WO2006037993A2 - Marqueurs cancereux - Google Patents

Marqueurs cancereux Download PDF

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Publication number
WO2006037993A2
WO2006037993A2 PCT/GB2005/003813 GB2005003813W WO2006037993A2 WO 2006037993 A2 WO2006037993 A2 WO 2006037993A2 GB 2005003813 W GB2005003813 W GB 2005003813W WO 2006037993 A2 WO2006037993 A2 WO 2006037993A2
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WIPO (PCT)
Prior art keywords
target protein
cancer
cyp51
cyp2s1
cyp4z1
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PCT/GB2005/003813
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English (en)
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WO2006037993A3 (fr
Inventor
Graeme Ian Murray
Colin Matheson Telfer
William Thomas Melvin
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Auvation Limited
The University Court Of The University Of Aberdeen
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Priority claimed from GB0421919A external-priority patent/GB0421919D0/en
Priority claimed from GB0421917A external-priority patent/GB0421917D0/en
Priority claimed from GB0421914A external-priority patent/GB0421914D0/en
Priority claimed from GB0421913A external-priority patent/GB0421913D0/en
Priority claimed from GB0421920A external-priority patent/GB0421920D0/en
Priority claimed from GB0421918A external-priority patent/GB0421918D0/en
Application filed by Auvation Limited, The University Court Of The University Of Aberdeen filed Critical Auvation Limited
Publication of WO2006037993A2 publication Critical patent/WO2006037993A2/fr
Publication of WO2006037993A3 publication Critical patent/WO2006037993A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to tumour diagnosis and therapy, and to materials and methods for use therein. More particularly, the invention is based on the identification of certain cytochrome P450 enzymes, for example CYP2S1 , CYP2U1 , CYP3A43, CYP4F11 , CYP4X1 , CYP4Z1, CYP26A1 and CYP51, that are over-expressed in tumours in comparison with normal tissue, and proposes the use of these enzymes as tumour markers, and as the basis of selective therapeutic approaches involving the design of drugs. For example this may include drugs that are activated to a cytotoxic form by the action of one or more of the P450 enzymes of the invention, or drugs that inhibit the activity of one or more of the P450 enzymes of the invention in order to deliver a therapeutic benefit.
  • cytochrome P450 enzymes for example CYP2S1 , CYP2U1 , CYP3A43, CYP4F11 , CYP4X
  • pathological conditions including cancer
  • these molecules are characterized by the abnormal expression of certain molecules, and these molecules thus serve as "markers" for a particular pathological condition.
  • diagnostic molecules i.e. abnormal components that can be identified to diagnose the pathological condition
  • such molecules can serve as reagents that can be used to generate diagnostic and/or therapeutic agents.
  • markers of cancer to produce antibodies specific to a particular marker.
  • a further non-limiting example is the use of a peptide which complexes with an MHC molecule, to generate cytotoxic T cells against cells expressing the marker.
  • the abnormal over-expression of a target protein within cancerous cells may indicate that the particular protein plays a role in the development or perpetuation of tumours. Therefore a further non-limiting example of the utility of such targets is there use as the basis of a mechanism, such as a compound screening assay, for the identification of compounds that inhibit the activity of the target and thereby disrupt its role in the biology of the cancerous cell to generate a therapeutic effect.
  • Colorectal cancers are the third most common malignancies in the world, and amongst men in the European Union it is the second most common cause of cancer death after lung cancer. Although more than 90% of cases are curable when diagnosed at an early stage in development, the majority of patients with colorectal cancer present clinically when the tumour is at an advanced, metastatic stage.
  • CEA is immunologically detectable in the serum of colorectal cancer patients, and detection of CEA mRNA by RT-PCR can identify lymph node micrometastases, which are a prognostic indicator of a reduced chance of survival in colorectal cancers (Liefers et al, N. Engl. J. Med. 339: 223-8, 1998).
  • Another promising marker for colorectal cancer is minichromosome maintenance protein 2 (MCM2), which is being developed as a target for diagnosis from stool samples (Davies et a/, Lancet 359: 1917-9, 2002).
  • the current routine diagnostic test for colorectal cancer is the FOBT (Faecal Occult Blood Test), which is lacking in sensitivity and specificity. Evaluation of the effectiveness of this test indicates that it may fail to detect as many as 76% of suspicious growths (Lieberman et a/, N. Engl. J. Med. 345: 555-60, 2001). It also results in a large number of false positives and these patients are required to undergo the unpleasant, invasive procedure of colonoscopy. Even when administered together these two procedures have been found to miss 24% of tumours and precancerous polyps. Currently the best candidates for new diagnostic tests are based on DNA analysis, but these have at best a 50% detection rate.
  • Ovarian cancer is the fifth most common cancer in women, accounting for 5% of all cancers in females and afflicting just under 2% of women at some point in their lives. The overall five-year survival rate for the disease is 42%, but early diagnosis improves prognosis. Patients who present with stage I or Il disease have a 5-year survival of 90% and 70% respectively, whilst the 80% of cases with advanced stage III or IV disease at first diagnosis have a 5-year survival of just 20% (Tortolero-Luna & Mitchell, J. Cell Biochem. Suppl. 23: 200-7, 1995). These statistics highlight the importance of early diagnosis of ovarian cancer, and of understanding the phenotype of this disease in premalignant stages. Ovarian cancer patients require earlier detection and more successful treatment of their illness, and to this end it is desirable to identify proteins whose expression is associated with cancerous cells, which may serve as diagnostic markers, prognostic indicators and therapeutic targets.
  • Serum CA125 is the most extensively evaluated marker for ovarian cancer.
  • the heavily glycosylated, high-molecular-weight protein antigen is absent from normal ovarian epithelium, but highly expressed in serous and mucinous papillary tumours (Kabawat et a/, Am. J. Clin. Pathol. 79: 98-104, 1983) and found at elevated levels in the serum of >80% of ovarian cancer patients.
  • a double determinant immunoassay has been used to determine the normal serum concentration of CA125 in healthy individuals as ranging between 30 and 60 U / ml (Bon et al, Am. J. Obstet. Gynecol. 174: 107-14, 1996).
  • CA125 alone does not have sufficient sensitivity or specificity to detect early ovarian cancers when used alone, but combination with other assays, such as measurement of OVX-1 or the reproductive hormone inhibitin, permits a detection rate of up to 90% (Robertson et a/, Clin. Chem. 45: 651-8, 1999), and CA125 levels are widely used in follow-up monitoring of ovarian cancer patients to evaluate treatment response and disease recurrence.
  • liver disease and other gynaecological conditions such as ovarian cysts and endometriosis also cause elevations in serum CA125 (Bergmann et a/, Clin. Chim.
  • CA125 assessment assisted in clinical disease management and produced a significant improvement in survival time in the screened cohort, but did not impact overall mortality.
  • the antigen is also expressed in other tissues, including epithelial cell of the lung, breast, conjunctiva and prostate (Nap, Int. J. Biol. Markers 13: 210-5, 1998), making CA125 less desirable as a therapeutic target.
  • Prominent examples include components of the phosphatidylinositol 3 kinase signalling pathway, and the G-protein coupled receptors LPAR1 , LPAR2 and LPAR3.
  • the LPAR receptors activate ovarian cells, promoting proliferation and motility and inhibiting apoptosis, in response to binding of lysophosphatidic acid, which is present at elevated levels in plasma from ovarian cancer patients (Xu et al, JAMA 280: 719-23, 1998).
  • these components are expressed in a variety of other pathologically normal tissue types, suggesting that their use as drug targets may give rise to side effects.
  • the cytochromes P450 are a multi-gene family of constitutive and inducible enzymes that play a central role in the oxidative metabolism of a wide range of xenobiotics (i.e. drugs and foreign chemicals) and biologically active endogenous compounds including steroids, retinoids, cholesterol, and arachidonic acid (reviewed by Nebert & Russell, Lancet 360: 1155-62, 2002; Danielson, Curr. Drug Metab. 3: 561-97, 2002).
  • the P450s are classified into families and sub-families based on nucleic acid homology and there are currently 57 known human P450s in 18 families (http://drnelson.utmem.edu/human.P450.table.html).
  • Families 1-3, and to a lesser extent family 4 are regulated by ligand-activated transcription factors and are central to the clearance of xenobiotics, catalysing hydroxylation that renders lipophilic substrate moiecules more reactive and ultimately leads to the excretion of water-soluble metabolites.
  • the remaining families are involved in endogenous processes including hormone biosynthesis, production of epoxyeicosatrienoic acids and metabolism of vitamin D and retinoic acid.
