WO2001002828A2 - Diagnostic du cancer par detection de polynucleotides ou de polypeptides ash2 - Google Patents

Diagnostic du cancer par detection de polynucleotides ou de polypeptides ash2 Download PDF

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WO2001002828A2
WO2001002828A2 PCT/US2000/018657 US0018657W WO0102828A2 WO 2001002828 A2 WO2001002828 A2 WO 2001002828A2 US 0018657 W US0018657 W US 0018657W WO 0102828 A2 WO0102828 A2 WO 0102828A2
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ash2
cancer
polypeptide
seq
polynucleotide
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PCT/US2000/018657
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WO2001002828A3 (fr
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Susan Erster
Scott Powers
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Tularik Inc.
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Priority to AU57889/00A priority Critical patent/AU5788900A/en
Publication of WO2001002828A2 publication Critical patent/WO2001002828A2/fr
Publication of WO2001002828A3 publication Critical patent/WO2001002828A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Definitions

  • colorectal cancer is the third most common cancer in men and women.
  • An estimated 56,600 deaths (47,900 from colon cancer, 8,700 from rectal cancer) are expected to occur in 1999.
  • Cancer is a genetic disease of single cell origin caused by the accumulation of inherited and acquired mutations in specific cancer genes which have normal cellular functions, but which contribute to cancer when mutated. Mutations that unlock the cancer- causing potential ' of cancer genes include, for example, gene amplification, i.e., where a specific chromosomal region (including the cancer gene) undergoes a relative increase in DNA copy number, so that more cancer gene copies are present and a correspondingly higher amount of mRNA and protein is produced, causing deleterious effects.
  • the identification of such amplified (or overexpressed) genes may provide important tools for the diagnosis and/or treatment of cancer. To date, several such overexpressed genes have been identified, such as HER2 and MYC. These genes, however, are not involved in all types of cancers, and many cancers contain amplified regions where the underlying cancer gene is not known.. Clearly, a large number of genes that are amplified and/or overexpressed in cancer cells remain to be identified.
  • RDA representational difference analysis
  • the ASH2 gene is a member of the "trithorax" group of genes whose products function to maintain active transcription of homeotic selector genes. Mutations in Drosophila ASH2 result in homeotic transformations as well as a variety of pattern formation defects ⁇ see, e.g., Genetics (1996) 144(2): 621-633; see also the entry for ASH2 at Flybase, a Drosophila gene database, at http://flybase.bio.indiana.edu/). Homologs of ASH2 have been identified in organisms other than Drosophila, such as humans, mice, and fish, but no function has been attributed to any of these non-Drosophila ASH2 homologs.
  • the present invention is based on the surprising discovery that ASH2 polynucleotide sequences are amplified and/or overexpressed in many types of cancer cells in mammals. As described infra, this invention thus provides novel and badly needed diagnostic, prognostic, and therapeutic tools for many types of cancers.
  • the present invention provides methods for diagnosing and treating cancer in an animal, e.g., a human.
  • the methods typically involve detecting the level of ASH2 nucleic acid or protein in a biological sample taken from the animal.
  • the presence of ASH2 nucleic acid and/or protein in the sample at a level that is greater than that expected for a non-cancerous sample is indicative of cancer in the sample, or in the animal from which the sample is derived.
  • the presence of a cancer in an animal will be assessed by detecting the presence or absence of a diagnostic presence of ASH2 polypeptide or polynucleotide in a biological sample taken from the animal.
  • the assay used to detect ASH2 in the sample will often be performed under conditions that would not detect ASH2 polypeptide or polynucleotide in a sample that is not cancerous.
  • a detection of a diagnostic presence of ASH2 indicates the presence of cancer in the animal.
  • a diagnostic presence represents at least about a 2, 5, 10, or greater fold increase in the ASH2 polypeptide or polynucleotide in the biological sample compared to a level expected in a sample from a control, cancer-free animal.
  • the present invention provides methods for monitoring the efficacy of a cancer treatment.
  • a level of ASH2 polypeptide or polynucleotide in a biological sample from an animal undergoing treatment for cancer is detected.
  • a reduced level of ASH2 polypeptide or polynucleotide in the biological sample compared to a level in a sample taken from the animal prior to, or earlier in, the treatment indicates that the treatment is efficacious.
  • the methods provided herein can be used to treat cancer.
  • the presence or absence of a diagnostic presence of ASH2 polynucleotide or polypeptide is detected in a biological sample taken from an animal with cancer.
  • the detection of a diagnostic presence of ASH2, indicating the presence of cancer in the biological sample is followed by the administration of one or more types of cancer therapy.
  • the detection of a presence or absence of a diagnostic presence of ASH2 polynucleotide or polypeptide is often repeated after, or during, the administration of the cancer therapy, thereby allowing a determination of the efficacy of the cancer therapy.
  • the proliferation is decreased using an inhibitor of ASH2 activity, such as antisense polynucleotides, ribozymes, antibodies, dominant negative ASH2 inhibitors, and small molecule inhibitors of ASH2 activity.
  • the present methods are performed by detecting an ASH2 gene, an ASH2 mRNA, an ASH2 polypeptide or ASH2 protein activity.
  • the animal is a mammal, e.g., a primate, canine, feline, murine, bovine, equine, ovine, porcine, lagomorph, etc.
  • the animal is a human.
  • a biological sample used for detection of ASH2 is a sample selected from the group consisting of tissue biopsy, blood sample, buccal scrape, saliva, nipple discharge, urine, etc.
  • the present methods can be used to diagnose, determine the prognosis for, or treat, any of a number of types of cancers.
  • the cancer is an epithelial cancer, e.g., breast, lung, colorectal, prostate, kidney, stomach, bladder, or ovarian cancer, or any cancer of the gastrointestinal tract.
  • the present invention also provides ASH2 polynucleotides and polypeptides.
  • the ASH2 polynucleotides and polypeptides provided herein represent novel splice variants that lack N-terminal and exon sequences found in previously described ASH2 sequences.
  • the ASH2 polypeptide sequences provided herein comprise novel subsequences not described previously.
  • the present invention provides ASH2 polynucleotides, and polypeptides encoded thereby, that encode ASH2 polypeptides including novel subsequences that are at least about 70% identical to the sequences shown as SEQ LD NO:3 or SEQ ID NO:9, or which lack the sequence shown as SEQ LD NO:4.
  • Kits for practicing the present invention are also provided.
  • Figure 1 shows a physical map of the 8pl l-pl2 amplified chromosomal region, indicated by the solid line marked with solid circles.
  • the present invention provides methods for assessing the presence of cancer in a biological sample taken from an animal.
  • the methods provided herein are based on the surprising discovery that the ASH2 gene, forms of which have been previously identified in mammals, but with no known function, is amplified and/or overexpressed in numerous types of cancers. Accordingly, it has been discovered that the detection of overexpression or amplification of ASH2 polynucleotides or polypeptides in a biological sample from an animal is diagnostic of cancer in the animal. Detection of ASH2 in a sample also provides a means for monitoring the efficacy of a cancer treatment.
