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WO2008086502A2 - Marqueur moléculaire pour carcinome du rein à cellules claires - Google Patents

Marqueur moléculaire pour carcinome du rein à cellules claires Download PDF

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Publication number
WO2008086502A2
WO2008086502A2 PCT/US2008/050812 US2008050812W WO2008086502A2 WO 2008086502 A2 WO2008086502 A2 WO 2008086502A2 US 2008050812 W US2008050812 W US 2008050812W WO 2008086502 A2 WO2008086502 A2 WO 2008086502A2
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Prior art keywords
spop
sample
level
patient
protein
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WO2008086502A3 (fr
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Kevin P. White
Jiang Liu
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University of Chicago
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University of Chicago
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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney

Definitions

  • the present invention relates generally to the fields of biochemistry, molecular biology, and medicine.
  • the invention is related to diagnosis and therapy for renal cell carcinoma. More particularly, it concerns the SPOP protein, SPOP nucleic acids, inhibitors of SPOP, and their relevance to cancer.
  • Renal cell carcinoma is a type of kidney cancer in which the cancerous cells are found in the lining of very small tubes (tubules) in the kidney.
  • RCC is the most common type of kidney cancer, accounting for about 9 out of 10 kidney cancers.
  • Clear cell RCC also known as conventional RCC, comprises 75% of all RCC.
  • the cancer can affect both kidneys and spreads easily, most often to the lungs and other organs.
  • RCC is the 7 th leading malignant condition among men and the 12 th among women, accounting for 2.6% of all cancers, with 36,000 new cases occurring per year. When detected early, radical nephrectomy can result in an excellent survival rate for RCC patients.
  • IL-2 recombinant human interleukin-2
  • IFN- ⁇ recombinant human interferon ⁇ -2b
  • VEGF vascular endothelial growth factor
  • mTOR mammalian target of rapamycin
  • the present invention is based on the finding that greater than 99% of tissue samples from renal clear cell carcinomas are positive for SPOP protein expression while no normal tissue was positive for SPOP protein expression. Therefore, the present invention concerns methods and compositions for diagnosing renal cell carcinoma (RCC) in a patient based on SPOP expression.
  • the invention also provides methods and compositions for treating RCC using an SPOP inhibitor and methods of screening for SPOP inhibitors.
  • kidney cell carcinoma a kidney cell
  • methods for diagnosing renal cell carcinoma (RCC) in a patient by evaluating the level of SPOP expression in a kidney cell, wherein expression of SPOP is indicative of renal cell carcinoma.
  • the kidney cell may be obtained from the patient in the form of a biological sample containing a kidney cell.
  • samples include, but are not limited to, a tissue sample.
  • the tissue sample is obtained from a biopsy, such as a biopsy of tissue that may be cancerous or tumorigenic or a metastasis.
  • SPOP expression may refer to mRNA expression or to protein expression.
  • the level of SPOP mRNA is evaluated, measured, and/or determined. This may be done using any method by which mRNA expression is levels are evaluated, measured, or determined. A variety of such methods are well known to those of skill in the art, and these include, but are not limited to, those involving complementary probes or primers, amplification primers, cDNAs, etc. Such methods may involve RT-PCR, in situ hybridization (ISH), and/or arrays or biochips for evaluating RNA expression.
  • ISH in situ hybridization
  • the level of SPOP protein is evaluated, measured, and/or determined. This is discussed in further detail below.
  • kidney cell carcinoma there are methods for diagnosing renal cell carcinoma (RCC) in a patient comprising evaluating the level of SPOP protein in a biological sample obtained from the patient, wherein a higher level of SPOP in the patient sample as compared to a level from a normal sample is indicative of renal cell carcinoma.
  • kidney cell carcinoma there are methods for diagnosing renal cell carcinoma (RCC) in a patient comprising: (a) obtaining a biological sample from the patient; and, (b) obtaining information regarding the level of SPOP protein in the patient sample, wherein a higher level of SPOP protein in the patient sample as compared to a level in a normal sample is indicative of renal cell carcinoma.
  • Further embodiments include methods for evaluating the level of SPOP protein in a biological sample comprising (a) screening the biological sample for the level of SPOP protein using an SPOP monoclonal antibody; and (b) reporting the level of SPOP protein in the sample to a medical professional.
  • methods for diagnosing renal cell carcinoma in a patient comprising determining the level of SPOP protein in a biological sample obtained from the patient, wherein a higher level of SPOP protein or diffused SPOP localization in the cells as compared to the level of SPOP protein or SPOP localization in normal cells is indicative of renal cell carcinoma.
  • SPOP localization in normal cells was confined to the nucleus, while in the clear cell carcinoma cells, SPOP was observed outside the nucleus. Consequently, SPOP localization outside the nucleus or throughout the cell is indicative of renal cell carcinoma.
  • Other methods of the invention include methods of identifying a metastasized tumor comprising determining the level of SPOP protein in a biological sample obtained from the patient, wherein a higher level of SPOP proteinor diffused SPOP localization in the cells as compared to the level of SPOP protein in normal neighboring cells is indicative renal cell carcinoma.
  • the normal neighboring cells refers to cell adjacent to or nearby cells suspected of being cancerous or tumorigenic.
  • a sample from a patient has a higher level of SPOP expression than the level of SPOP in a normal sample, the sample is indicative of renal cell carcinoma in the patient.
  • the SPOP expression level is higher than the level of a normal sample by virtue of expression being detected at any level.
  • the level of SPOP expression is at least about or at most about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300% or more, or any range derivable therein, compared the level in a normal sample.
  • the level of SPOP expression is at least about or at most about 2-, 3-, A-, 5-, 6-, 7-, 8-, 9-, 10-, H-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-fold or times, or any range derivable therein, greater than the level of a normal sample.
  • the level of expression refers to the level of detectable expression by any particular method of measuring or determining that level.
  • a biological sample is obtained from a patient.
  • the entity evaluating the sample for SPOP expression levels did not directly obtain the sample from the patient. Therefore, methods of the invention involve obtaining the sample indirectly or directly from the patient.
  • a doctor, medical practitioner, or their staff may obtain a biological sample for evaluation.
  • the sample may be analyzed by the practitioner or their staff, or it may be sent to an outside or independent laboratory.
  • the medical practitioner may be cognizant of whether the test is providing information regarding a quantitative level of SPOP expression, or the medical practitioner may be aware that the test indicates directly or indirectly that the test was positive or negative for SPOP expression.
  • the evaluation may indicate simply that a sample is positive or negative for SPOP protein.
  • the biological sample will contain an SPOP mRNA, SPOP protein, and/or cells that may express SPOP mRNA or SPOP protein.
  • tissue samples include blood, serum or any other blood component that does not include peripheral blood mononuclear cells (PBMC), semen saliva, tears, urine, fecal material, sweat, a buccal sample, tissue sample, skin and hair.
  • PBMC peripheral blood mononuclear cells
  • the tissue sample contains a kidney cell.
  • a sample contains one or more of the following kidney cells: clear cell, chromophobe, papiliary, oncocytic, metastatic, or rate type.
  • the evaluated kidney cell whose SPOP levels (protein or mRNA) is a renal clear cell.
  • the biological sample is from a biopsy of tissue that may or may not be cancerous, tumorigenic, and/or metastatic.
  • the biological sample containing kidneys cells was not obtained from the kidney. Such may be the case in patients who have a metastatic RCC tumor.
  • biological samples may be placed on a slide for histological analysis on either the protein or nucleic acid level.
  • the sample may be fixed or not fixed. Such methods are well known to those of skill in the art.
  • the medical practitioner may know the relevant information that will allow him or her to determine whether the patient can be diagnosed as having renal cell carcinoma based on quantitative or qualitative information about SPOP expression. It is contemplated that, for example, a laboratory conducts the test to determine whether and/or to what extent SPOP is expressed as an mRNA and/or protein. Laboratory personnel may report back to the practitioner with the specific result of the test performed or the laboratory may simply report that the patient is positive for SPOP expression. Evaluations of SPOP expression is also applicable to evaluations regarding SPOP protein localization in kidney cell.
  • the patient from whom a biological sample is obtained may be identified as at risk for RCC or be identified as having symptoms of RCC.
  • some methods of the invention involve identifying a patient as at risk for RCC or as having symptoms of RCC.
  • the level of SPOP expression may be evaluated quantitatively. In these cases, methods may involve comparing the level of SPOP expression in the biological sample of a patient to the level of expression in a normal sample.
  • the normal cells may be obtained from the patient, though they may also be from someone other than the patient. It is contemplated that the level of expression in a normal sample may be evaluated, determined, or measured at the same time as the patient's sample, or it may be a level previously determined based on one or more normal samples.
  • the level of SPOP expression in a normal sample may be a normalized value against which to compare the value from the patient. It is specifically contemplated that when levels of SPOP expression are compared to a normal sample that the normal sample may be from the same kind of tissue or be the same kind of sample as the patient's sample. In other words, the levels of expression in homologous samples are compared. For example, the level of SPOP protein in a biological sample obtained from a patient's kidney could be compared to the level of SPOP protein in normal kidney tissue. Moreover, it is assumed that amounts of biological material may be normalized when quantitative values are compared.
  • the normal sample is a sample from a non-cancerous subject or from non-cancerous tissue.
  • the normal sample is from normal kidney tissue or the normal sample comprises normal kidney cells.
  • the level of SPOP protein may be evaluated using a SPOP antibody.
  • the antibody is a polyclonal antibody, while in others it is a monoclonal antibody. It is also contemplated that the level of SPOP protein may be evaluated by immunohistochemistry (also known as immunohistology) .
  • a patient may also be treated for RCC after the level of SPOP expression (protein and/or mRNA) is evaluated in the patient if the level is indicative of RCC.
  • Methods may also involve further or previous tests for RCC such as a pathological evaluation of kidney cells. Consequently, methods may also involve treating the patient with renal cell carcinoma with a conventional cancer treatment.
  • the treatment is chemotherapy, radiotherapy, surgery, gene therapy, and/or immunotherapy.
  • treatment may involve administering to the patient an SPOP inhibitor.
  • An SPOP inhibitor refers to a substance that specifically inhibits SPOP activity or expression. This may be done separately or in conjunction with the other cancer treatments, hi certain embodiments, the SPOP inhibitor inhibits SPOP activity, while in others the SPOP inhibitor inhibits SPOP expression.
  • the SPOP inhibitor is an SPOP siRNA that inhibits SPOP expression.
  • Methods of the invention specifically include methods of treating renal cell carcinoma (RCC) comprising administering to an RCC patient an effective amount of an SPOP inhibitor.
  • RCC renal cell carcinoma
  • effective amount refers to the amount needed to inhibit SPOP and effect a therapeutic benefit relevant to cancer.
  • therapeutic benefit refers to anything that promotes or enhances the well-being of the subject with respect to cancer.
  • a list of nonexhaustive examples of this includes extension of the subject's life by any period of time, decrease or delay in the neoplastic development of the disease, decrease in hyperproliferation, reduction in tumor growth, delay of metastases, reduction in cancer cell or tumor cell proliferation rate, and a decrease in pain to the subject that can be attributed to the subject's condition.
