WO1996019244A1 - Adn codant une proteine inhibitrice de cdk6 de 20 kilodaltons - Google Patents

Adn codant une proteine inhibitrice de cdk6 de 20 kilodaltons Download PDF

Info

Publication number
WO1996019244A1
WO1996019244A1 PCT/US1995/016553 US9516553W WO9619244A1 WO 1996019244 A1 WO1996019244 A1 WO 1996019244A1 US 9516553 W US9516553 W US 9516553W WO 9619244 A1 WO9619244 A1 WO 9619244A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
nucleic acid
protein
dna
leu
Prior art date
Application number
PCT/US1995/016553
Other languages
English (en)
Inventor
Yue Xiong
Kun-Liang Guan
Original Assignee
The University Of North Carolina At Chapel Hill
The Regents Of The University Of Michigan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of North Carolina At Chapel Hill, The Regents Of The University Of Michigan filed Critical The University Of North Carolina At Chapel Hill
Priority to AU46023/96A priority Critical patent/AU4602396A/en
Publication of WO1996019244A1 publication Critical patent/WO1996019244A1/fr

Links

Classifications

    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to cyclin- dependent kinase (CDK) inhibiting proteins in general, and particularly relates to DNA encoding a 20 Kilodalton inhibitor of CDK6.
  • CDK cyclin- dependent kinase
  • CDKs cyclin-dependent kinases
  • CDKs may be controlled by a number of small proteins
  • a first aspect of the present invention is an isolated nucleic acid encoding a CDK inhibiting protein, particularly a CDK6 inhibiting protein.
  • the nucleic acid may be selected from the group consisting of:
  • nucleic acids which hybridize to DNA of (a) above e.g., under stringent hybridization conditions; e.g., nucleic acids that do not hybridize to DNA of SEQ ID NO:3 under the same hybridization conditions) and which encode a CDK inhibiting protein;
  • nucleic acids which differs from the nucleic acids of (a) or (b) above due to the degeneracy of the genetic code, and which encode a CDK inhibiting protein encoded by a nucleic acid of (a) or (b) above.
  • a second aspect of the present invention is a nucleic acid construct having a promoter and a heterologous nucleic acid operably linked to said promoter, wherein said heterologous nucleic acid is a nucleic acid as given above, along with cells containing such nucleic acid constructs (e.g., wherein the cell is one which expresses the encoded protein) .
  • a third aspect of the present invention is a protein encoded by a nucleic acid as given above. Such proteins may be isolated and/or purified in accordance with known techniques.
  • a fourth aspect of the present invention is an antibody (e . g. , a polyclonal antibody, a monoclonal antibody) which specifically binds to a protein as given above.
  • an antibody e . g. , a polyclonal antibody, a monoclonal antibody
  • a fifth aspect of the present invention is an antisense oligonucleotide complementary to a nucleic acid as given above and having a length sufficient to hybridize thereto under physiological conditions, along with DNA encoding such an antisense oligonucleotide, and a nucleic acid construct having a promoter and a heterologous nucleic acid operably linked to said promoter, wherein the heterologous nucleic acid is a DNA encoding such an antisense oligonucleotide.
  • a sixth aspect of the present invention is a method of inhibiting DNA synthesis in a human cell (e . g. , a tumor cell) which comprises providing to the cell a protein as given above in an amount effective to inhibit DNA synthesis therein.
  • the cell may be provided in any suitable form, such as in in vi tro culture.
  • the providing step may be carried out by any suitable means, such as by delivering the protein into the cell or by delivering into the cell a nucleic acid encoding the protein and which expresses the protein in the cell.
  • a seventh aspect of the present invention is a method for increasing DNA synthesis in a cell, wherein DNA synthesis in the cell is inhibited, by providing to the cell an antisense oligonucleotide as given above in an amount effective to increase DNA synthesis in said cell.
  • the cell may be a skin cell, such as a cell present in wound or burn tissue.
  • the providing step may be carried out by any suitable means, such as by delivering the antisense oligonucleotide into the cell, or by delivering into the cell a nucleic acid encoding an antisense oligonucleotide as given above and which transcribes the antisense oligonucleotide in the cell.
  • Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. Nucleotide sequences are presented herein by single strand only, in the 5' to 3 ' direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the
  • DNAs of the present invention include those coding for proteins homologous to, and having essentially the same biological properties as, the proteins disclosed herein, and particularly the DNA disclosed herein as SEQ ID NO:l and encoding the protein given herein SEQ ID NO:2. This definition is intended to encompass natural allelic variations therein.
