EP1155126A2 - Genes associes a des maladies du colon - Google Patents

Genes associes a des maladies du colon

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Publication number
EP1155126A2
EP1155126A2 EP00913330A EP00913330A EP1155126A2 EP 1155126 A2 EP1155126 A2 EP 1155126A2 EP 00913330 A EP00913330 A EP 00913330A EP 00913330 A EP00913330 A EP 00913330A EP 1155126 A2 EP1155126 A2 EP 1155126A2
Authority
EP
European Patent Office
Prior art keywords
gene
colon cancer
polynucieotide
sequence
disease
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00913330A
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German (de)
English (en)
Inventor
Michael G. Walker
Wayne Volkmuth
Tod M. Klingler
Preeti Lal
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Incyte Corp
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Incyte Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Incyte Pharmaceuticals Inc filed Critical Incyte Pharmaceuticals Inc
Publication of EP1155126A2 publication Critical patent/EP1155126A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to seven genes associated with diseases of the colon, particularly colon cancer, as identified by their coexpression with known colon cancer genes.
  • the invention also relates to the use of these biomolecules in diagnosis, prognosis, prevention, treatment, and evaluation of therapies for diseases of the colon.
  • Colon cancer is the third leading cause of cancer deaths in the United States. Each year over 100,000 new cases are diagnosed, and 50,000 patients die from the disease. In large part this death rate is due to the inability to diagnose the disease at an early stage (Wanebo (1993) Colorectal Cancer. Mosby, St Louis MO). Although some of the genes that participate in or regulate the growth of colon cells are known, many other genes remain to be identified. Identification of new genes with significant levels of expression in cells of the diseased colon will provide new diagnostics, opportunities for earlier patient diagnosis, and targets for the development of therapeutic agents.
  • the present invention satisfies a need in the art by providing new compositions, seven genes associated with diseases of the colon identified by their coexpression patterns with genes expressed in colon cancer, that are useful for diagnosis, prognosis, treatment, prevention, and evaluation of therapies for diseases of the colon.
  • the invention provides for a substantially purified polynucieotide comprising a gene that is coexpressed with one or more known colon cancer genes in a plurality of biological samples.
  • known colon cancer genes are selected from the group consisting of carbonic anhydrase I, II, and IV (CA I, II, and IV), carcinoembryonic antigen family of proteins (cea), colorectal carcinoma tumor- associated antigen (CO-029), down-regulated in adenoma (dra), fatty-acid binding protein (fabp), galectin
  • Preferred embodiments include: (a) a polynucieotide sequence selected from SEQ ID NOs: 1-7; (b) a polynucieotide sequence which encodes the polypeptide of SEQ ID NOs:8 or 9; (c) a polynucieotide sequence having at least 75% identity to the polynucieotide sequence of (a) or (b); (d) a polynucieotide sequence which is complementary to the polynucieotide sequence of (a), (b), or (c); (e) a polynucieotide sequence comprising at least 10, preferably at least 18, sequential nucleotides of the polynucieotide sequence of (a), (b), (c), or (d); or (f)
  • the invention provides an expression vector comprising any of the polynucleotides described above and host cells comprising the expression vector. Still further, the invention provides a method for treating or preventing a disease or condition associated with the altered expression of a gene that is coexpressed with one or more known colon cancer genes comprising administering to a subject in need a polynucieotide described above in an amount effective for treating or preventing the disease.
  • the invention provides a substantially purified polypeptide comprising the gene product of a gene that is coexpressed with one or more known colon cancer genes in a plurality of biological samples.
  • the known colon cancer gene may be selected from the group consisting of carbonic anhydrase I, II, and IV, carcinoembryonic antigen family of proteins, colorectal carcinoma tumor- associated antigen, down-regulated in adenoma, fatty-acid binding protein , galectin, glutathione peroxidase, guanylin, cytokeratin 8 and 20, cadherin, and intestinal mucin.
  • Preferred embodiments are (a) the polypeptide sequence of SEQ ID NOs: 8 and 9; (b) a polypeptide sequence having at least 85% identity to the polypeptide sequence of (a); and (c) a polypeptide sequence comprising at least 6 sequential amino acids of the polypeptide sequence of (a) or (b).
  • the invention provides antibodies that bind specifically to any of the above described polypeptides and a method for treating or preventing a disease or condition associated with the altered expression of a gene that is coexpressed with one or more known colon cancer genes comprising administering to a subject in need such an antibody in an amount effective for treating or preventing the disease.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucieotide of claim 2 or the polypeptide of claim 3 in conjunction with a suitable pharmaceutical carrier and a method for treating or preventing a disease or condition associated with the altered expression of a gene that is coexpressed with one or more known colon cancer genes comprising administering to a subject in need such a composition in an amount effective for treating or preventing the disease.
  • the invention provides a method for diagnosing a disease or condition associated with the altered expression of a gene that is coexpressed with one or more known colon cancer genes, wherein each known colon cancer gene is selected from the group consisting of carbonic anhydrase I, II, and IV, carcinoembryonic antigen family of proteins, colorectal carcinoma tumor- associated antigen, down-regulated in adenoma, fatty-acid binding protein, galectin, glutathione peroxidase, guanylin, cytokeratin 8 and 20, cadherin, and intestinal mucin.