  • the common structural element in every P450 is a ligand-binding heme group that absorbs light at 450 nm when bound to CO.
  • CYP3A4 is the most abundant isoform, accounting for approximately 30% of total hepatic P450 protein (Lamba et al, Pharmacogenetics j2: 121-32, 2002).
  • the principal hepatic isoforms (e.g. CYP1A2, CYP2D6, CYP3A4) show constitutive expression and have been very well characterised. In contrast little is known about the biology of some of the more recently identified P450 isoforms.
  • CYP1B1 mRNA and protein have been identified in a wide range of malignant tumours (Murray et al, Annu. Rev. Pharmacol. Toxicol. 41.: 297-316, 2001) and a high level of enhanced expression is also seen in metastasis (McFadyen et al, Br. J. Cancer 85: 242- 6, 2001).
  • CYP1B1 mRNA is expressed in a wide variety of normal tissue but the protein is generally not detected (Gibson et al, MoI. Cancer Ther. 2: 527-34, 2003; Gordan & Vonderheide, Cytotherapy 4: 317-27, 2002).
  • CYP1 B1 protein expression is consistently found from the earliest stages of neoplastic development (e.g. benign prostatic hyperplasia and metaplastic prostatic urothelium) through to fully-developed carcinoma (Carnell et al, Int. J. Radiation Oncology Biol. Phys. 58: 500-9, 2004).
  • CYP3A4/5 is responsible for the metabolism of a number of anticancer drugs including those used in the treatment of osteosarcomas (ifosfamide, vinblastine, etoposide and doxorubicin) and so this enzyme may play a role in the resistance of osteosarcomas to chemotherapy.
  • Colon cancer cells exhibit increased expression of CYP27B1 (vitamin 1- ⁇ hydroxylase) mRNA and protein in comparison with normal colon cells (Tangpricha et al, Lancet 357: 1673-4, 2001).
  • CYP2B6 is expressed in breast carcinomas at higher levels than in normal breast tissue (Hellmold et al, J. Clin. Endocrinol. Metab. 83: 886-95, 1998).
  • CYP1A and CYP3A forms have been detected in breast tumours, in 40% and 22% of test cases respectively, but not in corresponding normal breast tissue (Murray et al, Br. J. Cancer 63: 1021-3, 1991; Murray et al, J. Pathol.
  • tumour cells may be part of the process of tumour development or a response to it, either activating tumour promoting compounds such as 4-hydroxyoestradiol or inactivating anti-tumour compounds such as 2-methoxy-oestradiol (Kakhani et al, Pharmacotherapy 23: 165-72, 2003 ; Dawling et al, Cancer Res. 63: 3127-31 , 2003).
  • activating tumour promoting compounds such as 4-hydroxyoestradiol
  • anti-tumour compounds such as 2-methoxy-oestradiol
  • a number of anticancer drugs are also known to undergo oxidative metabolism by P450 enzymes, and P450 isoforms that are expressed in tumours are capable of influencing the response of those tumours to chemotherapeutics (Murray, J. Pathol. 192: 419-26, 2000; Martinez et al, Br. J. Cancer 87: 681-6, 2002). Whether this influence favours activation (i.e. cytotoxicity) or deactivation (i.e. resistance) is dependent upon the relative quantities and enzymatic reactivates of specific P450s in individual tumour cells.
  • CYP3A4 and CYP3A5 are involved in both the activation and inactivation of ifosphamide, taxanes and the vinca alkaloids (reviewed by Kivist ⁇ et al, Brit. J. Clin. Pharmacol. 40: 523-30, 1995).
  • CYP2C8 catalyses taxol 6 ⁇ -hydroxylation to an inactive metabolite (Sonnichsen et al, J. Pharmacol. Exp. Ther. 275: 566-75, 1995).
  • Tamoxifen a non-steroidal anti-oestrogen undergoes 4-hydroxylation to a primary active metabolite, frans-4-hydrotamoxifen, that is one hundred times more potent an oestrogen receptor antagonist than the parent molecule.
  • This bioactivation is mediated principally in the liver by CYP2D6, with contributions from CYP2C9 and CYP3A4 (Crewe et al, Drug Metab. Dispos. 30: 869-74, 2002; Notley et a/, Chem. Res. Toxicol. 15: 614-22, 2002).
  • Cyclophosphamide and ifosphamide are also activated by 4-hydroxylation, principally by the CYP2B6 and CYP3A4 isoforms (Chang et al, Cancer Res. 53: 5629-37, 1993).
  • Tegafur a prodrug treatment for head & neck, pulmonary and colorectal cancers, is bioactivated by CYP1A2, CYP2A6 and CYP2C to the thymidylate synthase inhibitor 5- fluorouracil ( Komatsu et a/, Drug Metab. Dispos.. 28:1457-63, 2000).
  • PCT international patent publication WO9712246 discloses the tumour-associated expression of CYP1B1 and claims use of this enzyme as a diagnostic marker and target for immunotherapeutics, prodrugs and inhibitors.
  • Hedley et al WO0242325 describes a polymer encapsulated DNA vaccine that encodes portions of the CYP1B1 molecule within a proprietary expression cassette. This vaccine is intended to prime the immune system to destroy cells that express the protein, thereby effecting a treatment for CYP 1B1 -positive cancers, and has been shown to induce a specific anti-CYP1B1 T cell response in healthy donors and cancer patients in early clinical trials (Maecker et al, Blood 102: 3287-94, 2003).
  • CYP2W1 is specifically expressed in certain tumours, and claims the use of this isoform as a drug target.
  • this application relies upon semi-quantitative PCR to demonstrate the presence of increased CYP2W1 message in certain cDNA samples prepared from human tumours compared with samples prepared from normal tissues, but does not verify the differential expression of CYP2W1 protein in human tissues. This may be important as, in the case of CYP1B1 , it has been demonstrated that P450 mRNA expression in human tissues does not bear a simple correlation with the presence of translated protein.
  • Phortress (2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole, disclosed by Hutchinson et al in WO0114354) is a prodrug cytotoxic with a unique, biphasic mechanism of action dependent on P450 activity (Hutchinson et a/, J. Med. Chem. 44: 1446-55, 2001; Bradshaw et al, Br. J. Cancer 86: 1348-54, 2002). Phortress binds to the aryl hydrocarbon receptor, which in turn stimulates expression of CYP1A1. CYP1A1 then catalyses the activation of Phortress to cytotoxic metabolites.
  • Recombinant human CYP1 B1 is also capable of metabolising Phortress, and as this enzyme is known to be up-regulated in tumour cells it may also play a role in the activity of Phortress in vivo (Leong et al, Br. J. Cancer 8: 470-7, 2003).
  • WO03029176 pyrrolo-indole and pyrrolo-quinoline derivatives
  • WO02068412 indoline and tetrahydro-quinolines
  • WO02067937 indoline and tetrahydro-quinolines
  • benz-indole and benzo-quinoline derivatives WO02067930.
  • GDEPT Gene directed pro-drug therapy
  • MetXia-P450 is a retroviral vector encoding the human CYP2B6 gene, and designed to be injected directly into the tumour in order to sensitise it to the effects of cyclophosphamide.
  • WO0158444 discloses the ability of CYP 1 B1 to interfere with the cytotoxicity of certain chemotherapeutic agents (most notably docetaxel) and claims the use of inhibitors of CYP1B1 to prevent the metabolism of anti-cancer drugs within the environment of the tumour.
  • chemotherapeutic agents most notably docetaxel
  • Chun & Kim, WO03018013 (Promeditech) describes a stilbene derivative capable of inhibiting CYP1B1 activity, and claims its use to decrease or prevent chemical carcinogenesis by blocking the CYP1B1 -mediated activation of procarcinogens.
  • WO0187286 discloses a strategy for improving the pharmacokinetics of a drug by co-administering antisense oligonucleotides targeting RNAs encoding drug-metabolizing enzymes.
  • AVI- 4557 a morpholino antisense construct that blocks expression of the major hepatic isoform CYP3A4
  • the present inventors have used immunohistochemical staining of tumour microarrays to search for proteins of the cytochrome P450 family that are overexpressed in tumour tissues in comparison with the corresponding normal tissues.
  • This study demonstrates that CYP2S1, CYP2U1 , CYP3A5, CYP3A7, CYP3A43, CYP4F11 , CYP4X1 , CYP4Z1, CYP26A1 and CYP51 exhibit a greater intensity and in some case higher frequency of immunohistochemical staining in colorectal and/or ovarian cancer compared with normal tissue.
