorders, and hepatitis, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of CBNAACOl polypeptide or CBNAACOl mRNA Decreased or increased
  • RNA 13 expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR. RNase protection, Northern blotting and other hybridization methods Assay techniques that can be used to determine levels of a protem, such as an CBNAACOl polypeptide, m a sample denved from a host are well-known to those of skill in the art Such assay methods mclude radioimmunoassays. competitive-binding assays Western Blot analysis and ELISA assays
  • the present invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly cancer, AIDS, metabolic disorders, and hepatitis which comprises (a) a CBNAACOl polynucleotide, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,
  • a CBNAACOl polypeptide preferably the polypeptide of SEQ ID NO 2. or a fragment thereof, or (d) an antibody to a CBNAACOl polypeptide, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that in any such kit. (a), (b) (c) or (d) may comprise a substantial component
  • the nucleotide sequences of the present mvention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hybndize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step m conelating 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, in V McKusick, Mendehan Inhentance m Man (available on lme tlirough Johns Hopl ⁇ ns Umversity Welch Medical Library)
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co nhentance 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
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressmg them can also be used as immunogens to produce antibodies lmmunospecific for the CBNAACO 1 polypeptides
  • immunogens to produce antibodies lmmunospecific for the CBNAACO 1 polypeptides
  • the term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pnor art
  • Antibodies generated agamst the CBNAACO 1 polypeptides can be obtained bv administenng the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routme protocols
  • any technique which provides antibodies produced by contmuous cell line cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tnoma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985) Techniques for the production of smgle cham antibodies (U S Patent No 4.946,778)
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to punfy the polypeptides by affinity chromatography .
  • Antibodies against CBNAACOl polypeptides may also be employed to treat cancer. AIDS, metabolic disorders, and hepatitis, among others
  • ./Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with CBNAACOl polypeptide. or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer, DS. metabolic disorders, and hepatitis, among others
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, dehvenng CBNAACOl polypeptide via a vector directing expression of CBNAACOl polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases
  • composition which, when introduced into a mammalian host, induces an immunological response in that mammal to a CBNAACOl polypeptide wherein the composition comprises a CBNAACOl
  • the vaccine formulation may further comprise a suitable carrier Since CBNAACOl polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc injection)
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation instonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the foimulation, such as oil-in water systems and other systems .known in the art The dosage will depend on the specific activity
  • the CBNAACOl polypeptide of the present invention may be employed a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBNAACO 1 polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonisrt: or antagonists from, for example, cells, cell-free preparations, chemical branes, and natural product mixtures
  • agonists or antagonists may be natural or modified substrates, ligands, 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 Coligan et al , Current Protocols m Immunology 1(2) Chapter 5 (1991)
  • CBNAACO 1 polypeptides are responsible for many biological functions, including many pathologies Accordmgly, it is desirous to find compounds and drugs which stimulate CBNAACOl polypeptide on the one hand and which
  • CBNAACOl polypeptide or respond to CBNAACOl polypeptide of the present mvention Such cells mclude cells from mamirals, yeast, Drosophila or E coh Cells which express the CBNAACO 1 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBNAACOl polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a
  • the assavs may simply test bmding of a candidate compound wherein adherence to the cells bearing the CBNAACOl polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assav involving competition with a labeled competitor Further, these assays may test whether the candidate compound results in a signal generated by activation of the CBNAACOl polypeptide.
  • Inhibitors of activation are generally assayed m the presence of a lcnown agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing a CBN.AAC01 polypeptide to form a mixture, measuring CBNAACOl activity in the mixture, and comparing the CBNAACOl activity of the mixture to a standard
  • the CBNAACO 1 cDNA, protein and antibodies to the protein may also be used to configure assay s for detecting the effect of added compounds on the production of CBNAACO 1 mRNA and protein in cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of CBNAACOl 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 CBNAACOl (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBNAACOl protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques lcnown in the art These include, but are not limited to, hgand binding and crosshnking assays in which the CBNAACOl is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or punfication, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy In addition to being used for purification and cloning of the receptor, these bmding assays can be used to identify agonists and antagonists of CBNAACOl which compete with the binding of CBNAACOl to its receptors, if any Standard methods for conducting screening assays are well understood in the art Examples of potential CBNAACO
  • the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc for CBNAACOl polypeptides, or compounds which decrease or enhance the production of CBNAACOl polypeptides.
  • This mvention provides methods of treating abnormal conditions such as, cancer, AIDS, metabolic disorders, and hepatitis, related to both an excess of and insufficient amounts of CBNAACOl polypeptide activity
  • CBNAACOl polypeptide If the activity of CBNAACOl polypeptide is m excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as heremabove descnbed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBNAACOl polypeptide, such as, for example, by blocking the binding of hgands. substrates, receptors, enzymes, etc .
  • soluble fo ⁇ ns of CBNAACO 1 polypeptides still capable of binding the hgand, substrate, en.zymes, receptors, etc in competition with endogenous CBNAACOl polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBNAACOl polypeptide
  • expression of the gene encoding endogenous CBNAACOl 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 Ohgodeoxynucleotides as ⁇ Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)
  • ohgonucleotides which form tnple helices with the gene can be supplied See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360
  • These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • a polynucleotide of the mvention may be engmeered for expression m a replication defective retroviral vector, as discussed above
  • the retroviral expression construct may then be isolated and mtroduced mto a packagmg cell transduced with a retroviral plasmid vector containmg RNA encodmg a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containmg the gene of mterest
  • Peptides such as the soluble form of CBNAACOl polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m combination with a suitable phaimaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a phairnaceutically acceptable earner or excipient Such earners mclude but are not limited to, saline, buffered salme, dextrose, water, glycerol.
  • 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 in conjunction with other compounds, such as therapeutic compounds
  • Prefened forms of systemic administration of the pharmaceutical compositions include injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used .
  • Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be used.
  • the dosage range required depends on the choice of peptide, the route of administration, the nature of the foimulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are in the range of 0 1-100 ⁇ g/kg of subject Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by mtravenous injection Vanations in these dosage levels can be adjusted usmg standard empincal routmes for opt ⁇ m ⁇ .zat ⁇ on, as is well understood in the art
  • Polypeptides used m treatment can also be generated endogenously m the subject, m treatment modalities often refened to as "gene therapy" as descnbed above
  • m treatment modalities often refened to as "gene therapy" as descnbed above
  • cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then mtroduced mto the subject
  • ADDRESSEE RATNER & PRESTIA
  • STREET P.O. BOX 980
  • NAME PRESTIA, PAUL F