  • the present invention provides numerous methods for treating cancer, including determining the most effective course of anti-cancer therapy based on ASH2 levels, and methods of inhibiting the proliferation of cancer cells by reducing ASH2 polypeptide or polynucleotide levels or inhibiting ASH2 protein activity. Also provided are novel ASH2 polynucleotide and polypeptide sequences.
  • detecting a cancer refers to the ascertainment of the presence or absence of cancer in an animal.
  • Detecting a cancer can also refer to obtaining indirect evidence regarding the likelihood of the presence of cancerous cells in the animal. Detecting a cancer can be accomplished using the methods of this invention alone or in combination with other methods, and can be aided by other information regarding the state of health of the animal.
  • a “cancer” in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may circulate in the blood stream as independent cells, such as leukemic cells.
  • an “animal” refers to a member of the kingdom Animalia, characterized by multicellularity, the possession of a nervous system, voluntary movement, internal digestion, etc.
  • An “animal” can be a human or any other mammal, including non-human primates, canines, felines, murines, bovines, ovines, equines, porcines, and lagomorphs.
  • "Providing a biological sample” means to obtain a biological sample for use in the methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo.
  • control sample refers to a sample of biological material representative of healthy, cancer-free animals.
  • the level of ASH2 in a control sample is desirably typical of the general population of normal, cancer-free animals. This sample can be removed from an animal expressly for use in the methods described in this invention, or can be any biological material representative of normal, cancer- free animals.
  • a control sample can also refer to an established level of ASH2, representative of the cancer-free population, that has been previously established based on measurements from normal, cancer- free animals.
  • a detection method that only detects ASH2 when a level higher than that typical of a normal, cancer-free animal is present, i.e., an immunohistochemical assay giving a simple positive or negative result, this is considered to be assessing the ASH2 level in comparison to the "control" level, as the "control" level is inherent in the assay.
  • a level of ASH2 polypeptide or polynucleotide that is "expected" in a control sample refers to a level that represents a typical, cancer- free sample, and from which an elevated, or diagnostic, presence of ASH2 polypeptide or polynucleotide can be distinguished.
  • an "expected" level will be controlled for such factors as the age, sex, medical history, etc. of the animal, as well as for the type of biological sample being tested.
  • an “increased,” or “elevated,” level of ASH2 refers to a level of ASH2 polynucleotide or polypeptide, that, in comparison with a control level of ASH2, is detectably higher.
  • the method of comparison can be statistical, using quantified values for the level of ASH2, or can be compared using non-statistical means, such as by a visual, subjective assessment by a human.
  • a "diagnostic presence" of ASH2 polynucleotides or polypeptides refers to any amount of ASH 2 in a biological sample, detected using any method, that represents an increase over a control level.
  • an "ASH2 polynucleotide” or “ASH2 nucleic acid” is a DNA or RNA sequence of at least about 50 nucleotides that is at least about 70% identical, preferably at least about 80% or more, identical over a region of at least about 50, 100, 200, 500, or more nucleotides to one or more ASH2 polynucleotide sequences (see, e.g., GenBank Accession Nos: AF056717, AB020982, AF056718, AB020983, SEQ LD NO:l, SEQ LD NO:5, SEQ LD NO:7, and others).
  • an "ASH2 polynucleotide” can comprise naturally occuring nucleotides, or any derivative or analog thereof, e.g., labeled or modified deoxyribo- or ribonucleotides.
  • the term “ASH2 polynucleotide” can refer to a mutated copy of any of the above sequences, or a fragment thereof.
  • An “ASH2 polynucleotide” can refer to a natural sequence derived from an ASH2 polynucleotide from any organism, or can be a sequence designed de novo.
  • ASH2 protein or "ASH2 polypeptide” refers to a polypeptide of at least about 20 amino acids that is typically about 70% identical, preferably at least about 80%, more preferably at least about 90%, or more, identical over a region of at least about 20, 50, 100 or more amino acids, to one or more of the ASH2 polypeptide sequences (see, e.g., GenBank Accession Nos: AF056717, AB020982, AF056718, AB020983, or SEQ ID NO:2, SEQ ID NO:6, SEQ LD NO:8), or to any derivative, variant, mutant, or fragment thereof.
  • An “ASH2 protein” can comprise naturally occurring or synthetic amino acids, e.g., labeled or otherwise modified amino acids or amino acid analogs.
  • An “ASH2 protein” will typically contain one or more characteristic protein motifs, e.g., zinc finger motif, any of which can be used independently of other elements normally present in a full-length ASH2 protein.
  • An “ASH2 protein” can refer to a natural sequence derived from an ASH2 polypeptide from any organism, or to a sequence designed de novo.
  • the "level of ASH2 mRNA" in a biological sample refers to the amount of mRNA transcribed from an ASH2 gene, or encoding an ASH2 polypeptide, that is present in a cell or a biological sample.
  • the mRNA generally encodes a functional ASH2 protein, although mutations or microdeletions may be present that alter or eliminate the function of the encoded protein.
  • a "level of ASH2 mRNA” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • the "level "of ASH2 "protein,” or “polypeptide” in a biological sample refers to the amount of polypeptide translated from an ASH2 mRNA, or to the amount of ASH2 protein as defined supra, that is present in a cell or biological sample.
  • the polypeptide may or may not have ASH2 protein activity, i.e., it may or may not bind to one or more DNA sequences or heterologous proteins.
  • a "level of ASH2 protein” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • a "full length" ASH2 protein or nucleic acid refers to an ASH2 polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, wild type ASH2 polynucleotide or polypeptide sequences.
  • a difference between a test sample and a control can be termed "statistically significant" when the probability of the test sample being abnormal can be any of a number of values, including 0.15, 0.1, 0.05, and 0.01. Numerous sources teach how to assess statistical significance, such as Freund, J.E. (1988) Modern elementary statistics, Prentice-Hall.
  • a level of ASH2 in a sample is said to be "at least about 2 fold greater" than the level of ASH2 in another sample or in a control, this signifies that the level of ASH2 in the first sample is at least about 2 times the value of ASH2 in the second sample or to a control value.
  • a level of ASH2 in a sample is said to be "at least about 5 fold greater" than the level of ASH2 in another sample or in a control, this signifies that the level of ASH2 in the first sample is at least about 5 times the value of ASH2 in the second sample or to a control value.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most prefe ⁇ ed embodiment, the sequences are substantially identical over the entire length of the coding regions.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • an ASH2 polypeptide is typically substantially identical to a second ASH2 polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two ASH2 nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • stringent conditions refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm,
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. III. Detecting ASH2 Polynucleotides and Polypeptides
  • the present invention is based on the discovery that a diagnosis or prognosis of cancer can be determined by detecting elevated levels of ASH2 polypeptides or polynucleotides in a biological sample from an animal.
  • any of a number of methods to detect the presence and/or levels of ASH2 can be used.
  • An ASH2 polynucleotide level can be detected by detecting the presence of any ASH2 DNA or RNA, including ASH2 genomic DNA, mRNA, and cDNA.