  • the study has further expanded on the significance of one of these P450s, namely CYP51. This was achieved by comparing the RT-PCR profile obtained from tumour tissue with that from the corresponding normal tissue and by investigating the frequency of CYP51 expression by analysing various different cancer cell lines with a broad spectrum of cellular origin.
  • CYP2S1 , CYP2U1 , CYP3A43, CYP4F11 , CYP4X1 , CYP4Z1 , CYP26A1 and CYP51 have not been previously shown to be up-regulated in tumour tissues, and therefore were not previously identified as cancer markers.
  • These eight Cytochrome P450 enzymes constitute the preferred target proteins of the present invention, and may hereinafter be referred to as such, or as "target proteins”.
  • the present invention describes the use of these target proteins as markers of cancer, and provides methods for their use in such applications.
  • the target proteins of the present invention are of particular use inter alia as diagnostic and prognostic markers of cancers. As with known markers, they may be used for example to assist in diagnosing the presence of cancer at an early stage in the progression of the disease and predicting the likelihood of clinically successful outcome, particularly with regard to the sensitivity or resistance of a particular patient's tumour to a chemotherapeutic agent or combinations of chemotherapeutic agents. Where tumour over-expression of one of the target proteins of the present invention is associated with a poor clinical prognosis, information on the expression status of tumours in individual patients may be used for example to inform clinical treatment decisions.
  • target proteins can be used for therapeutic intervention in colorectal, ovarian and other cancers, for example to specifically target neoplastic cells whilst limiting toxicity in healthy tissues, and to provide methods for the evaluation of the ability of candidate therapeutic compounds to modulate the biological activity of cancerous cells.
  • the present invention relates to the diagnosis, prognosis and treatment of cancer, and specifically to the discrimination of neoplastic cells from normal cells on the basis of over-expression of specific tumour antigens, the prognosis of clinical outcomes based on the observed pattern of expression of specific tumour antigens, and the targeting of treatment through exploitation of the differential expression of these antigens within neoplastic cells.
  • the invention specifically relates to the detection of over-expression of one or more of the target proteins of the invention in neoplastic cells compared with the expression in pathologically normal cells. Furthermore the invention provides evidence for up-regulation of expression of these targets in tumour cells where this has not previously been reported.
  • these proteins as well as nucleic acid sequences encoding these proteins, or sequences complementary thereto, can be used as cancer markers useful in diagnosing or predicting the onset or clinical course of a cancer such as colorectal cancer, monitoring the efficacy of a cancer therapy and/or as a target of such a therapy.
  • the invention in particular relates to the discrimination of neoplastic cells from normal cells on the basis of the over-expression of one or more target proteins of the present invention, or the gene that encodes this protein.
  • the invention provides a pattern of expression of one or more specific target proteins, the expression of which is increased in neoplastic cells in comparison to normal cells.
  • the invention provides a variety of methods for detecting this protein and the expression pattern of this target protein and using this information for the diagnosis and treatment of cancer.
  • novel therapeutics for treating cancer which include, for example: antibodies that can be administered to an individual that bind to and reduce or eliminate the biological activity of one or more target proteins in vivo; nucleic acid or peptidyl nucleic acid sequences which hybridize with genes or gene transcripts encoding one or more of the target proteins thereby to reduce expression of the target proteins in vivo; or small molecules, for example, organic molecules which interact with one or more target proteins of the invention or other cellular moieties that interact with the target proteins, thereby to reduce or eliminate the biological activity of the target protein.
  • the invention therefore further provides methods for targeting of therapeutic treatments for cancers by directing treatment against one or more of the over-expressed target proteins of the invention.
  • Methods for achieving this targeting may include, but are not limited to;
  • the present invention thereby provides a wide range of novel methods for the diagnosis, prognosis and treatment of cancers, including colorectal cancer and ovarian cancer, on the basis of the differential expression of one or more of the target proteins.
  • the study defined the expression profile for individual P450s, and discovered the prognostic significance of certain individual P450s in colorectal and/or ovarian cancer. The study has further analysed the significance of one of these P450s, namely CYP51 , through RT-PCR of tumour versus normal tissue and lmmunoblot analysis of various different cancer cell lines.
  • CYP3A4 was the isoform most frequently expressed and at the highest intensity in normal colon and this finding is consistent with previous studies which have found this P450 form to be the major P450 present in normal colon (McKay et al, Gut 34: 1234-9, 1993; McKinnon et al, Gut 36: 259-67, 1995).
  • the present study also establishes the statistically significant correlation of tumour expression of CYP2S1, CYP4Z1 and CYP51 with patient survival. Those cancer patients whose tumours exhibited negative, weak or moderate expression had a better prognosis than those who exhibited strong expression. These findings show that CYP2S1 , CYP4Z1 and CYP51 , the presence of which has not previously been reported in colorectal/ovarian tumours, are prognostic indicators and that information on the expression status of these proteins in tumours from individual cases would allow such patients to be assigned a
  • CYP51 As shown in example 4, the present investigation has shown the wide-spread expression of CYP51 in various cancer cell lines.
  • the expression of CYP51 was analysed in seventeen different cancer cell lines which have a broad spectrum of cellular origin, including five colon and one ovarian.
  • CYP51 was detected in all seventeen cancer cell lines examined which further emphasises the significance of this P450 protein as a tumour marker.
  • the target proteins of the invention are CYP51 , CYP2S1, CYP2U1 , CYP3A43, CYP4F11 , CYP4X1 , CYP4Z1, CYP26A1 , CYP3A7, CYP2F1, CYP4V2, and CYP39.
  • the preferred target proteins are CYP2S1 , CYP2U1 , CYP3A43, CYP4F11, CYP4X1 , CYP4Z1 , CYP26A1 and CYP51.
  • a first aspect of the present invention provides a method for the identification of cancer cells, which method comprises determining the expression of one or more target proteins of the invention in a sample of tissue from a first individual and comparing the pattern of expression observed with the pattern of expression of the same protein(s) in a second clinically normal tissue sample from the same individual or a second healthy individual, with the presence of tumour cells in the sample from the first individual indicated by a difference in the expression patterns observed.
  • the invention provides a diagnostic assay for characterising tumours and neoplastic cells, particularly human neoplastic cells, by the differential expression of one or more target proteins whereby the neoplastic phenotype is associated with, identified by and can be diagnosed on the basis thereof.
  • This diagnostic assay comprises detecting, qualitatively or preferably quantitatively, the expression level of one or more target proteins and making a diagnosis of cancer on the basis of this expression level.
  • determining the expression means qualitative and/or quantitative determinations, of the presence of one or more target proteins of the invention including measuring an amount of biological activity of one or more target proteins in terms of units of activity or units activity per unit time, and so forth.
  • the term “expression” generally refers to the cellular processes by which a polypeptide is produced from RNA.
  • the term “over-expression” generally refers to the production of a cellular component such as RNA or protein within a given abnormal tissue, such as a tumour, at a higher level than is demonstrable in the corresponding normal tissue.
  • cancer encompasses cancers in all forms, including polyps, neoplastic cells and preneoplastic cells and includes sarcomas and carcinomas.
  • Exemplary sarcomas and carcinomas include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma
  • acute lymphocytic leukaemia and acute myelocytic leukaemia myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukaemia
  • chronic leukaemia chronic myelocytic (granulocytic) leukaemia and chronic lymphocytic leukaemia
  • polycythemia vera lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstr ⁇ om's macroglobulinemia, and heavy chain disease.
  • this method may be applied to diagnosis of colorectal cancer.
  • colon cancer rectal cancer
  • colonrectal cancer colorectal cancer
  • this method may be applied to diagnosis of ovarian cancer.
  • Species variants are also encompassed by this invention where the patient is a non- human mammal, as are allelic or other variants of the target proteins of the invention and any reference to these target proteins herein will be understood to embrace, alleles, homologues or other naturally occurring variants.
  • variants of the naturally occurring sequences thereof are included within the definition of the target proteins of the invention.
  • Such variant sequences specifically include allelic forms such as those created by the occurrence of single nucleotide polymorphisms.
  • variant sequences are at least 75% homologous to the wild- type sequence, more preferably at least 80% homologous, even more preferably at least 85% homologous, yet more preferably at least 90% homologous or most preferably at least 95% homologous to at least a portion of the reference sequence supplied (SEQ ID NOs: 1-8).
  • the homology will be as high as 94 to 96 or 98%.
  • Homology in this context means sequence similarity or identity, with identity being preferred.
  • the candidate amino acid sequence and the reference amino acid sequence are first aligned using a standard computer programme such as are commercially available and widely used by those skilled in the art.
