WO1999046291A1 - Gene homologue humain du facteur de maturation des cellules gliales (gmf) type beta (cbfboe11) - Google Patents

Gene homologue humain du facteur de maturation des cellules gliales (gmf) type beta (cbfboe11) Download PDF

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Publication number
WO1999046291A1
WO1999046291A1 PCT/CN1998/000033 CN9800033W WO9946291A1 WO 1999046291 A1 WO1999046291 A1 WO 1999046291A1 CN 9800033 W CN9800033 W CN 9800033W WO 9946291 A1 WO9946291 A1 WO 9946291A1
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Prior art keywords
polypeptide
cbfboel
seq
polynucleotide
nucleotide sequence
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PCT/CN1998/000033
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English (en)
Inventor
Gang Fu
Yu Shen
Mao Mao
Yaxin Wang
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Shanghai Second Medical University
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Priority to CN98805074.9A priority Critical patent/CN1255927A/zh
Priority to PCT/CN1998/000033 priority patent/WO1999046291A1/fr
Publication of WO1999046291A1 publication Critical patent/WO1999046291A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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

  • GMF Human Glia Maturation Factor
  • CBFBOEl 1 Human Glia Maturation Factor
  • This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present invention relate to the Glia Maturation Factor family, hereinafter referred to as CBFBOEl 1 The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides
  • Glia Maturation Factor is involved in neuron development and maturation This indicates that the Glia Maturation Factor family has an established, proven history as therapeutic targets Clearly there is a need for identification and characterization of further members of the G a Maturation Factor family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer, neurological disease, and autoimmune disease
  • the invention relates to CBFBOEl 1 polypeptides and recombinant matenals and methods for their production Another aspect of the invention relates to methods for using such
  • CBFBOEl 1 polypeptides and polynucleotides Such uses include the treatment of cancer, neurological disease, and autoimmune disease, among others
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBFBOEl 1 imbalance with the identified compounds
  • Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropnate CBFBOEl 1 activity or levels
  • CBFBOEl 1 refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO 2 or an allehc variant thereof
  • CBFBOEl 1 activity or CBFBOEl 1 polypeptide activity or "biological activity of the CBFBOEl 1 or CBFBOEl 1 polypeptide” refers to the metabolic or physiologic function of said CBFBOEl 1 including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said CBFBOEl 1.
  • CBFBOEl 1 gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 2 or an allehc variant thereof
  • CBFBOEl 1 activity or CBFBOEl 1 polypeptide activity or "biological activity of the CBFBOEl 1 or CBFBOEl 1 polypeptide” refers to the metabolic or physiologic function of said CBFBOEl 1 including similar activities or improved activities or these activities with decreased undesirable side-effects.
  • 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. 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 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 oligomers, 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 posttranslational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the amino or carboxj 1 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 branching Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include acetylation, acylation, ADP- ⁇ bosylation, 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
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ m amino acid sequence by one or more substitutions, additions, deletions m any combination A substitute
  • 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 in (Computational Molecular Biology, Lesk, A.M., ed. , Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed.
  • 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 (BIAST 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:
  • 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: l , wherein said reference sequence may be identical to the sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO. l
  • y is 0.50 for 50% , 0.60 for 60% , 0.70 for 70% , 0.80 for 80% , 0.85 for 85 % , 0.90 for 90% , 0.95 for 95% , 0.97 for 97% or 1.00 for 100%
  • any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • Preferred polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity and subtracting that product from said total number of amino acids in SEQ ID NO:2, or:
  • 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% , and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • the present invention relates to CBFBOEl 1 polypeptides (or CBFBOEl 1 proteins).
  • the CBFBOEl 1 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.
  • CBFBOEl 1 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 its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO:2. Furthermore, those with at least 97-99% are highly preferred.
  • CBFBOEl 1 polypeptide exhibit at least one biological activity of CBFBOEl 1.
  • the CBFBOEl 1 polypeptides mav 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 CBFBOEl 1 polypeptides
  • fragments may be "free-standing,” or compnsed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of CBFBOEl 1 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 CBFBOE 1 1 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
  • fragments characte ⁇ zed by structural 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, 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 CBFBOEl 1 activity, including those with a similar activity or an improved activity, or
  • vanants are those that vary from the referents by conservative amino acid substitutions — l e , those that substitute a residue with another of like charactenstics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vanants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination
  • the CBFBOEl 1 pol peptides of the invention can be prepared in any suitable manner Such polypeptides include isolated naturally occurring polypeptides. recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such poly
  • CBFBOEl 1 polynucleotides include isolated polynucleotides which encode the CBFBOEl 1 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBFBOEl 1 polynucleotide of the invention include a polynucleotide composing the nucleotide sequence contained in SEQ ID NO 1 encoding a