  • SPOP inhibitor or other cancer treatment may be administered topically, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage.
  • SPOP activity is specific binding with Cullin 3, inducing apoptosis, inhibition of c-Jun phosphorylation, increase in JNK activity, and/or regulation of cell proliferation. Assays for evaluating this activity are contemplated as part of the invention. Also contemplated is the ability of SPOP to ubiquitinate a substrate. Methods may involve evaluating SPOP activity in the presence and absence of the candidate inhibitor to determine whether the candidate is an inhibitor.
  • the candidate inhibitor may be a nucleic acid, a protein, or a small molecule.
  • the candidate inhibitor is a nucleic acid that inhibits the expression of SPOP.
  • the candidate inhibitor is a small molecule. It is contemplated that such methods may be performed using highthroughput screening, arrays, or a biochip such that multiple candidates can be evaluated.
  • compositions such as an SPOP monoclonal antibody that immunologically reacts with human SPOP.
  • the antibody is SP0P-5G, which may be properly deposited if necessary.
  • FIG. 1 Identification of direct targets of even-skipped. Heatmap at left depicts log ⁇ -fold change, mutant vs. wild type, in expression of 98 putative direct targets of eve. Columns represent time points, labeled by hours post fertilization. Rows depict individual genes, sorted by hierarchical clustering. Genes with 'regulation of development' or 'transcriptional regulation' GO annotations are marked in the center panel. Locations of even-skipped binding sites, relative to each target gene, are represented at left.
  • FIG. 2 Genetic and biochemical network of ftz and eve interaction combining mutant expression microarray data, DNA binding sites mapping, yeast two-hybrid system data and literature mining data.
  • FIG. 3 Experimental confirmation of network predictions.
  • FIG. 3A RNA in situ localization of selected target genes following the microarray experiments fox ftz and eve. RNA localization for Impl2, Cad74a and Paxillin was performed in wild type, ftz and eve mutant backgrounds. Half the number of the parasegments seen in the wild type are missing in eve and ftz mutants.
  • FIG. 3B In vitro interaction between FTZ-Fl and Dichaete in a pull down assay. FTZ-Fl and ftz also interact, but ftz and Dichaete do not inteact. E, elution, F, flow- through, D, Dichaete. (FIG.
  • FTZ but not FTZ-Fl binds two regulatory elements in the 5" upstream non coding sequence of D- SPOP by gel shift analysis.
  • D-SPOPl and D-SP0P2 are two predicted obligos, sequence see the method.
  • FIG. 3F D-SPOP and EVE antibodies staining in wild type, eve mutant backgrounds. D-SPOP is stained with blue and EVE was stained with DAB brown. Wild type animal, SPOP was stained between segments. In eve mutant embryos, SPOP expression pattern was lost.
  • FIG. 4 D-SPOP promotes puckered ubiquitination and degradation.
  • FIG. 4A Light micrographs of Drosophil ⁇ adult eyes for wild type (GMR-Gal4/+).
  • FIG. 4B GMR>Egr triggered cell death and produces a small eye phenotype (GMR-Gal4 UAS-Egr/+).
  • FIG. 4C Deleting one copy of D-SPOP (GMR-Gal4 UAS-Egr/+; D- SPOP ⁇ 6/+) suppressed the phenotype of (GMR-Gal4 UAS-Egr/+).
  • FIG. 4D Co- expression of a D-SPOP RNAi (GMR-GaM UAS-Egr/+; UAS-D-SPOP-RNAi) suppressed the phenotype of (GMR-GaW UAS-Egr/+).
  • FIG. 4E Expression of D- SPOP (GMR-GaH UAS-O-SVO?/+) under the control of GMR promoter produced rough eyes with slightly reduced size.
  • FIG. 4F In vitro pull down assay to detect the interaction of puckered (puc) and D-SPOP. Puc and D-SPOP were tagged with or without polyHis and transcribed/translated in vitro. Proteins were incubated with MagZTM particles.
  • FIG. 4G In vivo immunoprecipitate to detect the interaction of puc and D-SPOP. Western blots of immunoprecipitates from S2 cells expressing indicated proteins.
  • FIG. 4H In vivo ubiquitination of puc promoted by D-SPOP. Myc-puc was immunoprecipitated from S2 cells expressing the indicated proteins treated with or without MGl 32 for 4 hours before cells lysis. Puc was detected by immunoblot with anti-myc antibody.
  • FIG. 41 Puc degradation was promoted by SPOP.
  • FIG. 4J Western blot of lysates from S2 cells expressing the indicated proteins.
  • FIG. 4J HEK293 cells were transfected with SPOP, and treated with 50ng/ml TNF at 0, 5, 15, 30, 60 minutes, followed by immunoblot with anti P-c-Jun.
  • HEK293 cells were transfected with empty pcDNA3.1.
  • FIG. 4K HEK293 cells are transfected with siRNA, and treated with 50ng/ml TNF at 0, 5, 15, 30, 60 minute, followed by immunoblot with anti P-c-Jun.
  • Control treatments are cells transfected with non-target control siRNA.
  • FIG. 5 Eiger (homolog human TNF) binds and activates Wengen (TNF receptor) to activate JNK (Bsk) dependent apoptosis pathway.
  • SPOP enhances Eiger induced apoptosis pathway by negative regulate Puc (MAKP phosphatase).
  • FIG. 6 Function of SPOP in mammalian TNF pathway and renal cell carcinoma.
  • FIG. 6A HEK293 cells were transfected with SPOP, and treated with 50ng/ml TNF at 0, 5, 15, 30, 60 minutes, followed by immunoblot with anti P-c-Jun. In the control treatments, HEK293 cells were transfected with empty pcDNA3.1.
  • FIG. 6B Histology analysis of renal tissue array from renal cell carcinoma patients. Tissue images from normal renal cell, oncocytic renal cell carcinoma and clear cell renal carcinoma. H&E staining was used to identify cell types. Brown staining tissues with SPOP monoclonal antibody were used to detect SPOP expression in cells.
  • FIG. 6A HEK293 cells were transfected with SPOP, and treated with 50ng/ml TNF at 0, 5, 15, 30, 60 minutes, followed by immunoblot with anti P-c-Jun. In the control treatments, HEK293 cells were transfected with empty pcDNA3.1.
  • FIG. 6B
  • Renal patients are classified into different categories depending on cell types. Only 1 clear cell renal carcinoma patient in 171 clear cell renal carcinoma patients is negative staining. According to the H&E image, cell type of this patient is not typical clear cell, its morphology between clear cell and oncocytic cell.
  • FIG. 7 H&E staining of HEK cells, HEK-SPOP cells and Caki-2 cells. Morphology of HEK-SPOP cells is more close to Caki-2 cells.
  • FIG. 8 FTZ and FTZ-Fl probe distribution in Adh region.
  • FIG. 9 Distribution of ChIPchip probe locations relative to direct target genes. All panels are plotted as histograms, distance plotted along x-axis, density of probes along y-axis. Probe-to-gene data are depicted for ftz probes to ftz differentially expressed genes, eve probes to eve differentially expressed genes, permuted probes to ftz differentially expressed genes and eve differentially expressed genes.
  • FIG. 9A Distribution of probe locations relative to target genes identified under the UWD lkb* parameter set.
  • X-axis depicts absolute distance from the TSS. Again note the enrichment at the TSS and the decreased variance of the experimental distributions relative to permuted probes.
  • FIG. 9D Distribution of probe locations relative to up or down regulated direct target genes***. Distances are plotted as in (FIG. 9A). Solid lines represent distributions relative to up regulated genes (mutant > wt), dashed lines represent distributions relative to down regulated genes. No obvious difference in the distributions of up or down regulated genes, but the data sets are getting relatively small at this point.
  • FIG. 10 SPOP involves in nervous system development.
  • FIG. 10A Center nervous system staining by using anti BP 102 antibody in RNAi GFP control embryos.
  • FIG. 10B Peripheral nervous system staining by using anti 22clO in GFP RNAi control embryos.
  • FIG. 10C Center nervous system staining by using anti BP 102 antibody in RNAi SPOP embryos. Center nervous system was corrupted.
  • FIG. 10D Peripheral nervous system staining by using anti 22clO in RNAi SPOP embryos. Peripheral nervous system was corrupted too.
  • FIG. 11 Genetics mapping of D-SPOP in fly Eiger signaling pathway.
  • HA Ectopic expression of dTAKl (sev-Gal4 UAS-dTAKl/+) in developing eyes under the control of sev promoter induces apoptosis and generates rough eyes with reduced size.
  • FIG. HB The apoptosis induced by dTAKl is suppressed by removing one wild-type copy of D-SPOP (B, sev-Gal4 UAS-dTAKl/+; D-SPOP ⁇ 6/+).
  • FIG. HC Ectopic expression HepCA (sev-Gal4 UAS-HepCA /+) in developing eyes under the control of sev promoter induces apoptosis and generates rough eyes with reduced size.
  • FIG. 12 SPOP cellular location is changed after kidney cell transformed into clear cell carcinoma. From the figure, we can find SPOP is overlapped with nuclear. This result indicates that SPOP in the normal kidney cells localizes in the nuclear, while SPOP spreads out into cytosol in the clear RCC. SPOP failing to transport into nuclear may result that it functions incorrectly.
  • D-SPOP contains two conserved domains, a MATH domain and a BTB/POZ domain (Xu, Wei et al. 2003).
  • MEL-26 the ortholog of human SPOP in Caenorhabditis elegans, was first identified as a BTB protein that serves as an adaptor of Cul3 based ubiquitin ligase (Xu, Wei et al. 2003).
  • SPOP was shown to mediate ubiquitination of death domain-associated protein (Daxx) (Kwon, La et al. 2006), the polycomb group BMI, the histone variant MacroH2A (Hernandez-Munoz, Lund et al.
  • JNK activation and c-Jun phosphorylation are required for cellular transformation induced by RAS, an oncogene that 30% of human cancers have hypermorphic mutations.
  • Over-expression of SPOP increases the level of phosphorylated JNK (P-JNK) and P- c-Jun.
  • P-JNK phosphorylated JNK
  • P-c-Jun Recent studies reported a significant upregulation of P-c-Jun levels in tumor cells from patients with myeloid leukemia, Hodgkin lymphoma, anaplastic large cell- lymphomas and renal cell carcinoma (Weiss and Bohmann 2004; Oya, Mikami et al. 2005; WO 2004/048933).
  • Phosphorylation of c-Jun which is a negative regulator of p53 expression, is essential for cellular transformation in several tumors (Schreiber, Kolbus et al. 1999; Shaulian, Schreiber et al. 2000).
  • the specific over-expression of SPOP in tissue samples of patients with clear cell RCC supports the involvment of SPOP in RCC.
  • SPOP In normal cells, SPOP is generally localized in the nucleus.
  • the inventors have also discovered that in RCC cells, SPOP diffuses throughout the cell. SPOP cellular location is changed after a kidney cell is transformed into clear cell carcinoma. SPOP (brown) staining overlaps with nuclear staining (blue).