  • isolated DNA or cloned genes of the present invention can be of any species of origin, including mouse, rat, rabbit, cat, porcine, and human, but are preferably of mammalian origin.
  • DNAs which hybridize to DNA disclosed herein as SEQ ID NO:l are also an aspect of this invention.
  • Conditions which will permit other DNAs which code on expression for a protein of the present invention to hybridize to the DNA of SEQ ID N0:1 disclosed herein can be determined in accordance with known techniques.
  • hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5x Denhardt' s solution, 0.5% SDS and lx SSPE at 37°C; conditions represented by a wash stringency of 40-45% Formamide with 5x Denhardt's solution, 0.5% SDS, and lx SSPE at 42°C; and conditions represented by a wash stringency of 50% Formamide with 5x Denhardt' s solution, 0.5% SDS and lx SSPE at 42°C, respectively) to DNA of SEQ ID NO:l disclosed herein in a standard hybridization assay. See, e . g. , J.
  • sequences which code for proteins of the present invention and which hybridize to the DNA of SEQ ID NO:l disclosed herein will be at least 75% homologous, 85% homologous, and even 95% homologous or more with SEQ ID NO:l.
  • DNAs which code for proteins of the present invention, or DNAs which hybridize to that of SEQ ID NO:l, but which differ in codon sequence from SEQ ID NO:l due to the degeneracy of the genetic code are also an aspect of this invention.
  • nucleotide sequence as disclosed herein in SEQ ID NO:l can be used to generate hybridization probes which specifically bind to the DNA of the present invention or to mRNA to determine the presence of amplification or overexpression of the proteins of the present invention.
  • oligonucleotide probes that are homologous to both DNA of SEQ ID N0:1 and to MTS1 or MTS2 DNA encoding pl6 as described in M. Serrano et al. , Nature 366, 704-707 (16 Dec. 1993) and A. Kamb, Science 264, 436 (1994) may be used to locate homologous DNAs of the same family in the same species or other species .
  • probes having the sequences given herein as SEQ ID NO:3 and SEQ ID NO:4.
  • the hybridization probes may be cDNA fragments or oligonucleotides, and may be labelled with a detectable group as discussed hereinbelow. Pairs of probes which will serve as PCR primers for the DNA sequences of the present invention, or portions thereof, may be used in accordance with the process described in
  • a vector is a replicable DNA construct.
  • Vectors are used herein either to amplify DNA encoding the proteins of the present invention or to express the proteins of the present invention.
  • An expression vector is a replicable DNA construct in which a DNA sequence encoding the proteins of the present invention is operably linked to suitable control sequences capable of effecting the expression of proteins of the present invention in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.
  • Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus) , phage, retroviruses and integratable DNA fragments (i.e., fragments integratable into the host, genome by recombination) .
  • the vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself.
  • Expression vectors should contain a promoter and RNA binding sites which are operably linked to the gene to be expressed and are operable in the host organism.
  • DNA regions are operably linked or operably associated when they are functionally related to each other.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase.
  • Transformed host cells are cells which have been transformed or transfected with vectors containing DNA coding for proteins of the present invention, constructed using recombinant DNA techniques .
  • Transformed host cells ordinarily express protein, but host cells transformed for purposes of cloning or amplifying DNA coding for the proteins of the present invention need not express protein.
  • Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic organism cells.
  • Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or Bacilli.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below.
  • Exemplary host cells are E. coli W3110 (ATCC 27,325) , E. coli B, E. coli X1776 (ATCC 31,537) , E. coli 294 (ATCC 31,446) .
  • a broad variety of suitable prokaryotic and microbial vectors are available.
  • E. coli is typically transformed using pBR322. See Bolivar et al . , Gene 2, 95
  • Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase
  • Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host . Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al . , Na ture 275, 615 (1978) ; and Goeddel et al . , Na ture 281, 544 (1979)) , a tryptophan (trp) promoter system (Goeddel et al . , Nuclei c Acids Res . 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H.
  • the promoter and Shine-Dalgarno sequence are operably linked to the DNA encoding the desired protein, i.e., they are posi ⁇ tioned so as to promote transcription of the protein messenger RNA from the DNA.
  • Eukaryotic microbes such as yeast cultures may be transformed with suitable protein-encoding vectors. See, e . g. , U.S. Patent No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available.
  • Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS) , a promoter, DNA encoding the desired protein, sequences for polyadenylation and transcription termination, and a selection gene.
  • An exemplary plasmid is YRp7,
  • This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example
  • Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phospho- glycerate kinase (Hitzeman et al . , J. Biol . Chem . 255, 2073 (1980) or other glycolytic enzymes (Hess et al . , J. Adv. Enzyme Reg. 7, 149 (1968) ; and Holland et al.
  • enolase such as enolase, glyceral- dehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used) , a polyadenylation site, and a transcriptional termination sequence.
  • transcriptional and translational control sequences in expression vectors to be used in trans ⁇ forming vertebrate cells are often provided by viral sources.
  • promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40
  • SV40 SV40
  • the early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. See Fiers et al . , Na ture 273, 113 (1978) .
  • the protein promoter, control and/or signal sequences may also be used, provided such control sequences are compatible with the host cell chosen.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV) , or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Host cells such as insect cells (e.g., cultured
  • baculovirus expression vector e.g., vectors derived from Autographa calif ornica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV
  • baculovirus expression vector e.g., vectors derived from Autographa calif ornica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV
  • baculovirus expression vector e.g., vectors derived from Autographa calif ornica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV
  • a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.
  • a selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase.
  • proteins generally enzymes, that enable the identification of transformant cells, i.e., cells which are competent to take up exogenous DNA. Generally, identification is by survival of transformants in culture medium that is toxic, or from which the cells cannot obtain critical nutrition without having taken up the marker protein.
  • ligand refers to a molecule that is recognized by a particular receptor protein.
  • a ligand is a molecule, such as a peptide, that is specifically binds to CDK6 at the site bound by the protein having the sequence given herein as SEQ ID NO:2 and competes with that protein for binding to that site.
  • an “inhibitory ligand” or an “inhibitory binding ligand” is a ligand which binds to and inhibits the normal activity of the receptor protein.
  • Receptor refers to a molecule that has an affinity for a given ligand.
  • Analogs of p20 are an aspect of the present invention.
  • an "analog” is a chemical compound similar in structure to a first compound, and having either a similar or opposite physiologic action as the first compound.
  • p20 analogs are those compounds which, while not having the amino acid sequences of native p20, are capable of binding to CDK6. Such analogs may be peptide or non-peptide analogs, including nucleic acid analogs, as described in further detail below.
  • Patent No. 4,863,857 to Blalock (applicants specifically intend that the dis ⁇ closures of all U.S. Patent references cited herein be incorporated by reference herein in their entirety) . See also Waldrop, Science, 247, 28029 (1990) ; Rossmann, Na ture, 333, 392-393 (1988) ; Weis et al . , Na ture, 333, 426-431 (1988) ; James et al . , Science, 260, 1937 (1993) (development of benzodiazepine peptidomimetic compounds based on the structure and function of tetrapeptide ligands) .
  • peptides containing such dele ⁇ tions or substitutions are a further aspect of the present invention.
  • one or more amino acids of a peptide sequence may be replaced by one or more other amino acids wherein such replacement does not affect the function of that sequence.
  • Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydro- phobicity/hydrophilicity, size and configuration, so that amino acids are substituted with other amino acids having essentially the same functional properties.
  • Ala may be replaced with Val or Ser; Val may be replaced with Ala, Leu, Met, or lie, preferably Ala or Leu; Leu may be replaced with Ala, Val or lie, preferably Val or lie; Gly may be replaced with Pro or Cys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replaced with Pro or Cys, preferably Cys; His may be replaced with Phe or Gin, preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe or Trp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may be replaced with Gin or Ser, preferably Gin; Gin may be replaced with His, Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced with Gin, Thr
  • Non-peptide mimetics of the peptides of the present invention are also an aspect of this invention.