  • each known colon cancer gene is selected from the group consisting of carbonic anhydrase I, II, and IV, carcinoembryonic antigen family of proteins, colorectal carcinoma tumor- associated antigen, down-regulated in adenoma, fatty-acid binding protein, galectin, glutathione peroxidase, guanylin, cytokeratin 8 and 20, cadherin, and intestinal mucin
  • the method comprises the steps of (a) providing a sample comprising one of more of the coexpressed genes; (b) hybridizing the polynucieotide of claim 2 to the coexpressed genes under conditions effective to form one or more hybridization complexes; (c) detecting the hybridization complexes; and (d) comparing the levels of the hybridization complexes with the level of hybridization complexes in a nondiseased sample, wherein altered levels of one or more of the hybridization complexes in a diseased sample compared with the level of hybridization complexes in a non-diseased sample correlates with the presence of the disease or condition.
  • the invention provides antibodies, antibody fragments, and immunoconjugates that exhibit specificity to any of the above described polypeptides and methods for treating or preventing diseases or conditions of the colon.
  • Sequence Listing provides exemplary colon cancer gene sequences including polynucieotide sequences, SEQ ID NOs: l-7, and the polypeptide sequences, SEQ ID NOs:8 and 9. Each sequence is identified by a sequence identification number (SEQ ID NO) and by the Incyte clone number with which the sequence was first identified. DESCRIPTION OF THE INVENTION
  • NEQ refers generally to a polynucieotide sequence of the present invention, including SEQ ID NOs: l-7.
  • PSEQ refers generally to a polypeptide sequence of the present invention, SEQ ID NOs:8 and 9.
  • a “fragment” refers to a nucleic acid sequence that is preferably at least 20 nucleic acids in length, more preferably 40 nucleic acids, and most preferably 60 nucleic acids in length, and encompasses, for example, fragments consisting of nucleic acids 1-50, 51-400, 401-4000, 4001-12,000, and the like, of SEQ ID NOs: 1-7.
  • Gene refers to the partial or complete coding sequence of a gene and to its 5' or 3' untranslated regions.
  • the gene may be in a sense or antisense (complementary) orientation.
  • Colon cancer gene refers to a gene whose expression pattern is similar to that of known colon cancer genes which are useful in the diagnosis, treatment, prognosis, or prevention of diseases of the colon, particularly colon cancer and other diseases associated with abnormal cell growth.
  • known colon cancer gene refers to a sequence which has been previously identified as useful in the diagnosis, treatment, prognosis, or prevention of diseases of the colon. Typically, this means that the known gene is expressed at higher levels (i.e., has more abundant transcripts) in diseased or cancerous colon tissue than in normal or non-diseased colon or any other tissue.
  • Polynucieotide refers to a nucleic acid molecule, nucleic acid sequence, oligonucleotide, nucleotide, or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein or other materials to perform a particular activity or form a useful composition. "Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligomer, element, and probe.
  • Polypeptide refers to an amino acid molecule, amino acid sequence, oligopeptide, peptide, or protein or portions thereof whether naturally occurring or synthetic.
  • a “portion” refers to peptide sequence which is preferably at least 5 to about 15 amino acids in length, most preferably at least 10 amino acids long, and which retains some biological or immunological activity of, for example, a portion of SEQ ID NOs:8 and 9.
  • sample is used in its broadest sense.
  • a sample containing nucleic acids may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA,
  • RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; and the like.
  • Substantially purified refers to a nucleic acid or an amino acid sequence that is removed from its natural environment and that is isolated or separated, and is at least about 60% free, preferably about 75% free, and most preferably about 90% free, from other components with which it is naturally present.
  • Substrate refers to any suitable rigid or semi-rigid support to which polynucleotides or polypeptides are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels, and pores.
  • a “ variant” refers to a polynucieotide whose sequence diverges from SEQ ID NOs: 1-7 or to a polypeptide who sequence diverges from SEQ ID NOs: 8 and 9, respectively.
  • Polynucieotide sequence divergence may result from mutational changes such as deletions, additions, and substitutions of one or more nucleotides; it may also be introduced to accommodate differences in codon usage. Each of these types of changes may occur alone, or in combination, one or more times in a given sequence.
  • Polypeptide variants include sequences that possess at least one structural or functional characteristic of SEQ ID NOs:8 and 9.
  • the present invention encompasses a method for identifying biomolecules that are associated with a specific disease, regulatory pathway, subcellular compartment, cell type, tissue type, or species.
  • the method identifies genes useful in diagnosis, prognosis, treatment, prevention, and evaluation of therapies for diseases of the colon including, but not limited, colon cancer, metastatic colon cancer, atrophic gastritis, cholecystitis, Crohns disease, irritable bowel syndrome, ulcerative colitis, and the like.
  • the method entails first identifying polynucleotides that are expressed in a plurality of cDNA libraries.
  • the identified polynucleotides include genes of known or unknown function which are known to be expressed in a specific disease process, subcellular compartment, cell type, tissue type, or species.
  • the expression patterns of the genes with known function are compared with those of the genes with unknown function to determine whether a specified coexpression probability threshold is met. Through this comparison, a subset of the polynucleotides having a high coexpression probability with the known genes can be identified.
  • the high coexpression probability correlates with a particular coexpression probability threshold which is preferably less than 0.001 and more preferably less than 0.00001.