  • SPOP in the normal kidney cells localizes in the nucleus, while SPOP spreads out into cytosol in the clear RCC. SPOP failing to transport into the nucleus may indicate abberrent function. This indicates that SPOP plays an important role in the tumorigensis of renal cell carcinoma.
  • the nucleic acid sequences disclosed herein have a variety of other uses. For example, they have utility as probes or primers for embodiments involving nucleic acid hybridization. They may be used in diagnostic or screening methods of the present invention. Detection of nucleic acids encoding SPOP or SPOP modulators are also encompassed by the invention. See WO 2004/048933.
  • the present invention concerns diagnosing renal cell carcinoma by determining the level of SPOP expression by determining the level of gene expression.
  • the invention provides for an inhibitor of SPOP, wherein the inhibitor may be a nucleic acid.
  • the present invention concerns polynucleotides and oligonucleotides, isolatable from cells, that are free from total genomic DNA and that are capable of expressing all or part of a protein or polypeptide.
  • the polynucleotides or oligonucleotides may be identical or complementary to all or part of a nucleic acid sequence encoding a SPOP amino acid sequence. These nucleic acids may be used directly or indirectly to assess, evaluate, quantify, or determine SPOP expression.
  • SPOP polynucleotide refers to a SPOP- encoding nucleic acid molecule that has been isolated essentially or substantially free of total genomic nucleic acid to permit hybridization and amplification, but is not limited to such. Therefore, a “polynucleotide encoding SPOP” refers to a DNA segment that contains wild-type, mutant, or polymorphic SPOP polypepti de-coding sequences isolated away from, or purified free from, total mammalian or human genomic DNA.
  • a SPOP oligonucleotide refers to a nucleic acid molecule that is complementary or identical to at least 5 contiguous nucleotides of a SPOP-encoding sequence, which is the cDNA sequence encoding human SPOP. It also is contemplated that a particular polypeptide from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein.
  • a polynucleotide comprising an isolated or purified wild-type, polymorphic, or mutant polypeptide gene refers to a DNA segment including wild- type, polymorphic, or mutant polypeptide coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences.
  • the term "gene” is used for simplicity to refer to a functional protein, polypeptide, or peptide-encoding unit. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • a nucleic acid encoding all or part of a native or modified polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide of the following lengths: about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120
  • Hybridization The use of a probe or primer of between 13 and 100 nucleotides, preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained. One will generally prefer to design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired.
  • nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs or to provide primers for amplification of DNA or RNA from samples.
  • relatively high stringency conditions For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50 0 C to about 70 0 C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions.
  • Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20 0 C to about 55°C.
  • Hybridization conditions can be readily manipulated depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 1.0 mM dithiothreitol, at temperatures between approximately 20 0 C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40 0 C to about 72°C.
  • indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.
  • the probes or primers described herein will be useful as reagents in solution hybridization, as in PCRTM, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
  • the conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art.
  • hybridization After washing of the hybridized molecules to remove non-specif ⁇ cally bound probe molecules, hybridization is detected, and/or quantified, by determining the amount of bound label.
  • Representative solid phase hybridization methods are disclosed in U.S. Patents 5,843,663, 5,900,481 and 5,919,626.
  • Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Patents 5,849,481, 5,849,486 and 5,851,772 and U.S. Patent Publication 2008/0009439. The relevant portions of these and other references identified in this section of the Specification are incorporated herein by reference.
  • ISH In situ hybridization
  • FISH Fluorescent DNA ISH
  • RNA ISH hybridization histochemistry
  • probe is either a labeled complementary DNA or, now most commonly, a complementary RNA (riboprobe).
  • riboprobe a complementary RNA
  • the probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of unhybridized, excess RNA probe).
  • Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e., only exact sequence matches will remain bound).
  • ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.
  • Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 2001). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template- dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.
  • Pairs of primers designed to selectively hybridize to nucleic acids corresponding to any sequence corresponding to a nucleic acid sequence are contacted with the template nucleic acid under conditions that permit selective hybridization.
  • high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers.
  • hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences.
  • the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Bellus, 1994).
  • a number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample.
  • One of the best known amplification methods is the polymerase chain reaction (referred to as PCRTM) which is described in detail in U.S. Patents 4,683,195, 4,683,202 and 4,800,159, and in Innis et al. , 1988, each of which is incorporated herein by reference in their entirety.
  • a reverse transcriptase PCRTM amplification procedure may be performed to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et al, 2001).
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641.
  • Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent 5,882,864.
  • RT Reverse transcription
  • RT-PCR quantitative PCR
  • the cycle number is on the X axis
  • the log of the concentration of the amplified target DNA is on the Y axis
  • a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • the concentration of the target DNA in the linear portion of the PCR amplification is directly proportional to the starting concentration of the target before the reaction began.
  • concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundances is only true in the linear range of the PCR reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCR for a collection of RNA populations is that the concentrations of the amplified PCR products must be sampled when the PCR reactions are in the linear portion of their curves.
  • a second condition for an RT-PCR experiment is to determine the relative abundances of a particular mRNA species. Typically, relative concentrations of the amplifiable cDNAs are normalized to some independent standard. The goal of an RT- PCR experiment is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
  • RT-PCR can be performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target.
  • This assay measures relative abundance, not absolute abundance of the respective mRNA species.
  • Another method for amplification is ligase chain reaction ("LCR”), disclosed in
  • U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
  • a method based on PCRTM and oligonucleotide ligase assy (OLA), disclosed in U.S. Patent 5,912,148, may also be used.
  • Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S.
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as an amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which may then be detected.
  • SDA Strand Displacement Amplification
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification
  • TAS transcription-based amplification systems
  • ssRNA RNA
  • ssDNA double-stranded DNA
  • dsDNA double-stranded DNA
  • PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and “one-sided PCR” (Frohman, 1990; Ohara et ai, 1989).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 2001). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
  • Separation of nucleic acids may also be effected by chromatographic techniques known in art.
  • chromatographic techniques There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion- exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the amplification products are integrally labeled with radio- or fiuorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al., 2001).
  • U.S. Patent 5,279,721, incorporated by reference herein discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids.
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • Various nucleic acid detection methods known in the art are disclosed in U.S.
  • chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al.
  • An array generally refers to ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of mRNA molecules or cDNA molecules and that are positioned on a support material in a spatially separated organization.
  • Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
  • Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
  • Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample. A variety of different array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art.
  • Useful substrates for arrays include nylon, glass and silicon Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non- covalent, and the like.
  • the labeling and screening methods of the present invention and the arrays are not limited in its utility with respect to any parameter except that the probes detect expression levels; consequently, methods and compositions may be used with a variety of different types of genes.
  • the arrays can be high density arrays, such that they contain 100 or more different probes. It is contemplated that they may contain 1000,
  • the probes can be directed to targets in one or more different organisms.
  • the oligonucleotide probes range from 5 to 50, 5 to 45, 10 to 40, or 15 to 40 nucleotides in length in some embodiments. In certain embodiments, the oligonucleotide probes are 20 to 25 nucleotides in length.
  • each different probe sequence in the array are generally known.
  • the large number of different probes can occupy a relatively small area providing a high density array having a probe density of generally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes per cm 2 .
  • the surface area of the array can be about or less than about 1, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm 2 .
  • a person of ordinary skill in the art could readily analyze data generated using an array.
  • the present invention concerns determining the expression level of the protein SPOP to diagnose renal cell carcinoma.
  • the invention provides for an inhibitor of SPOP, wherein the inhibitor may be a protein.
  • a "protein,” “proteinaceous molecule,” “proteinaceous composition,” “proteinaceous compound,” “proteinaceous chain” or “proteinaceous material” generally refers, but is not limited to, a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. All the "proteinaceous” terms described above may be used interchangeably herein.
  • the proteinaceous composition may comprise at least one antibody, for example, a SPOP.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single chain Fv
  • scFv single chain Fv
  • the techniques for preparing and using various antibody-based constructs and fragments are well known in the art.
  • Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Harlow et al, 1988; incorporated herein by reference). a. Immunodetection Methods
  • the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and/or otherwise detecting biological components such as antigenic regions on polypeptides and peptides.
  • the immunodetection methods of the present invention can be used to identify antigenic regions of a peptide, polypeptide, or protein that has therapeutic implications, particularly in reducing the immunogenicity or antigenicity of the peptide, polypeptide, or protein in a target subject.
  • Immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot, though several others are well known to those of ordinary skill.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoradiometric assay fluoroimmunoassay
  • fluoroimmunoassay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • Western blot though several others are well known to those of ordinary skill.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle et al, 1999; Gulbis et al, 1993; De Jager et al, 1993; and Nakamura et al. , 1987, each incorporated here
  • the immunobinding methods include obtaining a sample suspected of containing a protein, polypeptide and/or peptide, and contacting the sample with a first antibody, monoclonal or polyclonal, in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • these methods include methods for purifying a protein, polypeptide and/or peptide from organelle, cell, tissue or organism's samples.
  • the antibody removes the antigenic protein, polypeptide and/or peptide component from a sample.
  • the antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the protein, polypeptide and/or peptide antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody to be eluted.
  • the immunobinding methods also include methods for detecting and quantifying the amount of an antigen component in a sample and the detection and quantification of any immune complexes formed during the binding process.
  • detecting and quantifying the amount of an antigen component in a sample and the detection and quantification of any immune complexes formed during the binding process.
  • one would obtain a sample suspected of containing an antigen or antigenic domain and contact the sample with an antibody against the antigen or antigenic domain, and then detect and quantify the amount of immune complexes formed under the specific conditions.
  • the biological sample analyzed may be any sample that is suspected of containing an antigen or antigenic domain, such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, an organelle, separated and/or purified forms of any of the above antigen-containing compositions, or even any biological fluid that comes into contact with the cell or tissue, including blood and/or serum.
  • an antigen or antigenic domain such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, an organelle, separated and/or purified forms of any of the above antigen-containing compositions, or even any biological fluid that comes into contact with the cell or tissue, including blood and/or serum.
  • the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any antigens present.
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specif ⁇ cally bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed.
  • This system may provide for signal amplification if this is desired.
  • One method of immunodetection designed by Charles Cantor uses two different antibodies.
  • a first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin.
  • the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex.
  • the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
  • the amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
  • This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • a conjugate can be produced which is macroscopically visible.
  • Another known method of immunodetection takes advantage of the immuno-
  • PCR Polymerase Chain Reaction
  • the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. b. ELISAs
  • immunoassays in their most simple and/or direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and/or western blotting, dot blotting, FACS analyses, and/or the like may also be used.
  • antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the antigen, such as a clinical sample, is added to the wells. After binding and/or washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA.” Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the antigen are immobilized onto the well surface and/or then contacted with antibodies. After binding and/or washing to remove non-specifically bound immune complexes, the bound anti-antibodies are detected. Where the initial antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the antigens are immobilized involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against an antigen are added to the wells, allowed to bind, and/or detected by means of their label.
  • the amount of an antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against the antigen during incubation with coated wells.
  • the presence of an antigen in the sample acts to reduce the amount of antibody against the antigen available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against an antigen in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
  • ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non- specifically bound species, and detecting the bound immune complexes. These are described below.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • suitable conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • An example of a washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
  • the second or third antibody will have an associated label to allow detection.