  • the NCBI BLAST method is used (http://www.ncbi.nlm.nih.gov/BLAST/). Once the two sequences have been aligned, a percent similarity score may be calculated.
  • variants of the naturally- occurring sequence as detailed in SEQ ID NOs:1-8 herein, must be confirmed for their function as marker proteins. Specifically, their presence or absence in a particular form or in a particular biological compartment must be indicative of the presence or absence of cancer in an individual. This routine experimentation can be carried out by using standard methods known in the art in the light of the disclosure herein.
  • the target protein can be detected using a binding moiety capable of specifically binding the marker protein.
  • the binding moiety may comprise a member of a ligand-receptor pair, i.e. a pair of molecules capable of having a specific binding interaction.
  • the binding moiety may comprise, for example, a member of a specific binding pair, such as antibody-antigen, enzyme-substrate, nucleic acid-nucleic acid, protein-nucleic acid, protein-protein, or other specific binding pair known in the art. Binding proteins may be designed which have enhanced affinity for the target protein of the invention.
  • the binding moiety may be linked with a detectable label, such as an enzymatic, fluorescent, radioactive, phosphorescent, coloured particle label or spin label.
  • a detectable label such as an enzymatic, fluorescent, radioactive, phosphorescent, coloured particle label or spin label.
  • the labelled complex may be detected, for example, visually or with the aid of a spectrophotometer or other detector.
  • a preferred embodiment of the present invention involves the use of a recognition agent, for example an antibody recognising one or more target proteins of the invention, to contact a sample of tissues, cells, blood or body product, or samples derived therefrom, and screening for a positive response.
  • a recognition agent for example an antibody recognising one or more target proteins of the invention
  • the positive response may for example be indicated by an agglutination reaction or by a visualisable change such as a colour change or fluorescence, e.g. immunostaining, or by a quantitative method such as in use of radio-immunological methods or enzyme-linked antibody methods.
  • the method therefore typically includes the steps of (a) obtaining from a patient a tissue sample to be tested for the presence of cancer cells; (b) producing a prepared sample in a sample preparation process; (c) contacting the prepared sample with a recognition agent, such as an antibody, that reacts with the target protein of the invention; and (d) detecting binding of the recognition agent to the target protein, if present, in the prepared sample.
  • a recognition agent such as an antibody
  • the human tissue sample can be from the colon or any other tissue in which tumour-specific over-expression of the appropriate protein can be demonstrated.
  • the sample may further comprise sections cut from patient tissues or it may contain whole cells or it may be, for example, a body fluid sample selected from the group consisting of: blood; serum; plasma; faecal matter; urine; vaginal secretion; breast exudate; spinal fluid; saliva; ascitic fluid; peritoneal fluid; sputum; and colorectal exudate, or an effusion, where the sample may contain cells, or may contain shed antigen.
  • a preferred sample preparation process includes tissue fixation and production of a thin section. The thin section can then be subjected to immunohistochemical analysis to detect binding of the recognition agent to the target protein.
  • the immunohistochemical analysis includes a conjugated enzyme labelling technique.
  • a preferred thin section preparation method includes formalin fixation and wax embedding.
  • Alternative sample preparation processes include tissue homogenisation. When sample preparation includes tissue homogenisation, a preferred method for detecting binding of the antibody to the target protein is Western blot analysis.
  • an immunoassay can be used to detect binding of the antibody to the target protein.
  • immunoassays are antibody capture assays, two-antibody sandwich assays, and antigen capture assays.
  • sandwich immunoassay two antibodies capable of binding the target protein generally are used, e.g. one immobilised onto a solid support, and one free in solution and labelled with a detectable chemical compound.
  • chemical labels that may be used for the second antibody include radioisotopes, fluorescent compounds, spin labels, coloured particles such as colloidal gold and coloured latex, and enzymes or other molecules that generate coloured or electrochemically active products when exposed to a reactant or enzyme substrate.
  • the target protein When a sample containing the target protein is placed in this system, the target protein binds to both the immobilised antibody and the labelled antibody, to form a "sandwich" immune complex on the support's surface.
  • the complexed protein is detected by washing away non-bound sample components and excess labelled antibody, and measuring the amount of labelled antibody complexed to protein on the support's surface.
  • the antibody free in solution which can be labelled with a chemical moiety, for example, a hapten, may be detected by a third antibody labelled with a detectable moiety which binds the free antibody or, for example, the hapten coupled thereto.
  • the immunoassay is a solid support-based immunoassay.
  • the immunoassay may be one of the immunoprecipitation techniques known in the art, such as, for example, a nephelometric immunoassay or a turbidimetric immunoassay.
  • a nephelometric immunoassay or a turbidimetric immunoassay.
  • Western blot analysis or an immunoassay is used, preferably it includes a conjugated enzyme labelling technique.
  • the recognition agent will conveniently be an antibody, other recognition agents are known or may become available, and can be used in the present invention.
  • antigen binding domain fragments of antibodies such as Fab fragments
  • RNA aptamers may be used. Therefore, unless the context specifically indicates otherwise, the term "antibody” as used herein is intended to include other recognition agents. Where antibodies are used, they may be polyclonal or monoclonal.
  • the antibody can be produced by a method such that it recognizes a preselected epitope from the target protein of the invention.
  • the isolated target protein of the invention may be used for the development of diagnostic and other tissue evaluation kits and assays to monitor the level of the proteins in a tissue or fluid sample.
  • the kit may include antibodies or other specific binding moieties which bind specifically to the target protein which permit the presence and/or concentration of the cancer-associated proteins to be detected and/or quantified in a tissue or fluid sample.
  • the invention further provides for the production of suitable kits for detecting the target protein, which may for example include a receptacle or other means for receiving a sample to be evaluated, and a means for detecting the presence and/or quantity in the sample of the target protein of the invention and optionally instructions for performing such an assay.
  • a method of evaluating the effect of a candidate therapeutic drug for the treatment of cancer comprising administering said drug to a patient, removing a cell sample from said patient; and determining the expression profile of the target protein of the invention in said cell sample.
  • This method may further comprise comparing said expression profile to an expression profile of a healthy individual.
  • said patient is receiving treatment for ovarian or colorectal cancer and said cell sample is derived from tissues of the ovary or colon and/or rectum.
  • the present invention provides a method to determine the efficacy of a therapeutic regime at one or more time-points, said method comprising determining a baseline value for the expression of the protein being tested in a given individual within a given tissue such as a tumour, administering a given therapeutic drug, and then redetermining expression levels of the protein within that given tissue at one or more instances thereafter, observing changes in protein levels as an indication of the efficacy of the therapeutic regime.
  • one or more target proteins of the invention provide a mechanism for the selective targeting of anti-cancer drugs based on metabolism by the target protein within tumours.
  • the present invention therefore provides for the design of, or screening for, drugs that undergo specific metabolism in tumours mediated by a target protein of the invention, whereby this metabolism converts a non-toxic moiety into a toxic one, which kills or inhibits the tumour or makes it more susceptible to other agents.
  • a method of treating colorectal cancer is provided, said method comprising use of a drug that is specifically metabolised to an active form by contact with the target protein of the invention.
  • a further aspect of the invention provides for the targeting of cytotoxic drugs or other therapeutic agents, or the targeting of imaging agents, by virtue of their recognition of epitopes derived from the target protein of the invention on the surface of a tumour cell, whether as part of the complete target protein itself or in some degraded form such as in the presentation on the surface of a cell bound to a MHC protein.
  • a further embodiment of the present invention is the development of therapies for treatment of conditions which are characterized by over-expression of one or more target proteins of the invention via immunotherapeutic approaches.
  • the invention provides methods for stimulation of the immune system of cancer patients, for example by activating cytotoxic or helper T-cells which recognise epitopes derived from a target protein of the invention so as to implement a cell-mediated or humoral immune response against the tumour.
  • the activation of the immune system can be achieved by immunisation with sequences derived from the target protein of the invention in an amount sufficient to provoke or augment an immune response.
  • these may be administered as naked peptides, as peptides conjugated or encapsulated in one or more additional molecules (e.g. liposomes) such that a pharmacological parameter (e.g. tissue permeability, resistance to endogenous proteolysis, circulating half-life etc) is improved, or in a suitable expression vector which causes the expression of the sequences at an appropriate site within the body to provoke an immune response.
  • a pharmacological parameter e.g. tissue permeability, resistance to endogenous proteolysis, circulating half-life etc
  • the proteins or peptides may be combined with one or more of the known immune adjuvants, such as saponins, GM-CSF, interleukins, and so forth.