  • the polynucleotides originate from cDNA libraries derived from a variety of sources including, but not limited to, eukaryotes such as human, mouse, rat, dog, monkey, plant, and yeast, and prokaryotes such as bacteria; and viruses. These polynucleotides can also be selected from a variety of sequence types including, but not limited to, expressed sequence tags (ESTs), assembled polynucieotide sequences, full length gene coding regions, promoters, introns, enhancers, 5' untranslated regions, and 3' untranslated regions. To have statistically significant analytical results, the polynucleotides need to be expressed in at least three cDNA libraries.
  • ESTs expressed sequence tags
  • the cDNA libraries used in the coexpression analysis of the present invention can be obtained from adrenal gland, biliary tract, bladder, blood cells, blood vessels, bone marrow, brain, bronchus, cartilage, chromaffin system, colon, connective tissue, cultured cells, embryonic stem cells, endocrine glands, epithelium, esophagus, fetus, ganglia, heart, hypothalamus, immune system, intestine, islets of Langerhans, kidney, larynx, liver, lung, lymph, muscles, neurons, ovary, pancreas, penis, peripheral nervous system, phagocytes, pituitary, placenta, pleurus, prostate, salivary glands, seminal vesicles, skeleton, spleen, stomach, testis, thymus, tongue, ureter, uterus, and the like.
  • the number of cDNA libraries selected can range from as few as 3 to greater than 10,000.
  • genes are assembled to reflect related sequences, such as assembled sequence fragments derived from a single transcript. Assembly of the polynucieotide sequences can be performed using sequences of various types including, but not limited to, ESTs, extensions, or shotgun sequences. In a most preferred embodiment, the polynucieotide sequences are derived from human sequences that have been assembled using the algorithm disclosed in "System and Methods for Analyzing Biomolecular Sequences", USSN 09/276,534, filed March 25, 1999, incorporated herein by reference.
  • differential expression of the polynucleotides can be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational difference analysis, and transcript imaging. Additionally, differential expression can be assessed by microarray technology. These methods may be used alone or in combination.
  • known colon cancer genes can be selected based on the use of these genes as diagnostic or prognostic markers or as therapeutic targets.
  • the known colon cancer genes include carbonic anhydrase I, II, and IV, carcinoembryonic antigen family of proteins, colorectal carcinoma tumor- associated antigen, down-regulated in adenoma, fatty-acid binding protein, galectin, glutathione peroxidase, guanylin, cytokeratin 8 and 20, cadherin, intestinal mucin, and the like.
  • the procedure for identifying novel genes that exhibit a statistically significant coexpression pattern with known colon cancer genes is as follows.
  • a gene is present in a cDNA library when at least one cDNA fragment corresponding to that gene is detected in a cDNA sample taken from the library, and a gene is absent from a library when no corresponding cDNA fragment is detected in the sample.
  • the significance of gene coexpression is evaluated using a probability method to measure a due-to-chance probability of the coexpression.
  • the probability method can be the Fisher exact test, the chi-squared test, or the kappa test.
  • a Bonferroni correction (Rice, supra, page 384) can also be applied in combination with one of the probability methods for correcting statistical results of one gene versus multiple other genes.
  • the due-to-chance probability is measured by a Fisher exact test, and the threshold of the due-to-chance probability is set preferably to less than 0.001, more preferably to less than 0.00001.
  • occurrence data vectors can be generated as illustrated in Table 1. The presence of a gene occurring at least once in a library is indicated by a one, and its absence from the library, by a zero. Table 1. Occurrence data for genes A and B
  • Table 2 presents co-occurrence data for gene A and gene B in a total of 30 libraries. Both gene A and gene B occur 10 times in the libraries. Table 2 summarizes and presents: 1) the number of times gene A and B are both present in a library, 2) the number of times gene A and B are both absent in a library, 3) the number of times gene A is present and gene B is absent, and 4) the number of times gene B is present and gene A is absent.
  • the upper left entry is the number of times the two genes co-occur in a library, and the middle right entry is the number of times neither gene occurs in a library.
  • the off diagonal entries are the number of times one gene occurs and the other does not. Both A and B are present eight times and absent 18 times.
  • Gene A is present and gene B is absent two times; and gene B is present and gene A is absent two times.
  • the probability (“p-value”) that the above association occurs due to chance as calculated using a Fisher exact test is 0.0003. Associations are generally considered significant if a p-value is less than 0.01 (Agresti, supra; Rice, supra).
  • This method of estimating the probability for coexpression of two genes makes several assumptions. The method assumes that the libraries are independent and are identically sampled. However, in practical situations, the selected cDNA libraries are not entirely independent, because more than one library may be obtained from a single subject or tissue. Nor are they entirely identically sampled, because different numbers of cDNAs may be sequenced from each library. The number of cDNAs sequenced typically ranges from 5,000 to 10,000 cDNAs per library. In addition, because a Fisher exact coexpression probability is calculated for each gene versus 41,419 other assembled genes, a Bonferroni correction for multiple statistical tests is necessary. Using the method of the present invention, we have identified seven novel genes that exhibit strong association, or coexpression, with known genes that are specific to colon cancer.
  • colon cancer genes include carbonic anhydrase I, II, and IV, carcinoembryonic antigen family of proteins, colorectal carcinoma tumor-associated antigen, down-regulated in adenoma, fatty-acid binding protein, galectin, glutathione peroxidase, guanylin, cytokeratin 8 and 20, cadherin, and intestinal mucin.