  • CBFBOEl 1 polypeptide of SEQ ID NO 2 and polynucleotide having the particular sequence of SEQ ID NO 1 CBFBOEl 1 polynucleotides further include a polynucleotide composing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBFBOEl 1 polypeptide of SEQ ID NO 2, and a polynucleotide compnsmg a nucleotide sequence that is at least 80% identical to of SEQ ID NO 1 over its entire length
  • polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred
  • those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred
  • CBFBOEl 1 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO 1 to hy
  • CBFBOEl 1 of the invention is structurally related to other proteins of the Glia Maturation Factor family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human CBFBOEl 1
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 5 to 430) encoding a polypeptide of 142 amino acids of SEQ ID NO 2
  • the amino acid sequence of Table 2 (SEQ ID NO 2) has about 82 1% identity (using FASTA) in 140 amino acid residues with human and rat GMF-beta (R Kaplan, et al , J Neurochem 57 (2), 483-490, 1991)
  • the nucleotide sequence of Table 1 (SEQ ID NO 1) has about 70 2% identity (using FASTA) in 433 nucleotide residues with human and rat GMF-beta (R Kaplan, et al , J Neurochem 57 (2), 483-490, 1991
  • a nucleotide sequence of a human CBFBOEl 1 (SEQ ID NO: 1).
  • One polynucleotide of the present invention encoding CBFBOEl 1 may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651-1656; Adams, M.D. et al., Nature, (1992) 555:632-634; 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 CBFBOEl 1 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 5 to 430 of SEQ ID NO 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment 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 descnbed in Gentz et al , Proc Natl Acad Set USA ( 1989) 86 821 -824, or is an HA tag
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such
  • polynucleotides encoding CBFBOE 11 vanants compnse the amino acid sequence CBFBOEl 1 polypeptide of Table 2 (SEQ ID NO 2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination
  • the present invention further relates to polynucleotides that hybndize to the herein above-desenbed sequences
  • the present mvention especially relates to polynucleotides which hybndize under stringent conditions to the herein above-desenbed polynucleotides
  • stringent conditions means hybndization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO 1 or a
  • CBFBOEl 1 polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ID NO: 1 or a fragment thereof.
  • CBFBOEl 1 polypeptides are polypeptide comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition.
  • hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
  • the present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et 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.
  • mice such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • appropriate hosts 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; and plant cells.
  • 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
  • Such systems include, among others, 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.
  • viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmid
  • 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 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).
  • secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide be produced at the surface of the cell.
  • the cells may be harvested prior to use in the screening assay. If CBFBOEl 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.
  • CBFBOEl 1 polypeptides 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, phosphocellulose 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 CBFBOEl 1 polynucleotides for use as diagnostic reagents.
  • Detection of a mutated form of CBFBOEl 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 CBFBOE 11.
  • Individuals carrying mutations in the CBFBOEl 1 gene may be detected at the DNA level by a variety 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 amphfied enzymatically by using PCR or other amplification techniques poor to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions 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 CBFBOEl 1 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 See, e g , Myers et al , Science (1985) 230 1242 Sequence changes at specific locations may also be revealed by
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to cancer, neurological disease, and autoimmune disease, through detection of mutation in the CBFBOEl 1 gene by the methods descnbed
  • cancer, neurological disease, and autoimmune disease can be diagnosed by methods compnsing determining from a sample derived from a subject an abnormally decreased or increased level of CBFBOEl 1 polypeptide or CBFBOEl 1 mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods
  • Assay techniques that can be used to determine levels of a protein, such as an
  • CBFBOE 11 polypeptide, in a sample denved from a host are well-known to those of skill in the art
  • assay methods include 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, neurological disease, and autoimmune disease, which composes
  • a CBFBOEl 1 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,
  • CBFBOEl 1 polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • the nucleotide sequences of the present invention 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 invention is an important first step in correlating those sequences with gene associated disease
  • genetic map data are found, for example, in V McKusick, Mendelian Inheotance 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 (coinheotance of physically adjacent genes)
  • the differences m 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,
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as lmmunogens to produce antibodies lmmunospecific for the CBFBOEl 1 polypeptides
  • the term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the pnor art
  • Antibodies generated against the CBFBOEl 1 polypeptides can be obtained by administering the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols
  • any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybndoma technique (Kohler, G and Ivhlstein, C , ⁇ fa/wre (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)
  • the above-desenbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to punfy the polypeptides by affinity chromatograph ⁇
  • Antibodies against CBFBOEl 1 polypeptides may also be employed to treat cancer, neurological disease, and autoimmune disease, among others
  • Vaccines Another aspect of the invention relates to a method for inducing an lmmunological response in a mammal which comprises inoculating the mammal with CBFBOEl 1 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer, neurological disease, and autoimmune disease, among others
