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WO2004072285A1 - Polypeptides associes au cancer « goblin », reactifs associes, et procedes d'utilisation associes - Google Patents

Polypeptides associes au cancer « goblin », reactifs associes, et procedes d'utilisation associes Download PDF

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Publication number
WO2004072285A1
WO2004072285A1 PCT/AU2004/000169 AU2004000169W WO2004072285A1 WO 2004072285 A1 WO2004072285 A1 WO 2004072285A1 AU 2004000169 W AU2004000169 W AU 2004000169W WO 2004072285 A1 WO2004072285 A1 WO 2004072285A1
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Prior art keywords
seq
goblin
sequence
cancer
polypeptide
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Prudence STANFORD
Jessica Harris
Christopher Ormandy
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Garvan Institute of Medical Research
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Garvan Institute of Medical Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • This invention relates to novel nucleic acid molecules and polypeptides present in healthy and neoplastic cells, including fragments, variants and derivatives ofthe nucleic acids and polypeptides.
  • the nucleic acids and/or polypeptides ofthe present invention are expressed highly in malignant tissues and are particularly useful for the diagnosis of cancer or malignant tumors in human subjects.
  • the present invention also relates to antibodies to the polypeptides of the invention, as well as methods for identifying agonists and/or antagonists of the polypeptides of the invention.
  • the invention also relates to compositions comprising the nucleic acids, polypeptides, antibodies, variants, and derivatives of the invention and methods for the use of these compositions.
  • These uses include identifying, diagnosing, monitoring, staging, imaging and treating malignancies and non-cancerous disease states in human tissues,
  • the uses also include therapeutic applications of such compositions, including gene therapy, the production of transgenic animals and cells, and production of engineered human cells ( and tissues for treatment and research.
  • this invention relates to cancer and the use of nucleic acid or antibody probes to specifically detect over-expression of a gene encoding ⁇ a GOBLIN polypeptide in cancer cells, such as, for example, tumorigenic mammary epithelial cells (eg., cancer cells of the ectoderm, duct, lobuloalveolar compartment, ductule, lobule, alveolus, secretory epithelium, or myoepithelium of the mammary gland), which over- expression is found in cell lines derived from tumors.
  • tumorigenic mammary epithelial cells eg., cancer cells of the ectoderm, duct, lobuloalveolar compartment, ductule, lobule, alveolus, secretory epithelium, or myoepithelium of the mammary gland
  • the diagnostic and prognostic test of the present invention is particularly useful for the detection of ovarian cancer or mammary cancer or metastases thereof, and for monitoring the progress of disease, such as, for example, during remission or following surgery or chemotherapy.
  • the present invention is also directed to methods of therapy wherein GOBLIN gene expression is modulated.
  • nucleotide and amino acid sequence information prepared using Patentln Version 3.1, presented herein after the claims.
  • Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (e.g. ⁇ 210>1, ⁇ 210>2, ⁇ 210>3, etc).
  • the length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213>, respectively.
  • Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as ⁇ 400>1).
  • nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
  • derived from shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • the present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference:
  • Bodanszky M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer- Verlag, Heidelberg.
  • the mammary gland consists of two cellular compartments, the epithelium and surrounding stro a.
  • the epithelium is derived embryonically from ectoderm, and comprises: (i) a branched ductal system (ducts branching into ductules, and terminating in lobules comprising alveoli that consist of secretory epithelium, and surrounded by contractile myoepithelium) that mainly develops during puberty; and (ii) the lobuloalveolar compartment that develops during pregnancy.
  • Receptors for estrogen, progesterone and prolactin, the Stat5 transcription factors, cyclinDl, and the family of activins and inhibins are required for the establishment of functional mammary tissue. Mammary development is reviewed in detail by Hennighausen and Robinson, Devel. Cell, 1, 1-20, 2001, which is incorporated herein in its entirety by way of reference.
  • the secretory epithelium of the ductal system undergoes functional differentiation during parturition.
  • the secretory compartment arises from stem cells during each pregnancy, produces milk during lactation, and is fully remodelled after weaning ofthe young. This remodelling is accompanied by the loss ofthe entire secretory epithelium.
  • prolactin In healthy mammary glands, proliferation and differentiation ofthe secretory mammary epithelium requires prolactin, a prolactin receptor (PrlR) and an operable Jak2/Stat5 signalling pathway (Ormandy et al., Genes Devel. II, 167-178, 1997; Liu et al., Genes Devel. 11, 179-186, 1997). Briefly, binding of prolactin or placental lactogen to PrlR induces receptor dimerization, leading to tyrosine phosphorylation of PrlR by Jak2. Subsequently, the transcription factors Stat5a and Stat5b are recruited by their SH2 domains to the receptor where they are also phosphorylated by Jak2.
  • mice deficient in one of both Stat5 transcription factors have arrested mammary gland development, including impaired alveolar proliferation and functional differentiation (Liu et al, Genes Devel. 11, 179-186, 1997; Liu et al, Cell. Growth Differ. 9, 795-803, 1998; Miyoshi et al, J. Cell. Biol, 2001; Teglund et al, Cell 93, 841-850, 1998). Additionally, The effects of prolactin on cell growth are synergistic with the effects of progesterone, which appears to act, in part, by increasing the level of PrlR.
  • stage TO early stage
  • stage T4 the five-year survival rate
  • Breast cancers, or mammary gland tumors may consist of lobular lesions, stromal lesions, ductal carcinoma (non-invasive ductal carcinoma or invasive ductal carcinoma), proliferative fibrocystic changes, or epitheliosis.
  • Intraductal papilloma and/or atypical ductal hyperplasia are considered to be precursors to ductal carcinomas.
  • Atypical ductal hyperplasia predicts a 4 fold increased relative risk for subsequent invasive ductal adenocarcinoma.
  • the term "breast cancer” shall be taken to include any one or more of these lesions, carcinomas or precursors, or a metastases thereof internal or external to the mammary gland.
  • Cancer diagnosis generally depends upon recognition of large-scale morphologic alterations of cells and tissues of the mammary gland by microscopic techniques, particularly light microscopy.
  • proliferative fibrocystic changes, ductal hyperplasia, and epitheliosis are characterized generally by enhanced number of ductal cells, as a consequence of their proliferation as a multilayered epithelium.
  • intraductal papillomas the ductal cells appear to proliferate along with an increase in the stromal fibroblastic compartment.
  • atypical ductal hyperplasia can overlap with those of proliferative ductal lesions at one end of a spectrum, and with intraductal carcinoma at the other end, with the ductal cells appearing to have random relative orientation and similar cytology irrespective of their location (i.e. near the basal lamina or myoepithelium).
  • intraductal carcinoma also known as ductal carcinoma in situ (DCIS)
  • DCIS ductal carcinoma in situ
  • the ductal cells have no dependence on the basal lamina environment for trophic support and grow equally well at any polarity anywhere within the duct.
  • invasive ductal carcinoma the ductal cells appear able to induce a new trophic relationship with the stromal fibroblasts and blood vessels outside the duct.
  • Some detection techniques such as mammography and biopsy, involve increased discomfort, expense, and/or radiation, and are only prescribed only to patients with an increased risk of breast cancer.
  • a patient's risk of breast cancer has been positively associated with increasing age, nuUiparity, family history of breast cancer, personal history of breast cancer, early menarche, late menopause, late age of first full term pregnancy, prior proliferative breast disease, irradiation of the breast at an early age and a personal history of malignancy.
  • Lifestyle factors such as fat consumption, alcohol consumption, education, and socioeconomic status have also been associated with an increased incidence of breast cancer although a direct cause and effect relationship has not been established. While these risk factors are statistically significant, their weak association with breast cancer limited their usefulness. Most women who develop breast cancer have none of the risk factors listed above, other than the risk that comes with growing older.
  • the current methods of breast cancer prevention involve prophylactic mastectomy (mastectomy performed before cancer diagnosis) and chemoprevention (chemotherapy before cancer diagnosis) which are drastic measures that limit their adoption even among women with increased risk of breast cancer.
  • CEA carcinoembryonic antigen
  • Ovarian cancer is the fourth most frequent cause of cancer death in females and in the United States, and accounts for approximately 13,000 deaths annually. Furthermore, ovarian cancer remains the number one killer of women with gynaecological malignant hyperplasia and the incidence is rising in industrialized countries. The etiology of the neoplastic transformation remains unknown although there is epidemiological evidence for an association with disordered endocrine function. The incidence of ovarian carcinoma is higher in nulliparous females and in those with early menopause.
  • ovarian cancers are thought to arise from the ovarian surface of epithelium (OSE).
  • OSE epithelium
  • Epithelial ovarian cancer is seldom encountered in women less than 35 years of age. Its incidence increases sharply with advancing age and peaks at ages 75 to 80, with the median age being 60 years.
  • the single most important known risk factor is a strong familial history of breast or ovarian cancer. To date, little is known about the structure and function of the OSE cells. It is known that the OSE is highly dynamic tissue that undergoes morphogenic changes, and has proliferative properties sufficient to cover the ovulatory site following ovulation.
  • ovarian cancer as used herein shall be taken to include an early or developed tumor of the ovary, such as, for example, any one or more of a number of cancers of epithelial origin, such as serous, mucinous, endometrioid, clear cell carcinoma, papillary serous, Brenner cell or undifferentiated adenocarcinoma, non-invasive ovarian cancer such as borderline ovarian cancer or low- malignant potential ovarian cancer, or a mixed phenotype ovarian cancer, and optionally, any metastases outside the ovary (e.g., in a tissue selected from the group consisting of omentum, abdominal fluid, lymph nodes, lung, liver, brain, and bone) that occurs in a subject having a primary tumor of the ovary.
  • Carcinomas e.g., basal cell carcinoma or clear cell carcinoma, including those occurring within inclusion cysts, are also included within the generic term "ovarian cancer” as used herein.
  • Ovarian cancers are not readily detectable by diagnostic techniques (Siemens et al, J. Cell. Physiol, 134: 347-356, 1988). In fact, the diagnosis of carcinoma of the ovary is generally only possible when the disease has progressed to a late stage of development. Approximately 75% of women diagnosed with ovarian cancer are already at an advanced stage (III and IV) of the disease at their initial diagnosis. During the past 20 years, neither diagnosis nor five year survival rates have greatly improved for these patients. This is substantially due to the high percentage of high-stage initial detection of the disease. There is therefore a need to develop new markers that improve early diagnosis and thereby reduce the percentage of high-stage initial diagnoses.
  • oncogenes associated with ovarian cancers have been identified, including HER-2/neu (c-erbB-2) which is over- expressed in one-third of ovarian cancers (USSN 6,075,122 by Cheever et al, issued June 13, 2000), the fins oncogene, and abnormalities in ⁇ xep53 gene, which are seen in about half of ovarian cancers.
  • the instant invention is particularly useful in terms of providing a diagnostic or prognostic assay of hyperproliferative disease, since there is a significant distinction between GOBLIN gene expression levels in cancer cells and those of normal or healthy tissues and/or subjects.
  • GOBLIN gene expression is up- regulated in cancer cells, particularly in mammary gland epithelial transplants expressing the PrlR (eg., murine transplants), and in a range of breast cancer cell lines irrespective of whether or not they express the PrlR at high level .
  • PrlR eg., murine transplants
  • expression of GOBLIN in several cancer cells derived from mammary gland tumors was enhanced or up-regulated several-fold compared to the level observed in healthy cell lines, indicating that GOBLIN is a useful cancer marker, particularly for the detection of mammary cancer and metastases thereof, and preferably for the early detection of mammary cancer and metastases thereof.
  • metastases occur in organs and tissues outside the site ofthe primary tumor.
  • metastases commonly appear in a tissue selected from the group consisting of omentum, cervical tissue, abdominal fluid, lymph nodes, lung, liver, brain, and bone.
  • the term "mammary cancer” as used herein shall be taken to include an early or developed tumor ofthe mammary gland and any metastases outside the mammary gland that occurs in a subject having a primary tumor of the breast.
  • the detection of elevated or enhanced GOBLIN expression is particularly useful for detecting any stage of progression of a cancer, including early stages of the disease and metastases outside the primary tumor tissue.
  • the present invention clearly encompasses nucleic acid-based methods and protein-based methods for diagnosing cancer in humans and other mammals.
  • nucleic acid-based assays described herein rely upon the detection or relative quantification of RNA levels in samples using probes of at least about 12 or 15 or 18 or 20 nucleotides in length that hybridize specifically to RNA encoding the GOBLIN polypeptide, or alternatively, amplify cDNA from RNA encoding the GOBLIN polypeptide.
  • Such probes are derived from unique regions of any one or more of the GOBLLN-encoding genes described herein, such as, for example, any probe comprising 12 or 15 or 18 or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 or the protein-encoding region of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 or a complementary nucleotide sequence thereto, or homologous or identical sequence in any other mammalian GOBLLN-encoding gene.
  • full-length antisense cDNA or cRNA derived from a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 is also encompassed by the present invention.
  • one aspect of the present invention provides a method for detecting a cancer cell in a subject, said method comprising determining the level of expression of a GOBLIN gene in a sample of said subject wherein elevated expression of said gene is indicative of a primary cancer or a micrometastasis or metastasis thereof and wherein said GOBLIN gene comprises a nucleotide sequence selected from the group consisting of:
  • SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10;
  • the cancer is selected from the group consisting of squamous cell carcinoma, hepatocellular carcinoma, ovarian cancer, breast cancer, melanoma, head and neck cancer, adenocarcinoma, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, prostate cancer, non-squamous carcinoma, glioblastoma and medulloblastoma.
  • the cancer is an ovarian cancer, preferably an ovarian cancer selected from the group consisting of a basal cell carcinoma, a clear cell carcinoma, an endometrioid ovarian cancer, and a mutinous ovarian cancer.
  • the cancer is a breast cancer, preferably a breast cancer selected from the group consisting of a lobular lesion, stromal lesion, ductal carcinoma, ductal adenocarcinoma, proliferative fibrocystic change, epitheliosis, intraductal papilloma and atypical ductal hyperplasia.
  • the sample comprises mammary tissue, prostate tissue, kidney tissue, uterine tissue, placenta, a cervical specimen, mammary epithelium, rectal tissue, brain tissue, bone tissue, lung tissue, lymphatic tissue, urine, semen, blood, abdominal fluid, or serum, or a cell preparation or nucleic acid preparation derived there from. More preferably, the sample comprises serum, abdominal fluid or a tissue selected from the group consisting of brain, breast, bone, cervical tissue, colon, kidney, lymph node, omentum, prostate, skin, spleen, stomach, small bowel, salivary gland and testis.
  • ovarian cancer For diagnosing/prognosing ovarian cancer, it is preferred to test a sample comprising cervical tissue, ovarian surface of epithelium (OSE) or omentum.
  • OSE epithelium
  • breast cancer it is preferred to test lobular tissue and/or stroma and/or ducts of mammary gland tissue.
  • the sample is preferably prepared on a " solid matrix e.g., a histology slide or protein chip or antibody chip or nucleic acid chip or tissue chip.
  • the sample can be solubilized e.g., to produce an extract for hybridization or immunoassay purposes.
  • the subject method further comprises obtaining the sample from a subject.
  • the sample has been obtained previously from a subject.
  • the diagnostic/prognostic platforms described herein are suitable for diagnosing/prognosing hyperproliferative disorders from any mammalian or human subject.
  • the subject is a human.
  • expression of a GOBLIN gene is determined by a process comprising determining the level of a polypeptide encoded by the GOBLIN gene in a test sample from the subject. In a preferred embodiment, such detection comprises
  • the level of the polypeptide is determined by a process comprising contacting an antibody that binds specifically to a polypeptide encoded by the GOBLIN gene to the test sample for a time and under conditions sufficient for an antigen- antibody complex to form and then detecting the complex.
  • this diagnostic/prognostic platform comprises an immunohistochemical (IHC) detection means, an enzyme-linked immunosorbent assay (ELISA), or a Western blot immunoassay.
  • IHC immunohistochemical
  • ELISA enzyme-linked immunosorbent assay
  • Western blot immunoassay for performing such assays, a polyclonal antibody or monoclonal antibody that binds to the polypeptide encoded by the GOBLIN gene is employed.
  • Preferred antibodies bind to an epitope of the polypeptide encoded by the GOBLIN gene that is contained within an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
  • Antibodies prepared against SEQ ID NO: 19 and SEQ ID NO: 21 are exemplified herein.
  • the assay method comprises: (a) providing a cell that expresses the polypeptide; (b) incubating the cell in the presence and absence of a compound to be tested; (c) contacting an extract of the cell comprising the polypeptide with the antibody under conditions sufficient for an antigen-antibody complex to form thereby capturing the polypeptide; and
  • the assay method comprises:
  • the level of expression of a GOBLIN gene is determined by a process comprising determining the level of mRNA encoded by a GOBLIN gene in a test sample from the subject.
  • this diagnostic/prognostic platform comprises:
  • the mRNA is detected by contacting a nucleic acid probe to nucleic acid in the test sample for a time and under conditions sufficient for hybridization to occur and then detecting the hybridization.
  • the nucleic acid probe comprises a nucleotide sequence selected from the group consisting of: (a) a sequence encoding an amino acid sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NOS: 10-21;
  • (f) a sequence comprising at least about 20 contiguous nucleotides of any one of (a) to (e), including any one of SEQ ID Nos: 22-353 and more preferably any one of SEQ ID Nos: 22-45.
  • nucleic acid is detected in such nucleic acid- based diagnostic/prognostic assay platforms by labelling the nucleic acid probe with a reporter molecule and detecting hybridization by detecting the reporter molecule.
  • hybridization is detected by detecting nucleic acid amplified in a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Preferred primer pairs for such amplification reactions are provided herein, e.g., a pair of nucleic acid primers selected from the group consisting of:
  • a second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
  • the present invention further provides several allelic variants of the human GOBLIN gene e.g., wherein the codon encoding amino acid residue Gin 1050 is present or absent.