  • This may be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease glucose oxidase, alkaline phosphatase or hydrogen peroxidase- conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PB S -containing solution such as PBS-Tween).
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS), or H 2 O 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. c. Immunohistochemistry
  • the antibodies of the present invention may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • immunohistochemistry may be utilized to characterize SPOP or to evaluate the amount SPOP in a cell.
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al, 1990; Allred et al,
  • Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. It takes its name from the roots “immuno,” in reference to antibodies used in the procedure, and "histo,” meaning tissue. Immunohistochemical staining is widely used in the diagnosis and treatment of cancer. Specific molecular markers are characteristic of particular cancer types, such as SPOP for RCC.
  • an antibody-antigen interaction can be accomplished in a number of ways.
  • an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction.
  • the antibody can also be tagged to a fluorophore, such as FITC, rhodamine, Texas Red, Alexa Fluor, or DyLight Fluor.
  • FITC fluorophore
  • rhodamine such as Texas Red, Alexa Fluor
  • DyLight Fluor DyLight Fluor
  • frozen-sections may be prepared by rehydrating 50 mg of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70 0 C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.
  • the direct method is a one-step staining method, and involves a labeled antibody (e.g. FITC conjugated antiserum) reacting directly with the antigen in tissue sections.
  • FITC conjugated antiserum a labeled antibody
  • the indirect method involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) which reacts with the primary antibody.
  • first layer which reacts with tissue antigen
  • second layer which reacts with the primary antibody.
  • the secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised.
  • This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody.
  • the second layer antibody can be labeled with a fluorescent dye or an enzyme.
  • a biotinylated secondary antibody is coupled with streptavidin-horseradish peroxidase. This is reacted with 3,3'-Diaminobenzidine (DAB) to produce a brown staining wherever primary and secondary antibodies are attached in a process known as DAB staining.
  • DAB staining 3,3'-Diaminobenzidine
  • the reaction can be enhanced using nickel, producing a deep purple/gray staining.
  • the indirect method aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be generated.
  • a labeled secondary antibody raised against rabbit IgG which can be purchased "off the shelf," is useful with any primary antibody raised in rabbit.
  • the direct method it would be necessary to make custom labeled antibodies against every antigen of interest.
  • Antibodies Another embodiment of the present invention are antibodies, in some cases, a
  • SPOP antibody It is understood that antibodies can be used for inhibiting or modulating SPOP. It is also understood that this antibody is useful for screening samples from human patients for the purpose of detecting SPOP present in the samples. The antibody also may be useful in the screening of expressed DNA segments or peptides and proteins for the discovery of related antigenic sequences. In addition, the antibody may be useful in passive immunotherapy for cancer. All such uses of the said antibody and any antigens or epitopic sequences so discovered fall within the scope of the present invention.
  • the present invention involves antibodies.
  • a monoclonal, single chain, or humanized antibody may function as a modulator of SPOP.
  • Other aspects of the invention involve administering antibodies as a form of treatment or as a diagnostic to identify or quantify a particular polypeptide, such as SPOP.
  • antibodies in addition to antibodies generated against full length proteins, antibodies also may be generated in response to smaller constructs comprising epitopic core regions, including wild-type and mutant epitopes.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE.
  • IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • Monoclonal antibodies are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred.
  • the invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single chain Fv
  • scFv single chain Fv
  • a polyclonal antibody may be prepared by immunizing an animal with an immunogenic polypeptide composition in accordance with the present invention and collecting antisera from that immunized animal.
  • serum is collected from persons who may have been exposed to a particular antigen. Exposure to a particular antigen may occur within a work environment, such that those persons have been occupationally exposed to a particular antigen and have developed polyclonal antibodies to a peptide, polypeptide, or protein.
  • polyclonal serum from occupationally exposed persons is used to identify antigenic regions in the gelonin toxin through the use of immunodetection methods.
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat.
  • a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
  • albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • Suitable molecule adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
  • Adjuvants that may be used include IL-I, IL-2, IL-4, IL-7, IL- 12, ⁇ -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.
  • MHC antigens may even be used.
  • Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund'
  • BRM biologic response modifiers
  • CCM Cimetidine
  • CYP Cyclophosphamide
  • cytokines such as ⁇ -interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.
  • a second, booster injection also may be given.
  • the process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate monoclonal antibodies.
  • Monoclonal antibodies may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified polypeptide, peptide or domain, be it a wild-type or mutant composition.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Monoclonal antibodies may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.
  • a molecular cloning approach may be used to generate monoclonal antibodies.
  • combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells.
  • the advantages of this approach over conventional hybridoma techniques are that approximately 10 4 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.
  • Humanized monoclonal antibodies are antibodies of animal origin that have been modified using genetic engineering techniques to replace constant region and/or variable region framework sequences with human sequences, while retaining the original antigen specificity. Such antibodies are commonly derived from rodent antibodies with specificity against human antigens. Such antibodies are generally useful for in vivo therapeutic applications. This strategy reduces the host response to the foreign antibody and allows selection of the human effector functions.
  • Humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
  • the techniques for producing humanized immunoglobulins are well known to those of skill in the art.
  • US Patent 5,693,762 discloses methods for producing, and compositions of, humanized immunoglobulins having one or more complementarity determining regions (CDR' s). When combined into an intact antibody, the humanized immunoglobulins are substantially non-immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin to the antigen, such as a protein or other compound containing an epitope.
  • Protein array technology is discussed in detail in Pandey and Mann (2000) and MacBeath and Schreiber (2000), each of which is herein specifically incorporated by reference.
  • arrays typcially contain thousands of different proteins or antibodies spotted onto glass slides or immobilized in tiny wells, allow one to examine the biochemical activities and binding profiles of a large number of proteins at once.
  • a labeled protein is incubated with each of the target proteins immobilized on the slide, and then one determines which of the many proteins the labeled molecule binds.
  • such technology can be used to quantitate a number of proteins in a sample, such as SPOP.
  • the basic construction of protein chips has some similarities to DNA chips, such as the use of a glass or plastic surface dotted with an array of molecules. These molecules can be DNA or antibodies that are designed to capture proteins. Defined quantities of proteins are immobilized on each spot, while retaining some activity of the protein. With fluorescent markers or other methods of detection revealing the spots that have captured these proteins, protein microarrays are being used as powerful tools in high-throughput proteomics and drug discovery.
  • the earliest and best-known protein chip is the ProteinChip by Ciphergen Biosystems Inc. (Fremont, Calif.).
  • the ProteinChip is based on the surface-enhanced laser desorption and ionization (SELDI) process.
  • Known proteins are analyzed using functional assays that are on the chip.
  • chip surfaces can contain enzymes, receptor proteins, or antibodies that enable researchers to conduct protein- protein interaction studies, ligand binding studies, or immunoassays.
  • the ProteinChip system detects proteins ranging from small peptides of less than 1000 Da up to proteins of 300 kDa and calculates the mass based on time-of- flight (TOF).
  • TOF time-of- flight
  • the ProteinChip biomarker system is the first protein biochip-based system that enables biomarker pattern recognition analysis to be done. This system allows researchers to address important clinical questions by investigating the proteome from a range of crude clinical samples (i.e., laser capture microdissected cells, biopsies, tissue, urine, and serum). The system also utilizes biomarker pattern software that automates pattern recognition-based statistical analysis methods to correlate protein expression patterns from clinical samples with disease phenotypes. 3. Samples
  • the present invention provides a method of diagnosis or treatment of renal cell carcinoma on at least one sample obtained from an individual.
  • the individual may be any mammal, but is preferably a human.
  • the individual may be any individual, an individual predisposed of a disease or an individual suffering from a disease, wherein the disease is renal cell carcinoma.
  • a sample as defined herein refers to a sample obtained from an organism or form components (e.g., cells) of an organism.
  • the sample may be of any biological tissue or fluid.
  • the sample may be a "clinical sample” which is a sample derived from a patient.
  • Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
  • Biopsies are small pieces of tissue and may be fresh, frozen or fixed, such as formalin-fixed and paraffin embedded (FFPE). Samples may be removed surgically, by extraction i.e. by hypodermic or other types of needles, by microdissection or laser capture. Samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • FFPE formalin-fixed and paraffin embedded
  • the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode all or part of a wild- type, polymorphic, or mutant SPOP polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to a native polypeptide.
  • the term "recombinant" may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is the replicated product of such a molecule.
  • the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to the polypeptide.
  • nucleic acid segments used in the present invention may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid constructs of the present invention may encode full-length polypeptide from any source or encode a truncated version of the polypeptide, for example a truncated SPOP polypeptide, such that the transcript of the coding region represents the truncated version. The truncated transcript may then be translated into a truncated protein.
  • a nucleic acid sequence may encode a full-length polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post- translational modification, or for therapeutic benefits such as targetting or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.
  • one or more nucleic acid constructs may be prepared that include a contiguous stretch of nucleotides identical to or complementary to the a particular gene, such as the human SPOP gene.
  • a nucleic acid construct may be at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 30,000, 50,000, 100,000, 250,000, 500,000, 750,000, to at least 1,000,000 nucleotides in length, as well as constructs of greater size, up to and including chromosomal sizes (including all intermediate lengths and intermediate ranges), given the advent of nucleic acids constructs such as a yeast artificial chromosome are known to those of ordinary skill in the art. It will be readily understood that "intermediate lengths
  • DNA segments used in the present invention encompass biologically functional equivalent modified polypeptides and peptides, for example, a modified gelonin toxin.
  • biologically functional equivalent modified polypeptides and peptides for example, a modified gelonin toxin.
  • Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged.
  • Changes designed by human may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein, to reduce toxicity effects of the protein in vivo to a subject given the protein, or to increase the efficacy of any treatment involving the protein.
  • this invention is not limited to the particular nucleic acid and amino acid sequences of the polynucleotide encoding SPOP.
  • Recombinant vectors and isolated DNA segments may therefore variously include the SPOP-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include SPOP-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
  • DNA segments of the present invention encompass biologically functional equivalent SPOP proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein.
  • fusion proteins and peptides e.g., where the SPOP- or SPOP modulator-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
  • DNA segments encoding relatively small peptides such as, for example, peptides of from about 15 to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length; and also larger polypeptides up to and including proteins corresponding to the full-length published sequences for SPOP.
  • Native and modified polypeptides may be encoded by a nucleic acid molecule comprised in a vector.
  • vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • a vector may encode non-modified polypeptide sequences such as a tag or targetting molecule.
  • Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al, 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
  • GST glutathione S-transferase
  • a targetting molecule is one that directs the modified polypeptide to a particular organ, tissue, cell, or other location in a subject's body.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. 1. Promoters and Enhancers
  • a “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned,”
  • operatively linked means that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
  • a promoter may or may not be used in conjunction with an "enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • a recombinant or heterologous promoter refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference.