  • Non-protein drug design may be carried out using computer graphic modeling to design non-peptide, organic molecules which bind to sites bound by pl8 INK6 . See, e . g . , Knight, BIO /Technology, 8, 105 (1990) . Itzstein et al, Na ture , 363, 418 (1993) (peptidomimetic inhibitors of influenza virus enzyme, sialidase) .
  • Lam et al modeled the crystal structure of the sialidase receptor protein using data from x-ray crystallography studies and developed an inhibitor that would attach to active sites of the model; the use of nuclear magnetic resonance ( ⁇ MR) data for modeling is also known in the art. See also Lam et al, Science, 263, 380 (Jan. 1994) regarding the rational design of bioavailable nonpeptide cyclic ureas that function as HIV protease inhibitors. Lam et al used information from x-ray crystal structure studies of HIV protease inhibitor complexes to design nonpeptide inhibitors. The modeling of a protein kinase structure using the known structure of other kinases is reported by Knighton et al .
  • Analogs may also be developed by generating a library of molecules, selecting for those molecules which act as ligands for a specified target, and identifying and amplifying the selected ligands. See, e . g. , Kohl et al . , Science, 260, 1934 (1993) (synthesis and screening of tetrapeptides for inhibitors of farnesyl protein transferase, to inhibit ras oncoprotein dependent cell transformation) . Techniques for constructing and screen ⁇ ing combinatorial libraries of oligomeric biomolecules to identify those that specifically bind to a given receptor protein are known. Suitable oligomers include peptides, oligonucleotides, carbohydrates, nonoligonucleotides
  • Peptide libraries may be synthesized on solid supports, or expressed on the surface of bacteriophage viruses (phage display libraries) .
  • Known screening methods may be used by those skilled in the art to screen combi ⁇ natorial libraries to identify analogs of p20.
  • Techniques are known in the art for screening synthesized molecules to select those with the desired activity, and for labelling the members of the library so that selected active molecules may be identified. See, e . g. , Brenner and Lerner, Proc . Natl . Acad . Sci . USA, 89, 5381 (1992) (use of genetic tag to label molecules in a combinatorial library); PCT US93/06948 to Berger et al . , (use of recombinant cell transformed with viral transactivating element to screen for potential antiviral molecules able to inhibit initiation of viral transcription) ; Simon et al.
  • combinatorial library refers to collections of diverse oligomeric biomolecules of differing sequence, which can be screened simultaneously for activity as a ligand for a particular target.
  • Combinatorial libraries may also be referred to as "shape libraries", i . e . , a population of randomized polymers which are potential ligands.
  • shape libraries i . e .
  • the shape of a molecule refers to those features of a molecule that govern its interactions with other molecules, including Van der Waals, hydrophobic, electrostatic and dynamic.
  • Nucleic acid molecules may also act as ligands for receptor proteins. See, e . g. , Edgington, BIO/ Technology, 11, 285 (1993) .
  • U.S. Patent No. 5,270,163 to Gold and Tuerk describes a method for identifying nucleic acid ligands for a given target molecule by selecting from a library of RNA molecules with randomized sequences those molecules that bind specifically to the target molecule.
  • a method for the in vi tro selection of RNA molecules immunologically cross-reactive with a specific peptide is disclosed in Tsai, Kenan and Keene, Proc. Natl . Acad. Sci . USA, 89, 8864 (1992) and Tsai and Keene, J.
  • an antiserum raised against a peptide is used to select R ⁇ A molecules from a library of R ⁇ A molecules; selected R ⁇ A molecules and the peptide compete for antibody binding, indicating that the RNA epitope functions as a specific inhibitor of the antibody-antigen interaction.
  • the present invention provides isolated and purified p20 proteins, such as mammalian (or more preferably human) p20 proteins. Such proteins can be purified from host cells which express the same, in accordance with known techniques, or even manufactured synthetically.
  • DNAs of the present invention constructs containing the same and host cells that express the encoded proteins are useful for making proteins of the present invention.
  • Proteins of the present invention are useful as immunogens for making antibodies as described herein, and these antibodies and proteins provide a "specific binding pair.” Such specific binding pairs are useful as components of a variety of immunoassays and purification techniques, as is known in the art.
  • the proteins of the present invention are of known amino acid sequence as disclosed herein, and hence are useful as molecular weight markers in determining the molecular weights of proteins of unknown structure.
  • the DNAs, proteins and imetics of the present invention can be used in a similar manner as the DNA and proteins of U.S. Patent No. 5,302,706 to Smith, the disclosure of which is incorporated herein by reference in its entirety.