  • several substitutions have also been detected e.g., wherein the sequence set forth in SEQ ID NO: 1 or 3 encodes an amino acid sequence comprising Glu at position 127 of SEQ ID NO: 2 or 4 and/or wherein the sequence set forth in SEQ ID NO: 1 or 3 encodes an amino acid sequence comprising Lys at position 127 of SEQ ID NO: 2 or 4 and/or wherein the sequence set forth in SEQ ID NO: 1 or 3 encodes an amino acid sequence comprising Pro at position 448 of SEQ ID NO: 2 or 4 and/or wherein the sequence set forth in SEQ ID NO: 1 or 3 encodes an amino acid sequence comprising Ser at position 448 of SEQ ID NO: 2 or 4 and/or wherem the sequence set forth in SEQ ID NO: 1 or 3 encodes an amino acid sequence comprising Met at position 734 of SEQ
  • the isolated nucleic acid of the present invention is expressed in a range of tissues, such as, for example, a tissue selected from the group consisting of abdominal fluid, brain, breast, bone, cervical tissue, colon, kidney, lymph node, omentum, prostate, skin, spleen, stomach, small bowel, salivary gland and testis.
  • tissues such as, for example, a tissue selected from the group consisting of abdominal fluid, brain, breast, bone, cervical tissue, colon, kidney, lymph node, omentum, prostate, skin, spleen, stomach, small bowel, salivary gland and testis.
  • the isolated nucleic acid of the present invention is over-expressed or expressed more highly in a range of different malignant tissues. As will be apparent to the skilled person, such high expression of the nucleic acid of the invention in malignant or tumor tissue is readily detectable in a cell of that tissue.
  • the expression of the nucleic acid in mammary glands or breast tissue or a cell thereof is prolactin-modulated.
  • the present invention extends clearly to a vector comprising the isolated nucleic acid supra and/or the use of said isolated nucleic acid to detect a cancer cell.
  • Plasmids or expression vectors, including any viral vectors, comprising the nucleic acid described herein are preferred.
  • Such vectors may be introduced into suitable host cells, such as, for example, bacterial cells, yeast cells, insect cells, or mammalian cells, for the purpose of expressing a recombinant GOBLIN polypeptide or a functional fragment thereof, in particular an immunogenic peptide fragment.
  • a further aspect of the present invention provides an antibody e.g., a monoclonal or polyclonal antibody, that binds specifically to a polypeptide comprising an amino acid sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NOS: 10-21, in particular an antibody that binds to an epitope within any one of SEQ ID Nos: 19-21, more preferably to an epitope within the amino acid sequence set forth in SEQ ID NO: 19 or 21.
  • an antibody e.g., a monoclonal or polyclonal antibody, that binds specifically to a polypeptide comprising an amino acid sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NOS: 10-21, in particular an antibody that binds to an epitope
  • a further aspect of the present invention provides an isolated polypeptide comprising an amino acid sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NOS: 10-21, including any novel allelic variant or immunogenic epitope thereof.
  • This aspect of the present invention clearly extends to any fusion protein comprising the isolated polypeptide, such as, for example, a fusion protein comprising the isolated polypeptide linked to a detectable tag, e.g. a V5 loop, 6X His tag, green fluorescent protein tag, or FLAG epitope.
  • the isolated polypeptide or fusion protein of the invention is particularly useful for the production of antibodies or as control peptides in an assay platform described herein.
  • the present invention further provides a method of producing a GOBLIN polypeptide or a functional fragment thereof, said method comprising culturing a host cell comprising the nucleic acid of the invention in an expressible format under conditions sufficient for expression to occur and then recovering the expressed polypeptide.
  • Ligands, agonists, or antagonists of GOBLIN function are identified in a suitable assay, and further assessed for their therapeutic efficacy.
  • Antagonists of GOBLIN are used to inhibit (ie. reduce or diminish or prevent) GOBLIN-mediated effects in cells, such as, for example, breast cancer.
  • ligands and/or agonists of GOBLIN are useful for inducing or enhancing GOBLIN- mediated effects in cells.
  • a further aspect ofthe present invention provides a method of identifying a compound that reduces or antagonizes expression of a GOBLIN gene comprising:
  • GOBLIN gene comprises a nucleotide sequence selected from the group consisting of: (i) a sequence encoding a polypeptide comprising an amino acid sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10; (ii) a sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9;
  • the assay is preferably performed using a cancer cell in which expression of the GOBLIN gene is elevated e.g., a cell line set forth in Table 1 or Table 4.
  • Preferred cell lines for this purpose include T47-D, MCF-7, BT20, BT474, ZR-75-1, MDA-MB-175- VII, MDA-MB-134, SKBR3, MDA-MB-231, MDA-MB-468, MDA-MB-330, MDA- MB-436, BT549, HBL-100, Hs578,T, MDA-MB-157, MCF10A, 184 or MDA-MB- 361 cells.
  • the cell line is selected from the group consisting of MCF-7, BT20, BT474, MDA-MB-134, SKBR3, MDA-MB-231, BT549, and MDA-MB-361 that express GOBLIN at moderate-to-high levels.
  • the present invention further comprises obtaining the cancer cell.
  • the assay may be performed using a cell that over expresses a GOBLIN gene by virtue of having been stably transformed or transiently transfected with a nucleic acid comprising a GOBLIN gene.
  • the 293 cell line is particularly preferred for such purposes.
  • the present invention further comprises obtaining or producing the transformed or transfected cell.
  • test compounds for antagonizing expression of a GOBLIN gene include siRNA or shRNA comprising a nucleotide sequence set forth in any one of SEQ ID Nos: 46-353, antisense RNA, nucleic acid encoding a WW domain or C2 domain of a polypeptide encoded by the GOBLIN gene or antibodies. All such compounds can be tested using the assay platforms described in the following paragraphs.
  • the level of GOBLIN gene expression is determined by a process comprising determining the level of a polypeptide encoded by the gene in the presence of the compound relative to the level of the polypeptide in the absence of the compound, wherein a reduced level ofthe polypeptide in the presence ofthe compound indicates that the compound is an antagonist of expression ofthe gene.
  • the level ofthe polypeptide is determined by a process comprising:
  • Preferred antibodies for such applications bind to an epitope within an amino acid sequence selected from the group consisting of SEQ ID Nos: 19-21, preferably to an epitope within the amino acid sequence set forth in SEQ ID NO: 19 or 21.
  • the assay method comprises: (a) providing a cell that expresses the polypeptide;
  • the assay method comprises:
  • the level of GOBLIN gene expression is determmed by a process comprising determining the level of an mRNA transcription product ofthe gene in the presence ofthe compound relative to the level ofthe mRNA in the absence ofthe compound, wherein a reduced level of the mRNA in the presence of the compound indicates that the compound is an antagonist of expression ofthe gene.
  • the mRNA is detected by contacting a nucleic acid probe with nucleic acid in the cell or an extract thereof for a time and under conditions sufficient for hybridization to occur and then detecting the hybridization.
  • the probe may comprise a nucleotide sequence selected from the group consisting of:
  • (f) a sequence comprising at least about 20 contiguous nucleotides of any one of (a) to (e), including a sequence selected from the group consisting of SEQ ID Nos: 22-353 and preferably a sequence selected from the group consisting of SEQ ID Nos: 22-45.
  • the nucleic acid probe can be labeled with a reporter molecule and hybridization detected by detecting the reporter molecule, or the hybridization can be detected by detecting nucleic acid amplified in a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a further aspect of the present invention provides a process for identifying or determining a compound comprising: (a) performing a method described herein to identify or determine a compound that reduces or antagonizes expression of a GOBLIN gene; (b) optionally, determining the structure ofthe compound; and
  • the present invention provides a process for producing a compound said method comprising:
  • a further aspect of the present invention provides an isolated nucleic acid that antagonizes expression of a GOBLIN gene, wherein said nucleic acid comprises a nucleotide sequence selected from the group set forth in SEQ ID Nos: 46-353.
  • a further aspect ofthe present invention provides an isolated antisense nucleic acid that antagonizes expression of a GOBLIN gene, wherein said nucleic acid comprises a nucleotide sequence capable of selectively hybridizing to mRNA encoded by the isolated nucleic acid ofthe invention.
  • a further aspect of the present invention provides a process for monitoring the efficacy of treatment of a cancer in a subject comprising performing the diagnostic method supra on a sample from a subject suffering from the cancer wherein treatment commenced before the time when the sample was taken and wherein a reduced level of expression relative to the level of expression in a healthy or normal subject indicates that the subject has responded to treatment.
  • the present invention provides a process for monitoring the efficacy of treatment of a cancer in a subject comprising performing the diagnostic method supra on a sample from a subject suffering from the cancer wherein treatment commenced before the time when the sample was taken and wherein a similar or enhanced level of expression relative to the level of expression in a healthy or normal subject indicates that the subject has not responded to treatment.
  • the present invention provides a process for monitoring the efficacy of treatment of a cancer in a subject comprising performing the diagnostic method supra on samples from a subject suffering from the cancer taken at least two different time points wherein treatment commenced at or following the first of said time points and wherein a reduced level of expression at a later time point indicates that the subject has responded to treatment.
  • the present invention provides a process for monitoring the efficacy of treatment of a cancer in a subject comprising performing the diagnostic method supra on samples from a subject suffering from the cancer taken at least two different time points wherein treatment commenced at or following the first of said time points and wherein a similar or enhanced level of expression at a later time point indicates that the subject has not responded to treatment.
  • results of the diagnostic/prophylactic assays described herein are of particular use in designing and/or recommending effective or alternative therapeutic regimes for subjects suffering from cancer, based upon a primary diagnosis or assay result obtained following a primary diagnosis e.g., during primary treatment. Included within such recommendations are recommendations following surgical resection or chemotherapy or radiotherapy.
  • a further aspect of the present invention provides a method of treating a hyperproliferative disease, such as, for example, cancer, comprising administering an antagonist of GOBLIN function to an individual (e.g., a mammal) for a time and under conditions sufficient to reduce or prevent GOBLIN activity in said individual, thereby reducing or preventing one or more GOBLIN-mediated effects.
  • a hyperproliferative disease such as, for example, cancer
  • the present invention further provides a method of agonising or otherwise enhancing GOBLIN activity in an individual comprising administering a GOBLIN ligand or GOBLIN agonist to said individual for a time and under conditions sufficient to enhance GOBLIN activity.
  • Figure 1 is a graphical representation showing the expression of GOBLIN mRNA transcripts at: (i) day 2 of pregnancy in mice (left group of three columns); (ii) day 4 of pregnancy in mice (middle group of three .columns); and (iii) day 6 of pregnancy in mice (right group of three columns).
  • the samples consisted of epithelial transplants that were homozygous for the prolactin receptor (Prlr +/+ ; left column in each group); PrlR null epithelial transplants (Prlr 7" ; middle column in each group); or fat pads that were homozygous for the prolactin receptor but cleared of epithelium (right column in each group).
  • Data indicate prolactin-modulated expression of GOBLIN in mammary epithelium.
  • Figure 2 is a graphical representation showing the expression of GOBLIN mRNA transcripts at various stages of pregnancy, lactation and involution in mice.
  • Figure 3 is a graphical representation showing the expression of GOBLIN mRNA transcripts in several breast cancer cell lines as indicated in the left column.
  • Levels of the prolactin receptor (PRLR), estrogen receptor (ER) and progesterone receptor (PR) present in each cell line are indicated in columns 2-4 (very high expression, +++; high expression ++; moderate, +; expressed at RNA level only, m; or no detectable expression,-).
  • GOBLIN expression was detected in all cell lines tested.
  • Data also show high level GOBLIN expression in several cell lines having high levels of PrlR, including T-47D, MCF-7, BT474, and BT483 cells. However, data also indicate high level expression of GOBLIN mRNA in cells that do not express PrlR, including MDA-
  • Figure 4 is a schematic representation of GOBLIN protein structure showing the positions of two WW domains (WW1 and WW2) and a C2 domain.
  • Two peptide fragments of GOBLLN-encoding nucleic acid, encoding amino acids 1-623 (WW Fragment) or amino acids 348-1113 (C2 Fragment) were cloned for use in expression studies.
  • a and B denote the position of the peptides used to generate the GOBLIN polyclonal antibodies.
  • Figure 5 is a copy of a photographic representation of a Western blot showing binding of anti-GOBLIN polyclonal antibodies to recombinant GOBLIN protein.
  • Left-hand panel shows immunoblot analysis of whole cell lysates from 293 cells transfected with the pcDNA3.1 -GOBLIN gene construct wherein the antisera prepared against SEQ ID NO: 21 antibody detected a single immunoreactive band in 293 cell lysates transfected with GOBLLN-encoding nucleic acid (left lane) but not in non-transfected 293 cell lysates (right lane).
  • Upper right-hand panel shows immunoblot analysis of whole cell lysates from 293 cells transfected with the WW-V5-6xHis or C2-V5-6xHis gene constructs, wherein the antisera prepared against SEQ ID NO: 21 antibody detected a single immunoreactive band in 293 cell lysates transfected with the GOBLLN-encoding nucleic acids (lanes 1 and 3) but not in non-transfected 293 cell lysates (lanes 2 and 4).
  • Immunoblot analysis employed antibodies raised against a synthetic peptide comprising amino acids 584-600 of GOBLIN protein (SEQ ID NO: 21).
  • the lower right-hand panel shows immunoblot analysis of whole cell lysates from 293 cells transfected with the WW-V5-6xHis or C2-V5-6xHis gene constructs, wherein antibodies against the V5 domain detected immunoreactive bands in 293 cell lysates transfected with the GOBLLN-encoding WW-V5-6xHis or C2-V5-6xHis gene constructs (lanes 1 and 3) but not in non-transfected 293 cell lysates (lanes 2 and 4).
  • Molecular weights are indicated at the left-hand side of each panel
  • Figure 6 is a copy of photographic representations showing GOBLIN protein expression in various tissues of humans as determined by immunohistochemical staining of tissue sections using antibodies raised against a synthetic peptide comprising amino acids 584-600 of GOBLIN protein (SEQ ID NO: 21).
  • A. skin B. brain C. spleen D. stomach E. colon F. small bowel G. breast H. prostate I. testis J. lung K. liver and L. skeletal muscle. Brown staining indicates tissue positive for GOBLIN protein (magnification x400).
  • Figure 7 is a copy of a photographic representation of a Western blot showing expression of the GOBLIN protein in the breast cancer cell lines T47D, BT474, MDA- MB-134, SKBR3, MDA-MB-468, BT20, MDA-MB-330 and MDA-MB-436.
  • These cell lines include cell lines that represent a model of breast cancer in humans.
  • Whole cell lysates were separated by SDS-PAGE, transferred to membranes and probed using antibodies raised against a synthetic peptide comprising amino acids 584-600 of GOBLIN protein (SEQ ID NO: 21).
  • GOBLIN protein was present in all breast cancer cell lines examined.
  • As a positive control cell lysates from 293 cells transfected with the pcDNA3.1 -GOBLIN gene construct were treated similarly. Numbers at the left of the figure indicate molecular weights ofthe proteins.
  • Figure 8 is a copy of a photographic representation showing expression ofthe GOBLIN protein in the breast cancer cell lines BT20 (panel A), BT474 (panel B), SKBR3 (panel C), BT549 (panel D), MCF7 (panel E), MDA-MB-134 (panel F), MDA-MB-231 (panel A), BT20 (panel A), BT474 (panel B), SKBR3 (panel C), BT549 (panel D), MCF7 (panel E), MDA-MB-134 (panel F), MDA-MB-231 (panel
  • MDA-MB-361 panel H
  • MDA-MB-157 panel I
  • 184 panel J
  • BT483 panel K
  • MDA-MB-453 panel L
  • Figure 9 is a copy of a photographic representation showing expression ofthe GOBLIN protein in epithelial ovarian cancer tissue by immunohistochemistry, using antibodies raised against a synthetic peptide comprising amino acids 584-600 of GOBLIN protein (SEQ ID NO: 21).
  • Samples were as follows: (A) ovarian stroma; (B) ovarian surface epithelium; (C) ovarian inclusion cysts; (D) serous ovarian cancer; (E) mutinous ovarian cancer; (F) endometrioid ovarian cancer; and (G) clear cell carcinoma.
  • GOBLIN protein was identified in all samples except serous ovarian cancer tissue. (magnification x200).
  • FIG 10 is a copy of a photographic representation showing expression of the GOBLIN protein in breast cancer tissue, as determined by immunohistochemistry, using antibodies raised against a synthetic peptide comprising amino acids 584-600 of GOBLIN protein (SEQ ID NO: 21).
  • Samples were as follows: (A) Lung tissue, a negative control; (B) Brain tissue, a positive control for GOBLIN expression; (C) Healthy breast tissue; (D) and (E) representative breast cancer tissues.
  • GOBLIN protein was detected in healthy breast epithelium (panel C) and at elevated levels in both breast cancer samples (panels D and E). (magnification A-C x 200, D-E x 400).
  • One aspect of the present invention provides an isolated nucleic acid encoding a
  • GOBLIN polypeptide comprising an amino acid sequence having at least about 65-70% identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 or to a protein-encoding region or open reading frame thereof that encodes a GOBLIN polypeptide or fragment.
  • the amino acid sequence identity to any one of these exemplified GOBLIN polypeptides is about 80%, and more preferably at least about 85% or 90% or 95% or 99% identity.
  • the present invention provides an isolated nucleic acid encoding a GOBLIN polypeptide wherein said nucleic acid comprises a nucleotide sequence having at least about 65-70% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, or to a protein-encoding region or open reading frame thereof that encodes a GOBLIN polypeptide or fragment.
  • the sequence identity to any one of these exemplified nucleic acids is about 80%, and more preferably at least about 85% or 90% or 95% or 99% identity.
  • nucleic acid shall be taken to mean any single-stranded or double-stranded RNA, DNA, cDNA, cRNA, or synthetic oligonucleotide, or alternatively, an analog of RNA, DNA, cDNA, cRNA, or a synthetic oligonucleotide.
  • Nucleic acid also includes any genomic gene equivalents of a cDNA molecule.