  • Peptides that are too small to generate a sufficient immune response when administered alone can be coupled to one or more of the various conjugates used to stimulate such responses which are well known in the art.
  • peptides which form non-covalent complexes with MHC molecules within cells of the host immune system may be used to elicit proliferation of cytolytic T cells against any such complexes in the subject.
  • Such peptides may be administered endogenously or may be administered to isolated T-cells ex-vivo and then reperfused into the subject being treated.
  • the generation of a host immune response can be accomplished by administration of cells, preferably rendered non ⁇ proliferative by standard methods, which present relevant T cell or B cell epitopes to trigger the required response.
  • the present invention provides for the reduction of the expression level of one or more target proteins in tumour cells, for example by the use of inhibitors or by using antisense RNA or RNA interference methods to decrease the synthesis of the protein. Similarly, this reduction in expression levels could also be achieved by down-regulation of the corresponding gene promoter.
  • a preferred method comprises the step of administering to a patient diagnosed as having cancer, such as colorectal cancer, a therapeutically-effective amount of a compound which reduces in vivo the expression of the target protein.
  • the compound is a polynucleotide, for example, an anti-sense nucleic acid sequence or a peptidyl nucleic acid (PNA), more preferably from 10 to 100 nucleotides in length, capable of binding to and reducing the expression (for example, transcription or translation) of a nucleic acid encoding at least a portion of the target protein of the invention.
  • PNA peptidyl nucleic acid
  • the anti-sense nucleic acid sequence or the anti-sense PNA molecule binds to the nucleic acid sequences encoding, at least in part, the target protein thereby to reduce in vivo expression of the target protein.
  • constructs of the present invention capable of reducing expression of the target protein can be administered to the subject either as a naked polynucleotide or formulated with a carrier, such as a liposome, to facilitate incorporation into a cell.
  • Such constructs can also be incorporated into appropriate vaccines, such as in viral vectors (e.g. vaccinia), bacterial constructs, such as variants of the well known BCG vaccine, and so forth.
  • a particularly useful therapeutic embodiment of the present invention provides an oligonucleotide or peptidyl nucleic acid sequence complementary and capable of hybridizing under physiological conditions to part, or all, of the gene encoding the target protein or to part, or all, of the transcript encoding the target protein thereby to reduce or inhibit transcription and/or translation of the target protein gene.
  • Anti-sense oligonucleotides have been used extensively to inhibit gene expression in normal and abnormal cells. For a recent review, see Phillips, ed., Antisense Technology, in Methods in Enzymology, vols. 313-314, Academic Press; Hartmann, ed., 1999.
  • the synthesis and use of peptidyl nucleic acids as anti-sense-based therapeutics are described by Buchardt & Egholm M in WO9220702 . Accordingly, the anti-sense- based therapeutics may be used as part of chemotherapy, either alone or in combination with other therapies.
  • Double stranded RNA has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire et a/, Nature, 391.: 806-11 ,
  • RNA interference see also Fire, Trends Genet. 15: 358-63, 1999; Sharp, Genes Dev. 15: 485-90, 2001 ; Hammond et al, Nature Rev. Genes 2: 1110-9, 2001 and Tuschl, Chem. Biochem. 2: 239-45, 2001).
  • RNA interference is a two step process.
  • dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23 nucleotides in length with 5 1 terminal phosphate and 3 1 short overhangs ( ⁇ 2 nucleotides)
  • siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore, Nat. Struct. Biol. 8: 746-50, 2001)
  • the invention provides double stranded RNA comprising a sequence encoding a target protein of the present invention, which may for example be a "long" double stranded RNA (which will be processed to siRNA, e.g., as described above).
  • RNA products may be synthesised in vitro, e.g., by conventional chemical synthesis methods.
  • RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'-overhang ends (Zamore et a/, Cell 101: 25-33, 2000).
  • siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir et a/, Nature AVV. 494-8, 2001).
  • siRNA duplexes containing between 20 and 25 bps, more preferably between 21 and 23 bps, of the sequence encoding a target protein of the present invention form one aspect of the invention e.g. as produced synthetically, optionally in protected form to prevent degradation.
  • siRNA may be produced from a vector, in vitro (for recovery and use) or in vivo.
  • the vector may comprise a nucleic acid sequence encoding a target protein of the present invention (including a nucleic acid sequence encoding a variant or fragment thereof), suitable for introducing an siRNA into the cell in any of the ways known in the art, for example, as described in any of references cited herein, which references are specifically incorporated herein by reference.
  • the vector may comprise a nucleic acid sequence according to the invention in both the sense and antisense orientation, such that when expressed as RNA the sense and antisense sections will associate to form a double stranded RNA.
  • This may for example be a long double stranded RNA (e.g., more than 23 nucleotides) which may be processed in the cell to produce siRNAs (see for example Myers, Nat. Biotechnol. 21: 324-8, 2003).
  • the double stranded RNA may directly encode the sequences which form the siRNA duplex, as described above.
  • the sense and antisense sequences are provided on different vectors.
  • vectors and RNA products may be useful for example to inhibit de novo production of the protein of the present invention in a cell. They may be used analogously to the expression vectors in the various embodiments of the invention discussed herein.
  • double-stranded RNA which comprises an RNA sequence encoding a target protein of the present invention or a fragment thereof, which may be an siRNA duplex consisting of between 20 and 25 bps.
  • vectors encoding said dsRNA or siRNA duplexes.
  • methods of producing said siRNA duplexes comprising introducing such vectors into a host cell and causing or allowing transcription from the vector in the cell.
  • Separate vectors may encode: (i) the sense sequence of the siRNA duplex, and (ii) the anti-sense sequence of the siRNA duplex.
  • An additional DNA based therapeutic approach provided by the present invention is the use of a vector which comprises one or more nucleotide sequences, preferably a plurality of these, each of which encodes an immunoreactive peptide derived from the target protein of the invention.
  • a further method of the invention involves combining one or more of these nucleotide sequences encoding peptides derived from the target protein of the invention in combination with nucleotide sequences encoding peptides derived from other tumour markers known in the art to be expressed by cancer cells, and encompasses inclusion of such sequences in all possible variations, such as one from each protein, several from one or more protein and one from each of one or more additional proteins, and so forth.
  • a further aspect of the present invention provides novel methods for screening for compositions that modulate the expression or biological activity of the target protein of the invention.
  • biological activity means any observable effect resulting from interaction between the target protein and a ligand or binding partner.
  • Representative, but non-limiting, examples of biological activity in the context of the present invention include association of the target protein of the invention with a ligand.
  • biological activity also encompasses both the inhibition and the induction of the expression of the target protein of the invention.
  • biological activity encompasses any and all effects resulting from the binding of a ligand or other in vivo binding partner by a polypeptide derivative of the protein of the invention.
  • a method of screening drug candidates comprises providing a cell that expresses the target protein of the invention, adding a candidate therapeutic compound to said cell and determining the effect of said compound on the expression or biological activity of said protein.
  • the method of screening candidate therapeutic compounds includes comparing the level of expression or biological activity of the protein in the absence of said candidate therapeutic compound to the level of expression or biological activity in the presence of said candidate therapeutic compound. Where said candidate therapeutic compound is present its concentration may be varied, and said comparison of expression level or biological activity may occur after addition or removal of the candidate therapeutic compound.
  • the expression level or biological activity of said target protein may show an increase or decrease in response to treatment with the candidate therapeutic compound.
  • the candidate therapeutic compound is a substance capable of inhibiting the biological activity of a target protein of the invention, thereby preventing said protein from inactivating, detoxifying or otherwise modifying an anti-cancer drug (or a pro ⁇ drug form thereof, or a metabolic product of an anti-cancer drug or pro-drug) such that it loses some or all of its anti-cancer activity (e.g. cytotoxicity to cancer cells).
  • the candidate therapeutic compound is a substance capable of inhibiting the biological activity of a target protein of the invention such that it prevents that protein from catalysing the formation of carcinogenic metabolites of endogenous or exogenous procarcinogenic compounds.
  • the candidate therapeutic compound is a substance capable of inhibiting the biological activity of a target protein of the invention such that it blocks the contribution of that protein to an endogenous metabolic process that favours tumour formation or persistence.
  • Preferred inhibitors are selective, that is they inhibit the activity of a given target protein of the invention under the conditions extant within tumour cells while having a reduced inhibitory effect, or more preferably substantially no effect, on the function of endogenous enzymes present in normal cells.
  • Candidate therapeutic molecules of the present invention may include, by way of example, peptides produced by expression of an appropriate nucleic acid sequence in a host cell or using synthetic organic chemistries, or non-peptide small molecules produced using conventional synthetic organic chemistries well known in the art. Screening assays may be automated in order to facilitate the screening of a large number of small molecules at the same time.