  • Antibodies ⁇ Antibodies which specifically bind to the proteins of the present invention i.e., antibodies which bind to a single antigenic site or epitope on the proteins) are useful for a variety of diagnostic purposes. Such antibodies may be polyclonal or monoclonal in origin, but are preferably of monoclonal origin.
  • the antibodies are preferably IgG antibodies of any suitable species, such as mouse, rat, rabbit, or horse, but are generally of mammalian origin. Fragments of IgG antibodies which retain the ability to specifically bind the proteins of the present invention, such as F(ab') 2 ⁇ F(ab') , and Fab fragments, are intended to be encompassed by the term "antibody” herein. See generally E. Harlow and D. Lane, Antibodies: A Laboratory Manual (1988) (New York: Cold Spring Harbor Laboratory Press) . The antibodies may be chimeric, as described by M. Walker et al . , Molecular Immunol . 26, 403 (1989) .
  • Monoclonal antibodies which bind to proteins of the present invention are made by culturing a cell or cell line capable of producing the antibody under conditions suitable for the production of the antibody (e.g., by maintaining the cell line in HAT media) , and then collecting the antibody from the culture (e.g., by precipitation, ion exchange chromatography, affinity chromatography, or the like) .
  • the antibodies may be generated in a hybridoma cell line in the widely used procedure described by G. Kohler and C. Milstein, Na ture 256, 495 (1975) , or may be generated with a recombinant vector in a suitable host cell such as E ⁇ cheri chia col i in the manner described by W. Huse et al .
  • Immunoassavs. Assays for detecting expression of proteins of the present invention in a cell or the extent of expression thereof generally comprise the steps of, first, contacting cells or extracts of cells to antibodies capable of specifically binding the proteins, and determining the extent of binding of said antibodies to said cells.
  • the antibody is preferably labelled, as discussed above, to facilitate the detection of binding. Any suitable immunoassay procedure may be employed, such as radioimmunoassay, immunofluorescence, precipitation, agglutination, complement fixation, and enzyme-linked immunosorbent assay.
  • the antibodies are labelled with a radioactive detectable group and administered to the mammal, and the extent of binding of the antibodies to the cells is observed by external scanning for radioactivity.
  • a radioactive detectable group As discussed above, while any type of antibody may be employed for the foregoing diagnostic purposes, monoclonal antibodies are preferred.
  • monoclonal antibodies are preferred.
  • Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E Harlow and D. Lane, supra ; E. Maggio, Enzyme- Immunoassay, (1980) (CRC Press, Inc., Boca Raton, FL) ; see also U.S. Patent No. 4,727,022 to Skold et al .
  • Antibodies may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies may likewise be conjugated to detectable groups such as radiolabels (e.g., 35 S, :25 I , 131 I) , enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase) , and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • radiolabels e.g., 35 S, :25 I , 131 I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescent labels e.g., fluorescein
  • Kits for determining if a sample contains proteins of the present invention will include at least one reagent specific for detecting the presence or absence of the protein. Diagnostic kits for carrying out antibody assays may be produced in a number of ways.
  • the diagnostic kit comprises (a) an antibody which binds proteins of the present invention conjugated to a solid support and (b) a second antibody which binds proteins of the present invention conjugated to a detectable group.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates) , agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • a second embodiment of a test kit comprises (a) an antibody as above, and (b) a specific binding partner for the antibody conjugated to a detectable group. Ancillary agents as described above may likewise be included.
  • the test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test . Nucleic Acid Assays.
  • Assays for detecting pl8 iNK6 DNA or m RNA in a cell, or the extent of amplification thereof typically involve, first, contacting the cells or extracts of the cells containing nucleic acids therefrom with an oligonucleotide that specifically binds to pl8 INK6 DNA or mRNA as given herein
  • any suitable assay format may be employed (see, e . g. , U.S. Patent No. 4,358,535 to Falkow et al . ; U.S. Patent No. 4,302,204 to Wahl et al.; 4,994,373 to Stavrianopoulos et al; 4,486,539 to Ranki et al. ; 4,563,419 to Ranki et al . ; and 4,868,104 to Kurn et al . ) (the disclosures of which applicant specifically intends be incorporated herein by reference) .
  • Antisense Oligonucleotides may be made in accordance with conventional techniques. See, e . g. , U.S. Patent No. 5,023,243 to Tullis; U.S. Patent No. 5,149,797 to Pederson et al .