  • the results presented in Table 6 show that the expression of the seven novel genes have direct or indirect association with the expression of known colon cancer genes. Therefore, the novel genes can potentially be used in diagnosis, treatment, prognosis, or prevention of diseases of the colon or in the evaluation of therapies for diseases of the colon.
  • the present invention encompasses a polynucieotide sequence comprising the sequence of SEQ ID NOs: 1-7. These seven polynucleotides are shown by the method of the present invention to have strong coexpression association with known colon cancer genes and with each other.
  • the invention also encompasses a variant of the polynucieotide sequence, its complement, or 18 consecutive nucleotides of a sequence provided in the above described sequences.
  • Variant polynucieotide sequences typically have at least about 75%, more preferably at least about 85%, and most preferably at least about 95% polynucieotide sequence identity to NSEQ.
  • NSEQ or the encoded PSEQ may be used to search against the GenBank primate (pri), rodent (rod), mammalian (mam), vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt, BLOCKS (Bairoch et al. (1997) Nucleic Acids Res 25:217-221), PFAM, and other databases that contain previously identified and annotated motifs, sequences, and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5:35- 51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1993) J Mol Evol 36:290-300; Altschul et al. (1990) J Mol Biol 215:403-410), BLOCKS (Henikoff and Henikoff (1991) Nucleic Acids Research 19:6565-6572), Hidden Markov Models (HMM; Eddy (1996) Cur Opin
  • polynucieotide sequences that are capable of hybridizing to SEQ ID NOs: 1-7, and fragments thereof under stringent conditions.
  • Stringent conditions can be defined by salt concentration, temperature, and other chemicals and conditions well known in the art. Suitable conditions can be selected, for example, by varying the concentrations of salt in the prehybridization, hybridization, and wash solutions or by varying the hybridization and wash temperatures. With some substrates, the temperature can be decreased by adding formamide to the prehybridization and hybridization solutions.
  • Hybridization can be performed at low stringency, with buffers such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60° C, which permits complex formation between two nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45° C (medium stringency) or 68° C (high stringency), to maintain hybridization of only those complexes that contain completely complementary sequences. Background signals can be reduced by the use of detergents such as SDS, Sarcosyl, or Triton X-100, and/or a blocking agent, such as salmon sperm DNA. Hybridization methods are described in detail in Ausubel (supra, units 2.8-2.1 1, 3.18-3.19 and 4-6-4.9) and Sambrook et al. (1989: Molecular Cloning. A Laboratory
  • NSEQ can be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences such as promoters and other regulatory elements.
  • upstream sequences such as promoters and other regulatory elements.
  • primers may be designed using commercially available software, such as OLIGO 4.06 Primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 18 to 30 nucleotides in length, to have a GC content of about 50%, and to form a hybridization complex at temperatures of about 68°C to 72°C.
  • commercially available software such as OLIGO 4.06 Primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 18 to 30 nucleotides in length, to have a GC content of about 50%, and to form a hybridization complex at temperatures of about 68°C to 72°C.
  • NSEQ can be cloned in recombinant DNA molecules that direct the expression of PSEQ or structural or functional fragments thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express the polypeptide encoded by NSEQ.
  • the nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • NSEQ In order to express a biologically active protein, NSEQ, or derivatives thereof, may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions. Methods which are well known to those skilled in the art may be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook. supra: and Ausubel, supra).
  • NSEQ A variety of expression vector/host cell systems may be utilized to express NSEQ. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus vectors; plant cell systems transformed with viral or bacterial expression vectors; or animal cell systems. For long term production of recombinant proteins in mammalian systems, stable expression in cell lines is preferred.
  • NSEQ can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable or visible marker gene on the same or on a separate vector. The invention is not to be limited by the vector or host cell employed.
  • host cells that contain NSEQ and that express PSEQ may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of PSEQ using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • Host cells transformed with NSEQ may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transgenic cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing NSEQ may be designed to contain signal sequences which direct secretion of the protein through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa,
  • MDCK, HEK293, and WI38 are available from the American Type Culture Collection (ATCC, Manasas VA) and may be chosen to ensure the correct modification and processing of the expressed protein.
  • natural, modified, or recombinant nucleic acid sequences are ligated to a heterologous sequence resulting in translation of a fusion protein containing heterologous protein moieties in any of the aforementioned host systems.
  • heterologous protein moieties facilitate purification of fusion proteins using commercially available affinity matrices.
  • moieties include, but are not limited to, glutathione S-transferase, maltose binding protein, thioredoxin, calmodulin binding peptide, 6-His, FLAG, c-myc, hemaglutinin, and monoclonal antibody epitopes.
  • nucleic acid sequences are synthesized, in whole or in part, using chemical or enzymatic methods well known in the art (Caruthers et al. (1980) Nucl Acids Symp Ser (7)
  • peptide synthesis can be performed using various solid-phase techniques (Roberge et al. (1995) Science 269:202-204), and machines such as the ABI 431 A Peptide synthesizer (PE Biosystems) can be used to automate synthesis.
  • the amino acid sequence may be altered during synthesis and/or combined with sequences from other proteins to produce a variant protein.
  • the invention entails a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NOs: 8 and 9 or fragments thereof.
  • DIAGNOSTICS and THERAPEUTICS The polynucieotide sequences can be used in diagnosis, prognosis, treatment, prevention, and evaluation of therapies for diseases of the colon including, but not limited, colon cancer, metastatic colon cancer, atrophic gastritis, cholecystitis, Crohns disease, irritable bowel syndrome, ulcerative colitis, and the like.