  • candidate therapeutic compound refers to a substance that is believed to interact with a target protein of the invention (or a fragment thereof), and which can be subsequently evaluated for such an interaction.
  • candidate therapeutic compounds include "xenobiotics", such as drugs and other therapeutic agents, natural products and extracts, carcinogens and environmental pollutants, as well as “endobiotics” such as steroids, fatty acids and prostaglandins.
  • endobiotics such as steroids, fatty acids and prostaglandins.
  • candidate compounds that can be investigated using the methods of the present invention include, but are not restricted to, agonists and antagonists of the target protein of the invention, toxins and venoms, viral epitopes, hormones (e.
  • the present invention provides a method of drug screening utilising eukaryotic or prokaryotic host cells stably transformed with recombinant polynucleotides expressing a target protein of the invention or a fragment thereof, preferably in competitive binding assays.
  • Such cells either in viable or fixed form, can be used for standard binding assays.
  • the assay may measure the formation of complexes between a target protein and the agent being tested, or examine the degree to which the formation of a complex between the target protein or fragment thereof and a known ligand or binding partner is interfered with by the agent being tested.
  • the present invention provides methods of screening for drugs comprising contacting such an agent with a target protein of the invention or a fragment thereof or a variant thereof found in a tumour cell and assaying (i) for the presence of a complex between the agent and the target protein, fragment or variant thereof, or (ii) for the presence of a complex between the target protein, fragment or variant and a ligand or binding partner.
  • the target protein or fragment or variant is typically labelled.
  • Free target protein, fragment or variant thereof is separated from that present in a protein: protein complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to the target protein or its interference with binding of the target protein to a ligand or binding partner, respectively.
  • an assay of the invention may measure the influence of the agent being tested on a biological activity of a target protein of the invention.
  • the present invention provides methods of screening for drugs comprising contacting such an agent with the target protein of the invention or a fragment thereof or a variant thereof found in a tumour cell and assaying for the influence of such an agent on a biological activity of the target protein, by methods well known in the art.
  • the biological activity of the target protein, fragment or variant thereof is typically monitored by provision of a reporter system. For example, this may involve provision of a natural or synthetic substrate that generates a detectable signal in proportion to the degree to which it is acted upon by the biological activity of the target molecule.
  • rational drug design methodologies well known in the art may be employed to enhance their efficacy.
  • the goal of rational drug design is to produce structural analogues of biologically active polypeptides of interest or of small molecules with which they interact (e. g. agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, for example, enhance or interfere with the function of a polypeptide in vivo.
  • a protein of interest such as a target protein of the invention or, for example, of the target protein in complex with a ligand
  • computer modelling or most typically, by a combination of approaches.
  • the skilled artisan may use a variety of computer programmes which assist in the development of quantitative structure activity relationships (QSAR) that act as a guide in the design of novel, improved candidate therapeutic molecules.
  • QSAR quantitative structure activity relationships
  • useful information regarding the structure of a polypeptide may be gained by modelling based on the structure of homologous proteins.
  • peptides can be analysed by alanine scanning (Wells, Methods Enzymol.
  • each amino acid residue of the peptide is sequentially replaced by an alanine residue, and its effect on the peptide's activity is determined in order to determine the important regions of the peptide.
  • drugs based on a pharmacophore derived from the crystal structure of a target-specific antibody selected by a functional assay. It is further possible to avoid the use of protein crystallography by generating anti-idiotypic antibodies to such a functional, target-specific antibody, which have the same three-dimensional conformation as the original target protein. These anti-idiotypic antibodies can subsequently be used to identify and isolate peptides from libraries, which themselves act as pharmacophores for further use in rational drug design.
  • candidate therapeutic compounds so identified may be combined with a suitable pharmaceutically acceptable carrier, such as physiological saline or one of the many other useful carriers well characterized in the medical art.
  • a suitable pharmaceutically acceptable carrier such as physiological saline or one of the many other useful carriers well characterized in the medical art.
  • Such pharmaceutical compositions may be provided directly to malignant cells, for example, by direct injection, or may be provided systemically, provided the formulation chosen permits delivery of the therapeutically effective molecule to tumour cells containing a target protein of the invention. Suitable dose ranges and cell toxicity levels may be assessed using standard dose ranging methodology. Dosages administered may vary depending, for example, on the nature of the malignancy, the age, weight and health of the individual, as well as other factors.
  • a further aspect of the present invention provides for cells and animals which express one or more target proteins of the invention and can be used as model systems to study and test for substances which have potential as therapeutic agents.
  • Such cells may be isolated from individuals with mutations, either somatic or germline, in the gene encoding the target protein of the invention, or can be engineered to express or over-express the target protein or a variant thereof, using methods well known in the art. After a test substance is applied to the cells, any relevant trait of the cells can be assessed, including by way of example growth, viability, tumourigenicity in nude mice, invasiveness of cells, and growth factor dependence, assays for each of which traits are known in the art.
  • Animals for testing candidate therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. As discussed in more detail below, by way of example, such treatments can include insertion of genes encoding the target protein of the invention in wild-type or variant form, typically from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous target protein gene(s) of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques that are well known in the art. After test substances have been administered to the animals, the growth of tumours can be assessed.
  • test substance prevents or suppresses the growth of tumours
  • test substance is a candidate therapeutic agent for the treatment of those cancers expressing the target protein of the invention, for example of colorectal cancers.
  • target protein of the invention for example of colorectal cancers.
  • the present invention thus provides a transgenic non-human animal, particularly a rodent, which comprises an inactive copy of the gene encoding one or more target proteins of the present invention.
  • the invention further provides a method of testing a putative therapeutic of the invention which comprises administering said therapeutic to an animal according to the invention and determining the effect of the therapeutic.
  • reference to an inactive copy of the gene encoding a target protein of the present invention includes any non-wild-type variant of the gene which results in knock out or down regulation of the gene, and optionally in a cancer phenotype.
  • the gene may be deleted in its entirety, or mutated such that the animal produces a truncated protein, for example by introduction of a stop codon and optionally upstream coding sequences into the open reading frame of the gene encoding a target protein of the present invention.
  • the open reading frame may be intact and the inactive copy of the gene provided by mutations in promoter regions.
  • inactivation of the gene may be made by targeted homologous recombination.
  • a typical strategy is to use targeted homologous recombination to replace, modify or delete the wild-type gene in an embryonic stem (ES) cell.
  • a targeting vector comprising a modified target gene is introduced into ES cells by electroporation, lipofection or microinjection.
  • the targeting vector pairs with the cognate chromosomal DNA sequence and transfers the desired mutation carried by the vector into the genome by homologous recombination. Screening or enrichment procedures are used to identify the transfected cells, and a transfected cell is cloned and maintained as a pure population.
  • the altered ES cells are injected into the blastocyst of a preimplantation mouse embryo or alternatively an aggregation chimera is prepared in which the ES cells are placed between two blastocysts which, with the ES cells, merge to form a single chimaeric blastocyst.
  • the chimaeric blastocyst is surgically transferred into the uterus of a foster mother where the development is allowed to progress to term.
  • the resulting animal will be a chimera of normal and donor cells.
  • the donor cells will be from an animal with a clearly distinguishable phenotype such as skin colour, so that the chimaeric progeny is easily identified.
  • the progeny is then bred and its descendants cross-bred, giving rise to heterozygotes and homozygotes for the targeted mutation.
  • the production of transgenic animals is described further by Capecchi, Science 244: 1288- 92,1989; Valancius & Smithies, MoI. Cell. Biol. I V. 1402-8, 1991 ; and Hasty et al, Nature 350: 243-6, 1991 , the disclosures of which are incorporated herein by reference.
  • Homologous recombination in gene targeting may be used to replace the wild-type gene encoding a target protein of the present invention with a specifically defined mutant form (e.g. truncated or containing one or more substitutions).
  • the inactive gene may also be one in which its expression may be selectively blocked either permanently or temporarily. Permanent blocking may be achieved by supplying means to delete the gene in response to a signal.
  • An example of such a means is the cre-lox system where phage lox sites are provided at either end of the transgene, or at least between a sufficient portion thereof (e.g. in two exons located either side or one or more introns). Expression of a ere recombinase causes excision and circularisation of the nuclei acid between the two lox sites.