  • the present invention provides an isolated nucleic acid encoding a GOBLIN polypeptide wherein said nucleic acid comprises a nucleotide sequence that hybridizes under at least moderate stringency hybridization conditions to a sequence that is complementary to a sequence having at least about 65-70% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 or a protein-encoding region thereof that encodes a GOBLIN polypeptide or fragment.
  • the hybridizing sequence has at least about 80% identity to the complement of any one of these exemplified nucleic acids, and more preferably at least about 85% or 90% or 95% or 99% identity.
  • a high stringency hybridization is achieved using a hybridization buffer and/or a wash solution comprising the following: (i) a salt concentration that is equivalent to 0.1xSSC-0.2xSSC buffer or lower salt concentration; (i) a detergent concentration equivalent to 0.1% (w/v) SDS or higher; and (iii) an incubation temperature of 55°C or higher.
  • a moderate stringency hybridization is achieved using a hybridization buffer and/or a wash solution comprising the following: (i) a salt concentration that is equivalent to 0.2xSSC- 2xSSC buffer or lower salt concentration; (ii) a detergent concentration equivalent to 0.1 % (w/v) SDS or higher; and (iii) an incubation temperature between about room temperature and about 55°C.
  • the isolated nucleic acid of the invention is from humans (ie. it encodes a human GOBLIN polypeptide).
  • the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3 relate to nucleic acids encoding variants of a human GOBLIN polypeptide.
  • the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4 relate to variants of a human GOBLIN polypeptide.
  • nucleotide sequence set forth in SEQ ID NO: 1 lacks a codon encoding Gin 1050 that is present in SEQ ID NO: 3. Accordingly, the GOBLIN polypeptide set forth in SEQ IDS NO: 2 lacks Gin 1050 that is present in the allelic variant set forth in SEQ ID NO: 4.
  • SEQ ID NO: 1 and SEQ ID NO: 3 comprise nucleotide sequences encoding allelic variants of GOBLIN, as represented by the variable nucleotide residues at positions 391, 492, 1354, 1644, 2214, 2215, 3025 and 3988 of SEQ ID NO: 1, and at corresponding nucleotide positions 379, 480, 1342, 1632, 2202, 2203, 3013 and 3979 of SEQ ID NO: 3.
  • allelic variants at the gene level encode allelic variant GOBLIN polypeptides wherein, alternatively or in addition to the presence or absence of Gin 1050 , the amino acid residue at position 127 of a human GOBLIN polypeptide is Glu or Lys and/or the amino acid residue at position 448 of a human GOBLIN polypeptide is Pro or Ser and/or the amino acid residue at position 734 of a human GOBLIN polypeptide is Met or He and/or the amino acid residue at position 735 of a human GOBLIN polypeptide is Ala or Ser.
  • the isolated nucleic acid of the invention is from mice (ie. it encodes a murine GOBLIN polypeptide).
  • the nucleotide sequence set forth in SEQ ID NO: 5 relates to nucleic acid encoding a murine GOBLIN polypeptide.
  • the amino acid sequence set forth in SEQ ID NO: 6 relates to a murine GOBLIN polypeptide.
  • the isolated nucleic acid of the invention is from a non-human primate, specifically Macaca fascicularis (ie. it encodes a non-human primate GOBLIN polypeptide).
  • a non-human primate specifically Macaca fascicularis
  • the nucleotide sequence set forth in SEQ ID NO: 7 relates to nucleic acid encoding a partial M. fascicularis GOBLIN polypeptide.
  • the amino acid sequence set forth in SEQ ID NO: 8 relates to a partial M. fascicularis GOBLIN polypeptide.
  • the isolated nucleic acid of the invention is from a rat (ie. it encodes a rat GOBLIN polypeptide).
  • the nucleotide sequence set forth in SEQ ID NO: 9 relates to nucleic acid encoding a rat GOBLIN polypeptide.
  • the amino acid sequence set forth in SEQ ID NO: 10 relates to a rat GOBLIN polypeptide.
  • the GOBLIN polypeptide as exemplified herein by a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 and variants thereof, will comprise a cytosolic WW domain.
  • cytosolic WW domain or "WW domain” is meant an amino acid sequence comprising about 35 to about 40 amino acid residues in length that potentially folds into a three-stranded monomeric anti-parallel beta-sheet structure that is stable in the absence of disulfide bonds, cofactors or ligands, wherein said sequence preferably binds to proline-rich amino acid sequences, phosphoserine or phosphothreonine in proteins.
  • a "WW domain” as used herein will comprises the following: (i) two conserved tryptophan residues spaced apart by about 20 to about 30 amino acid residues; and
  • the structure ofthe encoded GOBLIN polypeptide of humans i.e. SEQ ID NO: 2 or SEQ ID NO: 4 or a variant thereof
  • the corresponding murine sequence i.e. SEQ ID NO: 6
  • the WW domain preferably comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11 to 18.
  • the GOBLIN polypeptide comprises two or three WW domains as exemplified in the Sequence Listing.
  • the WW domain of the GOBLIN polypeptide binds to a protein selected from the group consisting of Src-tyrosine kinase-like protein, sodium channel protein, ubiquitin ligase protein, and cytoskeletal protein (eg., dendrin).
  • the WW domain of the GOBLIN polypeptide binds to a proline-rich region of a protein selected from the group consisting of Src-tyrosine kinase-like protein, sodium channel protein, ubiquitin ligase protein, and cytoskeletal protein (eg., dendrin).
  • Preferred proline-rich regions comprise an amino acid sequence such as, for example, selected from the group consisting of PPPY, PPPNY, or PPLP.
  • the GOBLIN polypeptide as exemplified herein by a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 10, will comprise a C2 domain.
  • C2 domain is meant an amino acid sequence having at least about 65% identity, more preferably at least about 70% or 75% or 80% or 85% or 90% or 95% identity to amino acid residues from about position 601 to about position 801 of a human or murine sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 10.
  • C2 domain shall be taken to mean an amino acid sequence having at least about 65% identity, more preferably at least about 70% or 75% or 80% or 85% or 90% or 95% identity to amino acid residues from about position 811 to about position 1008 ofthe rat sequence set forth in SEQ ID NO: 10.
  • C2 domain is also included in the partial GOBLIN amino acid sequence from Macaca fascicularis, from position 1 to about position 119 of SEQ ID NO: 8. Accordingly, the term "C2 domain” as used herein also includes a peptide or polypeptide comprising a sequence having at least about 65% identity, more preferably at least about 70% or 75% or 80% or 85% or 90% or 95% identity to amino acid residues from position 1 to about position 119 of SEQ ID NO: 8.
  • Preferred C2 domains comprise an amino acid sequence that binds a phospholipid in a calcium-dependent manner.
  • a GOBLIN polypeptide of humans as exemplified herein by SEQ ID NO: 2 and/or SEQ ID NO: 4 will comprise an immunogenic domain or region, preferably a region that is unique to GOBLIN proteins e.g., of humans, positioned between amino acid residues from about Ala 373 to about Leu 398 and preferably from about Ala 384 to about Leu 398 .
  • an immunogenic domain or region, preferably a region that is unique to GOBLIN proteins e.g., of humans is positioned between amino acid residues from about Ser 584 to about Gin 600 .
  • an immunogenic domain or region preferably a region that is unique to GOBLIN proteins e.g., of humans, will comprise an amino acid sequence set forth in any one of SEQ ID Nos: 19-21.
  • the isolated nucleic acid ofthe present invention is expressed in a range of body fluids or tissues, such as, for example, serum or abdominal fluid, or a tissue selected from the group consisting of omentum, cervical tissue, prostate, mammary gland, ovary, placenta, lymph, lung, liver, brain, and bone, as determined by the appearance of RNA encoding said polypeptide in any one or more of said body fluids or tissues.
  • the GOBLIN polypeptide is expressed in epithelial cells.
  • the isolated nucleic acid of the present invention is over-expressed or expressed more highly in a malignant tissue or tumor, such as, for example, a malignancy or tumor of the prostate, a malignancy or tumor of the omentum, a malignancy or tumor of cervical tissue, a malignancy or tumor of the lymph node, a malignancy or tumor of the lung, a malignancy or tumor of the liver, a malignancy or tumor of the brain, or a malignancy or tumor of bone tissue.
  • a malignant tissue or tumor such as, for example, a malignancy or tumor of the prostate, a malignancy or tumor of the omentum, a malignancy or tumor of cervical tissue, a malignancy or tumor of the lymph node, a malignancy or tumor of the lung, a malignancy or tumor of the liver, a malignancy or tumor of the brain, or a malignancy or tumor of bone tissue.
  • a malignancy or tumor of the prostate such as, for example
  • the isolated nucleic acid of the invention is over-expressed or more highly expressed in a malignant tissue or tumor of the cervix, omentum, prostate, or breast or in a cell derived from said malignant tissue or tumor. More preferably, the isolated nucleic acid of the invention is over-expressed or more highly expressed in a malignant tissue or tumor ofthe cervix, omentum, or breast.
  • the nucleic acid of the present invention is over-expressed or more highly expressed in a malignant tissue or tumor of the mammary gland or breast.
  • the isolated nucleic acid ofthe invention is also more highly expressed in a cancer cell, such as, for example, a carcinoma of tissue selected from the group consisting of lung, prostate, breast, colon, pancreas, placenta, mammary epithelium and ovary, and/or in a cell of a brain anaplastic oligodendroglioma, as determined by the appearance of RNA encoding said polypeptide in the cell.
  • a cancer cell such as, for example, a carcinoma of tissue selected from the group consisting of lung, prostate, breast, colon, pancreas, placenta, mammary epithelium and ovary, and/or in a cell of a brain anaplastic oligodendroglioma, as determined by the appearance of RNA encoding said polypeptide in the cell.
  • tumor and cancer as used herein shall be taken to include an early or developed tumor of the primary tissue and any metastases outside the primary tissue that occurs in a subject.
  • tumor in the case of mammary cancer, metastases commonly appear in a tissue selected from the group consisting of omentum, cervical tissue, abdominal fluid, lymph nodes, lung, liver, brain, and bone.
  • mammary cancer as used herein shall be taken to include an " early or developed tumor of the mammary gland and any metastases outside the mammary gland that occurs in a subject having a primary tumor ofthe breast.
  • cancer cell includes any biological specimen or sample comprising a cancer cell irrespective of its degree of isolation or purity, such as, for example, tissues, organs, cell lines, bodily fluids, or histology specimens that comprise a cell in the early stages of transformation or having been transformed. Bodily fluids shall be taken to include whole blood, serum, peripheral blood mononuclear cells (PBMC), or buffy coat fraction.
  • PBMC peripheral blood mononuclear cells
  • the nucleic acid of the invention is expressed in a cell line selected from the group set forth in Table 1. It is well-established in the field that the array of breast cancer cell lines provided in Table 1, or a sub-set thereof, represent a suitable model of the different cellular phenotypes associated with tumor progression, from highly differentiated estrogen receptor (ER)-positive cell types (eg T- 47D, MCF-7) through to poorly differentiated ER-negative types eg MDA-MB-231 (Sutherland RL, Watts CKW, Lee CSL and Musgrove EA, 'Breast Cancer' in Masters JRW and Palsson B (Eds), Human Cell Culture Vol II, ⁇ 79-106). ER positive breast cancers generally have a better prognosis (Knight et al Cancer Res. 37, 4669-4671, 1977).
  • cancer cell is not to be limited by the stage of a cancer in the subject from which said cancer cell is derived (ie. whether or not the patient is in remission or undergoing disease recurrence or whether or not the cancer is a primary tumor or the consequence of metastases).
  • cancer cell is not to be limited by the stage ofthe cell cycle of said cancer cell or the stage of development of the tissue from which the cell is derived, subject to the proviso that the cell may be derived from a female subject at a particular stage of pregnancy, lactation, involution, or menopause, if desired.
  • the isolated nucleic acid is expressed at elevated levels in cancer cells compared to non-cancer cells, as detected by measuring the level of GOBLIN RNA or GOBLIN polypeptide.
  • the isolated nucleic acid ofthe invention is expressed at an elevated level in mammary cancer cells, such as, but not limited to, cancerous mammary epithelial cells, and metastases thereof, such as, for example, OSE, abdominal fluid, lymph nodes, lung, liver, brain, or bone.
  • the expression of the nucleic acid in mammary glands or breast tissue or a cell thereof is prolactin-modulated.
  • prolactin-modulated is meant that the level of expression of the GOBLIN protein-encoding gene and/or the level of the GOBLIN polypeptide is directly or indirectly enhanced or reduced in a cell or tissue of the mammary epithelium, in response to the action of prolactin.
  • the inventors have shown that the GOBLIN gene is expressed at higher levels in mammary epithelial transplants that express the PrlR at high levels compared to transplants that do not ( Figure 1).
  • allelic imbalance leading to gene amplification can occur in several cancers. Accordingly, the terms “over-expressed”, “more highly expressed”, “prolactin-modulated” or similar terms used herein shall be taken to include an enhancement of gene expression as a consequence of allelic imbalance. Similarly, because many cancers, such as certain tumors of epithelial origin, involve selective proliferation of particular cells relative to other cells, the terms “over-expressed”, “more highly expressed”, “prolactin-modulated” or similar terms used herein shall be taken to include an enhanced or reduced expression arising from or as a consequence of selective proliferation of a given cell type in a tissue.
  • the isolated nucleic acid of the present invention comprises a nucleotide sequence selected from the group consisting of: (i) the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9;
  • nucleotide residues 13 to 3348 of SEQ ID NO: 1 (ii) nucleotide residues 13 to 3348 of SEQ ID NO: 1 ; (iii) nucleotide residues 1 to 3339 of SEQ ID NO: 3; (iv) nucleotide residues 14 to 3325 of SEQ ID NO: 5; (v) nucleotide residues 3 to 1040 of SEQ ID NO: 7; (vi) nucleotide residues 1 to 4248 of SEQ ID NO: 9; (vii) a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10; (viii) a nucleotide sequence that comprises an allelic variant of any one of (i) to (vii); and (ix) a sequence that is complementary to any one of (i) to (vii).
  • HMEC184, HMEC184A1, HMEC184B5 and HMEC129-4 derived from human mammary epithelial cells (Sta pfer et al., Mol Biol Cell 8:12:2391-405, 1997).
  • IDC infiltrating ductal carcinoma
  • AC adenocarcinoma
  • TLC infiltrating lobular carcinoma
  • MC medullary carcinoma
  • FCBD fibrocystic breast disease
  • the present invention clearly extends to any derivative, homolog or analog of the subject isolated nucleic acid, such as, for example, a functionally or immunologically active derivative, mutant or variant sequence, or a fragment or portion that is useful as a diagnostic reagent to identify ligands of a GOBLIN polypeptide including an agonist or antagonist of GOBLIN or in the treatment of a condition in which GOBLIN expression or over expression is an indicator.
  • a functionally or immunologically active derivative, mutant or variant sequence or a fragment or portion that is useful as a diagnostic reagent to identify ligands of a GOBLIN polypeptide including an agonist or antagonist of GOBLIN or in the treatment of a condition in which GOBLIN expression or over expression is an indicator.
  • immunologically active derivative shall be taken to mean any peptide fragment of a GOBLIN polypeptide that is of a sufficient length and/or sufficiently antigenic to: (i) facilitate the production of antibodies that can detect GOBLIN in samples; and/or (ii) bind to antibodies against a GOBLIN polypeptide.
  • GOBLIN protein/peptide derivatives are immunologically or functionally equivalent to SEQ ID NO: 2, 4, 6, 8 or 10, or have enhanced activity or immunogenicity thereto.
  • Such active/enhanced derivatives will preferably comprise at least about 5-10 contiguous amino acids of the full-length amino acid sequence of a GOBLIN polypeptide or comprise a WW domain or C2 domain of the full-length protein.
  • Derivatives comprising at least about 10-20 contiguous amino acids in length of the base sequence, and even more preferably at least about 20-30 contiguous amino acids in length, are highly preferred.
  • Particularly preferred derivatives of a GOBLIN polypeptide include amino acid residues comprising a WW domain of a full-length GOBLIN polypeptide as set forth in any one of SEQ ID Nos: 11-18 or otherwise described herein. Similarly, a derivative comprising a C2 domain of a full-length GOBLIN polypeptide as described herein is particularly encompassed by the present invention.
  • the GOBLIN peptides exemplified as SEQ ID Nos: 19-21 are also particularly encompassed as derivatives of a full-length GOBLIN polypeptide ofthe present invention.
  • a length not exceeding about 30-50 amino acids in length is preferred, as longer peptides are difficult to produce at high efficiency.
  • Longer peptide fragments are readily achieved using recombinant DNA techniques wherein the peptide is expressed in a cell-free or cellular expression system comprising nucleic acid encoding the desired peptide fragment.
  • Such methods are particularly preferred for producing WW domain fragments, other GOBLLN-specific peptides (e.g., any one of SEQ ID Nos: 19-21) or C2 domain fragments or immunologically or functionally equivalent derivatives or derivatives having enhanced immunogenicity or function thereto.
  • any sufficiently antigenic region of at least about 5-10 amino acid residues in length derived from a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10, or of any one of SEQ ID NOS: 11-21 can be used to prepare antibodies that bind generally to human GOBLIN polypeptides.
  • antibodies against one GOBLIN polypeptide are contemplated to immunologically cross-react with antibodies against another GOBLIN polypeptide.
  • the derivative will comprise an amino acid sequence selected from the group consisting of:
  • WEEARDFDGKVYYIDHRNRTTSWIDP (SEQ ID NO: 15); (vi) the sequence of the WW domain peptide WEEAYDPQVGDYFIDHNTKTTQIEDPRVQW (SEQ ID NO: 16);
  • ARKRLEKDLQAARDTQSKALTERLKL (SEQ ID NO: 20); (xi) the sequence of the GOBLIN peptide SAQERYRLEEPGTEGKQ (SEQ ID NO: 21); (xii) the sequence of a C2 domain peptide comprising amino acid residues from about position 601 to about position 801 of SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6;
  • a peptide comprising a sequence that is specific to a GOBLIN polypeptide and any antibody raised against that peptide are particularly useful as diagnostic reagents for the specific detection of GOBLIN expression in a cell or tissue. Additionally, a peptide having a sequence derived from the WW domain of a
  • GOBLIN polypeptide and any antibodies that are raised against such a peptide are particularly useful as dominant negative mutants or antagonist compounds to block an interaction between GOBLIN and its binding partner, and/or to inhibit or prevent or otherwise reduce downstream signalling from GOBLIN.