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
  • regions include the human LIMK2 gene (Nomoto et al 1999), the somatostatin receptor 2 gene (Kraus et al, 1998), murine epididymal retinoic acid-binding gene (Lareyre et al, 1999), human CD4 (Zhao-Emonet et al, 1998), mouse alpha2 (XI) collagen (Tsumaki, et al, 1998), DlA dopamine receptor gene (Lee, et al, 1997), insulin-like growth factor II (Wu et al, 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al, 1996), and the SM22 ⁇ promoter.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5'- methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picornavirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • each open reading frame is accessible to ribosomes for efficient translation.
  • Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent 5,925,565 and 5,935,819, herein incorporated by reference).
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art.
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
  • RNA molecules will undergo RNA splicing to remove introns from the primary transcripts.
  • Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al., 1997, incorporated herein by reference.) 5. Termination Signals
  • the vectors or constructs of the present invention will generally comprise at least one termination signal.
  • a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
  • the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site.
  • RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
  • terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.
  • the terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.
  • Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
  • polyadenylation signal In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed.
  • Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport. 7. Origins of Replication
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously replicating sequence
  • cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • host cell refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
  • a host cell can, and has been, used as a recipient for vectors.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a modified protein-encoding sequence, is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Host cells may be derived from prokaryotes or eukaryotes, including yeast cells, insect cells, and mammalian cells, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences.
  • Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).
  • ATCC American Type Culture Collection
  • An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
  • a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
  • Bacterial cells used as host cells for vector replication and/or expression include DH5 ⁇ , JMl 09, and KC8, as well as a number of commercially available bacterial hosts such as SURE ® Competent Cells and SOLOPACKTM Gold Cells (STRATAGENE ® , La Jolla, CA).
  • bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
  • Appropriate yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.
  • eukaryotic host cells for replication and/or expression of a vector examples include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patent No. 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC ® 2.0 from INVITROGEN ® and BACPACKTM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH ® .
  • STRATAGENE ® 's COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone- inducible receptor, or its pET Expression System, an E. coli expression system.
  • INVITROGEN ® which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
  • INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
  • a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
  • the expression vector comprises a virus or engineered vector derived from a viral genome.
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kb of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells; they can also be used as vectors.
  • Other viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno- associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
  • nucleic acid delivery to effect expression of compositions of the present invention are believed to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art.
  • a nucleic acid e.g., DNA, including viral and nonviral vectors
  • Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S.
  • Patent 5,789,215 incorporated herein by reference
  • electroporation U.S. Patent No. 5,384,253, incorporated herein by reference
  • calcium phosphate precipitation Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe e? al, 1990
  • DEAE-dextran followed by polyethylene glycol
  • direct sonic loading Fechheimer et al, 1987
  • liposome mediated transfection Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau e/ al, 1987; Wong et al, 1980; Kaneda et al, 1989; Kato et al, 1991
  • microprojectile bombardment PCT Application Nos.
  • an "amino molecule” refers to any amino acid, amino acid derivative or amino acid mimic as would be known to one of ordinary skill in the art.
  • the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues.
  • the sequence may comprise one or more non-amino molecule moieties.
  • the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
  • proteinaceous composition encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid.
  • the proteinaceous composition comprises at least one protein, polypeptide or peptide.
  • the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide.
  • biocompatible refers to a substance which produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions.
  • biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or peptides or synthetic proteins or peptides each essentially free from toxins, pathogens and harmful immunogens.
  • Proteinaceous compositions may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials.
  • the nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.
  • One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (www.ncbi.nlm.nih.gov/).
  • Genbank and GenPept databases www.ncbi.nlm.nih.gov/
  • the coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art.
  • various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
  • a proteinaceous compound may be purified.
  • purified will refer to a specific or protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as would be known to one of ordinary skill in the art for the specific or desired protein, polypeptide or peptide.
  • SPOP When the present application refers to the function or activity of SPOP, it is meant that the molecule in question has the ability to bind Cullin 3. Determination of which molecules possess this activity may be achieved using assays familiar to those of skill in the art. For example, transfer of genes encoding products that inhibit SPOP, or variants thereof, into cells that have a functional SPOP product will identify, by virtue of an increased level of apoptosis, those molecules having a SPOP inhibitor function.
  • An endogenous SPOP polypeptide refers to the polypeptide encoded by the cell's genomic DNA.
  • a SPOP modulator may be a molecule that affects SPOP expression, such as by binding a SPOP-encoding transcript. Determination of which molecules are suitable modulators of SPOP may be achieved using assays familiar to those of skill in the art and may include, for example, the use of native and/or recombinant SPOP.
  • Amino acid sequence variants of the polypeptides of the present invention can be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the native protein that are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above.
  • Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
  • Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of a SPOP polypeptide or a modulator of a SPOP provided the biological activity of the protein is maintained.
  • functionally equivalent codon is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3 1 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • a specialized kind of insertional variant is the fusion protein.
  • This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C- terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
  • Other useful fusions include linking of functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals or transmembrane regions.
  • SPOP SPOP modulator
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacryl amide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • purified protein or peptide as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • molecules are eluted from the column in decreasing size, so long as the shape is relatively constant.
  • Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g., alter pH, ionic strength, and temperature).
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand also should provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • the present invention provides a method of screening for a SPOP modulator.
  • SPOP is adaptor for E3 ligase Cullin3. It can help deliver substrate, such as puc (MKP), to Cullin3 for ubiquitination and degradation.
  • substrate such as puc (MKP)
  • MKP puc
  • Compounds may be screened to find modulators that could inhibit SPOP binding with Cullin 3.
  • binding affinity assays and E3 liagase enzyme acitivity assays may be used for determining inhibitor effeciency.
  • the present invention further comprises methods for identifying modulators of
  • SPOP activity may comprise random screening of large libraries of candidate substances; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to modulate the function of SPOP.
  • a method generally comprises:
  • step (c) measuring one or more characteristics of the compound or cell in step (b); and (d) comparing the characteristic measured in step (c) with the characteristic of the compound or cell in the absence of said candidate modulator, wherein a difference between the measured characteristics indicates that said candidate modulator is, indeed, a modulator of the compound or cell.
  • Assays may be conducted in cell free systems, in isolated cells, or in organisms including transgenic animals.
  • the term “candidate substance” refers to any molecule that may be a "modulator” of SPOP, i.e., potentially affect SPOP activity, directly or indirectly.
  • a modulator may be a "SPOP inhibitor,” which is a compound that overall effects an inhibition of SPOP activity, which may be accomplished by inhibiting SPOP expression, translocation or transport, function, expression, post-translational modification, location, half-life, or more directly by preventing its activity, such as by binding SPOP.
  • the candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule.
  • An example of pharmacological compounds will be compounds that are structurally related to SPOP, or a molecule that binds SPOP such as Cullin 3.
  • Using lead compounds to help develop improved compounds is know as "rational drug design" and includes not only comparisons with know inhibitors and activators, but predictions relating to the structure of target molecules.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs, which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a target molecule, or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches. It also is possible to use antibodies to ascertain the structure of a target compound activator or inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based.
  • anti-idiotypic antibodies it is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody.
  • the binding site of anti-idiotype would be expected to be an analog of the original antigen.
  • the anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore.
  • Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen. On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds.
  • combinatorially generated libraries e.g., peptide libraries
  • Screening of such libraries is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity.
  • Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds.
  • Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man- made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators.
  • modulators include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule.
  • antisense molecules include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule.
  • Such compounds are well known to those of skill in the art.
  • an antisense molecule that bound to a translational or transcriptional start site, or splice junctions would be ideal candidate inhibitors.
  • the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the structure of the modulators.
  • Such compounds which may include peptidomimetics of peptide modulators, may be used in the same manner as the initial modulators.
  • An inhibitor according to the present invention may be one which exerts its inhibitory or activating effect upstream, downstream or directly on SPOP. Regardless of the type of inhibitor or activator identified by the present screening methods, the effect of the inhibition or activator by such a compound results in alteration in SPOP activity as compared to that observed in the absence of the added candidate substance.
  • a quick, inexpensive and easy assay to run is an in vitro assay.
  • Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time.
  • a variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.
  • a cell free assay is a binding assay. While not directly addressing function, the ability of a modulator to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions.
  • the target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determining of binding. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enhance binding.
  • methods of assaying whether the candidate inhibits SPOP activity may involve screening for the activity of SPOP.
  • SPOP activity may be evaluated using any of the methods and compositions disclosed herein, including assays involving evaluating SPOP 's binding activity or ability to inhibit apoptosis.
  • Other indications of SPOP inhibition can include the activation of JNK or a modulated level of c-Jun phosphorylation. Any other the compounds or methods described herein may be employed to implement these methods.
  • Assays to evaluate the level of expression of a polypeptide are well known to those of skill in the art. This can be accomplished also by assaying SPOP mRNA levels, mRNA stability or turnover, as well as protein expression levels. It is further contemplated that any post-translational processing of SPOP may also be evaluated, as well as whether it is being localized or regulated properly. In some cases an antibody that specifically binds SPOP may be used. Furthermore, it is contemplated that the status of the gene may be evaluated directly or indirectly, by evaluating genomic DNA sequence comprising the SPOP coding regions and noncoding regions (introns, and upstream and downstream sequences) or mRNA sequence. The invention also includes determining whether any polymorphisms exist in SPOP genomic sequences (coding and noncoding). Such assays may involve polynucleotide regions that are identical or complementary to SPOP genomic sequences, such as primers and probes described herein.
  • the invention provides compositions and methods for the diagnosis and treatment of RCC.
  • the invention provides a method of treating RCC comprising administering to a patient an effective amount of an inhibitor of SPOP. This treatment may be further combined with additional cancer treatments.
  • One of skill in the art will be aware of many treatments that may be combined with the methods of the present invention, some but not all of which are described below.
  • the invention provides a method of treating renal cell carcinoma comprising administering to a patient an effective amount of an inhibitor of SPOP.
  • an inhibitor of SPOP an inhibitor of SPOP.
  • it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient.
  • Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • compositions also are referred to as inocula.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions. Of particular interest is direct intratumoral administration, perfusion of a tumor, or administration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed (e.g., post-operative catheter). For practically any tumor, systemic delivery also is contemplated. This will prove especially important for attacking microscopic or metastatic cancer.
  • the active compounds may also be administered as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the actual dosage amount of a composition of the present invention administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • Treatment and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • therapeutic benefit refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • a “disease” can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, and/or environmental stress.
  • Prevention and “preventing” are used according to their ordinary and plain meaning to mean “acting before” or such an act.
  • those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition.
  • the subject can be a subject who is known or suspected of being free of a particular disease or health-related condition at the time the relevant preventive agent is administered.
  • the subject for example, can be a subject with no known disease or health-related condition ⁇ i.e., a healthy subject).
  • methods include identifying a patient in need of treatment.
  • a patient may be identified, for example, based on taking a patient history or based on findings on clinical examination.
  • the method further comprises treating a patient with renal cell carcinoma with a conventional cancer treatment.
  • a conventional cancer treatment One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy, such as by combining traditional therapies with other anti-cancer treatments.
  • this treatment could be, but is not limited to, chemotherapeutic, radiation, a polypeptide inducer of apoptosis or other therapeutic intervention. It also is conceivable that more than one administration of the treatment will be desired.
  • chemotherapeutic agents may be used in accordance with the present invention.