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBNAAC01 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 CBNAAC01 dans la conception de protocoles pour le traitement de cancers, du SIDA, de troubles métaboliques et d'hépatites, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000043 1998-03-18 1998-03-18 Gene humain homologue du proteolipide mitochondrial mp68: cbnaac01 WO1999047664A1 (fr)

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PCT/CN1998/000043 WO1999047664A1 (fr) 1998-03-18 1998-03-18 Gene humain homologue du proteolipide mitochondrial mp68: cbnaac01

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000043 WO1999047664A1 (fr) 1998-03-18 1998-03-18 Gene humain homologue du proteolipide mitochondrial mp68: cbnaac01

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WO1999047664A1 true WO1999047664A1 (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
JP2012184210A (ja) * 2011-03-08 2012-09-27 Japan Science & Technology Agency ヒトｍｌｑを免疫原として得られたポリクローナル抗体

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, 34(33), (1995), OHBA T. et al., "Human Sterol Carrier Protein X/Sterol Carrier Protein 2 Gene Has Two Promoters", pages 10660-10668. *
FEBS LETT., 260(1), (1990), TERZI E. et al., "Isolation and Amino Acid Sequence of a Novel 6.8kDa Mitochondrial Proteolipid from Beef Heart. Use of FAB-MS for Molecular Mass Determination", pages 122-126. *
GENOMICS, 24(2), (1994), OHBA T. et al., "The Structure of the Human Sterol Carrier Protein X/Sterol Carrier Protein 2 Gene(SCP2)", pages 370-374. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012184210A (ja) * 2011-03-08 2012-09-27 Japan Science & Technology Agency ヒトｍｌｑを免疫原として得られたポリクローナル抗体

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