  • An ASH2 polypeptide can be detected by detecting an ASH2 polypeptide itself, or by detecting ASH2 protein activity, e.g., DNA or protein binding activity, transcriptional regulation, etc.
  • Detection can involve quantification of the level of ASH2 (e.g., gDNA, cDNA, mRNA, protein, or protein activity), or, alternatively, can be a qualitative assessment of the level, or of the presence or absence, of ASH2, in particular in comparison with a control level. Any of a number of methods to detect any of the above can be used, as described infra. Such methods include, for example, hybridization, amplification, and other assays. In certain embodiments, a level of ASH2 in a biological sample will be compared with a control sample taken from a cancer-free animal, or, preferably, with a value expected for a sample taken from a cancer-free animal.
  • an assay will be performed under conditions where only a higher than normal amount of ASH2 polynucleotide or polypeptide will be detectable in the assay.
  • an elevated level of ASH2 can be detected in a sample using a simple assay giving a simple, positive or negative result, with no need for quantification of ASH2 levels or a direct comparison with a control sample.
  • the level of ASH2 polynucleotide, polypeptide, or protein activity will be quantified.
  • the difference between an elevated level of ASH2 and a normal, control level will preferably be statistically significant.
  • an elevated level of ASH2 polynucleotide, polypeptide, and/or protein activity will be at least about 2, 5, 10, or more fold greater than a control level.
  • the ASH2 polynucleotides or polypeptides detected herein will be at least about 70% identical, and preferably 80% or more identical, over a region of at least about 50, 100, 200, or more nucleotides, or 20, 50, 100, or more amino acids, to SEQ LD NO:l, 5, or 7, or SEQ ID NO:2, 6, or 8, or to one or more sequences available, e.g., from GenBank (see, e.g., GenBank Accession Nos: AF056717, AB020982, AF056718, AB020983, U73809, AB022785, and others).
  • Such polynucleotides or polypeptides can represent any of the ASH2 variants described herein, e.g., splice variants, and can indicate functional or non-functional forms of ASH2, or any variant, derivative, or fragment thereof.
  • a biological sample refers to a cell or population of cells or a quantity of tissue or fluid from an animal. Most often, the sample has been removed from an animal, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, i.e., without removal from the animal. Typically, a “biological sample” will contain cells from the animal, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine. Numerous types of biological samples can be used in the present invention, including, but not limited to, a tissue biopsy, blood sample, a buccal scrape, a saliva sample, or a nipple discharge.
  • tissue biopsy refers to an amount of tissue removed from an animal for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.
  • tissue biopsy can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.
  • a "buccal scrape” refers to a sample of cells removed from the inner lining of the mouth.
  • a “nipple discharge” refers to fluid originating from a nipple, which may contain cancerous cells or may contain elevated levels of ASH2 polypeptide indicating the presence of cancerous cells in the breast.
  • the presence of cancer is evaluated by determining the copy number of ASH2 genes.
  • the "copy number of ASH2 genes” refers to the number of DNA sequences in a cell encoding an ASH2 protein. Generally, for a given autosomal gene, an animal has two copies of each gene. The copy number can be increased, however, by gene amplification or duplication, e.g., in cancer cells, or reduced by deletion. Methods of evaluating the copy number of a particular gene are well known to those of skill in the art, and include, inter alia, hybridization and amplification based assays.
  • any of a number of hybridization based assays can be used to detect the copy number of ASH2 genes in the cells of a biological sample.
  • One such method is by Southern Blot.
  • genomic DNA is typically, fragmented, separated electrophoretically, and hybridized to an ASH2 specific probe. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DNA (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative ASH2 copy number.
  • An alternative means for determining the copy number of ASH2 genes in a sample is in situ hybridization, e.g., fluorescence in situ hybridization, or FISH.
  • In situ hybridization assays are well known (e.g., Angerer (1987) Meih. Enzymol 152: 649).
  • in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments.
  • the probes used in such applications are typically labeled, e.g., with radioisotopes or fluorescent reporters. Prefe ⁇ ed probes are sufficiently long, e.g., from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, so as to specifically hybridize with the target nucleic acid(s) under stringent conditions.
  • “comparative probe” methods such as comparative genomic hybridization (CGH) are used to detect ASH2 gene amplification.
  • CGH comparative genomic hybridization
  • a "test" collection of nucleic acids is labeled with a first label
  • a second collection e.g., from a healthy cell or tissue
  • the ratio of hybridization of the nucleic acids is determined by the ratio of the first and second labels binding to each fiber in the array. Differences in the ratio of the signals from the two labels, e.g., due to gene amplification in the test collection, is detected and the ratio provides a measure of the ASH2 gene copy number.
  • Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994), and elsewhere.
  • amplification-based assays are used to measure an ASH2 copy number.
  • the ASH2 nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR).
  • an amplification reaction e.g., Polymerase Chain Reaction, or PCR.
  • the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the copy number of the ASH2 gene.
  • Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • a TaqMan based assay is used to quantify ASH2 polynucleotides.
  • TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end.
  • the 5' nuclease activity of the polymerase e.g., AmpliTaq
  • the polymerase e.g., AmpliTaq
  • This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, literature provided by Perkin-Elmer, e.g., www2.perkin- elmer.com).
  • ligase chain reaction (LCR) (see, Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
  • LCR ligase chain reaction
  • ASH2 levels are characterized by detecting ASH2 gene expression by virtue of levels of ASH2 mRNA or cDNA in a biological sample.
  • one method for evaluating the presence, absence, or quantity of ASH2 cDNA involves a Southern Blot as described above. Briefly, ASH2 mRNA is isolated using standard methods and reverse transcribed to produce cDNA. The cDNA is then optionally digested, run on a gel, and transfe ⁇ ed to a membrane. Hybridization is then carried out using nucleic acid probes specific for ASH2 cDNA and detected using standard techniques (see, e.g., Sambrook et al., supra).
  • a Northern transfer may be used to detect an mRNA directly.
  • mRNA is isolated from a given biological sample, elecfrophoresed to separate the mRNA species, and transferred from the gel to a nitrocellulose membrane.
  • labeled ASH2 probes are used to identify and/or quantify the mRNA.
  • an ASH2 transcript (e.g., ASH2 mRNA) can be measured using amplification-based methods (e.g., PCR).
  • a transcript level is assessed by using reverse transcription PCR (RT-PCR). RT-PCR methods are well known to those of skill (see, e.g., Ausubel et al, supra).
  • ASH2 levels can also be detected and/or quantified by detecting or quantifying ASH2 polypeptide.
  • ASH2 polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like
  • immunological methods such as fluid or gel precipit
  • an ASH2 polypeptide is detected using an immunoassay such as an ELISA assay (see, e.g., Crowther, John R. ELISA Theory and Practice. Humana Press: New Jersey, 1995).
  • an "immunoassay” is an assay that utilizes an antibody to specifically bind to the analyte (i.e., the ASH2 polypeptide). The immunoassay is thus characterized by detection of specific binding of an ASH2 polypeptide to an anti-ASH2 antibody.