  • mice Various lines of transgenic animals, particularly mice, are currently available in the art which express ere recombinase in a developmentally or tissue restricted manner (see for example Tsien, Cell 87: 1317-26, 1996; Betz, Current Biology 6: 1307-16,1996). These animals may be crossed with lox transgenic animals of the invention to examine the function of the gene encoding a target protein of the present invention.
  • An alternative mechanism of control is to supply a promoter from a tetracycline resistance gene, tet, to the control regions of the target gene locus such that addition of tetracycline to a cell binds to the promoter and blocks expression of the gene encoding a target protein of the present invention.
  • GAL4, VP16 and other transactivators could be used to modulate gene expression including that of a transgene containing the gene encoding a target protein of the present invention.
  • the target gene could also be expressed in ectopic sites, that is, in sites where the gene is not normally expressed in time or space.
  • Transgenic targeting techniques may also be used to delete the gene encoding a target protein of the present invention. Methods of targeted gene deletion are described by Brenner et al, WO94/21787 (Cell Genesys), the disclosure of which is incorporated herein by reference.
  • a non-human animal which expresses the gene encoding a target protein of the present invention at a higher than wild-type level.
  • the gene encoding a target protein of the present invention is expressed at least 120-200% of the level found in wild-type animals of the same species, when cells which express the gene are compared.
  • this gene could be expressed in an ectopic location where the target gene is not normally expressed in time or space. Comparisons may be conveniently done by northern blotting and quantification of the transcript level.
  • the higher level of expression may be due to the presence of one or more, for example two or three, additional copies of the target gene or by modification to the gene encoding a target protein of the present inventions to provide over-expression, for example by introduction of a strong promoter or enhancer in operable linkage with the wild-type gene.
  • the provision of animals with additional copies of genes may be achieved using the techniques described herein for the provision of "knock-out" animals.
  • animals are provided in which the gene encoding a target protein of the present invention is expressed at an ectopic location.
  • the gene is expressed in a location or at a time during development which does not occur in a wild- type animal.
  • the gene may be linked to a developmentally regulated promoter such as Wnt-1 and others (Echeland et al, Development
  • Non-human mammalian animals include non-human primates, rodents, rabbits, sheep, cattle, goats, pigs. Rodents include mice, rats, and guinea pigs. Amphibians include frogs. Fish such as zebra fish, may also be used.
  • Transgenic non-human mammals of the invention may be used for experimental purposes in studying cancer, and in the development of therapies designed to alleviate the symptoms or progression of cancer. By “experimental” it is meant permissible for use in animal experimentation or testing purposes under prevailing legislation applicable to the research facility where such experimentation occurs.
  • Figure 1 Mean cytochrome P450 intensity scores in normal colon, colon cancer and lymph node metastasis.
  • Figure 2 lmmunohistochemical staining of CYP51 in colorectal tissues. lmmunolocalisation of cytochrome P450 CYP51 in normal colon (A), primary colorectal cancer showing strong CYP51 immunoreactivity (B), no CYP51 immunostaining (C) and strong CYP51 immunoreactivity in a lymph node metastasis (D).
  • Figure 3 Correlation of patient survival and CYP51 expression in colorectal tumours.
  • Figure 5 Mean Cytochrome P450 intensity scores in normal ovary, primary cancer and metastatic cancer.
  • Figure 6 Kaplan-Meier survival analysis of CYP4Z1 negative and positive patient cohorts. Comparison of survival in patients whose tumours exhibited detectable CYP4Z1 immunoreactivity (of strong, moderate or poor intensity) and those tumours in which
  • Figure 7 lmmunoblot (western blot) using the CYP51 polyclonal antibody.
  • the primary antibody was diluted 1/1000 with 1% skimmed milk/PBS/Tween and the secondary antibody diluted 1/2000 with 5% skimmed milk/PBS/Tween.
  • Figure 8 CYP51 RT-PCR results. RT-PCR results of A. CYP51(1) and B. CYP51(2) in colorectal tumours and paired normal tissue (normal text: normal tissue; bold text: tumour tissue); quantitative comparison of C. CYP51(1) and D. CYP51(2) expression normalized for the amount of RPS13 (black: normal tissue; grey: tumour tissue).
  • SEQ ID NO 1 human Cytochrome P450 51 (NCBI RefSeq NP_000777)
  • SEQ ID No 2 human Cytochrome P450 2S1 (NCBI RefSeq NP_085125)
  • SEQ ID No 3 human Cytochrome P450 2U1 (NCBI RefSeq NP_898898)
  • SEQ ID No 4 human Cytochrome P450 26A1 (NCBI RefSeq NP_000774)
  • SEQ ID No 5 human Cytochrome P450 4Z1 (NCBI RefSeq NP_835235)
  • SEQ ID No 6 human Cytochrome P450 3A43 (NCBI RefSeq NP_073731)
  • SEQ ID No 7 human Cytochrome P450 4F11 (NCBI RefSeq NPJ367010)
  • SEQ ID No 8 human Cytochrome P450 4X1 (NCBI RefSeq 8288
  • Cytochrome P450 isoenzymes exhibiting differential expression in clinically resected colorectal tumours and normal colon tissues were identified as follows;
  • Tumour samples Tissues from 264 donor patients were selected from the ACCRI colorectal tumour bank with the permission of the Grampian Research Ethics Committee. All cases had a diagnosis of primary colorectal cancer and had undergone elective surgery for colorectal cancer, in Aberdeen, between 1994 and 2003. All the tumour samples had been submitted to the Department of Pathology, University of Aberdeen for diagnosis. The tumour excision specimens had been fixed in formalin and representative blocks embedded in wax and sections stained with haematoxylin and eosin. The clinico- pathological characteristics (age, gender, site of primary tumour, degree of primary tumour differentiation, Dukes stage) of the patients included in this study are detailed in Table 1. Complete follow-up was available for all patients and ranged from 1 month to
  • the lymph node metastases were from the corresponding Dukes C cases (adequate nodal metastatic tissue to sample was not available in 2 cases).
  • a single representative 1.6 mm core of tissue was taken from each donor block using a steel Menghini needle and arrayed into the recipient wax block.
  • One section from each microarray was stained with haematoxylin and eosin to confirm the histopathological findings.
  • Antibodies A panel of 22 antibodies directed against P450 enzymes were used in this study, as shown in Table 2. Polyclonal antibodies to individual P450s (CYP2F1 , CYP2J2, CYP2R1 CYP2S1 , CYP2U1 , CYP3A43, CYP4V2, CYP4X1 , CYP4Z1 , CYP24, CYP39and CYP51) were produced by immunising rabbits with the relevant C-terminal peptide (detailed in Table 2) conjugated to ovalbumin. Animals received 2 booster immunisations at 4-6 week intervals after the initial immunisation. Animals were bled 7-10 days after the last injection and periodically thereafter and serum obtained by centrifugation of the clotted blood.
  • CYP3A4 Monoclonal antibodies to CYP2A6, CYP3A5, CYP3A7, CYP4F11 and CYP26A1 were produced by the same methods. In each case the appropriate C-terminal peptide conjugated to ovalbumin was used as the immunogen. Briefly, mice were immunised with the relevant peptide conjugate and received booster immunisations. Spleens from those mice which showed the highest antibody titres as assessed by ELISA using the peptide immunogen were fused with myeloma cells.
  • the CYP2A antibody recognised both CYP2A6 and CYP2B6 reflecting the very close sequence similarity of these two P450s and the almost identical C-terminal amino acid sequences of these P450s. This antibody has therefore been designated CYP2A/CYP2B.
  • Polyclonal antibodies to CYP1A1 and CYP2C9 were purchased from Chemicon Europe (Chandlers Ford, UK) while a monoclonal antibody to CYP2D6 was bought from Gentest and a polyclonal antibody to CYP2E1 was obtained from Oxford Biomedical Research (Oxford, Ml).
  • lmmunohistochemistry lmmunohistochemistry for each P450 was carried out using a Dako autostainer (DakoCytomation, Ely, UK). Sections of the tissue microarray were dewaxed and rehydrated according to standard methods and an antigen retrieval step was performed when required.
  • the antigen retrieval step consisted of microwaving the sections in 0.01 M citrate buffer at pH6.0 for 20 minutes in 800W microwave oven operated at full power. The sections were then allowed to cool to room temperature.
  • Primary antibody appropriately diluted (Table 2) was applied for 60 minutes at room temperature, following by washing with buffer (DakoCytomation) followed by peroxidase blocking for 5 minutes (Dako) followed by a single 2 minute buffer wash.