  • a method of inducing lmmunological response m a mammal which comprises, delivering CBFBOEl 1 polypeptide via a vector directing expression of CBFBOEl 1 polynucleotide in vivo in order to induce such an lmmunological response to produce antibody to protect said animal from diseases
  • composition which, when introduced into a mammalian host, induces an lmmunological response in that mammal to a CBFBOEl 1 polypeptide wherein the composition comprises a CBFBOEl 1 polypeptide or CBFBOEl 1 gene
  • the vaccine formulation may further comprise a suitable earner Since CBFBOEl 1 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 lnstomc 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 m unit-dose or multi-dose containers,
  • the CBFBOEl 1 polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBFBOEl 1 polypeptide of the present invention
  • polypeptides of the mvention may also be used to assess identify agomst or antagonists from, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
  • agonists or antagonists may be natural or modified substrates, hgands, 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 invention See Co gan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991) CBFBOE 11 polypeptides are responsible for many biological functions, including many pathologies
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer, neurological disease, and autoimmune disease
  • Antagonists may be employed for a vanety of therapeutic and prophylactic purposes for such conditions as cancer, neurological disease, and autoimmune disease
  • such screening procedures may involve using appropnate cells which express the CBFBOEl 1 polypeptide or respond to CBFBOEl 1 polypeptide of the present invention
  • Such cells include cells from mammals, yeast, Drosophila or E coll Cells which express the CBFBOEl 1 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBFBOEl 1 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 CBFBOEl 1 activity
  • the assays may simply test binding of a candidate compound wherein adherence to the cells beanng the CBFBOEl 1 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 m a signal generated by activation of the CBFBOEl 1 polypeptide, using detection systems appropriate to the cells bea ⁇ ng the CBFBOEl 1 polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the assays may simply compose the steps of mixing a candidate compound with a solution containing a CBFBOEl 1 polypeptide to form a mixture, measuring CBFBOEl 1 activity in the mixture, and comparmg the CBFBOEl 1 activity of the mixture to a standard
  • the CBFBOEl 1 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBFBOEl 1 mRNA and protein in cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of CBFBOEl 1 protein using monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents which may inhibit or enhance the production of
  • CBFBOEl 1 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBFBOEl 1 protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These include, but are not limited to, gand binding and crosshnking assays in which the CBFBOEl 1 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 binding assays can be used to identify agonists and antagonists of CBFBOEl 1 which compete with the bmdmg of CBFBOEl 1 to its receptors, if any
  • CBFBOEl 1 polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the CBFBOEl 1 polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypetide of the present invention but do not ehcit a response, so that the activity of the polypeptide is prevented
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for CBFBOEl 1 polypeptides, or compounds which decrease or enhance the production of CBFBOEl 1 polypeptides, which composes (a) a CBFBOEl 1 polypeptide, preferably that of SEQ ID NO 2,
  • This mvention provides methods of treating abnormal conditions such as, cancer, neurological disease, and autoimmune disease, related to both an excess of and insufficient amounts of CBFBOEl 1 polypeptide activity
  • CBFBOEl 1 polypeptide 17 If the activity of CBFBOEl 1 polypeptide is in excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove descnbed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBFBOEl 1 polypeptide, such as, for example, by blocking the bmdmg of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of CBFBOEl 1 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc in competition with endogenous CBFBOEl 1 polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBFBOEl 1 polypeptide
  • expression of the gene encoding endogenous CBFBOEl 1 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)
  • oligonucleotides which form triple 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 oligomers can be administered per se or the relevant oligomers can be expressed in vivo
  • 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 packagmg cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packagmg cell now produces infectious viral particles containing the gene of interest
  • producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in
  • Peptides such as the soluble form of CBFBOEl 1 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 compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient
  • earners include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art
  • the invention further relates to pharmaceutical packs and kits 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 lntrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetiants such as bile salts or fiisidic acids or other detergents.
  • oral administration may also be possible Admmistration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like
  • the dosage range required depends on the choice of peptide, the route of admmistration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are m the range of 0 1-100 ⁇ g/kg of subject Wide vanations m the needed dosage, however, are to be expected in view of the vanety of compounds available and the differing efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Vanations m these dosage levels can be adjusted using standard empincal routines for optimization, as is well understood m the art
  • Polypeptides used m treatment can also be generated endogenously in the subject, in treatment modalities often referred 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 plasmid vector The cells are then introduced to the subject
  • Lys Leu Arg Lys Phe Arg Phe Arg Lys Glu Thr Asp Asn Ala Ala lie 20 25 30 lie Met Lys Val Asp Lys Asp Arg Gin Met Val Val Leu Glu Glu Glu 35 40 45
  • Phe Gin Asn lie Ser Pro Glu Glu Leu Lys Met Glu Leu Pro Glu Arg