  • the term “chemotherapy” refers to the use of drugs to treat cancer.
  • a “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
  • alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, car
  • Radiotherapy also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.
  • Radiation therapy used according to the present invention may include, but is not limited to, the use of ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmuno therapy).
  • Antibodies are highly specific proteins that are made by the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large quantities of these antibodies can be made in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.
  • Conformal radiotherapy uses the same radiotherapy machine, a linear accelerator, as the normal radiotherapy treatment but metal blocks are placed in the path of the x-ray beam to alter its shape to match that of the cancer. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive a lower dose of radiation, so the possibility of side effects is reduced.
  • a device called a multi-leaf collimator has been developed and can be used as an alternative to the metal blocks.
  • the multi-leaf collimator consists of a number of metal sheets which are fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beams can be shaped to the treatment area without the need for metal blocks. Precise positioning of the radiotherapy machine is very important for conformal radiotherapy treatment and a special scanning machine may be used to check the position of your internal organs at the beginning of each treatment.
  • High-resolution intensity modulated radiotherapy also uses a multi-leaf collimator. During this treatment the layers of the multi-leaf collimator are moved while the treatment is being given. This method is likely to achieve even more precise shaping of the treatment beams and allows the dose of radiotherapy to be constant over the whole treatment area.
  • Radiosensitizers make the tumor cells more likely to be damaged, and radioprotectors protect normal tissues from the effects of radiation.
  • Hyperthermia the use of heat, is also being studied for its effectiveness in sensitizing tissue to radiation. 3. Immunotherapy
  • immunotherapeutics In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Trastuzumab (HerceptinTM) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • toxin chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • the combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.
  • Another immunotherapy could also be used as part of a combined therapy with gen silencing therapy discussed above.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL- 12, GM-CSF, gamma-IFN, chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand.
  • immune stimulating molecules either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al, 2000).
  • antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.
  • immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998), cytokine therapy, e.g., interferons ⁇ , ⁇ , and ⁇ ; IL-I, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998) gene therapy, e.g., TNF, IL-I, IL-2, p53 (Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S.
  • immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds
  • cytokine therapy e
  • Patents 5,830,880 and 5,846,945) and monoclonal antibodies e.g., anti-ganglioside GM2, anti-HER-2, anti- pl85 (Pietras et al, 1998; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the gene silencing therapies described herein.
  • an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine” is administered, generally with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al, 1992; Mitchell et al, 1990; Mitchell et al, 1993).
  • the patient's circulating lymphocytes, or tumor infiltrated lymphocytes are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al., 1988; 1989).
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as a SPOP targeting agent is administered. Delivery of a SPOP targeting agent in conjunction with a vector encoding one of the following gene products may have a combined anti-hyperproliferative effect on target tissues.
  • a variety of proteins are encompassed within the invention, some of which are described below. 1. Inducers of Cellular Proliferation
  • the proteins that induce cellular proliferation further fall into various categories dependent on function.
  • the commonality of all of these proteins is their ability to regulate cellular proliferation.
  • a form of PDGF the sis oncogene
  • Oncogenes rarely arise from genes encoding growth factors, and at the present, sis is the only known naturally-occurring oncogenic growth factor.
  • anti-sense mRNA or siRNA directed to a particular inducer of cellular proliferation is used to prevent expression of the inducer of cellular proliferation.
  • the proteins FMS and ErbA are growth factor receptors. Mutations to these receptors result in loss of regulatable function. For example, a point mutation affecting the transmembrane domain of the Neu receptor protein results in the neu oncogene.
  • the erbA oncogene is derived from the intracellular receptor for thyroid hormone.
  • the modified oncogenic ErbA receptor is believed to compete with the endogenous thyroid hormone receptor, causing uncontrolled growth.
  • the largest class of oncogenes includes the signal transducing proteins (e.g., Src, AbI and Ras).
  • Src is a cytoplasmic protein- tyrosine kinase, and its transformation from proto-oncogene to oncogene in some cases, results via mutations at tyrosine residue 527.
  • transformation of GTPase protein ras from proto- oncogene to oncogene results from a valine to glycine mutation at amino acid 12 in the sequence, reducing ras GTPase activity.
  • the proteins Jun, Fos and Myc are proteins that directly exert their effects on nuclear functions as transcription factors. 2. Inhibitors of Cellular Proliferation
  • the tumor suppressor oncogenes function to inhibit excessive cellular proliferation.
  • the inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation.
  • the tumor suppressors p53, mda-7, FHIT, pl6 and C-CAM can be employed.
  • another inhibitor of cellular proliferation is pi 6.
  • the major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK' s.
  • CDK cyclin-dependent kinase 4
  • CDK4 cyclin-dependent kinase 4
  • the activity of this enzyme may be to phosphorylate Rb at late G].
  • the activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pl6 INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al, 1995). Since the pl6 1NK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6.
  • pl6 INIC4 maps to 9 ⁇ 21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the p jg iNK4 g ene are fj. e q Uent m human tumor cell lines. This evidence suggests that the pl6 INK4 gene is a tumor suppressor gene.
  • Apoptosis or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al, 1972).
  • the Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems.
  • the Bcl-2 protein discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al, 1985; Cleary and Sklar, 1985; Cleary et al, 1986; Tsujimoto et al, 1985; Tsujimoto and Croce, 1986).
  • Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies.
  • Bcl-2 e.g., BCIX L , BcIw, BcIs, McI-I, Al, BfI-I
  • Bcl-2 counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
  • RNA Interference RNA interference
  • the SPOP inhibitor is a double-stranded RNA (dsRNA) directed to an mRNA for SPOP.
  • RNA interference also referred to as "RNA-mediated interference” or RNAi
  • dsRNA Double- stranded RNA
  • dsRNA has been observed to mediate the reduction, which is a multi- step process.
  • dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin and Avery et al, 1999; Montgomery et al, 1998; Sharp and Zamore, 2000; Tabara et al, 1999). Activation of these mechanisms targets mature, dsRNA-complementary mRNA for destruction. RNAi offers major experimental advantages for study of gene function.
  • RNAi acts post-transcriptionally, targeting RNA transcripts for degradation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher and Labouesse, 2000). 1. siRNA siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest.
  • siRNA target sequences i.e., those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes.
  • siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above (Montgomery et ah, 1998).
  • siRNA are well known in the art. For example, siRNA and double- stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Applications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.
  • RNA sequences having di-nucleotide overhangs may provide the greatest level of suppression.
  • These protocols primarily use a sequence of two (2'-deoxy) thymidine nucleotides as the di-nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA.
  • the literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight ( ⁇ 20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.
  • sense and antisense RNA are synthesized from DNA templates using T7 polymerase (MEGAscript, Ambion). After the synthesis is complete, the DNA template is digested with DNaseI and RNA purified by phenol/chloroform extraction and isopropanol precipitation. RNA size, purity and integrity are assayed on denaturing agarose gels. Sense and antisense RNA are diluted in potassium citrate buffer and annealed at 80°C for 3 min to form dsRNA. As with the construction of DNA template libraries, a procedures may be used to aid this time intensive procedure.
  • dsRNA library The sum of the individual dsRNA species is designated as a "dsRNA library.”
  • the making of siRNAs has been mainly through direct chemical synthesis; through processing of longer, double-stranded RNAs through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive.
  • Chemical synthesis proceeds by making two single- stranded RNA-oligomers followed by the annealing of the two single-stranded oligomers into a double-stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Patents 5,889,136, 4,415,723, and 4,458,066, expressly incorporated herein by reference, and in Wincott et al. (1995).
  • RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference.
  • the enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase ⁇ e.g. , T3, T7, SP6) via the use and production of an expression construct as is known in the art. For example, see U.S. Patent 5,795,715.
  • the contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene.
  • the length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
  • An important aspect of this reference is that the authors contemplate digesting longer dsRNAs to 21-25mer lengths with the endogenous nuclease complex that converts long dsRNAs to siRNAs in vivo. They do not describe or present data for synthesizing and using in vitro transcribed 21-25mer dsRNAs. No distinction is made between the expected properties of chemical or enzymatically synthesized dsRNA in its use in RNA interference.
  • RNA single-stranded RNA is enzymatically synthesized from the PCR products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides.
  • WO 01/36646 incorporated herein by reference, places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures.
  • RNA polymerase e.g., T3, T7, SP6
  • RNA polymerase e.g., T3, T7, SP6
  • RNA polymerase e.g., T3, T7, SP6
  • RNA polymerase e.g., T3, T7, SP6
  • RNA interference no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.
  • U.S. Patent 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized.
  • the templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence.
  • the templates are preferably attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences.
  • shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs. Subsequently, the ends of these shRNAs are processed, converting the shRNAs into —21 nt siRNA-like molecules (Brummelkamp et al, 2002). The siRNA-like molecules can, in turn, bring about gene-specific silencing in the transfected mammalian cells.
  • immunomodulatory agents agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents.
  • Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-I, MIP- lbeta, MCP-I, RANTES, and other chemokines.
  • cytostatic or differentiation agents can be used in combination with the present invention to improve the anti- hyerproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
  • hyperthermia is a procedure in which a patient's tissue is exposed to high temperatures (up to 106 0 F).
  • External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia.
  • Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.
  • a patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets.
  • some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated.
  • Whole-body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.
  • the amount of therapeutic agent to be included in the compositions or applied in the methods set forth herein will be whatever amount is pharmaceutically effective and will depend upon a number of factors, including the identity and potency of the chosen therapeutic agent.
  • concentration of the therapeutic agent in the compositions set forth herein can be any concentration.
  • the total concentration of the drug is less than 10%.
  • concentration of the drug is less than 5%.
  • the therapeutic agent may be applied once or more than once.
  • the therapeutic agent is applied once a day, twice a day, three times a day, four times a day, six times a day, every two hours when awake, every four hours, every other day, once a week, and so forth. Treatment may be continued for any duration of time as determined by those of ordinary skill in the art.
  • FTZ and EVE are homeobox proteins that regulate the segmentation process during Drosophila embryonic development. FTZ is required for specification of even numbered parasegments while EVE is responsible for odd numbered parasegments.
  • FTZ and EVE combined mutant animal expression profiling and transcription factor DNA binding site mapping analysis. Using D. melanogaster whole-genome microarrays, the inventors profiled global gene expression for wild type, ftz and eve null mutant embryos. The inventors analyzed genes expression changes between wild type and mutant embryos during a developmental time course with time points at 2, 3, 6, 7, 8, 9, and 10 hours after egg lying (FIG.1).