  • the polynucieotide sequences are used for diagnostic purposes to determine the absence, presence, and excess expression of the protein.
  • the polynucleotides may be at least 18 nucleotides long and consist of complementary RNA and DNA molecules, branched nucleic acids, and/or peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • the polynucleotides are used to detect and quantify gene expression in samples in which expression of NSEQ is correlated with disease.
  • NSEQ can be used to detect genetic polymorphisms associated with a disease. These polymorphisms may be detected in the transcript cDNA.
  • the specificity of the probe is determined by whether it is made from a unique region, a regulatory region, or from a conserved motif. Both probe specificity and the stringency of diagnostic hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring, exactly complementary sequences, allelic variants, or related sequences. Probes designed to detect related sequences should preferably have at least 75% sequence identity to any of the nucleic acid sequences encoding PSEQ.
  • hybridization probes include the cloning of nucleic acid sequences into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by adding appropriate RNA polymerases and labeled nucleotides.
  • Hybridization probes may incorporate nucleotides labeled by a variety of reporter groups including, but not limited to, radionuclides such as 32 P or 35 S, enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, fluorescent labels, and the like.
  • the labeled polynucieotide sequences may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; and in microarrays utilizing samples from subjects to detect altered PSEQ expression.
  • NSEQ can be labeled by standard methods and added to a sample from a subject under conditions suitable for the formation and detection of hybridization complexes. After incubation the sample is washed, and the signal associated with hybrid complex formation is quantitated and compared with a standard value. Standard values are derived from any control sample, typically one that is free of the suspect disease. If the amount of signal in the subject sample is altered in comparison to the standard value, then the presence of altered levels of expression in the sample indicates the presence of the disease. Qualitative and quantitative methods for comparing the hybridization complexes formed in subject samples with previously established standards are well known in the art.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual subject. Once the presence of disease is established and a treatment protocol is initiated, hybridization or amplification assays can be repeated on a regular basis to determine if the level of expression in the subject begins to approximate that which is observed in a healthy subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to many years.
  • the polynucleotides may be used for the diagnosis of a variety of diseases associated with the colon. These include, but are not limited to, colon cancer, metastatic colon cancer, atrophic gastritis, cholecystitis, Crohns disease, irritable bowel syndrome, ulcerative colitis, and the like.
  • the polynucleotides may also be used as targets in a microarray.
  • the microarray can be used to monitor the expression patterns of large numbers of genes simultaneously and to identify splice variants, mutations, and polymorphisms. Information derived from analyses of the expression patterns may be used to determine gene function, to understand the genetic basis of a disease, to diagnose a disease, and to develop and monitor the activities of therapeutic agents used to treat a disease.
  • Microarrays may also be used to detect genetic diversity, single nucleotide polymorphisms which may characterize a particular population, at the genome level.
  • polynucleotides may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • Fluorescent in situ hybridization FISH
  • FISH Fluorescent in situ hybridization
  • antibodies or antibody fragments comprising an antigen binding site that specifically binds PSEQ may be used for the diagnosis of diseases characterized by the over-or-under expression of PSEQ.
  • a variety of protocols for measuring PSEQ, including ELISAs, RIAs, and FACS, are well known in the art and provide a basis for diagnosing altered or abnormal levels of expression.
  • Standard values for PSEQ expression are established by combining samples taken from healthy subjects, preferably human, with antibody to PSEQ under conditions suitable for complex formation The amount of complex formation may be quantitated by various methods, preferably by photometric means. Quantities of PSEQ expressed in disease samples are compared with standard values. Deviation between standard and subject values establishes the parameters for diagnosing or monitoring disease. Alternatively, one may use competitive drug screening assays in which neutralizing antibodies capable of binding PSEQ specifically compete with a test compound for binding the protein. Antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PSEQ. In one aspect, the anti-PSEQ antibodies of the present invention can be used for treatment or monitoring therapeutic treatment for diseases of the colon, particularly colon cancer.
  • the NSEQ, or its complement may be used therapeutically for the purpose of expressing mRNA and protein, or conversely to block transcription or translation of the mRNA.
  • Expression vectors may be constructed using elements from retroviruses, adenoviruses, herpes or vaccinia viruses, or bacterial plasmids, and the like. These vectors may be used for delivery of nucleotide sequences to a particular target organ, tissue, or cell population. Methods well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences or their complements. (See, e.g., Maulik et al. (1997) Molecular Biotechnology, Therapeutic Applications and Strategies.
  • NSEQ may be used for somatic cell or stem cell gene therapy.
  • Vectors may be introduced in vivo, in vitro, and ex vivo.
  • vectors are introduced into stem cells taken from the subject, and the resulting transgenic cells are clonally propagated for autologous transplant back into that same subject.
  • Delivery of NSEQ by transfection, liposome injections, or polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman et al.
  • NSEQ expression may be inactivated using homologous recombination methods which insert an inactive gene sequence into the coding region or other appropriate targeted region of NSEQ. (See, e.g. Thomas et a
  • Vectors containing NSEQ can be transformed into a cell or tissue to express a missing protein or to replace a nonfunctional protein.
  • a vector constructed to express the complement of NSEQ can be transformed into a cell to downregulate the overexpression of PSEQ.
  • Complementary or antisense sequences may consist of an oligonucleotide derived from the transcription initiation site; nucleotides between about positions -10 and +10 from the ATG are preferred.
  • inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g.. Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches. Futura Publishing, Mt. Kisco NY, pp 163- 177.)
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the cleavage of mRNA and decrease the levels of particular mRNAs, such as those comprising the polynucieotide sequences of the invention.
  • Ribozymes may cleave mRNA at specific cleavage sites.
  • ribozymes may cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The construction and production of ribozymes is well known in the art and is described in Meyers (supra).
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages within the backbone of the molecule.
  • nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases, may be included.
  • an antagonist, or an antibody that binds specifically to PSEQ may be administered to a subject to treat or prevent a disease associated with colon cancer.
  • the antagonist, antibody, or fragment may be used directly to inhibit the activity of the protein or indirectly to deliver a therapeutic agent to cells or tissues which express the PSEQ.
  • An immunoconjugate comprising a PSEQ binding site of the antibody or the antagonist and a therapeutic agent may be administered to a subject in need to treat or prevent disease.
  • the therapeutic agent may be a cytotoxic agent selected from a group including, but not limited to, abrin, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudomonas exotoxin A and 40, radioisotopes, and glucocorticoid.
  • a cytotoxic agent selected from a group including, but not limited to, abrin, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudom
  • Antibodies to PSEQ may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, such as those which inhibit dimer formation, are especially preferred for therapeutic use. Monoclonal antibodies to PSEQ may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma, the human B-cell hybridoma, and the EBV-hybridoma techniques. In addition, techniques developed for the production of chimeric antibodies can be used. (See, e.g., Pound (1998) Immunochemical Protocols.
  • an agonist of PSEQ may be administered to a subject to treat or prevent a disease associated with decreased expression, longevity or activity of PSEQ.
  • compositions may consist of PSEQ or antibodies, mimetics, agonists, antagonists, or inhibitors of the polypeptide.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • a stabilizing compound such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a subject alone or in combination with other agents, drugs, or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutical ly-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating and contrasting the ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population) statistics. Any of the therapeutic compositions described above may be applied to any subject in need of such therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • the COLNTUT16 cDNA library in which Incyte clone 2790708 was discovered, was constructed from colon tumor tissue obtained from a 60 year-old Caucasian male during a left hemicolectomy. Pathology indicated an invasive grade 2 adenocarcinoma, a sessile mass located three cm from the distal margin. The tumor extended through the submucosa and superficially into the muscularis basement. The margins of resection were free of involvement. One of nine regional lymph nodes contained metastatic adenocarcinoma. The patient presented with blood in the stool and a change in bowel habits. Patient history included thrombophlebitis, inflammatory polyarthropathy, prostatic inflammatory disease, and depressive disorder.
  • the COLNNOT08 cDNA library in which Incyte clone 1843578 was discovered is from the same patient.
  • the frozen tissue was homogenized and lysed in TRIZOL reagent (1 gm tissue/ 10 ml TRIZOL; Life Technologies), a monoplastic solution of phenol and guanidine isothiocyanate, using a Polytron homogenizer (PT-3000; Brinkmann Instruments, Westbury NY). After a brief incubation on ice, chloroform was added (1 :5 v/v), and the lysate was centrifuged. The chloroform layer was removed to a fresh tube, and the RNA extracted with isopropanol, resuspended in DEPC-treated water, and treated with
  • the mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies).
  • the cDNAs were fractionated on a SEPHAROSE CL4B column
  • This kit enabled the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers.
  • the recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours; at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water, after which samples were transferred to a 96-well block for storage at 4° C.
  • cDNAs were prepared using a MICROLAB 2200 (Hamilton, Reno NV) in combination with DNA ENGINE thermal cycler (PTC200; MJ Research, Watertown MA). cDNAs were sequenced by the method of Sanger et aj. (1975, J. Mol. Biol. 94:44 I f) using ABI PRISM 377 DNA sequencing systems (PE Biosystems) or MEGABASE 1000 sequencing systems (Molecular Dynamics, Sunnyvale CA).
  • sequences used for coexpression analysis were assembled from EST sequences, 5' and 3' longread sequences, and full length coding sequences. Selected assembled sequences were expressed in at least three cDNA libraries.
  • the assembly process is described as follows. EST sequence chromatograms were processed and verified. Quality scores were obtained using PHRED (Ewing et al. (1998) Genome Res 8: 175-185; Ewing and Green (1998) Genome Res 8: 186-194), and edited sequences were loaded into a relational database management system (RDBMS). The sequences were clustered using BLAST with a product score of 50. All clusters of two or more sequences created a bin, and each bin with its resident sequences represents one transcribed gene.
  • RDBMS relational database management system
  • Bins were annotated by screening the consensus sequence in each bin against public databases, such as GBpri and GenPept from NCBI.
  • the annotation process involved a FASTn screen against the gbpri database in GenBank. Those hits with a percent identity of greater than or equal to 75% and an alignment length of greater than or equal to 100 base pairs were recorded as homolog hits.
  • the residual unannotated sequences were screened by FASTx against GenPept. Those hits with an E value of less than or equal to 10 "8 were recorded as homolog hits.
  • colon cancer genes Fourteen known colon cancer genes were selected to identify novel genes that are closely associated with diseases of the colon. These known genes were carbonic anhydrase I, II, and IV, carcinoembryonic antigen family of proteins, colorectal carcinoma tumor-associated antigen, down- regulated in adenoma, fatty-acid binding protein, galectin, glutathione peroxidase, guanylin, cytokeratin 8 and 20, cadherin, and intestinal mucin. The colon cancer genes which were examined in this analysis and brief descriptions of their functions are listed in Table 4.