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBFBOE11 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 CBFBOE11 dans la conception de protocoles pour le traitement de cancers, d'affections neurologiques et de maladies auto-immunes, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000033 1998-03-12 1998-03-12 Gene homologue humain du facteur de maturation des cellules gliales (gmf) type beta (cbfboe11) WO1999046291A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN98805074.9A CN1255927A (zh) 1998-03-12 1998-03-12 人神经胶质成熟因子(GMF)β同系基因(CBFBOE11)
PCT/CN1998/000033 WO1999046291A1 (fr) 1998-03-12 1998-03-12 Gene homologue humain du facteur de maturation des cellules gliales (gmf) type beta (cbfboe11)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000033 WO1999046291A1 (fr) 1998-03-12 1998-03-12 Gene homologue humain du facteur de maturation des cellules gliales (gmf) type beta (cbfboe11)

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WO1999046291A1 true WO1999046291A1 (fr) 1999-09-16

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8609090B2 (en) 2003-07-18 2013-12-17 Amgen Inc. Specific binding agents to hepatocyte growth factor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMBL: LOCUS RNGMFB, Accession Z11558, 04 Feb. 1992, ZAHEER A., FINK B.D., LIM R., "R. Norvegicus mRNA(GMF-beta) for Glia Maturation Factore beta"; & BIOCHIM. BIOPHYS. ACTA, (1991), In Press. *
GENBANK: LOCUS HUMGMFB, Accession M86492, KAPLAN R., ZAHEER A., et al., "Molecular Cloning and Expression of Biologically Active Human Glia Maturation Factor-beta"; & J. NEUROCHEM., 57(2), Aug. 1991, pages 483-490. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8609090B2 (en) 2003-07-18 2013-12-17 Amgen Inc. Specific binding agents to hepatocyte growth factor

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