  • the length of the antisense oligonucleotide i.e., the number of nucleotides therein is not critical so long as it binds selectively to the intended location, and can be determined in accordance with routine procedures.
  • the antisense oligonucleotide will be from 8, 10 or 12 nucleotides in length up to 20, 30, or 50 nucleotides in length.
  • Such antisense oligonucleotides may be oligonucleotides wherein at least one, or all, of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioate s , phosphoromorpho1 idat e s , phosphoropiperazidates and phosphoramidates .
  • every other one of the internucleotide bridging phosphate residues may be modified as described.
  • such antisense oligonucleotides are oligonucleotides wherein at least one, or all, of the nucleotides contain a 2' loweralkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl) .
  • every other one of the nucleotides may be modified as described. See also P. Furdon et al . , Nuclei c Acids Res . 17, 9193-9204 (1989) ; S. Agrawal et al., Proc.
  • Transformants were plated on yeast drop-out media lacking leucine, tryptophan, and histidine, and containing 30 mM 3-amino-1, 2, 4 triazole (3-AT) . An estimated 5 X 10 6 transformants were screened. After 6 days of growth, histidine positive (His+) colonies were tested for b-galactosidase activity. Forty- five colonies were positive for b-galactosidase staining and were further purified on selective media. Plasmid D ⁇ A was recovered from positive colonies and introduced into Escherichia coli strain JM101 (J. Messing, Recomb . DNA Tech . bull . 2(2) : 43 (1979)) .
  • the cD ⁇ As from plasmids recovered from E. coli were excised from pGADGL vector and inserted into pBluescript (Stratagene Inc.) . The first three hundred nucleotide sequences were determined for each clone. Most clones corresponded to the previously reported P16 I ⁇ K4/MTSI_ In addition to pl6 INK4 ' a clone (6H10) was isolated that encoded a novel protein that was not present in the current data base ((GenBank release 82.00 April 1994) . A cDNA insert from clone 6H10 was used as probe to screen a human HeLa cDNA library constructed in ⁇ ZAP II vector (STRATAGENE Inc.) to obtain full length sequences.
  • Anti-CDK6 peptide antibodies were as described by, and were provided by, Meyerson and Harlow, Mol . Cell . Biol . 14, 2077-2086 (1994) .
  • the anti-pl8 antibody reacts strongly with human pl8 protein as tested by immunoblotting with purified pl8 protein and by immunoprecipitation with in vitro translated pl8 protein.
  • the antibody also cross- reacts very weakly with human pl6 protein in immunoprecipitation but not with any of the CDKs tested.
  • An acute lymphoblastic leukemia cell line, CEM was chosen for the analysis of pl8 protein interaction in vivo because it has a homozygous deletion of the pl6 locus and expresses pl8 mRNA at a high level.
  • CEM chronic lymphoblastic leukemia cell line
  • the anti-pl8 antibody precipitated several cellular proteins that were not seen in the corresponding precipitation with preimmune serum from the rabbit used to generate the anti-pl8 antibody.
  • AN 18-kD protein that comigrated with in vitro-translated pl8 antibody was later confirmed as pl8 by immunoblotting.
  • the anti-pl ⁇ antibody also coprecipitated two polypeptides that correspond to CDK4 and CDK6, respectively, as determined by gel mobility and immunoblotting.
  • anti- CDK6 antibody coprecipitates an 18-kD protein with CDK6 , which comigrates with the 18-kD protein present in the anti-pl ⁇ immunocomplex and is effectively competed by the preincubation of the antibody with an antigen peptide.
  • both anti-pl8 and anti-CDK6 antisera precipitated a 20-kD polypeptide, p20.
  • Partial V8 peptide mapping of p20 gave rise to a different pattern from both pl8 and pl6. This band was effectively competed by a CDK6 antigen peptide in anti- CDK6 immunoprecipitates and was not seen in immunoprecipitation with preimmune serum, indicating that p20 is a CDK-6 associated protein.
  • a human cDNA encoding the protein p20 described in Example 1 above was isolated as follows. A pair of degenerate oligonucleotides were chemically synthesized based on the conserved region of pl6 and pl8 protein sequence and used as primers in polymerase chain reaction (PCR) to amplify potential sequences using cDNA template prepared from a human cDNA library. DNA fragments with approximately the size of interest were excised from agarose gel and ligated into a cloning vector and sequenced.
  • PCR polymerase chain reaction
  • a cDNA insert was isolated from this screening that showed considerable similarity to pl6 and pi8 and was used as a probe to screen a human cDNA library to obtain full length cDNA encoding the p20 gene as shown in SEQ ID NO:l.
  • the amino acid sequence of the encoded protein, p20 is given as SEQ ID NO:2.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • GCT GTC A ⁇ CAT GAT GCG GCC AGA GCA GGT ⁇ C CTG GAC ACT ⁇ A CAG 354