  • ASH2 polypeptide in an immunoassay, can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
  • Immunoassays typically rely on direct or indirect labeling methods to detect antibody-analyte binding.
  • an anti-ASH2 antibody can be directly labeled, thereby allowing detection.
  • the anti-ASH2 antibody may itself be unlabeled, but may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second or third antibodies can also be modified with a detectable moiety, e.g., as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • other antibody-binding molecules can be used, e.g., labeled protein A or G (see, generally Kronval, et al. (1973) J Immunol, 111: 1401-1406, and Akerstrom (1985) J. Immunol, 135: 2589-2542).
  • Immunoassays for detecting an ASH2 polypeptide can be competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of captured analyte is directly measured.
  • "sandwich" assays will be used, for example, wherein anti-ASH2 antibodies are bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the ASH2 protein present in a test sample. The ASH2 thus immobilized is then bound by a labeling agent, such as a second anti-ASH2 antibody bearing a label.
  • the amount of ASH2 protein present in a sample is measured indirectly, e.g., by measuring the amount of added (exogenous) ASH2 displaced (or competed away) from an anti ASH2 antibody by ASH2 protein present in a sample. For example, a known amount of labeled ASH2 polypeptide is added to a sample and the sample is then contacted with an anti-ASH2 antibody. The amount of labeled ASH2 polypeptide bound to the antibody is inversely proportional to the concentration of ASH2 polypeptide present in the sample.
  • any of a number of labels can be used in any of the immunoassays of this invention, including fluorescent labels, radioisotope labels, or enzyme-based labels, wherein a detectable product of enzyme activity is detected (e.g., peroxidase, alkaline phosphatase, ⁇ - galactosidase, etc.).
  • Antibodies for use in the various immunoassays described herein can be produced according to standard methods (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY.)
  • ASH2 can exist in one or more splice variants, described infra, which can be distinguished in a variety of ways, e.g., electrophoretic separation based on size, detection using variant specific antibodies, etc.
  • ASH2 polypeptide levels are determined by virtue of the ASH2 protein activity in a biological sample.
  • protein activity can be easily measured using standard techniques.
  • the ASH2 protein has a putative DNA binding domain, i.e., a zinc finger, that likely mediates specific binding to a particular DNA sequence.
  • ASH2 levels can be indirectly detected in a biological sample by detecting the amount of binding activity to that particular sequence that is present in the sample. Methods of detecting, and quantifying, DNA binding activity are well known to those of skill and are described, e.g., in Sambrook et al, Ausubel et al., etc. Other ASH2 activities, e.g., protein binding, can also be assessed using standard methods.
  • ASH2 is required for the maintenance of gene expression in cells. Accordingly, in vitro or in vivo transcription based assays (e.g., examining expression of ASH2 target genes) can also be used. Such assays are well known to those of skill in the art.
  • the present invention provides numerous methods for diagnosing any of a number of types of cancer, including, but not limited to, determining whether or not an animal has a cancer, whether or not a biological sample contains cancerous cells, estimating the likelihood of an animal developing cancer, and monitoring the efficacy of anti-cancer treatment in an animal with cancer.
  • Such methods are based on the surprising result that cancer cells have an elevated level of ASH2 polynucleotides (i.e., gene copy number and/or mRNA) and/or polypeptide level. Accordingly, by determining whether or not a cell contains elevated levels of ASH2 polynucleotide or polypeptide, it is possible to determine whether or not the cell is cancerous.
  • cancerous cells can be determined indirectly, i.e., a biological sample that does not itself contain cancerous cells, but which has been taken from an animal with cancerous cells elsewhere in its body, may contain elevated levels of ASH2 reflecting the presence of the cancerous cells.
  • the level and/or presence or ASH2 polynucleotide or polypeptide is detected in a biological sample, thereby detecting the presence or absence of cancerous cells in the biological sample, or, in certain embodiments, in the animal from which the biological sample was removed.
  • the biological sample comprises a tissue sample from a tissue suspected of containing cancerous cells. For example, in a woman suspected of having breast cancer, breast tissue can be removed. Often, such methods will be used in conjunction with additional diagnostic methods, e.g., detection of other cancer markers, mammography, etc.
  • a tissue sample known to contain cancerous cells e.g., from a tumor
  • the amount of ASH2 polynucleotide or polypeptide used to determine the presence of a cancer will depend on numerous factors, including the type of cancer, the age, sex, medical history, etc., of the patient, the cell type, the assay format, etc.
  • a level of ASH2 in a biological sample will not be quantified or directly compared with a control sample, but will rather be detected relative to a "diagnostic presence" of ASH2, wherein a "diagnostic presence” refers to the amount of ASH2 polynucleotide or polypeptide that indicates the presence of cancer, or the likelihood of cancer, in a particular sample.
  • a "diagnostic presence” will be detectable in a simple assay giving a positive or negative result, where a positive “detection” of a "diagnostic presence” of ASH2 polynucleotide or polypeptide indicates the presence of cancer in the animal.
  • the ASH2 level need not be quantified for a "diagnostic presence” to be detected, merely any method of determining whether ASH2 is present at levels higher than in a normal, cancer free cell, sample, or animal.
  • a "diagnostic presence” does not refer to any absolute quantity of ASH2, but rather on an amount that, depending on the biological sample, cell type, assay conditions, medical condition, etc., is sufficient to distinguish the level in a cancerous, or pre-cancerous sample, from a normal, cancer-free sample.
  • ASH2 polynucleotide or polypeptide is normally present, or "expected” to be present, in a particular control sample.
  • ASH2 may not be expressed in certain cell types, resulting in a complete absence of ASH2 in a control biological sample consisting of such cell types.
  • a "diagnostic presence” refers to any detectable amount of ASH2.
  • a detectable level of ASH2 present in normal, cancer-free cells there may be a detectable level of ASH2 present in normal, cancer-free cells, and a "diagnostic presence” represents a level that is higher than the normal level, preferably representing a "statistically significant” increase over the normal level.
  • a "diagnostic presence" of ASH2 polynucleotide, polypeptide, and/or protein activity in a biological sample will be at least about 2, 5, 10, or more fold greater than a level expected in a sample taken from a normal, cancer-free animal.
  • the present methods can also be used to assess the efficacy of a course of treatment.
  • the efficacy of the treatment can be assessed by monitoring ASH2 levels over time. For example, a reduction in ASH2 polynucleotide or polypeptide levels in a biological sample taken from an animal following a treatment, compared to a level in a sample taken from the animal before the treatment, indicates efficacious treatment.
  • the level of ASH2 can be used to determine the prognosis of an animal with cancer. For example, if a cancer is detected using a technique other than by detecting ASH2, e.g., tissue biopsy, then the presence or absence of ASH2 can be used to determine the prognosis for the animal with the cancer. For example, an animal with a cancer that has elevated levels of ASH2 may have a reduced survival expectancy compared to an animal with a cancer, but which has normal levels of ASH2, where "survival expectancy" refers to a prediction regarding the severity, duration, or progress of a disease, condition, or any symptom thereof.
  • ASH2 levels or the presence or absence of ASH2, with a survival expectancy, likelihood of recu ⁇ ence of a cancer, and other prognostic factors are well known and can be readily practiced.