  • Hydrophobic ligand-binding protein expressed in liver and intestines differentially expressed in colon and other cancers (Davidson et al.
  • Neoplasma 40 107-109; Bravard et al.
  • SEQ ID NO:l (Incyte clone 1580553) is 219 nucleotides in length and has about 74% identity to the nucleic acid sequence of a mouse mucin glycoprotein (g2583092).
  • SEQ ID NO:2 (Incyte clone 2296694) is 252 nucleotides in length and has no known homologs in any of the public databases described in this application.
  • SEQ ID NO:3 (Incyte clone 2516888) is 285 nucleotides in length and has no known homologs in any of the public databases described in this application.
  • SEQ ID NO:4 (Incyte clone 2790708) is 1010 nucleotides in length and about 56% identity to the nucleic acid sequence from nucleotide 107789 to nucleotide 108777 of human chromosome 9 (g2564750).
  • SEQ ID NO:5 (Incyte clone 3235282) is 2616 nucleotides in length and has about 64% identity to the nucleic acid sequence encoding a mouse calcium sensitive chloride conductance protein (g3925280) and 70% identity to a partial cDNAs of a colon specific gene, CSG5, which is 878 nucleotides long.
  • SEQ ID NO:6 (Incyte clone 1843578) is 795 nucleotides in length and has about 64% identity to a nucleic acid sequence encoding a mouse calcium sensitive chloride conductance protein (g3925280).
  • SEQ ID NO:7 (Incyte clone 1961467) is 2225 nucleotides in length and has about 6% identity to human gene signature
  • SEQ ID NO:8 has 1 15 amino acids which are encoded by SEQ ID NO:6 and has no known homologs in any of the public databases described in this application. Motif analysis of SEQ ID NO:8 shows a potential phosphorylation site at S83.
  • SEQ ID NO:9 has 90 amino acids which are encoded by SEQ ID NO:7 and has no known homologs in any of the public databases described in this application. Motif analysis of SEQ ID NO:9 shows five potential phosphorylation sites at T10, T6, T21 ,
  • polypeptide sequences SEQ ID NOs: 1-7, and polypeptide sequences, SEQ ID NOs:8 and 9, were queried against databases derived from sources such as GenBank and SwissProt. These databases, which contain previously identified and annotated sequences, were searched for regions of similarity using BLAST (Altschul, supra). BLAST searched for matches and reported only those that satisfied the probability thresholds of 10 "25 or less for nucleotide sequences and 10 "8 or less for polypeptide sequences. The polypeptide sequences were also analyzed for known motif patterns using MOTIFS,
  • SPSCAN SPSCAN, BLIMPS, and HMM-based protocols.
  • MOTIFS Genetics Computer Group, Madison WI
  • SPSCAN Genetics Computer Group searches for potential signal peptide sequences using a weighted matrix method (Nielsen et al. (1997) Prot Eng 10: 1-6). Hits with a score of 5 or greater were considered.
  • BLIMPS uses a weighted matrix analysis algorithm to search for sequence similarity between the polypeptide sequences and those contained in BLOCKS, a database consisting of short amino acid segments, or blocks of 3-60 amino acids in length, compiled from the PROSITE database (Henikoff, supra; Bairoch, supra), and those in PRINTS, a protein fingerprint database based on non-redundant sequences obtained from sources such as SwissProt, GenBank, PIR, and NRL-3D (Attwood et al. (1997)
  • Polynucieotide sequences are isolated from a biological source and applied to a solid matrix (a blot) suitable for standard nucleic acid hybridization protocols by one of the following methods.
  • a mixture of target nucleic acids is fractionated by electrophoresis through an 0.7% agarose gel in lx TAE [40 mM Tris acetate, 2 mM ethylenediamine tetraacetic acid (EDTA)] running buffer and transferred to a nylon membrane by capillary transfer using 20x saline sodium citrate (SSC).
  • the target nucleic acids are individually ligated to a vector and inserted into bacterial host cells to form a library.
  • Target nucleic acids are arranged on a blot by one of the following methods.
  • bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane.
  • the membrane is placed on bacterial growth medium, LB agar containing carbenicillin, and incubated at 37°C for 16 hours.
  • Bacterial colonies are denatured, neutralized, and digested with proteinase K.
  • Nylon membranes are exposed to UV irradiation in a STRATALINKER UV-crosslinker (Stratagene, La Jolla)
  • target nucleic acids are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert.
  • Amplified target nucleic acids are purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified target nucleic acids are robotically arrayed onto a glass microscope slide. The slide was previously coated with 0.05% aminopropyl silane (Sigma-Aldrich, St Louis MO) and cured at 110°C. The arrayed glass slide (microarray) is exposed to UV irradiation in a STRATALINKER UV-crosslinker (Stratagene). Probe Preparation cDNA probe sequences are made from mRNA templates.