  • Table 1 1310 target genes for FTZ expression profile during developmental stages
  • FBgn0040010 FBgn0029661, FBgn0001224, FBgn0004516, HSP23, FBgn0031446, FBgn0032629, FBgn0031632, FBgn0028877, FBgn0024150, FBgn0031431,
  • FBgn0031452 FBgn0026324, FBgnOO31O65, FBgn0027779, FBgn0011230,
  • FBgnOO35781 FBgnOO36735, FBgn0032456, FBgn0039895, L71-1, FBgn0035174,
  • FBgn0038701 FBgnOO31368, FBgn0031427, FBgn0036408, FBgn0034432, FBgnOO33613, FBgn0015946, FBgn0032881, FBgn0033089, FBgn0017482,
  • FBgn0033965 FBgn0031456, FBgn0031281, FBgn0032491, FBgn0031621,
  • FBgn0040077 FBgnOO37153, FBgn0032348, FBgn0014848, FBgn0037825, FBgn0023184, FBgn0024732, FBgn0000606, FBgn0031004, FBgn0028648,
  • FBgn0040754 FBgn0031476, FBgn0032590, FBgn0033588, FBgn0033974,
  • FBgn0004242 FBgn0038070, FBgn0020368, FBgn0000299, FBgn0037419, FBgn0038803, FBgn0035548, FBgnOO385O9, FBgn0034075, FBgn0026562,
  • FBgn0039445 FBgn0027580, FBgn0039244, FBgn0002638, FBgn0035798,
  • FBgnOO3556O FBgn0021872, FBgn0010470, FBgn0030547, FBgn0028704, FBgn0032622, FBgn0019662, FBgn0037977, FBgn0034259, FBgn0034689,
  • FBgn0034421 FBgnOOlOOH, FBgn0036567, FBgn0030052, FBgn0034765,
  • FBgn0036920 FBgnOOO4363, FBgn0039142, SGS-7, FBgn0035209, FBgn0028487,
  • FBgn0030745 FBgn0031590, FBgnOO33239, FBgn0031323, FBgn0026398,
  • FBgn0028412 FBgn0034396, FBgn0037048, FBgn0028485, FBgn0037295,
  • FBgn0038922 FBgn0032153, FBgn0036517, FBgn0029889, FBgn0040541,
  • FBgn0036804 FBgn0037397, FBgn0033769, FBgn0035435, FBgn0020764, FBgn0004108, FBgnO03O083, FBgn0033663, FBgnOOO3715, FBgn0033948,
  • FBgn0035432 FBgnOO38275, FBgn0033610, FBgn0031364, FBgn0031343,
  • FBgn0035948 FBgn0026012, FBgn0015831, FBgn0031931, FBgn0035713, FBgn0034970, FBgn0035758, Br-CZ2, FBgn0039140, FBgnOO36861, FBgn0037472,
  • FBgnOO32393 FBgn0029172, FBgn0010105, FBgn0026176, FBgn0035622, FBgn0039726, FBgn0031942, FBgn0003525, FBgnOO31365, FBgn0002989,
  • FBgnOO3O851 FBgn0036001, FBgn0000489, FBgnOO33O28, FBgn0027948, FBgn0036102, FBgn0034602, FBgn0033605, FBgn0022943, FBgn0037936,
  • FBgn0034542 FBgnOO3336O, FBgn0031470, FBgnOO31334, FBgn0036674,
  • FBgn0034881 FBgn0039263, FBgn0004456, FBgn0035710, FBgn0034615,
  • FBgnOO33393 FBgn0028738, FBgn0037661, FBgn0037721, FBgn0033308, FBgn0039156, FBgn0000330, FBgn0030948, FBgn0035743, FBgn0035592,
  • FBgn0040594 FBgn0011291, FBgn0016641, FBgnOO39283, FBgn0037261,
  • FBgnOO32833 FBgn0032756, FBgn0023000, FBgn0039032, FBgn0015239,
  • FBgn0003733 FBgn0002566, FBgn0033068, FBgn0032322, FBgn0034694,
  • FBgn0033862 FBgn0025742, FBgn0037619, FBgn0040637, FBgnOO3O514,
  • FBgn0038462 FBgn0035729, FBgn0030180, FBgn0037204, FBgn0004908, FBgn0000462, FBgn0020611, FBgn0004066, FBgn0039748, FBgn0036660,
  • FBgn0004381 FBgn0031117, FBgn0020300, FBgn0032012, FBgn0037099, FBgn0036178, FBgn0031442, FBgn0030233, FBgn0005674, FBgn0038461,
  • FBgn0024832 FBgn0029123, FBgn0035675, FBgn0020641, FBgn0034804,
  • FBgn0033781 FBgnOO31157, FBgn0030108, FBgnOO35O85, FBgn0032719,
  • FBgn0032112 FBgn0029855, FBgn0039326, FBgn0038524, FBgnOO33578,
  • FBgnOO33138 FBgn0020305, FBgn0004571, SGS-8, FBgn0010241, FBgn0037705, FBgn0025693, FBgn0040872, FBgn0036295, FBgn0001250, FBgn0030418,
  • FBgn0035785 FBgnOOO36OO, FBgn0040692, FBgn0030287, FBgn0036727,
  • FBgnOO3O558 FBgn0031566, FBgn0003159, FBgn0033540, FBgn0027094,
  • FBgn0036054 FBgn0037632
  • FBgn0036722 FBgn0033793
  • FBgn0036622 FBgn0036054, FBgn0037632, FBgn0036722, FBgn0033793, FBgn0036622,
  • FBgn0010408 FBgnOO33611, FBgn0040755, FBgnOO37186, FBgnOO32O95, FBgn0034260, FBgnOO13955, FBgnOOOO639, FBgn0020236, FBgn0031167,
  • FBgn0030433 FBgnOOO1138, FBgnOO38O63, FBgn0001257, FBgn0042138,
  • FBgn0036705 FBgn0031419, FBgn0038410, FBgn0000228, FBgn0035254, FBgn0035391, FBgn0037476, FBgn0033927, FBgn0033636, FBgn0033868,
  • FBgnOO3OO88 Eip63E
  • FBgn0026361 FBgn0036577, FBgnOO27786, FBgn0032021, FBgn0030341,
  • FBgn0036901 FBgn0028683, FBgn0031029, FBgn0036869, FBgn0034792,
  • Table 2 1074 target genes for eve expression profile during developmental stages ProbelD, DHR78, IMPL2, FBgn0040010, FBgn0032629, LSPbeta, FBgn0036472,
  • FBgn0015621 FBgnOO39293, FBgn0011230, FBgn0039633, DIAP2, FBgn0027534, FBgn0010105, FBgn0003031, FBgn0020251, FBgn0013343, FBgn0015269,
  • FBgn0020369 FBgn0040232, FBgnOO3516O, FBgn0030882, FBgn0026262,
  • FBgn0010410 FBgn0003067, FBgn0035404, FBgn0036001, FBgn0036909,
  • FBgn0036002 FBgn0027779, FBgn0035854, FBgnOO37282, FBgn0038146, FBgn0032881, FBgn0014007, FBgn0004516, FBgn0001224, FBgnOO31759,
  • FBgn0040077 FBgn0011666, FBgnOO31187, FBgn0030602, FBgnOO38196, FBgn0004373, Cdc2c, FBgn0031609, FBgn0037569, FBgn0022073, FBgn0023526, FBgn0019664, FBgn0015806, FBgn0004390, FBgn0035347, FBgn0030054, HSP83, FBgnOO33189, FBgn0013756, FBgn0030871, FBgn0027865, FBgn0004893, FBgn0036517, FBgn0036290, FBgn0022097, DIAPl, FBgn0030851, FBgn0003659, FBgn0027842, Neuroglian
  • FBgnOO37636 FBgn0041789, FBgn0031485, FBgnOOOO581, FBgn0026611,
  • FBgn0035995 FBgnOOOO283, FBgn0034054, FBgn0036207, FBgn0035692, FBgn0003013, FBgn0031143, FBgn0017397, FBgn0000163, FBgn0039894,
  • FBgn0026611 FBgn0025776, FBgn0035760, FBgn0040207, FBgn0017565,
  • FBgn0035642 FBgn0027535, FBgn0036173, FBgn0038554, FBgn0000462,
  • FBgnOO38532 FBgnOO35869, FBgn0039610, FBgn0037771, FBgn0025117,
  • FBgnOO34O58 FBgn0026376, FBgn0033988, FBgn0004429, FBgn0039904, FBgn0036511, FBgn0001122, FBgn0015283, FBgn0023175, FBgn0023097,
  • FBgn0031832 FBgn0035693, FBgn0031001, FBgn0029006, FBgn0003308,
  • FBgn0031781 FBgnOOO3659, FBgn0000273, FBgn0038222, FBgnOO39233,
  • FBgn0034602 FBgn0034724, FBgn0035411, L71-1, FBgn0030856, FBgn0040021,
  • FBgn0035872 FBgn0035061, FBgn0038620, FBgnOO3556O, FBgn0003048,
  • FBgnOO37516 FBgn0021906, FBgn0035655, FBgnOOOO253, FBgn0037094,
  • FBgn0037340 FBgn0030250, FBgn0000566, FBgn0014391, FBgn0015269, Dronc
  • FBgn0040900 FBgn0026085, FBgn0022764, FBgnOO29833, FBgn0037370,
  • FBgn0002611 FBgn0022023, FBgn0024921, FBgn0030486, FBgn0027609, FBgn0033385, FBgn0029584, FBgn0004861, FBgnOO39258, FBgn0038504,
  • FBgn0033030 FBgn0036406, FBgn0028982, FBgn0024814, FBgn0002633,
  • FBgn0001977 FBgn0031990, FBgn0013765, FBgn0029764, FBgn0029003, FBgn0004456, FBgn0036291, FBgn0036301, FBgn0039014, FBgn0039627,
  • FBgnOO361O3 FBgn0034240, FBgn0032982, FBgn0025802, FBgn0036630,
  • FBgn0034774 FBgnOO3O328, FBgn0038446, FBgnOO38355, FBgn0002973,
  • FBgn0030306 FBgn0015286, FBgn0032742, FBgn0035807, FBgn0015402,
  • FBgn0003416 FBgn0010460, FBgn0015279, FBgnOO39891, FBgn0027890, FBgn0037344, FBgn0035746, FBgn0037443, FBgn0004655, FBgn0029887,
  • FBgn0034282 FBgn0039277, FBgnOO345O3, FBgn0033243, FBgn0030092,
  • FBgn0033250 FBgnOO35586, FBgn0003177, FBgn0032736, FBgnOO36845, FBgnOO36O36, FBgn0031639, FBgn0013759, FBgn0023179, FBgn0033062,
  • FBgn0035644 FBgn0030518, FBgnOOO338O, FBgnOO38557, FBgn0010408,
  • FBgn0036445 FBgn0015624, FBgn0033357, FBgn0001168, FBgn0004579, FBgnOO31993, FBgn0031170, FBgn0038071, FBgn0035570, FBgn0011260,
  • FBgn0035203 FBgn0036538, FBgn0015773, FBgn0032799, FBgn0035237,
  • FBgnOO37351 FBgn0026577, FBgn0030462, FBgn0030180, FBgn0010213,
  • FBgn0034881 FBgn0016797, FBgn0039669, FBgn0004465, FBgn0031842,
  • FBgn0038103 FBgn0004509, FBgn0040874, FBgn0035654, FBgn0038516, FBgnOO33528, FBgn0014026, FBgn0036279, FBgnOOO3396, FBgn0038553,
  • FBgn0038271 FBgnOO31599, FBgn0011716, FBgn0030856, FBgn0033225,
  • FBgn0039672 FBgn0035558, FBgnOO19968, FBgn0039140, FBgn0026787,
  • FBgn0038275 FBgn0035574, FBgn0022787, FBgn0040755, FBgn0024195, FBgnOO39936, FBgn0036398, FBgnOO34861, FBgn0038660, FBgn0030733,
  • FBgn0032422 FBgn0004586, FBgnOO39878, FBgnOO38715, FBgn0036764,
  • FBgn0033754 FBgn0003091, FBgn0030809, FBgn0000121, FBgn0029131, FBgn0038140, FBgn0042213, FBgnOO323O5, FBgn0037295, FBgn0035827,
  • FBgn0032595 FBgn0031698, FBgn0037427, FBgn0039909, FBgn0032214,
  • ChIP chromatin immunoprecipitation
  • Chip custom designed high density DNA microarrays
  • Fig 9 shows distribution of ChIP-chip binding probe locations relative to target genes. This parameter set yields 969 FTZ ChIP-chip target genes and 932 EVE ChIP-chip target genes (Table 3, 4), 137 FTZ direct target genes and 98 EVE direct target genes (FIG. 1, Table 5, 6).