  • alternative prognostic indicators e.g., the level or presence of other marker levels, are also detected in conjunction with the detection of ASH2.
  • the present methods can be used to determine the optimal course of treatment in an animal with cancer. For example, the detection of an elevated level of ASH2 can indicate a reduced survival expectancy of an animal with a cancer, thereby indicating a more aggressive treatment for the animal. In addition, a co ⁇ elation can readily be established between levels of ASH2, or the presence or absence of an elevated level of ASH2, and the relative efficacy of one or another anti-cancer agent.
  • Such analyses can be performed, e.g., retrospectively, i.e., by detecting ASH2 levels in samples taken previously from animals that have subsequently undergone one or more types of anti-cancer therapy, and co ⁇ elating the ASH2 levels with the known efficacy of the treatment.
  • the present invention provides numerous methods for treating an animal with a cancer.
  • methods are provided for treating a cancer by inhibiting the growth and/or proliferation of a cancer cell.
  • such methods are directed at reducing the level of ASH2 polypeptide levels, polynucleotide levels, or protein activity in a cancerous cell.
  • more than one of the methods described infra can be performed on a given animal, and may also be administered in conjunction with one or more traditional, well known anti-cancer therapies, e.g., chemotherapy, radiation therapy, surgery, hormone therapy, immunotherapy, etc.
  • a "method of treating cancer” refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in an animal, or to alleviate the symptoms of a cancer.
  • a method of treating cancer does not necessarily mean that the cancer cells will, in fact, be eliminated, that the number of cells will, in fact, be reduced, or that the symptoms of a cancer will, in fact, be alleviated.
  • a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an animal, is deemed an overall beneficial course of action.
  • the present invention provides methods for treating cancer by detecting the level and/or a diagnostic presence of ASH2 polynucleotide or polypeptide in a biological sample, and, when a diagnostic presence is detected, administering one or more of the above-listed anti-cancer therapies.
  • chemotherapy refers to the administration of chemical compounds to an animal with cancer that is aimed at killing or reducing the number of cancer cells within the animal.
  • chemotherapeutic agents a ⁇ est the growth of or kill cells that are dividing or growing, such as cancer cells.
  • examples of chemotherapeutic agents include doxirubicin, vinblastine, genistein, taxol, vincristine, etc.
  • Radiation therapy refers to the administration of radioactivity to an animal with cancer. Radiation kills or inhibits the growth of dividing cells, such as cancer cells.
  • the administration of radiation may be from an external source (e.g., a gamma source, a proton source, a molecular beam source, etc.), or may be through an implantable radioactive material.
  • a tissue found to be cancerous using the present methods will be removed using surgery.
  • “Surgery” refers to the direct removal or ablation of cells, e.g., cancer cells, from an animal. Most often, the cancer cells will be in the form of a tumor (e.g., a mammary tumor), which is removed from the animal.
  • Hormone therapy can also be used to treat cancers, e.g., breast cancer.
  • hormone therapy refers to the administration of compounds that counteract or inhibit hormones, such as estrogen or androgen, that have a mitogemc effect on cells. Often, such hormones act to increase the cancerous properties of cancer cells in vivo.
  • hormone therapy can also include methods of reducing or eliminating the production of hormones in an animal, e.g., the surgical removal of ovaries to prevent estrogen production.
  • immunotherapy will be used to treat cancer, e.g., a cancer detected using the present methods.
  • Immunotherapy refers to methods of enhancing the ability of an animal's immune system to destroy cancer cells within the animal. Numerous such methods are well known to those of skill in the art. This can involve the treatment with polyclonal or monoclonal antibodies (e.g., Herceptin) that bind to particular molecules located on, produced by, or indicative of, tumor cells. See, e.g., Pastan et al( ⁇ 992) Ann. Rev. Biochem., 61: 331-354, Brinkman and Pastan (1994) Biochimica Biphysica Acta, 1198: 27-45, etc. A. Reducing ASH2 Levels in Cells
  • this invention provides methods of treating a cancer by reducing ASH2 levels in a cell.
  • such methods are used to reduce an elevated level of ASH2, e.g., an elevated level in a cancerous cell.
  • "reducing the level of ASH2 activity” refers to inhibiting ASH2 protein activity in the cell, lowering the copy number of ASH2 genes, or decreasing the level of ASH2 mRNA or protein in the cell.
  • the level of ASH2 activity is lowered to the level typical of a normal, cancer-free cell, but the level may be reduced to any level that is sufficient to decrease the proliferation of the cell, including in certain cases to levels below those typical of normal cells.
  • inhibitors of ASH2 where an "inhibitor of ASH2" is a molecule that acts to reduce ASH2 polynucleotide levels, ASH2 polypeptide levels and/or ASH2 protein activity.
  • an inhibitor can include antisense polynucleotides, ribozymes, antibodies, dominant negative ASH2 forms, and small molecule inhibitors of ASH2.
  • ASH2 levels will be reduced so as to reduce the proliferation of a cancer cell with elevated ASH2 levels.
  • the "proliferation" of a cell refers to the rate at which the cell or population of cells grows and divides, or to the extent to which the cell or population of cells grows, divides or increases in number.
  • Proliferation can reflect multiple factors, including the rate of cell growth and division and the rate of cell death.
  • a method of "decreasing" the proliferation of a cell means to reduce the rate or extent of growth or division of a cell or population of cells. Such methods can involve preventing cell division or cell growth, and may also include cell killing, and can be practiced in vivo or in vitro.
  • ASH2 levels will be reduced in a tumor cell, a hyperproliferative cell, and/or a metastatic cell.
  • a “tumor cell” is a cancer cell, in vitro or in vivo, that is part of a tumor, has been isolated from a tumor, or which is capable of forming a tumor.
  • a “hyperproliferative cell” is a cell with an abnormally high rate of proliferation, or a cell that proliferates to an abnormally great extent, i.e., gives rise to a population of cells that increases in number over time.
  • cancer cells are metastatic, i.e., capable of leaving their normal anatomical location and moving to, and proliferating in, another part of an animal.
  • such "metastatic" cells have acquired the ability to cross basal laminae so as to leave their normal tissue, enter the circulation, leave the circulation, and proliferate in a new location.
  • ASH2 activity is downregulated, or entirely inhibited, by the use of antisense polynucleotide.
  • An "antisense polynucleotide” is a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g, ASH2 mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the ASH2 mRNA reduces the translation and/or stability of the ASH2 mRNA.
  • antisense polynucleotides can comprise naturally-occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. All such analogs are comprehended by this invention so long as they function effectively to hybridize with ASH2 mRNA. Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
  • ribozymes can be used to target and inhibit transcription of ASH2.
  • a ribozyme is an RNA molecule that catalytically cleaves other RNA molecules.
  • Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes (see Castanotto et al. (1994) Adv. in Pharmacology 25: 289-317 for a general review of the properties of different ribozymes).