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un acide nucléique codant une protéine inhibitrice de kinases cyclino-dépendantes (CDK), notamment une protéine inhibitrice de CDK6, est sélectionné dans le groupe comprenant: a) de l'ADN dont la séquence nucléotidique est représentée par le N° ID SEQ 1 (qui code la protéine dont la séquence d'acides aminés est représentée par le N° ID SEQ 2) et qui porte le nom de p20; b) des acides nucléiques qui s'hybrident à l'ADN précité et qui codent pour une protéine inhibitrice de CDK; et c) des acides nucléiques qui diffèrent de ceux précités en (a) et (b) en raison de la dégénérescence du code génétique et qui codent une protéine inhibitrice de CDK codée par un des acides nucléiques précités en (a) et (b). L'invention concerne également des produits de recombinaison contenant ces acides nucléiques, des cellules contenant ces produits de recombinaison, des protéines codées par ces acides nucléiques, des anticorps s'y fixant, des oligonucléotides antisens correspondant à ces acides nucléiques, ainsi que leur mode d'emploi.
PCT/US1995/016553 1994-12-22 1995-12-19 Adn codant une proteine inhibitrice de cdk6 de 20 kilodaltons WO1996019244A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46023/96A AU4602396A (en) 1994-12-22 1995-12-19 Dna encoding a 20 kd cdk6 inhibiting protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US36223594A 1994-12-22 1994-12-22
US08/362,235 1994-12-22