  • a peptide comprising a sequence derived from the C2 domain of a GOBLIN polypeptide and any antibodies that are raised against such a peptide are particularly useful as dominant negative mutants or antagonist compounds to block a calcium-dependent interaction between GOBLIN and a phospholipid, and/or to inhibit or prevent or otherwise reduce downstream signalling from GOBLIN.
  • nucleotide insertion derivatives of the protein-encoding region of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9, or an equivalent thereof are produced by making 5' and 3' terminal fusions, or by making infra-sequence insertions of single or multiple nucleotides or nucleotide analogues.
  • Insertion nucleotide sequence variants are produced by introducing one or more nucleotides or nucleotide analogues into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed.
  • Deletion variants are produced by removing one or more nucleotides from the nucleotide sequence.
  • substitutional nucleotide variants are produced by substituting at least one nucleotide in the sequence with a different nucleotide or a nucleotide analogue in its place. Such mutant derivatives will preferably have at least about 65% identity with the base amino acid sequence from which they are derived (ie. SEQ ID NO: 1, 3, 5, 7 and 9).
  • analog is meant nucleic acid that encodes GOBLIN or a derivative of GOBLIN and includes one or more nucleotide or non-nucleotide substituents not normally present in said isolated nucleic acid, such as, for example a carbohydrate, radiochemical, fluorescent molecule, biotin, DIG, alkaline phosphatase, horseradish peroxidase, or other reporter molecule.
  • Preferred reporter molecules include radioactively-labelled nucleotide triphosphates and biotinylated molecules. Analogs are generally produced to facilitate detection of the nucleic acid.
  • homologs or orthologs of the exemplified GOBLLN-encoding nucleic acids provided herein are readily identified using standard means known to those skilled in the art, including bioinformatics approaches, nucleic acid hybridization and polymerase chain reaction.
  • the homolog or ortholog is isolated by hybridizing a probe or primer derived from any one or more of SEQ ID Nos: 1, 3, 5, 7 or 9, or any one of SEQ ID Nos: 22-45, or a complementary sequence thereto to cDNA, mRNA or genomic DNA from another species and detecting the hybridization using a detection means.
  • detection of the hybridization is performed preferably by labelling a probe with a reporter molecule capable of producing an identifiable signal, prior to hybridization, and then detecting the signal after hybridization.
  • reporter molecules include radioactively-labelled nucleotide triphosphates and biotinylated molecules.
  • variants of the genes exemplified herein, including genomic equivalents are isolated by hybridisation under moderate stringency or more preferably, under high stringency conditions, to the probe.
  • hybridization may be detected using any format of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • two non-complementary nucleic acid primer molecules comprising at least about 20 nucleotides in length, and more preferably at least about 20 nucleotides in length are hybridized to different strands of a nucleic acid template molecule, and specific nucleic acid molecule copies of the template are amplified enzymatically.
  • PCR polymerase chain reaction
  • a second aspect of the present invention encompasses any plasmids or expression vectors, including any viral vectors, comprising the nucleic acid described herein.
  • Such vectors may be introduced into suitable host cells, such as, for example, bacterial cells, yeast cells, insect cells, or mammalian cells, for the purpose of expressing a recombinant GOBLIN polypeptide or a functional fragment thereof, in particular an immunogenic peptide fragment.
  • a protein-encoding region comprising at least about 15 contiguous nucleotides ofthe protein-encoding region of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9, or nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID Nos: 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 or an equivalent region from another GOBLLN-encoding gene, is placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in a cell-free system or cellular system.
  • promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • promoter is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably connected, and which encodes the polypeptide or peptide fragment.
  • Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression ofthe said nucleic acid molecule.
  • Placing a nucleic acid molecule under the regulatory control of, i.e., "in operable connection with”, a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the coding sequence that they control.
  • 'Gateway refers to the Invitrogen Gateway cloning protocols and vectors that are used to amplify and clone nucleic acids
  • the promoter is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived.
  • the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • the prerequisite for producing intact polypeptides and peptides in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site.
  • Typical promoters suitable for expression in bacterial cells such as E. coli include, but are not limited to, the lacz promoter, temperature-sensitive ⁇ L or ⁇ R promoters, T7 promoter or the IPTG-inducible tac promoter.
  • a number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047150338, 1987) or Sambrook et al (In: Molecular cloning.
  • Typical promoters suitable for expression in viruses of eukaryotic cells and eukaryotic cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst others.
  • CMV cytomegalovirus
  • CMV IE cytomegalovirus immediate early promoter amongst others.
  • WAP mouse whey acidic protein
  • the WAP gene is expressed almost exclusively in mammary tissue (Pittius et al, Proc Natl Acad Sci USA 85, 5874-5878, 1988), and its transcription is induced several thousand-fold at mid-pregnancy and remains high throughout lactation (Pittius et al, Mol Endocrinol 2, 1027-1032, 1988). Induction and maintenance of WAP gene expression is mediated to a large extent through the prolactin and glucocorticoid signalling pathways.
  • the distal Stat5-binding site of the WAP promoter is required for high level and prolactin-modulated (i.e. prolactin- induced) expression (Li and Rosen, Mol Cell Biol 15, 2063-2070, 1995).
  • the distal NF1 site also appears to be required for WAP gene expression (Li and Rosen, Mol Cell Biol 15, 2063-2070, 1995), and the promoter proximal Ets site mediates transcription in late pregnancy but not for high expression throughout lactation (McKnight et al, Mol Endocrinology 9, 717-724, 1995). Elements that confer glucocorticoid responsiveness on the WAP promoter have also been mapped in the promoter distal region. Binding sites for transcription factors belonging to the NF1 and Ets family have been located within 200 bp ofthe transcriptional start site.
  • Preferred vectors for expression in mammalian cells include, but are not limited to: (i) the pcDNA vector suite supplied by Invitrogen, in particular pcDNA 3.1 myc-His-tag comprising the CMV promoter and encoding a C- terminal 6xHis and MYC tag; and (ii) the retrovirus vector pSR ⁇ tkneo (Muller et al, Mol. Cell. Biol, 11, 1785, 1991).
  • the vector pcDNA 3.1 myc-His (Invitrogen) is particularly preferred for expressing a secreted form of GOBLIN or a derivative thereof in 293T cells, wherein the expressed peptide or protein can be purified free of conspecific proteins, using standard affinity techmques that employ a Nickel column to bind the protein via the His tag.
  • GOBLIN polypeptides or immunological derivatives thereof are available publicly, and described, for example, in Sambrook et al (In: Molecular cloning. A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
  • Means for introducing the isolated nucleic acid molecule or a gene construct comprising same into a cell for expression are well-known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into animal cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG- mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
  • a further aspect of the present invention provides a method of producing a GOBLIN polypeptide or a functional fragment thereof, said method comprising culturing a host cell comprising the nucleic acid of the invention in an expressible format under conditions sufficient for expression to occur and then recovering the expressed polypeptide.
  • the expressed polypeptide is directed to the cell surface.
  • the recovered polypeptide is rendered substantially free of conspecific proteins using known protein isolation/purification techniques.
  • Preferred cell lines for this purpose are insect or mammalian cells. Baculovirus cell expression systems using Sf9 or Sf21 cells, or vaccinia virus expression systems using COS cells, CHO cells, or HEK 293 cells, are especially preferred.
  • a further aspect of the present invention clearly extends to an isolated GOBLIN polypeptide or an immunologically active derivative thereof, in particular a derivative polypeptide comprising a WW domain and/or C2 domain of a full length GOBLIN polypeptide.
  • the isolated polypeptide of the invention is substantially free of conspecific proteins.
  • purity can be assessed by standard procedures, such as, for example, SDS/polyacrylamide gel electrophoresis, 2- dimensional gene electrophoresis, chromatography, amino acid composition analysis, or amino acid sequence analysis.
  • GOBLIN polypeptides or fragments standard protein purification techniques may be employed. For example, gel filtration, ion exchange chromatography, reverse phase chromatography, or affinity chromatography, or a combination of any one or more said procedures, may be used. High pressure and low pressure procedures can also be employed, such as, for example, FPLC, or HPLC.
  • a suitable cellular expression system in combination with a suitable affinity tag, such as a 6xHis tag, and to purify the polypeptide using an affinity step that bonds it via the tag (supra).
  • the tag may then be cleaved from the expressed polypeptide.
  • a proline-rich ligand such as, for example, a peptide comprising the amino acid sequence PPPY or tandem repeats thereof is particularly useful for isolating a GOBLIN polypeptide or the WW domain thereof.
  • proline-rich ligands will comprise one or more repeats of the amino acid sequence RX ⁇ PPX 2 Y wherein X, is any amino acid and preferably an amino acid selected from the group consisting of Q, D, M, P, A, E and N and wherein X 2 is any amino acid and preferably an amino acid selected from the group consisting of A, E, P, S and R.
  • a phospholipid-containing ligand is also useful for isolating a GOBLIN polypeptide or the C2 domain thereof.
  • the isolated polypeptide of the invention will comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or any one of SEQ ID NOS: 11-21 or an immunologically active derivative thereof.
  • a third aspect ofthe present invention provides a nucleic acid probe for detecting RNA encoding a GOBLIN polypeptide in a sample.
  • a "nucleic acid probe” is any nucleic acid described herein that is useful for detecting RNA encoding a GOBLIN polypeptide or a derivative or analog thereof in a sample.
  • Nucleic acid probes can comprise inosine, adenine, guanine, thymidine, cytidine or uracil residues or functional analogues or derivatives thereof that are capable of being incorporated into a polynucleotide molecule, provided that the resulting probe or primer is capable of hybridizing under at least low stringency conditions to GOBLLN-encoding RNA or DNA.
  • probes may comprise double-stranded or single-stranded nucleic acid
  • single-stranded probes are preferred because they do not require melting prior to use in hybridizations.
  • longer probes are also preferred because they can be used at higher hybridization stringency than shorter probes and may produce lower background hybridization than shorter probes.
  • oligonucleotide probes are particularly preferred by the present invention.
  • the nucleotide sequence ofthe probe is carefully selected to maximize the Tm at which hybridizations can be performed, reduce non-specific hybridization, and to reduce self-hybridization. Such considerations may be particularly important for applications involving high throughput screening using microarray technology.
  • nucleotide sequence of an oligonucleotide probe is selected such that it is unique to GOBLIN RNA or GOBLIN protein-encoding sequence, has a low propensity to form secondary structure, low self-complementary, and is not highly A/T-rich.
  • probes are cross-hybridize to nucleic acid encoding GOBLIN or the complementary nucleotide sequence thereto and are sufficiently unique in sequence to generate high signaknoise ratios under specified hybridization conditions.
  • long nucleic acid probes are preferred because they tend to generate higher signa noise ratios than shorter probes and/or the duplexes formed between longer molecules have higher melting temperatures (i.e. Tm values) than duplexes involving short probes.
  • Tm values melting temperatures
  • full-length DNA or RNA probes are contemplated by the present invention, as are specific probes comprising the sequence of the 3 '-untranslated region or complementary thereto.
  • the nucleotide sequence of an oligonucleotide probe has no detectable nucleotide sequence identity to a nucleotide sequence in a BLAST search (Altschul et al, J. Mol. Biol.
  • nucleotide sequence of an oligonucleotide probe has the following properties:
  • the self-complementarity of a nucleotide sequence can be dete ⁇ nined by aligning the sequence with its reverse complement, wherein detectable regions of identity are indicative of potential self-complementarity.
  • sequences may not necessarily form secondary structures during hybridization reaction, and, as a consequence, successfully identify a target nucleotide sequence.
  • reaction conditions can be modified to reduce the adverse consequences of such structure formation. Accordingly, a potential for self-complementarity should not necessarily exclude a particular candidate oligonucleotide from selection. In cases where it is difficult to determine nucleotide sequences having no potential self-complementarity, the uniqueness of the sequence should outweigh a consideration of its potential for secondary structure formation.
  • the nucleic acid probe may comprise a nucleotide sequence that is within the coding strand of the GOBLLN-encoding gene (ie. it is comprised within the nucleotide sequence of RNA encoding GOBLIN).
  • Such "sense" probes are useful for detecting RNA encoding GOBLIN by amplification procedures, such as, for example, polymerase chain reaction (PCR), and more preferably, quantitative PCR or reverse transcription polymerase chain reaction (RT-PCR).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • "sense" probes may be expressed to produce GOBLIN polypeptides or immunologically active derivatives thereof that are useful for detecting the expressed GOBLIN protein in samples.
  • the nucleic acid probe may comprise a nucleotide sequence that is within the antisense strand of said gene (i.e. it is complementary to RNA encoding GOBLIN).
  • antisense probes are useful for directly hybridizing to RNA encoding GOBLIN, or alternatively, for detecting RNA encoding GOBLIN by amplification, as described supra (eg. quantitative PCR or RT-PCR).
  • nuclei acid probes will comprise a nucleotide sequence set forth in any one of SEQ ID Nos: 22-45 (Table 2) or any one of SEQ ID Nos: 46-353 (Table
  • a further aspect of the present invention provides a method of identifying a compound that reduces or antagonizes GOBLIN expression, such as, for example, in the treatment of a hyperproliferative disease, said method comprising: (a) administering a candidate compound to a cancer cell that expresses a GOBLIN polypeptide at an elevated level relative to a non-cancer cell; and (b) determining the level of a GOBLIN polypeptide in the presence of the compound relative to the level of expression in the absence of the compound, wherein reduced level of a GOBLIN polypeptide in the presence of the compound indicates that the compound is an antagonist of GOBLIN expression.
  • the method according to this embodiment further comprises obtaining the cancer cell.
  • the cell in which the assay is conducted is a cell that over expresses a non-endogenous GOBLIN polypeptide by virtue of having been stably transformed or transiently transfected with a nucleic acid encoding a GOBLIN polypeptide as described herein.
  • a further embodiment of the present invention provides a method of identifying a compound that reduces or antagonizes GOBLIN expression, such as, for example, in the treatment of a hyperproliferative disease, said method comprising: (a) administering a candidate compound to a transformed or transfected cell that expresses a non-endogenous GOBLIN polypeptide at an elevated level relative to a non- transformed or non-transfected cell; and (b) determining the level of a GOBLIN polypeptide in the presence of the compound relative to the level of expression in the absence of the compound, wherein reduced level of a GOBLIN polypeptide in the presence of the compound indicates that the compound is an antagonist of GOBLIN expression.
  • the method according to this embodiment further comprises obtaining or providing or producing the transformed or transfected cell.
  • the assay comprises an immunoassay wherein the level of GOBLIN protein is determined by contacting the cell or a protein extract thereof with an antibody that binds to a GOBLIN polypeptide under conditions sufficient for an antigen-antibody complex to form and detecting the antibody bound.
  • the antibody bound is generally detected by contacting the antibody with a secondary antibody that is capable of producing a detectable signal.
  • an immunoassay is performed wherein the amount of GOBLIN is determined by contacting the GOBLIN protein with a primary and secondary antibody that each bind to GOBLIN under conditions sufficient for antigen- antibody complexes to form and detecting an antibody bound to the GOBLIN protein.
  • the antibody bound to the GOBLIN protein is generally detected by contacting the antibody with a secondary antibody that is capable of producing a detectable signal.
  • the primary and secondary antibody will generally bind to different epitopes on the GOBLIN protein.
  • the amount of GOBLIN protein in a cell is detected by an immunoassay comprising:
  • the amount of GOBLIN protein in a cell is detected by an immunoassay comprising:
  • the antibody is generally detected by contacting the antibody with an antibody that is capable of producing a detectable signal.
  • nucleic acid-based detection system is employed.
  • the present invention provides a method of identifying a compound that reduces or antagonizes GOBLIN expression, such as, for example, in the treatment of a hyperproliferative disease, said method comprising: (a) administering a candidate compound to a cancer cell that expresses mRNA encoding a GOBLIN polypeptide at an elevated level relative to a non-cancer cell; and (b) determining the level of mRNA encoding a GOBLIN polypeptide in the presence of the compound relative to the level of expression in the absence of the compound, wherein reduced level of mRNA encoding a GOBLIN polypeptide in the presence of the compound indicates that the compound is an antagonist of GOBLIN expression.
  • the method according to this embodiment further comprises obtaining the cancer cell.
  • the cell in which the assay is conducted is a cell that over expresses a non-endogenous GOBLLN-encoding mRNA by virtue of having been stably transformed or transiently transfected with a nucleic acid encoding a GOBLIN polypeptide as described herein.
  • a further embodiment of the present invention provides a method of identifying a compound that reduces or antagonizes GOBLIN expression, such as, for example, in the treatment of a hyperproliferative disease, said method comprising: (a) administering a candidate compound to a transformed or transfected cell that expresses mRNA encoding a non-endogenous GOBLIN polypeptide at an elevated level relative to a non-transformed or non- transfected cell; and (b) determining the level of mRNA encoding a GOBLIN polypeptide in the presence of the compound relative to the level of expression in the absence of the compound, wherein reduced level of mRNA encoding a GOBLIN polypeptide in the presence of the compound indicates that the compound is an antagonist of GOBLIN expression.
  • the method according to this embodiment further comprises obtaining or providing or producing the transformed or transfected cell.
  • the phenotype of the animal subject is determined in the presence and absence of the compound.
  • a test compound is administered to mammary gland tissue of the animal subject.
  • nucleic acids it is particularly preferred that these are provided by injection.