  • mRNA Five micrograms of mRNA is mixed with 1 ⁇ g random primer (Life Technologies), incubated at 70°C for 10 minutes, and lyophilized. The lyophilized sample is resuspended in 50 ⁇ l of lx first strand buffer (cDNA Synthesis system; Life Technologies) containing a dNTP mix, [ ⁇ - 32 P]dCTP, dithiothreitol, and MMLV reverse transcriptase (Stratagene), and incubated at 42°C for 1-2 hours. After incubation, the probe is diluted with 42 ⁇ l dH 2 0, heated to 95°C for 3 minutes, and cooled on ice. mRNA in the probe is removed by alkaline degradation. The probe is neutralized, and degraded mRNA and unincorporated nucleotides are removed using a
  • Hybridization is carried out at 65°C in a hybridization buffer containing 0.5 M sodium phosphate
  • the blot is incubated in hybridization buffer at 65°C for at least 2 hours, the buffer is replaced with 10 ml of fresh buffer containing the probe sequences. After incubation at 65°C for 18 hours, the hybridization buffer is removed, and the blot is washed sequentially under increasingly stringent conditions, up to 40 mM sodium phosphate, 1% SDS, 1 mM EDTA at 65°C. To detect signal produced by a radiolabeled probe hybridized on a membrane, the blot is exposed to a
  • SEQ ID NOs: 8-9, or portions thereof, substantially purified using polyacrylamide gel electrophoresis or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols as described in Pound (supra).
  • the amino acid sequence is analyzed using LASERGENE software (DNASTAR,
  • oligopeptides 15 residues in length are synthesized using an ABI 431 A Peptide synthesizer (PE Biosystems) using Fmoc-chemistry and coupled to keyhole limpet hemocyanin

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Abstract

L'invention concerne de gènes du cancer du côlon ainsi que des polypeptides encodés par ces gènes. L'invention concerne également des vecteurs d'expression, des cellules hôtes et des anticorps. L'invention concerne enfin des procédés pour le diagnostic, le traitement ou la prévention de maladies du côlon.
EP00913330A 1999-02-22 2000-02-01 Genes associes a des maladies du colon Withdrawn EP1155126A2 (fr)

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US20020192666A1 (en) * 2000-10-31 2002-12-19 Yongming Sun Compositions and methods relating to colon specific genes and proteins
AU2002243444A1 (en) 2000-10-31 2002-06-24 Diadexus, Inc. Compositions and methods relating to colon specific genes and proteins
DE10061110B4 (de) * 2000-12-07 2006-12-21 Schaller, Gerhard, Prof. Dr. med. Expression der Keratingene 8 und 18 zur Therapie von Tumoren, insbesondere des Mammakarzinoms
SI1513934T1 (sl) 2002-06-06 2011-06-30 Oncotherapy Science Inc Geni in polipeptidi, povezani s humanimi raki kolona
DK1513934T3 (da) 2002-06-06 2011-05-02 Oncotherapy Science Inc Gener og polypeptider relateret til humane coloncancersygdomme
EP1380644A1 (fr) * 2002-07-08 2004-01-14 Kylix B.V. Utilisation de gènes cible spécifiques de TCF pour identifier des medicaments pour le traitement du cancer, en particulier le cancer colorectal, dans lequel TCF/beta-catenin/WNT signalisation joue un rôle central
WO2005080597A1 (fr) * 2004-02-24 2005-09-01 Oncotherapy Science, Inc. Procede pour diagnostiquer des cancers colorectaux
FR2919062B1 (fr) 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de l'aminoacylase 1 pour le diagnostic in vitro du cancer colorectal.
FR2919063B1 (fr) 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage du leucocyte elastase inhibitor pour le diagnostic in vitro du cancer colorectal.
FR2919065B1 (fr) 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de l'apolipoproteine ai pour le diagnostic in vitro du cancer colorectal
FR2919061B1 (fr) 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de la plastine-i pour le diagnostic in vitro du cancer colorectal.
FR2919060B1 (fr) 2007-07-19 2012-11-30 Biomerieux Sa Procede de dosage de l'ezrine pour le diagnostic in vitro du cancer colorectal.
EP2171467B1 (fr) 2007-07-19 2016-01-13 bioMérieux Procede de dosage de la liver fatty acid-binding protein, de l'ace et du ca19-9 pour le diagnostic in vitro du cancer colorectal
FR2919064B1 (fr) 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de l'apolipoproteine all pour le diagnostic in vitro du cancer colorectal
FR2933773B1 (fr) 2008-07-10 2013-02-15 Biomerieux Sa Procede de dosage de la proteine disulfide isomerase pour le diagnostic in vitro du cancer colorectal
WO2010094499A1 (fr) 2009-02-20 2010-08-26 Ganymed Pharmaceuticals Ag Methodes et compositions de diagnostic et de traitement du cancer
EP2221063A1 (fr) * 2009-02-20 2010-08-25 Ganymed Pharmaceuticals AG Procédé et compositions pour le diagnostic et le traitement du cancer
KR102317098B1 (ko) 2009-11-11 2021-10-25 가니메드 파마슈티칼스 게엠베하 클라우딘 6 특이적 항체
EP2404936A1 (fr) 2010-07-06 2012-01-11 Ganymed Pharmaceuticals AG Thérapie du cancer utilisant des anticorps in vivo dirigés sur la cible CLDN6
PL2707390T3 (pl) 2011-05-13 2016-06-30 Ganymed Pharmaceuticals Ag Przeciwciała do leczenia raka z ekspresją klaudyny 6
WO2015014376A1 (fr) 2013-07-31 2015-02-05 Biontech Ag Diagnostic et thérapie du cancer impliquant des cellules souches cancéreuses

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