  • hairpin ribozymes The general features of hairpin ribozymes are described, e.g., in Hampel et al (1990) Nucl Acids Res. 18: 299-304; Hampel et al. (1990) European Patent Publication No. 0 360 257; U.S. Patent No. 5,254,678.
  • Methods of preparing ribozymes are well known to those of skill in the art (see, e.g., Wong-Staal et al., WO 94/26877; Ojwang et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6340-6344; Yamada et al. (1994) Human Gene Therapy 1: 39-45; Leavitt et al.
  • ASH2 activity can also be decreased by the addition of an inhibitor of the ASH2 polypeptide.
  • This can be accomplished in any of a number of ways, including by providing a dominant negative ASH2 polypeptide, e.g., a form of ASH2 that itself has no activity and which, when present in the same cell as a functional ASH2, reduces or eliminates the ASH2 activity of the functional ASH2.
  • inactive polypeptide variants muteins
  • Methods of making muteins are well known to those of skill (see, e.g., U.S. Patent Nos. 5,486,463, 5,422,260, 5,116,943, 4,752,585, 4,518,504).
  • this invention provides methods of screening for agents that modulate and preferably downregulate ASH2 protein activity or expression.
  • screening methods of this invention involve (i) contacting an ASH2-expressing cell (e.g., a cell capable of expressing ASH2) with a test agent; and (ii) detecting the level of ASH2 activity (e.g., as described above), where a decreased level of ASH2 activity as compared to the level of ASH2 activity in a cell not contacted with the test agent indicates that the test agent inhibits or downregulates ASH2.
  • any agent can be tested in such an assay.
  • agents include, but are not limited to, natural or synthetic nucleic acids, natural or synthetic polypeptides, natural or synthetic lipids, natural or synthetic small organic molecules, and the like.
  • test agents are provided as members of a combinatorial library.
  • a collection of small molecule inhibitors are tested for ASH2 inhibiting ability.
  • a "small molecule inhibitor" of ASH2 is any molecule, e.g., a carbohydrate, nucleotide, amino acid, oligonucleotide, oligopeptide, lipid, inorganic compound, etc. that inhibits ASH2 protein activity.
  • Such molecules can inhibit ASH2 protein activity by any of a number of mechanisms, e.g., by binding to an ASH2 protein and competitely inhibiting its interaction with DNA or with other proteins.
  • such "small molecule inhibitors" are smaller than about 10 kD. More preferably, such inhibitors are smaller than about 5, 2, or 1 kD or even smaller.
  • Test agents can also be screened based on functional properties of ASH2 protein. For example, loss of ASH2 function in the fruitfly Drosophila melanogaster results in "homeotic transformations," i.e., alterations in the identity of one or more parts, i.e., segments, of the Drosophila body plan. Accordingly, molecules that reduce or eliminate ASH2 function in Drosophila will reproduce these same alterations, even in the presence of wild type ASH2 function.
  • a candidate molecule or agent will be administered to, e.g., a Drosophila embryo (e.g., applying a compound to an embryo, or expressing a candidate molecule in cells of a Drosophila embryo), and the appearance of one or more homeotic transformations scored. Any treatment that results in homeotic transformations similar to those seen in ASH2 mutants can be further analyzed for their ability to directly inhibit ASH2 expression or activity.
  • a Drosophila embryo e.g., applying a compound to an embryo, or expressing a candidate molecule in cells of a Drosophila embryo
  • Any treatment that results in homeotic transformations similar to those seen in ASH2 mutants can be further analyzed for their ability to directly inhibit ASH2 expression or activity.
  • combinatorial libraries of potential ASH2 modulators will be screened for ASH2 -inhibiting ability.
  • new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, e.g., ASH2 inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • a chemical compound called a "lead compound”
  • HTS high throughput screening
  • high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop et al. (1994) J. Med. Chem. 37(9): 1233-1251). Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88), peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No.
  • a number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.
  • any of the assays to identify compounds capable of modulating ASH2 levels described herein are amenable to high throughput screening. Prefe ⁇ ed assays thus detect enhancement or inhibition of ASH2 gene transcription, inhibition or enhancement of ASH2 polypeptide expression, and inhibition or enhancement of ASH2 polypeptide activity.
  • high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay.
  • These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems.
  • Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
  • an ASH2 inhibiting compound i.e., a compound that reduces levels of ASH2 mRNA, polypeptide and/or protein activity
  • Such compounds can be administered by a variety of methods including, but not limited to, parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges.
  • the ASH2 modulators e.g., antibodies, antisense constructs, ribozymes, small organic molecules, etc.
  • the ASH2 modulators when administered orally, must be protected from digestion. This is typically accomplished either by complexing the molecule(s) with a composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the molecule(s) in an appropriately resistant carrier, such as a liposome. Means of protecting agents from digestion are well known in the art.
  • compositions for administration will commonly comprise an ASH2 modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 m per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980). The compositions containing modulators of ASH2 can be administered for therapeutic or prophylactic treatments.
  • compositions are administered to a patient suffering from a disease (e.g., a cancer) in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • a disease e.g., a cancer
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient.
  • epithelial cancers will be diagnosed and/or treated.
  • breast, ovarian, colorectal, kidney, stomach, bladder, lung, and any other epithelial cancer can be treated or diagnosed.
  • a cancer at any stage of progression can be detected, such as primary, metastatic, and recurrent cancers.
  • Information regarding numerous types of cancer can be found, e.g., from the American Cancer Society (www3.cancer.org), or from, e.g., Wilson et al. (1991) Harrison's Principles of Internal Medicine, 12 th Edition, McGraw-Hill, Inc.
  • the present invention provides novel variants of ASH2.
  • the ASH2 polynucleotides and polypeptides provided herein have numerous uses, including for diagnosing cancer, determining the prognosis of an animal with cancer, determining the efficacy of a cancer treatment, inhibiting the proliferation of a cell in vitro or in vivo, and others.
  • the present ASH2 polynucleotides and polypeptides can be synthesized and purified using standard techniques (see, e.g., Sambrook, Ausubel, both supra).
  • ASH2 comprises a number of splice variants, any of which can be used in the present invention.
  • novel ASH2 variants provided herein were identified, inter alia, as novel cDNA clones isolated from a human peripheral blood leukocyte cDNA library.
  • SEQ LD NO:l which comprises 1627 nucleotides and encodes a protein sequence of 305 amino acids (shown as SEQ LD NO:2)
  • SEQ LD NO:2 is similar to several previously described human ASH2 sequences (e.g., GenBank Accession Nos: AF056717, AB020982, and AF056718) that were of unknown function.
  • GenBank Accession Nos: AF056717, AB020982, and AF056718 are similar to several previously described human ASH2 sequences (e.g., GenBank Accession Nos: AF056717, AB020982, and AF056718) that were of unknown function.
  • certain of the ASH2 polynucleotides provided herein comprise a different translation start site than most previously described ASH2 sequences.
  • the human ASH2 polypeptide sequence shown as SEQ LD NO:2 lacks the first 94 amino acids of other ASH2 sequences, e.g., GenBank Accession No. AB022785.