  • Pre-diluted peroxidase-polymer labelled goat anti-mouse/rabbit secondary antibody (EnvisionTM, DakoCytomation) was applied for 30 minutes at room temperature, followed by further washing with buffer to remove unbound antibody. Sites of peroxidase activity were then demonstrated with diaminobenzidine as the chromogen applied for 3 successive 5 minute periods. Finally the sections were washed in water, lightly counterstained with haematoxylin, dehydrated and mounted. Negative controls were performed by omitting the primary antibody.
  • the sections were evaluated by microscopic examination and the intensity of immunostaining in each section assessed by two clinical pathologists. Discrepancies were resolved by simultaneous re-evaluation. The intensity of immunostaining was scored as zero (negative), 1 (weak), 2 (moderate) or 3 (strong).
  • CYP2E1 commercial PAb Expressed human
  • CYP2F1 own lab PAb C-terminal peptide, RPFQLCLRPR 20 min, 1/1000
  • CYP2R1 own lab PAb C-terminal peptide, QPYLICAERR 20 min, 1/1000
  • CYP2S1 own lab PAb C-terminal peptide, TDLHSTTQTR 20 min, 1/1000
  • CYP2U1 own lab PAb C-terminal peptide, HPPFNITISRR 20 mm, 1/1000
  • CYP3A4 own lab MAb Purified human CYP3A4 20 min, undiluted supernatant
  • CYP3A5 own lab MAb C-terminal peptide, DSRDGTLSGE 20 min, undiluted supernatant
  • CYP3A7 own lab MAb C-terminal peptide, ESRDETVSGA 20 min, undiluted supernatant
  • CYP4V2 own lab PAb C-terminal peptide, KLKRRNADER 20 min , 1/1000
  • CYP4X1 own lab PAb C-terminal peptide, NGMYLHLKKL 20 min , 1/1000
  • CYP4Z1 own lab PAb C-terminal peptide, NGIHVFAKKV 20min, 1/1000
  • Cytochrome P450 isoenzymes exhibiting differential expression in clinically resected ovarian tumours and normal ovary tissues was identified as follows;
  • the ovarian tissue assessed in this study was submitted to the Department of Pathology, University of Aberdeen, over a 5-year period from 1993 to 1998. Analysis was restricted to 115 patients with endometrioid, mucinous and serous histological classifications. Information regarding age, FIGO stage of disease, survival, and patient status was available for each case. Specimens had been fixed in formalin, embedded in wax and paraffin sections were stained with haematoxylin and eosin. The clinico-pathological characteristics (age, tumour type and stage) of the patients included in this study are detailed in Table 8. At the time of the last follow-up, 62.5% of patients had died from their disease. The median survival of the patients was 32 months, with the survival range between 0 to 126 months.
  • TMAs Tissue Microarrays
  • Core tissue specimens [1.6mm] were removed from selected areas of tumour and normal donor blocks and precisely arrayed into recipient paraffin blocks. The presence of tumour tissue arrayed was verified on a haematoxylin and eosin stained section. The blocks contained 9 positive controls [lung, liver and kidney], 99 ovarian tumour tissue samples, 22 metastatic ovarian cases and 13 normal ovarian tissue cores.
  • ovarian tissues used the same panel of antibodies as described in experiment 2.
  • Microarray blocks were sectioned (5 ⁇ M) onto glass slides. Slides were de-waxed in xylene and antigen retrieval carried out for 20 minutes in citrate buffer (0.01 M, pH 6) in an 800W microwave oven, lmmunohistochemistry was carried out using the DAKO flatbed autostainer (DakoCytomation, Ely, UK). Immunoreactivity was identified using the high sensitivity EnVision method (DakoCytomation). Negative controls used antibody diluent (DakoCytomation) in place of primary antibody. Sections were lightly counterstained with haematoxylin, dehydrated, mounted and examined using bright field light microscopy. Immunoreactivity within the sections was assessed as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong).
  • the present invention discloses the over-expression of eight cytochrome P450 isoenzymes in colorectal and/or ovarian cancer in comparison with comparable normal tissue. To determine whether these enzymes have potential use as cancer markers in tumours derived from other tissues, their expression was examined in a tumour tissue microarray containing samples from a range of cancers.
  • the aim of this study was to analysis various different cell lines for the presence of CYP51 protein.
  • the smaller splice form of CYP51 appears to be present in all the cell lines examined ( Figure 7).
  • the concentration of this splice form appears to vary between the cell lines with, cell line A498 expressing the lowest concentration amount and A2058 the highest amount. It would also appear that the larger splice form of CYP51 is absent from all the cells lines examined.
  • EXAMPLE 5 The aim of this study was to validate CYP51 , which had been shown to be expressed in colorectal tumour samples, using semi-quantitative RT-PCR.
  • First strand cDNA was used as a template in RT-PCR reactions to detect transcription levels of CYP51 in these tissues.
  • Gene-specific primers (0.01 nM) were designed and used in the separate reactions (Table 14). 0.5 ⁇ l_ of the forward and reverse primers for each gene was added to a PCR 'mastermix' containing 20 ⁇ L of ABgene Reddymix PCR Master Mix (ABgene), 1.25 ⁇ L DMSO (Sigma), 1.75 ⁇ L dH 2 O (Sigma) and 1 ⁇ L of the corresponding cDNA template.
  • PCR analysis of CYP51 transcription levels of two variants of CYP51 was carried out.
  • CYP51 (2) differs from CYP51 (1) in its 5' untranslated region.
  • Variant 2 codes for a protein missing 105 amino acids, and these missing amino acids correspond to a transmembrane region.
  • Primers CYP51F1 and CYP51R1(1264) were used to amplify variant 1 transcripts in a PCR reaction with a denaturing step at 95 0 C for 30 s, an annealing step at 52 0 C for 30 s and an extention step at 72 0 C for 1 min 15 s for 52 cycles.
  • Primers CYP51 F1 , CYP51 R(1099) and CYP51 R(1502) were used to amplify variant 2 in a PCR reaction of 44 cycles with parameters only differing from those for variant 1 in the extention time of 1 min.
  • RPS13 was used as an internal control to normalise samples. Denaturing occurred at
  • CYP51 (1) /RPS13 and CYP51 (2)/RPS13 was calculated using Gene Tools version 3.05 from SynGene. ldentification of CYP51 expression in colorectal carcinoma
  • Hoxb2 in rhombomere 4 is regulated by Hoxbl Genes & Dev. IJ.: 1885-95, 1997
  • RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.

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Abstract

L'invention concerne des marqueurs jusque-là non caractérisés de cancers, par exemple des cancers de l'ovaire et colorectal, ainsi que leurs utilisations en tant que marqueurs diagnostiques et pronostiques de cancers, en particulier des cancers de l'ovaire et colorectal. Ces marqueurs sont les enzymes cytochromes p450: CYP51, CYP2S1, CYP2U1, CYP3A43, CYP4F11, CYP4X1, CYP4Z1, CYP26A1, CYP3A7, CYP2F1, CYP4V2 et CYP39. L'invention concerne également des méthodes et des matériaux associés d'utilisation desdits marqueurs dans le cadre d'une intervention thérapeutique pour les cancers de l'ovaire, colorectal et autres, par exemple pour cibler spécifiquement des cellules néoplasiques sans causer de toxicité significative dans les tissus sains, et pour fournir des méthodes permettant d'évaluer la capacité de composés thérapeutiques candidats à moduler l'activité biologique de cellules cancéreuses provenant de l'ovaire, du colon, du rectum ou d'autres tissus.
PCT/GB2005/003813 2004-10-02 2005-10-03 Marqueurs cancereux WO2006037993A2 (fr)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
GB0421919A GB0421919D0 (en) 2004-10-02 2004-10-02 Cytochrome P450 prodrug target
GB0421913.5 2004-10-02
GB0421917A GB0421917D0 (en) 2004-10-02 2004-10-02 Cancer diagnosis and therapy
GB0421919.2 2004-10-02
GB0421914A GB0421914D0 (en) 2004-10-02 2004-10-02 Drug metabolising enzyme in cancer
GB0421913A GB0421913D0 (en) 2004-10-02 2004-10-02 Tumour-specific pro drug target
GB0421917.6 2004-10-02
GB0421914.3 2004-10-02
GB0421920A GB0421920D0 (en) 2004-10-02 2004-10-02 Cytochrome P450 enzymes and cancer
GB0421918.4 2004-10-02
GB0421918A GB0421918D0 (en) 2004-10-02 2004-10-02 Cancer therapeutic target
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WO2009003706A3 (fr) * 2007-06-29 2009-02-26 Bayer Schering Pharma Ag Procédés, trousses et composés pour la détermination d'une sensibilité à un traitement d'un trouble pathologique par épothilones
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