  • the nucleic acids are contained within a virus vector, in particular an adenovirus. As will be known to those skilled in the art, such adenoviruses tend to remain localized to the injection site. Accordingly, the metabolism-associated phenotype will generally be determined in muscle tissue of the animal subject to which the virus vector is administered.
  • the present invention clearly encompasses the use of any in silico analytical method and/or industrial process for carrying the screening methods described herein into a pilot scale production or industrial scale production of an inhibitory compound identified in such screens.
  • This invention also provides for the provision of information for any such production.
  • a further aspect of the present invention provides a process for identifying or determining a compound or modulator supra, said method comprising:
  • step (a) supra determination of the structure of the compound is implicit in step (a) supra. This is because the skilled artisan will be aware ofthe name and/or structure ofthe compound at the time of performing the screen.
  • the term "providing the compound or modulator” shall be taken to include any chemical or recombinant synthetic means for producing said compound or modulator or alternatively, the provision or a compound or modulator that has been previously synthesized by any person or means.
  • the compound or modulator or the name or structure ofthe compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.
  • a further aspect of the present invention provides a process for producing a compound or modulator supra, said method comprising:
  • the synthesized compound or modulator or the name or structure ofthe compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.
  • a further aspect ofthe present invention provides an antagonist of GOBLIN expression or activity.
  • the compound has been isolated by a process comprising performing a method of identifying a compound that reduces or antagonizes GOBLIN expression and/or determining a compound or modulator supra and/or producing a compound or modulator supra. All such screening embodiments apply mutatis mutandis to the present embodiment ofthe invention.
  • the antagonist of GOBLIN expression comprises nucleic acid such as, for example, an antisense nucleic acid, peptide nucleic acid (PNA), ribozyme, or small interfering RNA (siRNA), short hairpin RNA (shRNA) which is complementary, in whole or in part, to a target molecule comprising a sense strand, and can hybridize with the target molecule, in particular, GOBLLN-encoding RNA.
  • nucleic acid such as, for example, an antisense nucleic acid, peptide nucleic acid (PNA), ribozyme, or small interfering RNA (siRNA), short hairpin RNA (shRNA) which is complementary, in whole or in part, to a target molecule comprising a sense strand, and can hybridize with the target molecule, in particular, GOBLLN-encoding RNA.
  • PNA peptide nucleic acid
  • shRNA small interfering RNA
  • shRNA short hairpin RNA
  • nucleic acid capable of forming a triple helix eg. Helene,
  • the antisense nucleic acid, ribozyme, PNA, siRNA or shRNA comprises a sequence that is complementary to at least about 12 or 15 or 18 or 20 contiguous nucleotides of a sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9 (ie. it is complementary to GOBLIN RNA) and can hybridize thereto.
  • such antagonistic nucleic acid can be complementary to a target nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9 or a portion thereof sufficient to allow hybridization.
  • Longer molecules, comprising a sequence that is complementary to at least about 25, or 30, or 35, or 40, or 45, or 50 contiguous nucleotides of GOBLIN RNA are also encompassed by the present invention.
  • antisense nucleic acid shall be taken to mean DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990) and capable of interfering with a post-transcriptional event such as mRNA translation.
  • Antisense oligomers complementary to at least about 15 contiguous nucleotides of GOBLIN-encoding mRNA i.e., any one of SEQ ID NOs: 1, 3, 5, 7, or 9 are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target GOBLLN- producing cell.
  • the use of antisense methods is well known in the art (Marcus-Sakura, Anal. Biochem.
  • Preferred antisense nucleic acid will comprise a nucleotide sequence that is complementary to at least 15 contiguous nucleotides of a sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8 or 10-21, or a part thereof, including any one or more ofthe antisense nucleic acids set forth in Tables 2 or 3.
  • a "ribozyme” is a nucleic acid molecule having nuclease activity for a specific nucleic acid sequence.
  • a ribozyme specific for GOBLIN-encoding mRNA for example, binds to and cleaves specific regions of the mRNA, thereby rendering it untranslatable.
  • preferred ribozymes will comprise a nucleotide sequence that is complementary to at least about 12-15 contiguous nucleotides of a sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8 or 10-21, or a part thereof.
  • RNAi small interfering RNA
  • 'siRNA short interfering RNA
  • shRNA short hairpin RNA
  • RNAi homologous double stranded RNA
  • the dsRNA comprises two short nucleotide sequences derived from the target RNA encoding GOBLIN and having self-complementarity such that they can anneal, and interfere with expression of a target gene, presumably at the post- transcriptional level.
  • RNAi molecules are described by Fire et al., Nature 391, 806- 811, 1998, and reviewed by Sharp, Genes & Development, 13, 139-141, 1999).
  • short hairpin RNA is similar to siRNA, however comprises a single strand of nucleic acid wherein the complementary sequences are separated an intervening hairpin loop such that, following introduction to a cell, it is processed by cleavage of the hairpin loop into siRNA. Accordingly, each and every embodiment described herein is equally applicable to siRNA and shRNA.
  • Preferred siRNA or shRNA molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA.
  • the target sequence in GOBLIN mRNA commences with the dinucleotide AA, comprises a GC- content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than a GOBLIN gene in the genome ofthe animal in which it is to be introduced, e.g., as determined by standard BLAST search.
  • the siRNA or shRNA is preferably capable of down regulating expression of human GOBLIN in a cell.
  • this should not be taken to indicate a requirement for the siRNA or shRNA to be specific for human GOBLIN-encoding genes.
  • the siRNA or shRNA molecules it is possible and appropriate in certain circumstances for the siRNA or shRNA molecules to reduce expression of both endogenous murine GOBLIN, as well as ectopically expressed human GOBLIN in the cell.
  • Confirmation of a specific activity of an antagonist against human GOBLIN is preferably determined by assessing the activity of an inhibitor in a cell derived from a GOBLIN " ' " mouse that has been engineered to express human GOBLIN.
  • preferred siRNA against a GOBLIN encoding gene comprises a 21 -nucleotide sequence set forth in any one of SEQ ID Nos: 46-353. TABLE 3 Nucleotide sequences of oligonucleotides for producing siRNA and/or shRNA that targets GOBLIN mRNA expression.
  • GGTCTACTACATAGACCACtt 48 GTGGTCTATGTAGTAGACCtt 49
  • CAAGCTGAAGAGAGAGATGtt 80 CATCTCTCTCTTCAGCTTGtt 81
  • GCTGAAGAGAGATGGTTtt 82 AACCATCTCTCTCTTCAGCtt 83
  • GAGAAGGATCGCC AACCTGtt 118 CAGGTTGGCGATCCTTCTCtt 119
  • GGATCGCCAACCTGAAGATtt 120 ATCTTCAGGTTGGCGATCCtt 121
  • GGTGGAGTTCCTGCTCCTGtt 158 CAGGAGCAGGAACTCCACCtt 159
  • GGCAGAGGGAGGTGGCCGCtt 162 GCGGCCACCTCCCTCTGCCtt 163
  • TAAGCAATTTGCAATATTAtt 200 TAATATTGCAAATTGCTTAtt 201
  • GCAATTTGCAATATTAATCtt 202 GATTAATATTGCAAATTGCtt 203
  • TCATCCAGCTGAGTAACCTtt 208 AGGTTACTCAGCTGGATGAtt 209
  • GAGTGCCTGGGAGGCGCCCtt 230 GGGCGCCTCCCAGGCACTCtt 231
  • GCGGCCTTCCTCGGTCAAGtt 268 CTTGACCGAGGAAGGCCGCtt 269
  • CAAGCCAAGAGCCACGGGGtt 282 CCCCGTGGCTCTTGGCTTGtt 283
  • CTAAAGCAGTATTGAGTTGtt 326 CAACTCAATACTGCTTTAGtt 327
  • GTAATGCCTGGGGGACGGTtt 334 ACCGTCCCCCAGGCATTACtt 335
  • the even-numbered SEQ ID Nos: in Table 3 each comprise (i) a 19-nucleotide sequence corresponding to a human GOBLIN mRNA target sequence adjacent and downstream of a dinucleotide AA in said mRNA target; and (ii) a 3 '-extension dinucleotide TT.
  • the odd-numbered SEQ ID Nos: in Table 3 each comprise (i) a 19- nucleotide sequence complementary to a human GOBLIN mRNA target sequence contained within SEQ ID NOS: 46-353; and (ii) a 3 '-extension dinucleotide TT.
  • the sense and antisense strands are positioned such that they flank an intervening loop sequence.
  • Preferred loop sequences are selected from the group consisting of:
  • CTCGAG SEQ ID NO: 357
  • AAGCTT SEQ ID NO: 358
  • sequence set forth in SEQ ID NO: 360 is particularly preferred for modulating human GOBLIN expression in a cell, tissue (eg., cancer cell or animal model of cancer, including a GOBLIN knock-out mouse expressing a non- endogenous GOBLIN mRNA and/or protein).
  • tissue eg., cancer cell or animal model of cancer, including a GOBLIN knock-out mouse expressing a non- endogenous GOBLIN mRNA and/or protein.
  • Preferred siRNA molecules that are selectively active against human GOBLIN expression compared to murine GOBLIN expression are derived from the sequence of the 5'-non-coding and/or 3'-non-coding region ofthe human GOBLIN gene or mRNA encoding the GOBLIN unique peptides set forth in SEQ ID Nos: 19-21.
  • the antisense RNA, ribozyme, siRNA or shRNA can be introduced directly to a cell or cell-free extract capable of expressing GOBLIN as naked DNA.
  • DNA encoding a nucleic acid inhibitory molecule can be introduced into a cell in operable connection with a suitable promoter and transcription terminator sequence to facilitate expression of the inhibitory nucleic acid.
  • Preferred promoters for expression in mammalian cells that express GOBLIN include the CMV promoter, ubiquitin promoter, U6 small nuclear RNA promoter (Lee et al, Nature Biotech. 20, 500-505, 2002; Miyagishi et a;., Nature Biotech 20, 497-500, 2002; Paul et al, Nature Biotech.
  • the DNA encoding the inhibitory nucleic acid is operably connected to promoter and terminator regulatory sequences by cloning into a suitable vector that comprises the necessary promoter and transcriptional terminator sequences, and the recombinant vector is then introduced to the cell, tissue or organ by transient transfection of plasmid DNA, by establishing permanent cell lines or in infection with retroviral expression vectors (Barton et al, Proc. Natl Acad. Sci USA 99, 14943-14945, 2002; Devroe etal, BMC Biotech. 2, pl5, 2002).
  • Preferred retroviral vectors suitable for transiently transfecting into isolated cells e.g., by calcium phosphate precipitation (Ketteler et al, Gene Ther. 9, 477-487, 2002) in high throughput screens, or for the production of transducing supernatants (Ketteler et al, Gene Ther. 9, 477-487, 2002) for lower-throughput screening or validation of primary screen results, include pBABE (Morgenstern et al, Nuc. Acids Res. 18, 3587- 3596, 1990) and JZenNeo.
  • the pBabe retroviral vector constructs transmit inserted genes at high titres and express them from the Mo MuLV Long Terminal Repeat (LTR).
  • the pBabe vectors comprise one of four different dominantly-acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin.
  • the high titre ecotropic helper free packaging cell line, omega E reduces the risk of generation of wild type Mo MuLV via homologous recombination events.
  • the pBabe vectors and omega E cell line provide high frequency gene transfer, and/or concomitant expression of GOBLIN with one or more other genes in a single cell (e.g., a GOBLIN ligand), with minimal risk of helper virus contamination.
  • the adenoviral vectors pAdTrack and pAdTrack-CMV He et al, Proc. Natl Acad. Sci USA 95, 2509-2514, 1998; pAdTrack-HP (Zhao et al, Gene 316, 137-141, 2003)
  • an Ad5CMV-based vector e.g., Ad5CMV-GFP (Suoka et al, Am. J. Respir. Cell Mol. Biol. 23, 297-303, 2000)
  • pSilencer adeno 1.0-CMV are preferred for delivery and expression in specific organs or tissues, in particular muscle tissue of a mouse model.
  • the pAdTrack and pAdTrack-CMV vectors are particularly preferred for applications which require standardization for transfection or transduction efficiency eg., injection of adenovirus into hindlimb muscles of transgenic mouse models.
  • the pAdTrack vector is used for production of GFP-trackable viruses containing transgenes under the control of a chosen promoter. It contains the gene encoding enhanced GFP, a polylinker for insertion of exogenous transgenes surrounded by adenoviral sequences ("arms”) that allow homologous recombination with pAdEasy-1.
  • the left arm contains Ad5 nucleotides 34,931-35,935, which mediate homologous recombination with pAdEasy vectors in E. coli, plus inverted terminal repeat (ITR) and packaging signal sequences (nucleotides 1-480 of Ad5) required for viral production in mammalian cells.
  • the right arm contains Ad5 nucleotides 3,534-5,790, which mediate homologous recombination with pAdEasy vectors. Artificially created Pad sites surround both arms.
  • the AdTrack plasmid also contains a kanamycin resistance gene from pZero 2.1 (Invitrogen) and the origin of replication from pBR322 (Life Technologies).
  • the relatively low copy number of plasmids generated with this origin is essential for the stability of large constructs in E. coli.
  • the pAdTrack-CMV vector is identical to pAdTrack except for the addition of a cytomegalovirus (CMV) promoter and polyadenylation site (both from pEGFP-Cl, Clontech).
  • CMV cytomegalovirus
  • polyadenylation site both from pEGFP-Cl, Clontech.
  • a polylinker is present between the CMV promoter and polyadenylation site.
  • adenoviral vectors are also suitable for transfection of cell lines.
  • Antisense nucleic acids, ribozymes, PNAs, siRNAs or shRNAs are useful for a variety of purpose, including research and therapeutic applications.
  • a constract comprising an antisense nucleic acid, ribozyme, PNA, siRNA or shRNA can be introduced into a suitable cell to inhibit GOBLIN expression and/or activity therein.
  • a suitable cell provides a valuable control cell, for instance in assessing the specificity of the GOBLIN-ligand interaction with the parent cell or other related cell types.
  • such a construct can be introduced into some or all of the cells of a mammal.
  • the antisense nucleic acid, ribozyme, PNA, or interfering RNA inhibits receptor expression, and any cancer or hyperproliferative process mediated by GOBLIN in the cells containing the construct are inhibited.
  • a cancer or other hyperproliferative disease or condition can be treated using an antisense nucleic acid, ribozyme, PNA, siRNA or shRNA ofthe present invention.
  • an antagonist of GOBLIN activity is an antibody molecule, such as, for example, a polyclonal antibody or monoclonal antibody directed against a functional epitope of the protein.
  • Antibodies that can inhibit one or more functions characteristic of a GOBLIN protein, such as a binding activity, a signalling activity, and/or stimulation of a cellular response are clearly encompassed by the present invention.
  • antibodies of the present invention can inhibit binding of a ligand (i.e., one or more ligands) to a mammalian GOBLIN protein and/or can inhibit one or more functions mediated by a mammalian GOBLIN protein in response to ligand binding.
  • the antibodies can inhibit (reduce or prevent) the interaction of receptor with a natural ligand.
  • the "antibodies” contemplated herein are immunoreactive with GOBLIN polypeptides or functional fragments thereof. Antibodies that consist essentially of pooled monoclonal antibodies with different epitope specificities, as well as distinct monoclonal antibody preparations are contemplated. Monoclonal antibodies are produced from fragments of the GOBLIN protein that comprise one or more B cell epitopes by methods well known to those skilled in the art (Kohler et al, Nature 256:495, 1975). The term “antibody” as used herein includes intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , Fv and single chain antibody fragments capable of binding an epitopic determinant of GOBLIN.
  • Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.
  • An "Fab' fragment” of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule freated in this manner.
  • F(ab') 2 fragment of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • a (Fab')2 fragment is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • Fv fragment is a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.
  • a “single chain antibody” is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.
  • the antibody is produced against a synthetic peptide that comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19- 21 and more preferably, SEQ ID NO: 19 or 21.
  • Anti-GOBLLN antibodies or antibody fragments are generated using the entire GOBLIN polypeptide or an immunogenic fragment thereof (alone or linked to a suitable carrier or hapten) to immunize a subject (e.g., a mammal including, but not limited to a rabbit, goat, mouse or other mammal).
  • a subject e.g., a mammal including, but not limited to a rabbit, goat, mouse or other mammal.
  • a mammal including, but not limited to a rabbit, goat, mouse or other mammal.
  • the mammal being immunized does not contain endogenous GOBLIN (e.g., a GOBLLN-defitient genetically modified animal).
  • the immunogenic preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • an adjuvant such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic proteolytic or synthetic GOBLIN peptide preparation induces a polyclonal anti- GOBLLN antibody response.
  • the immunogenically effective amounts of the immunizing peptides are determined empirically. Factors to be considered include the immunogenicity ofthe native polypeptide, whether or not the polypeptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, the route of adminisfration for the composition, i.e., intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.
  • the anti-GOBLIN antibody titer in the immunized subject is generally monitored over time by standard techniques, such as with an enzyme linked ⁇ nmunosorbent assay (ELISA) using immobilized GOBLIN. Subsequently, the sera from the immunized subjects can be tested for GOBLIN inhibitory activity.
  • ELISA enzyme linked ⁇ nmunosorbent assay
  • nucleic acid expressing GOBLIN using DNA immunization technology, such as that disclosed in U.S. Pat. No. 5,795,872 to Ricigliano et al., or alternatively, in U.S. Pat. No. 5,643,578 to Robinson et al.
  • the antibody molecules directed against GOBLIN can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare e.g., monoclonal antibodies by standard techniques, such as the hybridoma technique originally described in the following disclosures: Kohler and Milstein Nature 256:495-497, 1975; Brown et al. J. Immunol. 127:53946, 1981; Brown et al. J. Biol Chem.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds GOBLIN.
  • the immortal cell line e.g., a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or S ⁇ 2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind GOBLIN, e.g., using a standard ELISA assay. The antibodies can then be tested for GOBLIN inhibitory activity.
  • an antagonist of GOBLIN activity comprises a peptide, protein or polypeptide, including a dominant-negative mutant of a GOBLIN polypeptide.