  • all previously described ASH2 sequences contain an exon, shown as SEQ ID NO:4 and representing, e.g., amino acids 389-510 in AB022785, that is not present in the sequences provided herein.
  • the terminal 11 amino acids of the ASH2 polypeptide shown as SEQ LD NO:2 (the 11 amino acid sequence is shown as SEQ LD NO:3) comprises a novel ASH2 subsequence not previously described in any protein.
  • the subsequence present in SEQ ID NO:8, shown alone as SEQ ID NO:9 also comprises a novel ASH2 subsequence never before described.
  • the ASH2 protein has a putative double zinc-finger domain, called a PHD finger (see, e.g., Adamson et al, Genetics 1996 Oct; 144(2): 621-33. Zinc fingers are generally thought to mediate DNA binding, suggesting that the ASH2 protein binds to DNA, e.g., to regulate the expression of one or more target genes.
  • the ASH2 polypeptide or polynucleotides sequences provided herein can represent fragments of a full length ASH2. Typically, such fragments will represent one or more discrete subdomains of ASH2, e.g., one or both zinc finger domains can be used independently of the entire protein.
  • any of the polynucleotides or proteins described herein can be accomplished using standard molecular biological techniques, as described, e.g., in Ausubel et al. (ed.) (1990) Cu ⁇ ent Protocols in Molecular biology, Greene Publishing and Wiley-Interscience, New York, Glover (ed.) (1987) DNA Cloning: A Practical Approach, vols 1-3, LRL Press, Oxford, or Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2d Ed., vols 1-3, Cold Spring Harbor Press, New York.
  • the ASH2 polynucleotides and polypeptides of this invention include isolated ASH2 polynucleotides and polypeptides, wherein "isolated” indicates that the polynucleotides or polypeptides are substantially free of other polypeptides and other cellular components with which they are naturally associated. 1. Introducing Nucleic Acids into Cells
  • one or more nucleic acids e.g., ASH2 polynucleotides, such as antisense polynucleotides or ribozymes, will be introduced into cells, in vitro or in vivo.
  • the present invention provides methods, reagents, vectors, and cells useful for expression of ASH2 and other polypeptides and nucleic acids using in vitro (cell-free), ex vivo or in vivo (cell or organism-based) recombinant expression systems.
  • the particular procedure used to introduce the nucleic acids into a host cell for expression of a protein or nucleic acid is application specific. Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger), F.M. Ausubel et al, eds., Cu ⁇ ent Protocols, a joint venture between Greene Publishing
  • nucleic acids encoding ASH2 polypeptides, or inhibitors thereof will be inserted into vectors using standard molecular biological techniques.
  • Vectors may be used at multiple stages of the practice of the invention, e.g., for subcloning nucleic acids encoding ASH2 polypeptides, ,or ASH2 inhibitors, e.g., ASH2 ribozymes or antisense sequences, or for subcloning additional elements used to control protein or mRNA expression, vector selectability, etc.
  • Vectors may also be used to maintain or amplify the nucleic acids, for example, by inserting the vector into prokaryotic or eukaryotic cells and growing the cells in culture.
  • vectors may be used to introduce and express ASH2 nucleic acids, or ASH2-inhibiting nucleic acids, e.g., ASH2 ribozymes or antisense sequences, into cells for therapeutic or experimental purposes.
  • a variety of commercially or commonly available vectors and vector nucleic acids can be converted into a vector of the invention by cloning a polynucleotide of this invention into the commercially or commonly available vector.
  • a variety of common vectors suitable for this purpose are well known in the art.
  • common vectors include pBR322-derived vectors such as pBLUESCRIPTTM, and bacteriophage derived vectors.
  • vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2.
  • Expression in mammalian cells can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculo virus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retro viruses).
  • lytic virus vectors e.g., vaccinia virus, adeno virus, and baculo virus
  • episomal virus vectors e.g., bovine papillomavirus
  • retroviral vectors e.g., murine retro viruses.
  • a nucleic acid subsequence encoding an ASH2 polypeptide is placed under the control of a promoter.
  • a nucleic acid is "operably linked" to a promoter when it is placed into a functional relationship with the promoter.
  • a promoter or enhancer is operably linked to a coding sequence if it increases or otherwise regulates the transcription of the coding sequence.
  • a "recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences.
  • Expression cassettes include promoters and, optionally, introns, polyadenylation signals, and transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.
  • an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.
  • promoters are well known, and can-be used in the vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included. For E. coli, example control sequences include the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal.
  • control sequences typically include a promoter which optionally includes an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, a retrovirus (e.g., an LTR based promoter) etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
  • kits are also provided by the invention.
  • such kits may include any or all of the following: assay reagents, buffers, ASH2 specific nucleic acids or antibodies, hybridization probes and/or primers, antisense polynucleotides, ribozymes, dominant negative ASH2 polypeptides or polynucleotides, small molecules inhibitors of ASH2, etc.
  • a therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
  • kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention.
  • Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
  • Such media may include addresses to internet sites that provide such instructional materials.
  • RDA representational difference analysis
  • FIG. 1 shows a physical map of the 8pl l-pl2 amplified chromosomal region, indicated by the solid line marked with solid circles. Underneath are solid lines representing individual YACs, and a dashed line respresent ⁇ ng genomic sequence from GenBank (Accession numbers: AP000065 through AP000084).
  • STSs on this map were derived from RDA probes or from the genomic sequence or public databases at the Whitehead Institute, Stanford University, and the Sanger Center.
  • the STSs are ordered on the basis of genomic sequence; or by determination by PCR of their presence on these YACs and others not shown, and confirmed by radiation hybrid mapping using the GB3 panel (Stanford University RH server). 92 cell lines were scanned for copy-number increases using Q-PCR and fluorogenic TaqMan probes to the RDA probe-derived STS, RDA-534.
  • 220 primary tumors were also surveyed for copy number increases using a TaqMan probe derived from the sequence of STS NLB1979 (3' UTR of ASH2). 21 of the 220 tumors were amplified 2.5-fold or greater, and of these 21, 10 were amplified five-fold or greater.
  • TaqMan probes (ABI, Foster City, Calif.) for the other 12 STSs were used to measure additional copy-number increases in six amplified primary tumors, and the results of these analyses are noted in Figure 1 (-Fold Amplification).
  • the ASH2 gene was initially identified as amplified in tumors by searching databases for gene identities of the public ESTs that mapped near to the RDA probe.
  • the public ESTs that were examined had been previously mapped nearby at low resolution (as part of the HGP gene-mapping effort).
  • the EST NIB 1979 mapped nearby RDA probe 534 and is derived from the 3' UTR of ASH2.
  • ASH2 was found to be expressed as a ⁇ 3.0 kb transcript at similar levels in all human tissues examined (pancreas, kidney, muscle, liver, lung, placenta, brain, and heart). ASH2 was significantly overexpressed in all cancer cell lines in which it is amplified, as well as in three breast cancer cell lines in the absence of gene amplification (Table 1). On the basis of this analysis, it was determined that ASH2 is overexpressed in -25% of breast cancer cell lines.
  • MDAMB330 Breast lx lOx Northern analysis of total RNA was quantitated with a Fuji phosphorimager, and the expression level is the ratio of the test gene to GAPDH normalized to human mammary epithelial cell (HMEC) levels.
  • HMEC human mammary epithelial cell
  • SE 707 shown as SEQ ID NO:5 (cDNA) and SEQ LD NO:6 (amino acid sequence), is identical to AB020982 (see, GenBank) except that it skips exons 11 and 12, co ⁇ esponding to nucleotides 1177-1538 Of AB020982.
  • This alternate splice produces a frameshift, resulting in a termination codon (the stop codon is shown as an asterix in the amino acid SEQ ID NO:6) 11 amino acids downstream of the splice.
  • SEQ ID NO:5 The nucleotides at the novel splice junction are underlined in SEQ ID NO:5, as is the novel C- terminal peptide in SEQ LD NO:6 (the novel 11 amino acid peptide is shown separately as SEQ LD NO:3).
  • Isoform SE 691 (the cDNA of which is shown as SEQ LD NO: 7 and the amino acid sequence as SEQ ID NO:8) is identical to SE 707, except that it has an insertion of 109 nucleotides between exons 1 and 2 (between nucleotides 199 and 200 of AB020982), resulting in a frameshift and a premature termination codon.
  • This nucleotide insertion, and the resulting novel polypeptide sequence are underlined in SEQ LD NO:7 and SEQ LD NO: 8, respectively.
  • the novel polypeptide sequence is also shown separately as SEQ LD NO:9.
  • ASH2 cDNA-isoform SE 707 ATGGCGGCGGCAGGAGCAGGACCTGGCCAGGAAGCGGGTGCCGGGCCTGGCCC AGGAGCGGTCGCAAATGCAACAGGGGCAGAAGAGGGGGAGATGAAGCCGGTG GCAGCGGGAGCAGCCGCTCCTCCTGGAGAGGGGATCTCTGCTGCTCCGACAGTT GAGCCCAGTTCCGGGGAGGCTGAAGGCGGGGAGGCAAACTTGGTCGATGTAAG CGGTGGCTTGGAGACAGAATCATCTAATGGAAAAGATACACTAGAAGGTGCTG GGGATACATCAGAGGTGATGGATACTCAGGCGGGCTCCGTGGATGAAGAGAAT GGCCGACAGTTGGGTGAGGTAGCTGCAATGTGGGATTTGTACAAAATGGTTC ACGGCTGACACATTTGGCATAGATACCTCATCCTGTCTACCTTTCATGACCAACT ACAGTTTTCATTGCAACGTCTGCCATCACAGTGGGAATACCTATTTCCTCCGGAA GCAAGC
  • MAAAGAGPGQEAGAGPGPGAVANATGAEEGEMKPVAAGAAAPPGEGIS AAPTVEP SSGEAEGGEANLVDVSGGLETESSNGKDTLEGAGDTSEVMDTQAGSVDEENGRQL GEVELQCGICTKWFTADTFGIDTSSCLPFMTNYSFHCNVCHHSGNTYFLRKQANLK EMCLSALANLTWQSRTQDEHPKTMFSKDKDILPFLDKYWECMTTRQRPGKMTWPN NIVKTMSKERDVFLVKEHPDPGSKDPEEDYPKFGLLDQDLSNIGPAYDNQKQSSAV STSGNLNGGIAAGSSGKGRGAKRKQQDGGTTGTTKKARSDPLFSAQRLPPHGYPLE HPFT ⁇ KDGYRYILAEPDPHAPDPEKLELDCWAGKPIPGDLYRACLYERVLLALHDRG FDKJOELFVF*GKRLCG*SREEPEADSP**DNIL*KWCQSRCGLQRYF*GGLLPS

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Abstract

La présente invention concerne des procédés permettant de déterminer la présence d'un cancer chez un animal. Les procédés décrits dans cette invention reposent sur la découverte surprenante selon laquelle le gène ASH2 est amplifié et/ou surexprimé dans divers types de cellules cancéreuses. C'est ainsi que l'on a découvert que la détection de la surexpression ou de l'amplification du ASH2 dans un échantillon biologique prélevé chez un animal permettait de diagnostiquer le cancer dans cet échantillon. De plus, l'invention concerne de nombreux procédés permettant de traiter le cancer, y compris par détermination de l'évolution la plus efficace d'une thérapie contre le cancer à partir des niveaux d'ASH2, et par inhibition de la prolifération des cellules cancéreuses par inhibition ou réduction des niveaux de polynucléotides ou de polypeptides ASH2.
PCT/US2000/018657 1999-07-07 2000-07-07 Diagnostic du cancer par detection de polynucleotides ou de polypeptides ash2 WO2001002828A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002066506A2 (fr) * 2001-02-21 2002-08-29 Glaxosmithkline Biologicals S.A. Nouvelle utilisation
WO2002092627A2 (fr) * 2001-05-16 2002-11-21 Glaxosmithkline Biologicals S.A. Nouvelle utilisation
EP2079484A2 (fr) * 2006-09-07 2009-07-22 Stemline Therapeutics, Inc. Surveillance de cellules souches cancéreuses
US7803379B2 (en) 2000-02-23 2010-09-28 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
US7811574B2 (en) 2000-02-23 2010-10-12 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
EP2266603A1 (fr) * 2000-10-18 2010-12-29 GlaxoSmithKline Biologicals S.A. Vaccins contre le cancer
US8916514B2 (en) 2009-05-27 2014-12-23 Glaxosmithkline Biologicals, S.A. CASB7439 constructs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] FERBUS ET AL.: 'Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas', XP002936627 Database accession no. 1999:69614 & INTERNATIONAL JOURNAL OF CANCER vol. 80, no. 3, 29 January 1999, pages 369 - 372 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7803379B2 (en) 2000-02-23 2010-09-28 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
US7811574B2 (en) 2000-02-23 2010-10-12 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
US8207123B2 (en) 2000-02-23 2012-06-26 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
EP2266603A1 (fr) * 2000-10-18 2010-12-29 GlaxoSmithKline Biologicals S.A. Vaccins contre le cancer
WO2002066506A2 (fr) * 2001-02-21 2002-08-29 Glaxosmithkline Biologicals S.A. Nouvelle utilisation
WO2002066506A3 (fr) * 2001-02-21 2002-12-19 Glaxosmithkline Biolog Sa Nouvelle utilisation
WO2002092627A2 (fr) * 2001-05-16 2002-11-21 Glaxosmithkline Biologicals S.A. Nouvelle utilisation
WO2002092627A3 (fr) * 2001-05-16 2003-02-06 Glaxosmithkline Biolog Sa Nouvelle utilisation
EP2079484A2 (fr) * 2006-09-07 2009-07-22 Stemline Therapeutics, Inc. Surveillance de cellules souches cancéreuses
EP2079484A4 (fr) * 2006-09-07 2010-03-17 Stemline Therapeutics Inc Surveillance de cellules souches cancéreuses
US8916514B2 (en) 2009-05-27 2014-12-23 Glaxosmithkline Biologicals, S.A. CASB7439 constructs

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