  • FBgn0036630 FBgn0024150, FBgn0013591, FBgn0003507, FBgn0027660, FBgn0038192, FBgn0020251, FBgn0031759, FBgn0001222, FBgnOO 15278,
  • FBgn0035340 FBgn0033103, FBgn0035174, FBgn0036826, FBgn0035574, FBgn0038457, FBgn0003175, FBgn0030306, FBgn0000575, FBgn0000546,
  • FBgn0027655 FBgn0031236, FBgn0003607, FBgn0033559, FBgn0003656,
  • FBgn0002973 FBgnOO29933, FBgn0037443, FBgn0035172, FBgn0032629, FBgn0031684, FBgn0032002, FBgn0034606, FBgnOO36398, FBgn0038853,
  • FBgn0002284 FBgn0037198, FBgn0037971, FBgn0010389, FBgn0028914, FBgnOOO3371, FBgnOOO3715, FBgnOO388O3, FBgnOO33218, FBgn0036461,
  • FBgnOO38887 FBgn0032646, FBgn0029577, FBgnOO357O6, FBgn0032426, FBgn0032450, FBgn0032001, FBgn0038891, FBgnOO33758, FBgnOO39181,
  • FBgnOO38898 FBgn0000157, FBgn0000480, FBgnOO3O3OO, FBgnOO38372, FBgn0002921, FBgn0034042, FBgnOO35O3O, FBgn0003499, FBgn0037992,
  • FBgnOO337O8 FBgn0013997, FBgn0032225, FBgn0025390, FBgn0004102, FBgn0034041, FBgnOOO345O, FBgn0037034, FBgn0030344, FBgn0037160,
  • FBgn0034957 FBgn0030022, FBgnOO38374, FBgn0038247, FBgn0039754, FBgnOO39166, FBgn0002522, FBgn0034141, FBgn0032476, FBgn0034044,
  • FBgnOO332O3 FBgnOO34888, FBgn0040520, FBgn0034695, FBgn0030307, FBgn0034408, FBgn0027552, FBgnOO34831, FBgn0029881, FBgn0031496,
  • FBgnOO3O458 FBgn0037847, FBgnOO3OO23, FBgn0034431, FBgn0039206,
  • FBgn0029502 FBgnOO25111, FBgnOO35573, FBgn0040991, FBgn0030964,
  • FBgnOO35392 FBgn0032735, FBgn0019948, FBgnOO39831, FBgn0040948, FBgnOO3O3O5, FBgn0031046, FBgn0000568, FBgn0035395, FBgn0041627,
  • FBgn0035220 FBgn0010348, FBgn0031032, FBgn0015818, FBgn0036994,
  • FBgn0036241 FBgn0027339, FBgn0033095, FBgn0013548, FBgn0039177,
  • FBgn0037702 FBgn0002989, FBgn0031495, FBgn0033000, FBgn0028496,
  • FBgn0037070 FBgn0031022, FBgn0015904, FBgn0029906, FBgnOO37189, FBgn0033534, FBgn0001991, FBgn0031149, FBgn0038193, FBgn0036798,
  • FBgnOO38937 FBgn0035425, FBgn0032710, FBgn0004396, FBgn0034429,
  • FBgn0040379 FBgn0022131, FBgn0035087, FBgn0032916, FBgnOO39178, FBgnOO23518, FBgn0000317, FBgn0015609, FBgn0001215.
  • FBgn0030478 FBgn0039732, FBgn0040889, FBgnOO13733, FBgn0030452, FBgn0030451, FBgn0031596, FBgn0030064, FBgnOOOO163, FBgn0040754,
  • FBgn0020236 FBgn0029653, FBgn0039263, FBgn0037048, FBgn0035073, FBgn0027524, FBgn0003984, FBgn0037639, FBgnOO16O81, FBgn0037153,
  • FBgn0038881 FBgn0039014, FBgn0000489, FBgn0030958, FBgn0035569,
  • FBgnOO35O59 FBgn0037748, FBgnOOOO636, FBgn0035728, FBgn0034270, FBgnOOO3328, FBgn0037526, FBgnOO3987O, FBgn0023172, FBgnOOO3O87,
  • FBgn0027066 FBgn0020912, FBgn0002440, FBgn0033749, FBgn0039738, FBgn0037160, FBgn0037468, FBgnOO35118, FBgn0026875, FBgnOO37253,
  • FBgn0024252 FBgn0029924, FBgn0035117, FBgn0039172, FBgn0030567,
  • FBgn0035422 FBgn0035459, FBgn0038858, FBgn0031849, FBgn0016797,
  • FBgnOO338O3 FBgn0031879, FBgn0020245, FBgn0032717, FBgn0032885, FBgn0031017, FBgn0032406, FBgn0037682, FBgn0011592, FBgn0035440,
  • FBgn0030479 FBgn0011217, FBgn0028703, FBgn0034060, FBgn0031374,
  • FBgn0013469 FBgn0010247, FBgn0037346, FBgnOO3O488, FBgn0036864,
  • FBgn0033575 FBgn0041702, FBgn0019809, FBgn0003118, FBgn0003022, FBgn0034370, FBgn0004914, FBgn0037392, FBgn0020496, FBgn0011763,
  • FBgn0034044 FBgnOOO3165, FBgn0030564, FBgn0031820, FBgn0032489,
  • FBgn0033992 FBgn0032440, FBgn0029798, FBgn0040689, FBgn0035976, FBgn0028494, FBgn0030337, FBgnOO31576, FBgnOO31O9O, FBgnOO39281,
  • FBgn0028692 FBgn0030521, FBgn0031285, FBgnOO23511, FBgn0034619, FBgn0039962, FBgn0001197, FBgn0013269, FBgnOO36678, FBgn0039123,
  • FBgn0038622 FBgn0019650, FBgn0028495, FBgn0030058, FBgn0037171,
  • FBgn0010909 FBgn0034181, FBgn0026741, FBgn0035595, FBgn0031232, FBgn0038831, FBgn0004834, FBgn0029576, FBgn0021953, FBgn0020386,
  • FBgn0037195 FBgn0034276, FBgn0032963, FBgnOO32217, FBgn0034180, FBgn0030018, FBgn0000557, FBgn0039324, FBgn0011739, FBgn0030400,
  • FBgn0040079 FBgn0037920, FBgn0037306, FBgn0035867, FBgn0039545, FBgn0011260, FBgn0039287, FBgn0038755, FBgn0036372, FBgn0040071,
  • FBgn0005671 FBgn0035127, FBgnOO38OO8, FBgn0020360, FBgn0036268, FBgn0038452, FBgn0000486, FBgn0015954, FBgn0030865, FBgn0036216,
  • FBgn0038904 FBgn0016120, FBgn0039167, FBgn0024432, FBgn0034121,
  • FBgn0032073 FBgnOO39285, FBgn0029948, FBgn0001332, FBgn0030942,
  • FBgn0030334 FBgn0030866, FBgnOO3899O, FBgn0030383, FBgn0000028,
  • FBgn0011708 FBgnOO33357, FBgn0034974, FBgn0033356, FBgn0039350, FBgn0033380, FBgn0039137, FBgn0015778, FBgn0039883, FBgn0033159,
  • FBgn0034535 FBgn0031889, FBgn0037642, FBgn0000259, FBgn0035712, FBgn0032431, FBgn0020556, FBgn0003380, FBgn0013973, FBgn0033094,
  • FBgn0026188 FBgn0037670, FBgnOO3O731, FBgn0035656, FBgn0033946,
  • FBgn0039412 FBgn0039549, FBgn0030166, FBgn0035894, FBgn0033549, FBgn0004646, FBgn0020445, FBgnOO159O3, FBgn0034217, FBgn0000179,

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Abstract

L'invention concerne des procédés et compositions pour diagnostiquer, traiter et détecter un carcinome du rein à cellules claires (RCC) chez un patient. Dans certains modes de réalisation, cela implique l'évaluation du taux d'expression SPOP, les taux des transcrits ou bien de protéine SPOP, de manière à déterminer si une cellule rénale est une cellule cancérigène ou tumorale. Dans d'autres modes de réalisation, cela implique d'évaluer si d'autres échantillons biologiques ont un taux élevé de protéine SPOP pour comparaison à un échantillon normal de manière à identifier le RCC chez le patient.
PCT/US2008/050812 2007-01-10 2008-01-10 Marqueur moléculaire pour carcinome du rein à cellules claires Ceased WO2008086502A2 (fr)

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US60/879,662 2007-01-10

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014185661A1 (fr) * 2013-05-14 2014-11-20 재단법인 의약바이오컨버젼스연구단 Méthode de suivi de métastase de cellules cancéreuses à l'aide de cellules cultivées dans un environnement tridimensionnel de collagène
US10443102B2 (en) 2011-02-24 2019-10-15 Cornell University Recurrent SPOP mutations in prostate cancer
RU2805811C1 (ru) * 2022-06-09 2023-10-24 Федеральное государственное бюджетное учреждение науки Институт теоретической и экспериментальной биофизики Российской академии наук (ИТЭБ РАН) Способ диагностики почечно-клеточной карциномы по наличию зрительных белков аррестина и рековерина в моче

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003512857A (ja) * 1999-11-05 2003-04-08 ザ バーナム インスティチュート Ｔｒａｆファミリータンパク質
JP2006514554A (ja) * 2002-11-21 2006-05-11 ワイス 腎細胞癌および他の固形腫瘍の診断法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10443102B2 (en) 2011-02-24 2019-10-15 Cornell University Recurrent SPOP mutations in prostate cancer
WO2014185661A1 (fr) * 2013-05-14 2014-11-20 재단법인 의약바이오컨버젼스연구단 Méthode de suivi de métastase de cellules cancéreuses à l'aide de cellules cultivées dans un environnement tridimensionnel de collagène
RU2805811C1 (ru) * 2022-06-09 2023-10-24 Федеральное государственное бюджетное учреждение науки Институт теоретической и экспериментальной биофизики Российской академии наук (ИТЭБ РАН) Способ диагностики почечно-клеточной карциномы по наличию зрительных белков аррестина и рековерина в моче

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