  • Preferred peptidyl GOBLIN inhibitors are chemically or recombinantly synthesized as oligopeptides (approximately 10-25 amino acids in length) spanning the GOBLIN protein sequence (SEQ ID NO: 2, 4, 6, 8 or 10-21).
  • GOBLIN fragments are produced by digestion of native or recombinantly produced GOBLIN by, for example, using a protease, e.g., trypsin, thermolysin, chymotrypsin, or pepsin.
  • Computer analysis using commercially available software, e.g. MacVector, Omega, PCGene, Molecular Simulation, Inc. is used to identify proteolytic cleavage sites.
  • the proteolytic or synthetic fragments can comprise as many amino acid residues as are necessary to partially or completely inhibit GOBLIN function.
  • Preferred fragments will comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.
  • peptides are selected which contain a sufficient number of B cell epitopes to elicit antibodies when administered to a mammal.
  • Such peptides are identified by immunizing a mammal with the peptide alone, or in combination with an adjuvant, or linked to an adjuvant (e.g., a hapten). Sera from the immunized animal are tested for anti-GOBLIN antibodies.
  • Preferred peptides generate anti-GOBLIN antibodies that inhibit a GOBLIN function.
  • Preferred peptidyl GOBLIN inhibitors will also not comprise a sufficient number of T cell epitopes to induce T-cell mediated (e.g., cytokine) responses when determined using any of a number of well known techniques, such as epitope prediction using algorithms (see e.g., Rothbard and Taylor EMBO J. 7: 93-100, 1988; Berzofsky, Philos Trans R. Soc. Lond. 323: 535-544, 1989; Rothbard, 1st Forum in Virology, Annals of the Pasteur Institute, pp 518-526, Dec. 1986; Rothbard and Taylor, Embo, 7: 93-100, 1988; EP 0 304 279; and Margalit et al., J. Immunol., 138: 2213-2229, 1987); or screening of peptide inhibitors for human T cell stimulating activity or T cell proliferation assays (e.g. Proc. Natl. Acad. Sci USA, 86:1333, 1989).
  • GOBLIN preferred peptide inhibitors of GOBLIN are located on the surface of the GOBLIN proteins, e.g., hydrophilic regions, as well as regions with high antigenicity or fragments with high surface probability scores can be identified using computer analysis programs well known to those of skill in the art (Hopp and Wood, (1983), Mol. Immunol., 20, 483-9, Kyte and Doolittie, (1982), J. Mol. Biol, 157, 105-32, Corrigan and Huang, (1982), Comput. Programs Biomed, 3, 163-8).
  • dominant-negative mutant refers to a GOBLIN polypeptide that has been mutated from its natural state and that interacts with a protein that GOBLIN normally interacts with thereby preventing endogenous native GOBLIN from forming the interaction.
  • Preferred dominant negative mutants will lack the WW domain or C2 domain of GOBLIN that is required for interaction with a binding partner of GOBLIN.
  • Preferred dominant-negative mutants comprise variants of the native GOBLIN protein, such as, for example, substitution or deletion mutants, that act as dominant-negative mutants of GOBLIN function.
  • a dominant-negative mutant may comprise one or more amino acid substitutions within the GOBLIN polypeptide such that, when expressed in a cell, the dominant-negative mutant protein competes with native endogenous GOBLIN for the cognate ligand, however has reduced or no activity.
  • Means for producing mutated nucleic acid are well known to those skilled in the art and may be achieved readily e.g., using the Quick Change Mutagenesis kit supplied by Stratagene, La Jolla, California USA according to the manufacturer's instructions.
  • Dominant negative mutant proteins are produced by expression of nucleic acid encoding the mutant protein, essentially as described herein above for expression of peptides in cells.
  • Peptides comprising a WW domain or C2 domain of a GOBLIN polypeptide that act as dominant negative mutants to prevent or inhibit the binding of a GOBLIN polypeptide to one of its cognate proteinaceous or phospholipid-containing binding partners in vivo, to thereby antagonize a signalling activity mediated by GOBLIN and/or stimulation of a cellular response mediated by GOBLIN, are also encompassed by the present invention.
  • a peptide comprising a GOBLIN WW domain inhibits binding of a ligand (i.e., one or more ligands) to a mammalian GOBLIN protein and/or inhibits one or more functions mediated by a mammalian GOBLIN protein in response to such ligand binding.
  • a ligand i.e., one or more ligands
  • the WW domain peptides inhibit (reduce or prevent) the interaction of GOBLIN with a naturally occurring protein in the mammary epithelium.
  • a peptide comprising a GOBLIN C2 domain inhibits binding of a phospholipid-rich ligand to a mammalian GOBLIN protein and/or inhibits one or more functions mediated by a mammalian GOBLIN protein in response to such ligand binding.
  • the C2 domain peptide inhibits (reduces or prevents) the interaction of GOBLIN with a phospholipid-containing ligand in the mammary epithelium.
  • an antagonist of GOBLIN activity comprises a small organic molecule.
  • a small organic molecule inhibitor or antagonist of GOBLIN is a compound that reduces the expression or activity of GOBLIN.
  • the small organic molecule belongs to a class of compounds that binds to the WW domain or C2 domain of GOBLIN.
  • a fourth aspect of the invention provides a probe that binds to a GOBLIN polypeptide of the invention.
  • the probe is an antibody molecule.
  • Preferred antibodies will selectively bind to a GOBLIN polypeptide or an immunological derivative thereof and will not bind, or will only bind weakly, to non-GOBLLN polypeptides or peptides.
  • Antibodies prepared against any one of SEQ ID Nos: 11-21 are preferred for this purpose.
  • Anti-GOBLIN antibodies that are particularly contemplated by the present invention include monoclonal and polyclonal antibodies as well as fragments thereof comprising the antigen-binding domain and/or one or more complementarity determining regions of the native antibody.
  • antibody fragment shall be taken to mean a portion of the variable region of the immunoglobulin molecule that binds to its target, i. e., the antigen-binding region.
  • Preferred antibodies for use in diagnostic imaging are those which react with an epitope on an external loop or external domain of the GOBLIN polypeptide as expressed in a cancer cell.
  • Such antibodies may be generated by using the complete human GOBLIN polypeptide as an immunogen.
  • a peptide fragment derived from a predicted external domain can be used.
  • regions ofthe polypeptide are selected and screened for their ability to elicit the production of specific anti-GOBLIN antibodies using standard immunoassays, such as, for example, ELISA.
  • the anti-GOBLIN antibodies of the invention are particularly useful in diagnostic and prognostic assays for cancer, particularly the early or later detection of mammary cancer or a metastasis thereof (eg. metastases in the mammary epithelium), by standard immunoassay or imaging methodologies. Similarly, such antibodies may be useful diagnosis and/or prognosis of any cancer in which GOBLIN is expressed at a level that differs from normal or healthy cells or tissue. As exemplified herein, antibodies have been prepared against the amino acid sequences set forth in SEQ ID Nos: 19 and 21 which identify a GOBLIN polypeptide by immunohistochemistry or Western blot detection methods. •
  • the probe is a ligand for a GOBLIN polypeptide, such as, for example, a ligand that binds to the WW domain or C2 domain of a GOBLIN polypeptide.
  • a further aspect ofthe present invention provides methods for detecting a cancer cell in a subject, said method comprising determining the level of GOBLIN-encoding nucleic acid or a GOBLIN polypeptide in a sample of said subject.
  • the detection of elevated or enhanced GOBLIN expression is particularly useful for detecting any stage of progression of a cancer, including early stages ofthe disease and metastases outside the primary tumor tissue.
  • the present invention clearly encompasses nucleic acid-based methods and protein-based methods for diagnosing cancer in humans and other mammals.
  • nucleic acid-based assays described herein rely upon the detection or relative quantification of RNA levels in samples using probes of at least about 12 or 15 or 18 or 20 nucleotides in length that hybridize specifically to RNA encoding the GOBLIN polypeptide, or alternatively, amplify cDNA from RNA encoding the GOBLIN polypeptide.
  • Such probes are derived from unique regions of any one or more of the GOBLIN-encoding genes described herein, such as, for example, any probe comprising 12 or 15 or 18 or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9 or the protein-encoding region of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9 or a complementary nucleotide sequence thereto, or homologous or identical sequence in any other mammalian GOBLIN-encoding gene.
  • full-length antisense cDNA or cRNA derived from a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9 is also encompassed by the present invention.
  • Particularly preferred probes for detecting GOBLIN-encoding mRNA are exemplified herein by SEQ ID Nos: 22-45.
  • any hybridization assay format can be used to detect GOBLIN-encoding RNA in samples, such as, for example, high-throughput screening using microarray technology, or conventional northern hybridization or reverse transcription polymerase chain reaction (i.e. RT-PCR). In situ localization can also be employed using histology specimens.
  • Probes for use in these assays including any allele-specific probes that selectively hybridize to one or more GOBLIN-encoding alleles, are not to be excluded and are readily identified by those skilled in the art based upon the nucleotide sequences and/or the amino acid sequence alignment, provided herein.
  • Protein-based assays are preferably immunoassays employing antibodies.
  • the immunoassays described herein utilize antibodies, including monoclonal and polyclonal antibodies, or a Fab fragment, F(ab') 2 fragment, or scFv fragment, that binds to a unique peptide region comprising at least about 5-10 contiguous amino acid residues of a human GOBLIN polypeptide.
  • antibodies against a WW domain of the GOBLIN polypeptide, or mimotopes of a WW domain that can act as dominant-negative molecules of the full-length GOBLIN protein are particularly preferred.
  • Antibodies against a C2 domain of the GOBLIN polypeptide or against a mimotope of a C2 -domain are also preferred.
  • the diagnostic methods described herein for the detection of GOBLIN-encoding nucleic acid or GOBLIN polypeptides or a derivative thereof in a sample are suitable for the diagnosis or prognosis of any hyperproliferative disease, such as, for example, a cancer selected from the group consisting of squamous cell carcinoma, hepatocellular carcinoma, ovarian cancer, breast cancer, melanoma, head and neck cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, prostate cancer, non-squamous carcinoma, glioblastoma and medulloblastoma, or a metastasis or micrometastasis thereof.
  • the cancer is other than ovarian cancer.
  • the cancer is an ovarian cancer selected from the group consisting of a basal cell carcinoma, a clear cell carcinoma, an endometrioid ovarian cancer, and a mutinous ovarian cancer. More preferably, the ovarian cancer is selected from the group consisting of an endometrioid ovarian cancer and a clear cell carcinoma.
  • the diagnostic methods described herein for the detection of GOBLLN-encoding nucleic acid or GOBLIN polypeptides or a derivative thereof in a sample are particularly useful for the diagnosis or prognosis of mammary cancer or a metastasis or micrometastasis thereof.
  • the term “diagnosis”, and variants thereof, such as, but not limited to “diagnose”, “diagnosed” or “diagnosing” shall not be limited to a primary diagnosis of a clinical state, however should be taken to include any primary diagnosis or prognosis of a clinical state.
  • the "diagnostic assay” formats described herein are equally relevant to assessing the remission of a patient, or monitoring disease recurrence, or tumor recurrence, such as following surgery or chemotherapy, or determining the appearance of metastases of a primary tumor. All such uses of the assays described herein are encompassed by the present invention.
  • control samples eg., mammary fat pads depleted of epithelium
  • the control may be derived from an established data set that has been generated from healthy or normal cells, tissues or individuals.
  • the term "healthy cell” or “healthy tissue” or “healthy individual” shall be taken to mean a non-cancerous cell or tissue or an individual who is known not to suffer from cancer, such knowledge being derived from clinical data on the individual, including, but not limited to, a different cancer assay to that described herein. It is preferred that the healthy individual is asymptomatic with respect to the early symptoms associated with a particular cancer. In the case of mammary cancer, the absence of any abnormalities of the mammary gland is indicated for a control sample of healthy individuals.
  • the term "normal tissue” or “normal individual” shall be taken to mean a tissue or an individual having an average level of GOBLIN expression characteristic of a population.
  • GOBLIN As will be known to those skilled in the art, data obtained from a sufficiently large sample of the population will normalize, allowing the generation of a data set for determining the average level of a particular parameter. Accordingly, the level of expression of GOBLIN can be determined for any population of individuals, and for any sample derived from said individual, for subsequent comparison to GOBLIN levels determined for a sample being assayed. Where such normalized data sets are relied upon, internal controls are preferably included in each assay conducted to control for variation.
  • the present invention provides a method for detecting a cancer cell in a subject, said method comprising: (a) determining the level of GOBLIN mRNA expressed in a test sample from said subject; and (b) comparing the level of GOBLIN mRNA determined at (i) to the level of
  • GOBLIN mRNA expressed in a comparable sample from a healthy or normal individual wherein a level of GOBLIN mRNA at (i) that is enhanced in the test sample relative to the comparable sample from the normal or healthy individual is indicative of the presence of a cancer cell in said subject.
  • the test is carried out ex vivo.
  • the method further comprises obtaining the test sample and/or the comparable sample e.g., from a subject.
  • the test sample and/or the comparable sample has(have) been previously obtained from a subject.
  • GOBLIN mRNA is meant mRNA encoding a GOBLIN polypeptide of mammals that has at least about 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10, and more particularly, mRNA comprising a nucleotide sequence that has at least about 75% identity or 80% or 85% or 90% or 95% or 99% identity to the nucleotide sequence set forth in a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the status of GOBLIN mRNA in patient samples may be analyzed by a variety protocols that are well known in the art including in situ hybridization, northern blotting techniques, RT-PCR analysis (such as, for example, performed on laser capture microdissected samples), and microarray technology, such as, for example, using tissue microarrays probed with nucleic acid probes, or nucleic acid microarrays (ie. RNA microarrays or amplified DNA microarrays) microarrays probed with nucleic acid probes. All such assay formats are encompassed by the present invention.
  • microarray technology is a preferred assay format.
  • the level of GOBLIN mRNA in the test sample is determined by hybridizing a GOBLIN probe to GOBLIN-encoding RNA in the test sample under low or moderate stringency hybridization conditions and detecting the hybridization using a detection means.
  • the level of GOBLIN mRNA in the comparable sample from the healthy or normal individual is preferably determined by hybridizing a GOBLIN probe to GOBLLN-encoding RNA in said comparable sample under low or moderate stringency hybridization conditions and detecting the hybridization using a detection means.
  • the sample comprises mammary tissue, prostate tissue, kidney tissue, uterine tissue, placenta, a cervical specimen, mammary epithelium, rectal tissue, brain tissue, bone tissue, lung tissue, lymphatic tissue, urine, semen, blood, abdominal fluid, or serum, or a cell preparation or nucleic acid preparation derived therefrom. More preferably, the sample comprises serum or abdominal fluid, or a tissue selected from the group consisting of: brain, breast, bone, cervical tissue, colon, kidney, lymph node, omentum, prostate, skin, spleen, stomach, small bowel, salivary gland and testis. Even more preferably, the sample comprises mammary epithelium or ovarian epithelium.
  • the sample can be prepared on a solid matrix for histological analyses, or alternatively, in a suitable solution such as, for example, an extraction huffer or suspension buffer, and the present invention clearly extends to the testing of biological solutions thus prepared.
  • the GOBLIN probe may be any nucleic acid probe described herein. As will be known to those skilled in the art, shorter probes are hybridized at lower stringency hybridization (ie. reduced temperature and/or higher salt concentration and/or higher detergent concentration) than longer nucleic acid probes. Generally, hybridization is carried out well below the calculated melting temperature (Tm) of a DNA duplex comprising the probe. Riboprobes are particularly preferred for applications utilizing oligonucleotides as RNA/RNA duplexes are more stable.
  • oligonucleotide probes having calculated Tm values in the range of about 55°C to about 60°C are hybridized to samples at a temperature in the range of ambient temperature to about 45° C, and more preferably between about 40°C to about 45°C (ie. low stringency to moderate stringency conditions). This contrasts with standard hybridization temperatures of about 65°C for nucleic acid probes of about 100 nucleotides or longer (ie. moderate to high stringency hybridization conditions).
  • a low stringency is defined herein as being a hybridization and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at 28°C, or equivalent conditions.
  • a moderate stringency is defined herein as being a hybridization and/or washing carried out in 2xSSC buffer, 0.1% (w/v) SDS at a temperature in the range 45°C to 65°C, or equivalent conditions.
  • a high stringency is defined herein as being a hybridization and/or wash carried out in O.lxSSC buffer, 0.1% (w/v) SDS, or lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions.
  • Reference herein to a particular level of stringency encompasses equivalent conditions using wash/hybridization solutions other than SSC known to those skilled in the art.
  • the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridization and/or wash.
  • concentration of SSC buffer and/or increasing the concentration of SDS and/or increasing the temperature of the hybridization and/or wash.
  • the conditions for hybridization and/or wash may vary depending upon the nature of the hybridization matrix used to support the sample RNA, or the type of hybridization probe used.
  • the sample or the probe is immobilized on a solid matrix or surface (e.g., nitrocellulose).
  • the sample or probe will generally comprise an array of nucleic acids on glass or other solid matrix, such as, for example, as described in WO 96/17958.
  • Techniques for producing high density arrays are described, for example, by Fodor et al, Science 767-773, 1991, and in U.S. Pat. No. 5,143,854.
  • Typical protocols for other assay formats can be found, for example in Current Protocols In Molecular Biology, Unit 2 (Northern Blotting), Unit 4 (Southern Blotting), and Unit 18 (PCR Analysis), Frederick M. Ausubul et al. (ed)., 1995.
  • the detection means according to this aspect ofthe invention may be any nucleic acid- based detection means such as, for example, nucleic acid hybridization or amplification reaction (eg. PCR), a nucleic acid sequence-based amplification (NASBA) system, inverse polymerase chain reaction (iPCR), in situ polymerase chain reaction, or reverse transcription polymerase chain reaction (RT-PCR), amongst others.
  • nucleic acid hybridization or amplification reaction eg. PCR
  • NASBA nucleic acid sequence-based amplification
  • iPCR inverse polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • the probe can be labelled with a reporter molecule capable of producing an identifiable signal (e.g., a radioisotope such as 32 P or 35 S, or a fluorescent or biotinylated molecule).
  • a reporter molecule capable of producing an identifiable signal
  • a radioisotope such as 32 P or 35 S, or a fluorescent or biotinylated molecule.
  • the detection of said reporter molecule provides for identification ofthe probe and that, following the hybridization reaction, the detection of the corresponding nucleotide sequences in the sample is facilitated. Additional probes can be used to confirm the assay results obtained using a single probe.
  • the detection means is an amplification reaction such as, for example, a polymerase chain reaction or a nucleic acid sequence-based amplification (NASBA) system or a variant thereof, one or more nucleic acid probes molecules of at least about 12 or 15 or 18 or 20 contiguous nucleotides in length is hybridized to mRNA encoding GOBLIN, or alternatively, hybridized to cDNA or cRNA produced from said mRNA, and nucleic acid copies ofthe template are enzymically-amplified.
  • amplification reaction such as, for example, a polymerase chain reaction or a nucleic acid sequence-based amplification (NASBA) system or a variant thereof
  • NASBA nucleic acid sequence-based amplification
  • the stringency conditions can be selected to promote hybridization.
  • PCR provides for the hybridization of non-complementary probes to different strands of a double-stranded nucleic acid template molecule (ie. a DNA/RNA, RNA/RNA or DNA/DNA template), such that the hybridized probes are positioned to facilitate the 5 '-to 3' synthesis of nucleic acid in the intervening region, under the control of a thermostable DNA polymerase enzyme.
  • a double-stranded nucleic acid template molecule ie. a DNA/RNA, RNA/RNA or DNA/DNA template
  • one sense probe and one antisense probe as described herein would be used to amplify DNA from the hybrid RNA/DNA template or cDNA.
  • the cDNA would generally be produced by reverse transcription of mRNA present in the sample being tested (ie. RT-PCR).
  • RT-PCR is particularly useful when it is desirable to determine expression of a GOBLIN-encoding gene.
  • niRNA/DNA hybrid molecules as a template for such amplification reactions, and, as a consequence, first strand cDNA synthesis is all that is required to be performed prior to the amplification reaction.
  • the amplification reaction detection means described supra can be further coupled to a classical hybridization reaction detection means to further enhance sensitivity and specificity of the inventive method, such as by hybridizing the amplified DNA with a probe which is different from any ofthe probes used in the amplification reaction.
  • hybridization reaction detection means described supra can be further coupled to a second hybridization step employing a probe which is different from the probe used in the first hybridization reaction.
  • the comparison to be performed in accordance with the present invention may be a visual comparison of the signal generated by the probe, or alternatively, a comparison of data integrated from the signal, such as, for example, data that have been corrected or normalized to allow for variation between samples. Such comparisons can be readily performed by those skilled in the art.
  • a further embodiment of the invention provides a method for detecting a cancer cell in a subject, said method comprising:
  • the method further comprises obtaining a sample from a subject.
  • the diagnostic method is carried out ex vivo.
  • the test sample has been previously obtained from a subject.
  • the subject is human.
  • Samples from the subject will be those samples that are suitable for screening using nucleic acid probes, however histological specimens are particularly amenable to antibody-based detection.
  • the level of GOBLIN polypeptide in the test sample is determined by a process comprising:
  • the method further comprises obtaining a sample from a subject.
  • the diagnostic method is carried out ex vivo.
  • the sample has been previously obtained from a subject.
  • the level of GOBLLN protein in the comparable sample from the healthy or normal individual is preferably determined by a process comprising:
  • the method further comprises obtaining a sample from a subject.
  • the diagnostic method is carried out ex vivo.
  • the sample has been previously obtained from a subject.
  • Standard assays are used to determine binding of the antibody to the GOBLIN polypeptide in the samples, such as, for example, ELISA, radioimmunoassay, western blot immunoassay, amongst others. Protocols are provided, for example, by Ausubel- et al (supra).
  • Modulation of GOBLIN function according to the present invention through the inhibition or promotion of at least one function characteristic of a mammalian GOBLIN protein, provides an effective and selective way of inhibiting or promoting GOBLLN- mediated functions in a cell.
  • a compound that inhibits one or more functions of a mammalian GOBLIN protein is administered to an individual to prevent, inhibit, or delay tumor growth, particularly in the treatment of carcinoma, such as, for example, an epithelial mammary carcinoma.
  • a mammalian GOBLIN protein e.g., a human GOBLIN
  • anti-GOBLIN antibodies of the present invention or antagonistic nucleic acid (antisense nucleic acid, PNA, interfering RNA, ribozyme, etc) or peptides comprising a WW domain or C2 domain of a full-length GOBLIN polypeptide, can be used in the method.
  • mammals including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species.
  • one or more agents can be administered to the host by an appropriate route, either alone or in combination with another drug.
  • An effective amount of a nucleic acid or antibody or peptide agent having antagonist activity is administered.
  • An effective amount is an amount sufficient to achieve the desired therapeutic or prophylactic effect, under the conditions of administration, such as an amount sufficient for inhibition of GOBLIN function.
  • routes of administration are possible including, but not necessarily limited to oral, dietary, topical, parenteral (e.g., intravenous, infra-arterial, intramuscular, subcutaneous injection), and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration.
  • Formulation of an agent to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule).
  • An appropriate composition comprising the agent to be administered can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils, for instance.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers and the like (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985).
  • the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
  • the agent can be administered via in vivo expression of the recombinant protein.
  • In vivo expression can be accomplished via somatic cell expression according to suitable methods (see, e.g. U.S. Pat. No. 5,399,346).
  • nucleic acid encoding the protein can be incorporated into a retroviral, adenoviral or other suitable vector (preferably, a replication deficient infectious vector) for delivery, or can be introduced into a transfected or transformed host cell capable of expressing the protein for delivery.
  • the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the protein in a therapeutically effective amount.
  • Another aspect of the present invention provides methods for isolating a GOBLIN- binding protein from a suitable cellular source, such as, for example, a mammary epithelial cell or ER-negative breast cancer cell.
  • a suitable cellular source such as, for example, a mammary epithelial cell or ER-negative breast cancer cell.
  • a full-length GOBLIN polypeptide or WW domain peptide or C2 domain peptide derived therefrom is used as an affinity probe to bind a protein in a cellular extract from a mammary epithelial cell and unbound protein removed, thereby isolating a GOBLIN-binding protein substantially free of conspecific proteins.
  • affinity purification methods can also be adapted to the isolation of a protein complex comprising GOBLIN or a portion of GOBLIN and its cognate binding partner from ER-negative breast cancer cells.
  • the protein or complex is provided substantially free of conspecific proteins, meaning that it is at least about 1-5% pure as determined by an analysis of proteins by SDS/PAGE. More preferably, the protein is at least about 10%, even more preferably at least about 20% pure, even more preferably at least about 25% pure, even more preferably at least about 30% pure, and even more preferably at least about 50% pure, and still more preferably substantially pure.
  • a protein complex comprising a GOBLIN polypeptide may be isolated from a cellular source that expresses GOBLIN and its binding partner(s) endogenously or ectopically. It is within the scope of this embodiment that the binding partners are expressed as a fusion protein or as distinct polypeptides.
  • Preferred cellular sources of the GOBLIN-binding protein or a protein complex comprising GOBLIN include any mammalian cell, and preferably, a mammalian cell that is known to express GOBLIN or can be engineered to express said protein(s).
  • Exemplary cells for such a purpose include cancer cells (e.g. carcinoma cells, breast cancer cells such as ER-negative breast cancer cells, or squamous epithelial carcinoma cells), epithelial cells, cells ofthe central nervous system, kidney cells, T cells, NIH3T3 cells, murine 10T fibroblasts, MDA-MB-231 cells, MDCK cells, COS cells, CHO cells, HeLa cells, or HEK 293 cells.
  • the use of other cells e.g. insect sf9 or sf21 cells, chick embryo cells and the like is not excluded, particularly for isolation of a non-naturally occurring peptide, polypeptide or complex expressed by recombinant means.
  • the GOBLIN-binding protein or a protein complex comprising GOBLIN is isolated from cell line that naturally or endogenously expresses one or both binding partners, such as, for example, a cancer cell selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian cancer), prostate cancer, squamous cell carcinoma, non-squamous carcinoma, glioblastoma and medulloblastoma. More preferably, the cell will be a breast cancer cell.
  • a cancer cell selected from the group consisting of head and neck cancer, breast cancer, adenocarcinoma, squamous lung cancer, gastrointestinal cancer (eg. gastric, colon, or pancreatic cancer), renal cell cancer, bladder cancer, a gynecological carcinoma (eg. ovarian
  • the cell will be an ER-negative breast cancer cell or ER-negative breast cancer cell line.
  • the cells listed in Table 1 are particularly preferred.
  • Means for isolating the peptide, polypeptide, or protein binding partners, or the protein complex include any means of protein isolation known to the skilled protein chemist, such as, for example, size exclusion chromatography, ion-exchange (anion or cation exchange) chromatography, reverse phase chromatography, or affinity chromatography. Both high pressure (e.g. HPLC, FLPC, MALDI) and low pressure systems can be used.
  • Affinity methods using ligands or antibodies that bind to one or both of the binding partners to the protein-protein interaction are particularly preferred.
  • the WW domain peptide, or antibodies against a proline rich region are also useful for isolating GOBLIN-binding polypeptides. The use of other peptide domains is not excluded.
  • Naturally-occurring or recombinant protein is purified free of conspecific proteins by providing a matrix comprising antibody coupled to activated chromatographic resin (eg. CNBr-activated Sepharose, Pharmacia), blocking the resin and washing to remove unbound antibody and blocking agent, contacting the resin with a protein extract comprising a peptide or polypeptide to which the antibody binds under conditions sufficient to allow binding of said peptide or polypeptide (e.g., high ionic strength buffers in the presence of detergent), and eluting said peptide or polypeptide under conditions that disrupt the antibody antigen binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion).
  • activated chromatographic resin eg. CNBr-activated Sepharose, Pharmacia
  • a protein extract comprising a peptide or polypeptide to which the antibody binds under conditions sufficient to allow binding of said peptide or polypeptide (e.
  • small molecules, or proteins capable of binding to one ofthe binding partners can also be used to isolate one or both binding partners, or the protein complex per se, by affinity means. Conditions to permit such isolation can be readily determined by those skilled in protein chemistry. Selection of buffer pH, ionic strength, and temperature, sufficient to maintain the binding partners in solution are generally preferred.
  • one or more protease inhibitors e.g. papain, PMSF, leupeptin
  • one or more protease inhibitors are included to prevent proteolytic digestion or degradation of the isolated polypeptides.
  • naturally-occurring or recombinant protein is purified free of conspecific proteins by providing a matrix comprising a small molecule or protein binding partner coupled to activated chromatographic resin (eg.
  • ligands, agonists, or antagonists of GOBLIN function are identified in a suitable assay, and further assessed for their therapeutic efficacy.
  • Antagonists of GOBLIN are used to inhibit (ie. reduce or diminish or prevent) GOBLIN-mediated effects in cells, such as, for example, breast cancer.
  • ligands and/or agonists of GOBLIN are useful for inducing or enhancing GOBLIN- mediated effects in cells.
  • a further aspect of the present invention provides a method of treating a hyperproliferative disease, such as, for example, cancer, comprising administering an antagonist of GOBLIN function to an individual (e.g., a mammal) for a time and under conditions sufficient to reduce or prevent GOBLIN activity in said individual, thereby reducing or preventing one or more GOBLIN-mediated effects.
  • the antagonist comprises nucleic acid, such as, for example, antisense nucleic acid, a ribozyme, siRNA, shRNA, or nucleic acid that forms a triple helical structure, capable of reducing GOBLIN expression in a cell of the individual.
  • the expression can be reduced at the RNA level or the protein level. Accordingly, antibodies that bind GOBLIN and. inhibit its activity are also useful in this context.
  • a further aspect of the present invention provides a method of monitoring the efficacy of a therapeutic treatment for a hyperproliferative disease comprising: (a) providing a biological sample, from a patient undergoing the therapeutic treatment; and (b) determining the level of GOBLIN-encoding mRNA in the biological sample by a process comprising contacting the biological sample with a polynucleotide that selectively hybridizes to a sequence having at least about 80% identity to a sequence as shown in any one of SEQ ID Nos: 1, 3, 5, 7, 9, or 22-353, thereby monitoring the efficacy ofthe therapy.
  • the method further comprises comparing the level of GOBLIN-encoding mRNA to a level of GOBLLN-encoding in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
  • the present invention provides a method of monitoring the efficacy of a therapeutic treatment for a hyperproliferative disease comprising: (a) providing a biological sample from a patient undergoing the therapeutic treatment; and (b) determining the level of a GOBLIN polypeptide comprising an amino acid sequence having at least about 80% identity to a sequence as shown in any one of SEQ ID Nos: 2, 4, 6, 8, or 10-21 in the biological sample by a process comprising contacting the biological sample with an antibody that binds specifically to said polypeptide, thereby monitoring the efficacy ofthe therapy.
  • the method further comprises comparing the level of the GOBLIN polypeptide to a level of the GOBLIN polypeptide in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
  • the present invention further provides a method of agonising or otherwise enhancing GOBLIN activity in an individual comprising administering a GOBLIN ligand or GOBLIN agonist to said individual for a time and under conditions sufficient to enhance GOBLIN activity.
  • GOBLIN epithelial WW domain-containing and C2 domain-containing protein
  • Mammary recombination used 1mm 3 portions from the fourth mammary gland of nine week old PrlR “ ' " and PrlR + + mice (C57BL6xl 29S VPas), implanted to a fourth mammary fat pad, previously excised from prepubescent 3-5 week old PrlR “ ' “ and PrlR + + mice and cleared of endogenous epithelium.
  • the recombined gland was then transplanted onto the abdominal wall (between the second and fourth endogenous mammary glands) of a Ragl " ' " mouse (C57BL6). These mice are able to accept the transplant as they are deficient in T cells and B cells.
  • the animals were then aged for 12 weeks to allow the epithelium to invade the fat pad. Animals were mated and checked for vaginal plugs in the morning. At 2, 4 and 6 days after observation of a plug the mammary glands were collected, frozen in liquid nitrogen and stored at -70°C.
  • the cRNA targets were generated as recommended by Affymetrix (Santa Clara, CA).
  • RNA was prepared from frozen mammary glands by homogenisation with a Polytron in Trizol for 30 seconds. Chloroform extraction and isopropanol precipitation ofthe RNA was performed as per manufacturers instructions and further purified using Qiagen RNeasy Mini Kit (Qiagen, Chats worth, CA). RNA diluted 1:50 in water was quantified using spectrophotometer at 260nM, pure samples having an A260/280 ratio of between 1.8 and 2.0. Integrity of the RNA was determined by running l ⁇ g of sample on a 1% (w/v) agarose gel containing ethidium bromide and observance of intact 18S and 28S ribosomal RNA bands.
  • RNA pooled from each transplant was performed using Superscript polymerase (Life Technologies) and an oligo-(dT)2 -T7 RNA polymerase promoter primer.
  • the double stranded cDNA was cleaned up by phenol-chloroform-isopropanol extraction and ethanol precipitation.
  • Biotin labeled cRNA was generated in the presence of biotinylated UTP and CTP (Sigma) by in vitro translation using T7 Megascript Kit (Ambion, Austin, TX).
  • the resulting cRNA was purified with an RNeasy Mini Kit (Qiagen) and integrity of the cRNA checked on a 1% agarose gel.
  • 15 ⁇ g of cRNA was fragmented by incubating for 35 min at 94°C, pH 8.1 and stored at - 80°C before application to the Gene Chip.
  • the target consisted of 15 ⁇ g of fragmented cRNA, oligo B2 and controls bioD, C and ere. This was hybridised to MG-U74A Gene Chips (Affymetrix) for 16 hours at 45°C. Following hybridisation the target was removed and the Gene Chips washed using the Affymetrix fluidics station and the EukGE-WS2 washing and staining protocol. This involved a number of stringent and non-stringent washes followed by staining with SAPE (R-Phycoerythrin Streptavidin, Molecular Probes) and an amplification of this stain using a biotinylated anti-streptavidin antibody (Vector Laboratories). The Gene Chips were scanned using the Gene Array Scanner and the hybridisation intensities and fold change between experiments obtained using Microarray Suite 4.0 (Affymetrix) and the MGU74A mask.
  • SAPE R-Phycoerythrin Streptavidin, Molecular Probes
  • Genes that were epithelial and decreasing in the PrlR " ' " transplants at one or more time points were chosen as candidates for searching the public databases.
  • Known genes were searched in PubMed for relevance to mammary gland development.
  • ESTs were associated with known genes or other ESTs by querying the UniGene database of the National Center for Biotechnology Information of the National Institutes of Health, located at the National Library of Medicine, Building 38 A, Bethesda, MD 20894, USA.
  • RNA from the PrlR " ' " and PrlR +/+ transplants and from PrlR “ ' “ and PrlR + + mammary glands at one day post-partum was prepared using the Trizol method.
  • RNA was reverse transcribed using AMV reverse transcriptase (Promega).
  • PCR primers were designed for GOBLIN using Macvector. To be sure the reaction was specific for cDNA and not genomic DNA the primers were designed so that the product spanned an intron.
  • PCR reactions were performed in a Light Cycler (Roche) using 1 ⁇ L of the cDNA diluted 1 :2, 5pmol of primers and the FastStart DNA master SYBR Green I enzyme mix (Roche) in a lO ⁇ L reaction volume. Absolute quantification was performed by comparing transcript levels in samples to a standard curve constructed by performing serial dilutions of PCR product purified using QIAquick Gel Extraction Kit (Qiagen) and analysed using the Second Derivative Maximum method (Roche). Efficiency of the PCR reaction in the standard was the same as efficiency of the reaction containing cDNA. All data were normalised to expression ofthe housekeeping gene ⁇ -actin.
  • Prolactin acts exclusively via the epithelium to promote lobuloalveolar development.
  • Both the epithelium and stroma of the murine mammary gland express the PrlR.
  • Glands were formed from recombined PrlR " ' " epithelium and/or PrlR " ' " stroma and transplanted to 3-week-old C57BL6 Ragl " ' " host animals, allowing mammary gland development to occur in a normal endocrine environment.
  • the transplants were examined at 12 weeks of age or the animals were mated at 12 weeks of age and the transplants examined lday post-partum.
  • Time points early in pregnancy ie., at 2 days, 4 days and 6 days
  • results were analysed using Affymetrix MicroArray Suite 4.0 (MAS 4.0). Approximately 50% of transcripts represented on the MGU74A Gene Chip were detected (called present by MAS 4.0). We defined epithelial transcripts as those that had an absolute call of present in the PrlR + + epithelial transplants and absent in PrlR+/+ fat pads devoid of epithelium. Prolactin modulated transcripts were identified as decreasing or increasing between PrlR +/+ and PrlR " ' " transplants when compared using MAS 4.0. The maximum number of detectable epithelial transcripts occurred at day 4, corresponding to the peak in cell proliferation in the mammary gland at that time and reflecting the endocrine changes during early pregnancy.
  • PC analysis identifies the major sources of variance in the data set. We ascribed biological meaning to each component by incorporating the Affymetrix calls of increasing or decreasing between genotypes or between time points. This analysis identified the first PC as representing the differences in overall expression level, as is common in PC analysis of transcript profiling experiments. The second PC represented the variance due to genotype and the third PC the variance with time. Mammary gland gene expression at day 4 of pregnancy appeared more closely related to expression at day 6 than day 2, and this was confirmed by hierarchical clustering.
  • the genes of interest (those that decreased in the PrlR “ ' " epithelium when compared to PrlR +/+ epithelium in at least two of the three days of pregnancy examined) were sorted into functional groups depending on their Gene Ontology. Many of these genes were cDNAs of unknown function or expressed sequence tags (ESTs) that are not discussed here. As expected the expression of a number of milk protein genes (casein alpha, casein beta, casein kappa and WDNMl) and the prolactin receptor decreased in the PrlR " ' " epithelium at days two, four and six of pregnancy. The decrease in these markers of epithelial differentiation in the PrlR " ' " epithelial transplants confirms that our model is able to detect epithelial transcripts induced by prolactin during lobuloalveolar development.
  • ESTs expressed sequence tags
  • GOBLIN One gene, designated GOBLIN, was identified on the basis that expression of this gene was up regulated at all three stages examined in PrlR + + epithelial transplants compared to the PrlR " ' " epithelial transplants ( Figure 1). We also confirmed that GOBLIN expression was low in PrlR + + mammary fat pads cleared of epithelium by quantitative RT-PCR analysis ( Figure 1).
  • GOBLIN expression was related to the expression of the estrogen receptor (ER) and progesterone receptor (PR) and prolactin receptor (PRLR). Data presented in Figure 3 indicate that GOBLIN expression is low in healthy human mammary epithelial cell lines human mammary epithelial cell lines (HMECs), and is expressed at elevated levels in a number of cancer cell lines.
  • HMECs human mammary epithelial cell lines
  • the data presented in Figures 1 and 2 indicate an association of the GOBLIN-encoding EST AI0850846 present on the Affymetrix gene chip array described supra and prolactin modulated gene expression in mammary epithelial transplants. Additionally, the GOBLIN-encoding EST was over expressed in a number of cancer cell lines ( Figure 3). To confirm that GOBLIN expression is modulated by prolactin, expression in primary cultures of mouse mammary epithelial cells (MECS) that are responsive to prolactin is compared to expression in MECS that are not responsive to prolactin (i.e. prolactin receptor null MECS).
  • MECS mouse mammary epithelial cells
  • this approach identified the full-length human and murine GOBLIN encoding nucleic acids set forth in SEQ ID Nos: 1, 3 and 5.
  • Additional GOBLIN-encoding genes were isolated by searching databases using the BLAST program. Alignment of the nucleotide and amino acid sequences of sequences identified by BLAST searching with the deduced human and murine sequences identified the M. fascicularis and rat nucleotide sequences presented in SEQ ID Nos: 7 and 9, respectively.
  • the GOBLIN cDNA was cloned from 293 cells by PCR using primers GOBLIN- Forward and GOBLIN-Reverse (SEQ ID Nos: 38 and 39; Table 2) and subcloned into pGEM-T-Easy (Promega).
  • the GOBLIN cDNA was then ligated into pcDNA3.1 at the Notl sites and also cloned into the Gateway entry vector pDO ⁇ R221 (Invitrogen Life Technologies) by PCR using primers attB-GOBLIN-Forward and attB-GOBLIN- Reverse (SEQ ID NOs: 43 and 44; Table 2).
  • Regions of the GOBLIN gene that encode specific domains of the GOBLIN protein were also amplified using specific primers presented in Table 2.
  • two overlapping fragments (WW and C2) of the GOBLIN cDNA ( Figure 4) were cloned into the vector pDONR221 by PCR.
  • Fragment 1 encoding two WW domains of GOBLIN, was amplified using primers attB-GOBLIN-Forward (SEQ ID NO: 42; Table 2) and attB-WW-Reverse (SEQ ID NO: 44; Table 2).
  • Fragment 2 encoding the C2 domain of GOBLIN protein, was amplified using primers attB-C2-Forward (SEQ ID NO: 45; Table 2) and attB-GOBLIN-Reverse (SEQ ID NO: 43; Table 2).
  • the GOBLIN cDNA and fragments encoding the WW and C2 domains were then cloned into mammalian Gateway expression vectors (invitrogen) pcDNA-DEST40 (CMV/C-terminal V5-6xHis) and pcDNA-DEST47 (CMV/C-terminal GFP) and the E.coli Gateway expression vectors pET-DEST42 (T7 llac promoter, C-terminal V5- 6xHis) and pDEST24 (T7/C-terminal GST) by recombination.
  • mammalian Gateway expression vectors invitrogen
  • pcDNA-DEST40 CMV/C-terminal V5-6xHis
  • pcDNA-DEST47 CMV/C-terminal GFP
  • E.coli Gateway expression vectors pET-DEST42 T7 llac promoter, C-terminal V5- 6xHis
  • pDEST24 T7/C-terminal GST
  • the Gateway vector pENTR/SD/D-TOPO is used, This vector contains a Shine- Dalgamo sequence for efficient sequence initiation of native expression or N-terminal fragments or C-terminal fusions in E. coli. Accordingly, amplified nucleic acid is cloned directionally into this vector and transformed into E. coli. Recombinant plasmids are selected on media comprising kanamycin. Peptide fragments are produced in E. coli in accordance with the protocol provided by Invitrogen.
  • the gene-specific primers Goblin 5' (SEQ ID NO: 38) and WW Domain R (SEQ ID NO: 40) are used to amplify a 1904 bp nucleic acid product comprising a sequence that encodes the two WW domains of human GOBLIN.
  • the gene-specific primers Goblin 3' (SEQ ID NO: 39) and C2 Domain F (SEQ ID NO: 41) are used to amplify a 2355 bp nucleic acid product comprising a sequence that encodes the C2 domain of human GOBLIN.
  • Goblin 5'F primer SEQ ID NO: 38
  • Goblin 3'R primer SEQ ID NO: 39
  • 293 cells were transfected with pcDNA3.1- GOBLLN, WW-V5-6xHis or C2-V5-6xHis expression vectors using FuGENE (Roche). 48 h after transfection, cells were lysed using normal lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% (v/v) Triton X-100, 1.5 mM MgCl 2 , 1 mM EDTA, 10 mM pyrophosphate, 100 mM NaF) containing protease inhibitors.
  • normal lysis buffer 50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% (v/v) Triton X-100, 1.5 mM MgCl 2 , 1 mM EDTA, 10 mM pyrophosphate, 100 mM NaF
  • Blots were incubated overnight at 4°C with primary antibodies raised against SEQ ID NO: 21 (1:500 (v/v) dilution) and antibodies against the V5 loop sequence (1:5000 (v/v) dilution; Invitrogen). Bound antibodies were detected using horseradish peroxidase-linked secondary antibodies and enhanced chemiluminescent detection (Perkin-Elmer, Boston, MA, USA).
  • the human GOBLIN transcript produced by the expression vector pcDNA3.1 -GOBLIN encodes a protein of 1113 amino acids (SEQ ID Nos: 2 or 4) with a predicted molecular mass of about 125.3 kDa.
  • the encoded WW and C2 peptide fragments produced from the vectors WW-V5-6xHis and C2-V5-6xHis ( Figure 4) are 623 amino acids and 765 amino acids in length respectively. These WW domain and C2 domain peptides have predicted molecular masses of about 70.8 kDa and about 87 kDa, respectively.
  • Immunoblot analysis of whole cell lysate from 293 cells transfected with the expression vector pcDNA3.1 -GOBLIN detected a single immunoreactive band in the region of 116-180 kDa ( Figure 5), consistent with the predicted mass ofthe full-length GOBLIN polypeptide.
  • Tissue sections (4 ⁇ m) of human brain, liver, kidney, spleen, small bowel, stomach, lung, skeletal muscle, salivary gland, breast, prostate and testis were mounted on Superfrost Plus adhesion slides (Lomb Scientific, Sydney, Australia) and heated in a convection oven at 75°C for 2 h to promote adherence. Sections were de-waxed and rehydrated according to standard protocols. Antigen retrieval was performed using citrate EDTA buffer (DAKO Corporation, Carpinteria, CA) boiled under pressure and endogenous peroxidase activity was inhibited with 3% H 2 O 2 .
  • citrate EDTA buffer DAKO Corporation, Carpinteria, CA
  • Sections were incubated with a primary antibody comprising antisera prepared against SEQ ID NO: 21 [1:200 dilution (v/v)] for 60 min and bound antibody was detected using DAKO LINK/LABEL and 3,3'-diaminobenzidine Plus (DAKO) as substrate. With each run, diluent and rabbit IgG without antibody, were the negative technical controls. Counterstaining was performed with haematoxylm and 1% acid alcohol.
  • GOBLIN protein was detected in skin, brain, spleen, stomach, colon, small bowel, breast, prostate and testis (Figure 6). GOBLIN protein was also present in the kidney and salivary gland, however non-specific staining with diluent and rabbit IgG controls was also evident. GOBLIN staining in all cases was cytoplasmic. GOBLIN protein was not evident in liver, lung or skeletal muscle ( Figure 6).
  • Formalin-fixed paraffin-embedded cell pellets of breast cancer cell lines (BT474, MDA-MB-134, SKBR3, BT483, BT20, BT549, MCF7, 184, MDA-MB-361, MDA- MB-231, MDA-MB-453, MDA-MB-157) were examined for GOBLIN expression by IHC. 4 ⁇ m sections of cell cultures were mounted on Superfrost Plus adhesion slides (Lomb Scientific, Sydney, Australia) and heated in a convection oven at 75°C for 2 h to promote adherence. Sections were de-waxed and rehydrated according to standard protocols.
  • Antigen retrieval was performed using citrate EDTA buffer (DAKO Corporation, Carpinteria, CA) boiled under pressure and endogenous peroxidase activity was inhibited with 3% H 2 O 2 . Sections were incubated with the antisera prepared against SEQ ID NO: 21 (1:200) primary antibody for 60 min and bound antibody was detected using DAKO LLNK/LABEL and 3,3'-diaminobenzidine Plus (DAKO) as substrate. With each ran, diluent and rabbit IgG without antibody, were the negative technical controls. Counterstaining was performed with haematoxylin and 1% acid alcohol. Results
  • GOBLIN expression in an array of breast cancer cell lines was investigated by Western immunoblot analysis using the antisera prepared against SEQ ID NO: 21 ( Figure 7).
  • Cell lines T47D and MDA-MB-134 expressed two immunoreactive GOBLIN protein bands having molecular weights of about 170 kDa and about 160 kDa ( Figure 7).
  • the cell lines SKBR3 and MDA-MB-330 each expressed a single immunoreactive band having a molecular weight of about 170 kDa
  • the cell lines BT474 and MDA- MB-436 each expressed a single immunoreactive band having a molecular weight of about 160 kDa ( Figure 7).
  • GOBLIN expression in breast cancer cell lines was also examined by IHC using the antisera prepared against SEQ ID NO: 21 ( Figure 8). GOBLIN expression levels were classified as absent, low, moderate and high (Table 4).
  • GOBLIN expression in breast and ovarian cancer tissues Immunohistochemistry GOBLIN expression levels, in breast and epithelial ovarian cancer samples, were investigated by IHC. Fresh/frozen and formalin fixed paraffin-embedded samples (4 ⁇ m tissue sections) of breast and epithelial ovarian cancer tissue, including ovarian inclusion cysts, serous ovarian cancer, mutinous ovarian cancer, endometrioid ovarian cancer and clear cell carcinoma, were mounted on Superfrost Plus adhesion slides (Lomb Scientific, Sydney, Australia) and heated in a convection oven at 75°C for 2 h to promote adherence. Sections were de- waxed and rehydrated according to standard protocols.
  • Antigen retrieval was performed using citrate EDTA buffer (DAKO Corporation, Carpinteria, CA) boiled under pressure and endogenous peroxidase activity was inhibited with 3% H 2 O 2 . Sections were incubated with the antisera prepared against SEQ ID NO: 21 (1:200) primary antibody for 60 min and bound antibody was detected using DAKO LINK/LABEL and 3,3'-diaminobenzidine Plus (DAKO) as substrate. With each run, diluent and rabbit IgG without antibody, were the negative technical controls. Counterstaining was performed with haematoxylin and 1% acid alcohol. Results
  • GOBLIN was present in both healthy and cancerous ovarian tissue ( Figure 9) with elevated expression in ovarian inclusion cysts, endometrioid ovarian cancer and clear cell carcinoma, relative to expression in healthy, tissues (i.e., ovarian stroma or ovarian surface epithelium, OSE) (Table 5). Expression was not detected using this antibody in serous ovarian cancer tissue.
  • GST fusion proteins of the amino terminus (including the two WW domains of human GOBLIN) and the carboxyl terminus (including the C2 domain of human GOLBIN) are produced as described in the preceding example. Affinity columns are then used to isolate the fusion proteins via the GST tags.
  • Cell lysates from a number of cell lines eg., breast cancer cells, HC11 (mouse mammary epithelial cells), HEK293, are contacted with the isolated fusion protein bound to the affinity matrix, to isolate proteins that interact with GOBLIN WW domains and C2 domains, particularly in cancer cells.
  • the interacting proteins are identified by mass spectrophotometry.

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Abstract

L'invention concerne des gènes GOBLIN et des polypeptides codés par ceux-ci. Les gènes GOBLIN selon l'invention sont fortement exprimés dans les tissus malins. L'invention concerne également des procédés diagnostiques permettant de détecter le cancer ou des tumeurs malignes chez des patients humains. Le test diagnostique et pronostique est particulièrement utile pour la détection précoce du cancer mammaire ou de métastases de celui-ci et pour surveiller l'évolution de la maladie, par exemple durant une rémission ou à la suite d'une intervention chirurgicale ou d'une chimiothérapie.
PCT/AU2004/000169 2003-02-14 2004-02-12 Polypeptides associes au cancer « goblin », reactifs associes, et procedes d'utilisation associes Ceased WO2004072285A1 (fr)

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DATABASE GENBANK [online] 12 July 2001 (2001-07-12), "Unknown (protein for IMAGE:3635709)[Homo sapiens]", Database accession no. (AAH04394) *
DATABASE GENBANK [online] 16 June 1999 (1999-06-16), "KIAA0869 protein (homo sapiens)", Database accession no. (BAA74892) *
DATABASE GENBANK [online] 2 January 2003 (2003-01-02), "KIBRA protein (homo sapiens)", Database accession no. (AAO15881) *
DATABASE GENBANK [online] 26 August 2002 (2002-08-26), "BC037006 protein (mus muscularis)", Database accession no. (AAH37006) *
DATABASE GENBANK [online] 27 October 2000 (2000-10-27), "Hypothetical protein [Macaca fascicularis]", Database accession no. (BAB17278) *
DATABASE GENBANK [online] 27 October 2000 (2000-10-27), "Unknown (protein for IMAGE:4413016)[Homo sapiens]", Database accession no. (AAH17746) *
DATABASE GENBANK [online] 30 April 2001 (2001-04-30), "BC037006 protein (mus muscularis)", Database accession no. (AAH06733) *
DATABASE GENBANK [online] 30 November 2001 (2001-11-30), "BC037006 protein (mus muscularis)", Database accession no. (AAH17638) *
DATABASE GENBANK [online] MONAHAN J.E. ET AL.: "Assessing whether a patient is afflicted with ovarian cancer", Database accession no. (ABG96353) *
DATABASE GENBANK [online] OTA T. ET AL.: "Primer sets for synthesising polynucleotides", Database accession no. (AAB93267) *
DATABASE GENBANK [online] SPYTECK K.A. ET AL.: "New cytoplasmic, nuclear membrane bound or secreted polypeptides(NOVX)", Database accession no. (ABO14757) *
DATABASE GENBANK [online] TANG Y.T. ET AL.: "Human proteins and DNA encoding sequences", Database accession no. (ABB11742) *
DATABASE GENBANK [online] YUE H. ET AL.: "New cytoskeletal-associated proteins and polynucleotides", Database accession no. (AAU74354) *
STRAUSBERG R.L. ET AL.: "Generation and initial analysis of more than 15 000 full-lenght human and mouse cDNA sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, vol. 99, no. 26, 11 December 2002 (2002-12-11), pages 16899 - 16903 *
STRAUSBERG R.L. ET AL.: "Generation and initial analysis of more than 15 000 full-length human and mouse cDNA sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, vol. 99, no. 26, 11 December 2002 (2002-12-11), pages 16899 - 16903, XP003024624, DOI: doi:10.1073/pnas.242603899 *

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