Publications (1)

Publication Number Publication Date
WO1996019244A1 true WO1996019244A1 (fr) 1996-06-27

Family

ID=23425259

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/016553 WO1996019244A1 (fr) 1994-12-22 1995-12-19 Adn codant une proteine inhibitrice de cdk6 de 20 kilodaltons

Country Status (2)

Country Link
AU (1) AU4602396A (fr)
WO (1) WO1996019244A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6489305B1 (en) 1998-05-08 2002-12-03 Canji, Inc. Methods and compositions for the treatment of ocular diseases
US7465442B2 (en) 2003-11-24 2008-12-16 Canji, Inc. Reduction of dermal scarring

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOLECULAR AND CELLULAR BIOLOGY, Volume 15, Number 5, issued May 1995, CHAN et al., "Identification of Human and Mouse p19, a Novel CDK4 and CDK6 Inhibitor With Homology to p16ink4", pages 2682-2688. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 15, Number 5, issued May 1995, HIRAI et al., "Novel INK4 Proteins, p19 and p18, are Specific Inhibitors of the Cyclin D-dependent Linases CDK4 and CDK6", pages 2672-2681. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6489305B1 (en) 1998-05-08 2002-12-03 Canji, Inc. Methods and compositions for the treatment of ocular diseases
US7465442B2 (en) 2003-11-24 2008-12-16 Canji, Inc. Reduction of dermal scarring
US8329671B2 (en) 2003-11-24 2012-12-11 Canji, Inc. Reduction of dermal scarring

Also Published As

Publication number Publication date
AU4602396A (en) 1996-07-10

Similar Documents

Publication Publication Date Title
US5629407A (en) DNA encoding an 18 KD CDK6 inhibiting protein and antibodies thereto
O'Connell et al. Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase
Yokoyama et al. A giant nucleopore protein that binds Ran/TC4
CA2119443C (fr) Cycline e humaine
JP2003274984A (ja) ＩＫＫ−βタンパク質、核酸及び方法
US5744313A (en) Assay employing novel protein domain which binds tyrosine phosphorylated proteins
AU6691398A (en) IkB kinases
CA2255143A1 (fr) Partenaires liants pour inhibiteurs de kinases cyclino-dependantes et leur utilisation dans la recherche d'inhibiteurs et de diagnostics ou d'une therapie de maladie
Lau et al. Two-hybrid cloning identifies an RNA-binding protein, GRY-RBP, as a component of apobec-1 editosome
US5585269A (en) Isolated DNA encoding c-mer protooncogene
US5618693A (en) Interleukin-2 signal transducers and binding assays
WO1996019244A1 (fr) Adn codant une proteine inhibitrice de cdk6 de 20 kilodaltons
WO1995005392A1 (fr) Nouvelles proteines se liant a la proteine du retinoblastome et sequences d'adn de codage de ces dernieres
US6307035B1 (en) BRCA1 associated polynucleotide (BAP-1) and uses therefor
US6242587B1 (en) DNA molecules encoding a calcium-integrin binding protein
US5891719A (en) IKAP nucleic acids
WO1998005968A9 (fr) Proteine bap-1 associee a la brca1 et ses utilisations
EP0938555B1 (fr) Constructions et complexes de cycline e
US6753413B1 (en) P35NCK5A binding proteins
AU731570B2 (en) Tyrosine phosphorylated cleavage furrow-associated proteins (PSTPIPs)
US6531296B1 (en) Nuclear tyrosine kinase Rak
US6040437A (en) Tyrosine phosphorylated cleavage furrow-associated proteins (PSTPIPS)
US5710025A (en) Cell-type specific transcription factor
AU758077B2 (en) Human proteins responsible for NEDD8 activation and conjugation
AU696986C (en) Novel proteins which bind to retinoblastoma protein and their encoding DNA equences

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA