WO2005047536A2 - Detection d'amplification et de deletion genomiques dans le cancer - Google Patents

Detection d'amplification et de deletion genomiques dans le cancer Download PDF

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WO2005047536A2
WO2005047536A2 PCT/EP2004/012876 EP2004012876W WO2005047536A2 WO 2005047536 A2 WO2005047536 A2 WO 2005047536A2 EP 2004012876 W EP2004012876 W EP 2004012876W WO 2005047536 A2 WO2005047536 A2 WO 2005047536A2
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antibody
pnmt
gene
cancer
expression
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WO2005047536A3 (fr
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Marlene Michelle Dressman
Rachel Helene Malinowski
Mihael Hristos Polymeropoulos
Alex Baras
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Novartis Ag
Novartis Pharma Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates generally to the analytical testing of tissue samples in vitro, and more particularly to methods for detecting genomic amplification and/or deletion in cancer tissue and to compositions and methods for the diagnosis and treatment of tumour.
  • this invention relates to detecting genomic amplification by using the combination of gene expression profiling and gene mapping (GEM) and to the use of specific genes found to be co-amplified with the gene c-erb B2 in breast cancer.
  • GEM gene expression profiling and gene mapping
  • Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumour mass, followed by the invasion of adjacent tissues by these neoplastic tumour cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
  • tumour suppression genes which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth.
  • tumour suppression genes which would normally function to prevent malignant cell growth
  • tumour suppression genes which would normally function to prevent malignant cell growth
  • oncogenes which act to promote malignant growth.
  • tumour suppression genes which would normally function to prevent malignant cell growth
  • tumour suppression genes which would normally function to prevent malignant cell growth
  • oncogenes such as oncogenes
  • HERCEPTINTM (trastuzumab). The identification of patients whose cancers overexpress c- erb B2 now has profound prognostic and therapeutic consequences. These cancers include, but are not limited to, breast and ovarian cancer.
  • tumours overexpress c-erb B2 for both diagnostic, prognostic and therapeutic reasons since these tumour cells may themselves be targeted by therapeutic antibodies.
  • tumour cells may co-amplify and /or co-overexpress other genes along with c-erb B2 for similar diagnostic, prognostic and therapeutic reasons since these tumour cells also, may be targeted by therapeutic antibodies specific to these other genes products.
  • the present invention meets these needs, in part, by using the novel technique of combining gene expression and gene mapping to identify co-amplified or co-deleted genes in cancer tissue. This technique can be used to identify small or large gene amplification or deletions within a given chromosome location.
  • this invention provides two genes which are co-amplified and over- expressed with c-erbB-2 (SEQ ID NO:1 ) in some breast cancer patients.
  • the identification of these two genes provides surrogate markers for identifying c-erbB-2 amplification and overexpression and therefore surrogate markers for the known diagnostic and prognostic consequences of c-erbB-2 amplification and overexpression.
  • the co-amplification of these two genes provides additional targets for antibody based therapeutic strategies.
  • This embodiment provides a method of treating breast cancer in a human, wherein the cancer expresses the phenylethanolamine N- methyltransferase (PNMT) gene (SEQ ID NO:3), comprising administering to the human a therapeutically effective amount of an antibody which binds the PNMT gene polypeptide expression product (SEQ ID NO:4).
  • PNMT phenylethanolamine N- methyltransferase
  • Additional embodiments of this method would include administering, in addition to the antibody, a chemotherapeutic agent to the human, including but not limited to those selected form the group consisting of; an anthracycline antibiotic, cyclophosphomide, a taxane, navelbine, xeloda, mitomycin C, oxaliplatin, gemcitabine, cisplatin, carboplatin, 5- fluorouracil (5-FU), leucovorin (LV), CPT- 11 , levamisole and a platinum compound.
  • the antibody may be an antibody fragment such as an Fab fragment and/or may additionally be conjugated with a cytotoxic agent.
  • PNMT phenylethanolamine N- methyltransferase
  • the polypeptide gene product of PNMT is phenylethanolamine N-methyltransferase, an enzyme that catalyzes the last step in the biosynthesis of catecholamines, i.e., changing norepinephrine to epinephrine.
  • This enzyme catalyzes the transfer of a methyl group from S-adenosylmethionine to the amino group on norepinephrine to form epinephrine.
  • Phenylethanolamine N-methyltransferase could catalyze a similar reaction to convert a nontoxic or minimally toxic prodrug to an effective cytotoxic agent. Since only tumour cells overproduce this enzyme, the cytotoxic action of the altered prodrug would be limited to the immediate area of the tumour cells.
  • the present invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The present invention is based on the identification of genes that are amplified in the genome of tumour cells.
  • Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumourigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act of predictors of the prognosis of tumour treatment.
  • the present invention concerns an isolated antibody which binds a MLN64 (SEQ ID NO:6) or PNMT (SEQ ID NO:4) polypeptide.
  • the antibody induces death of a cell overexpressing a MLN64 or PNMT polypeptide.
  • the antibody is a monoclonal antibody, which preferably has non-human complementarity determining region (CDR) residues and human framework region (FR) residues.
  • CDR complementarity determining region
  • FR human framework region
  • the antibody may be labelled and may be immobilized on a solid support.
  • the antibody is an antibody fragment, a single-chain antibody, or an anti- idiotypic antibody.
  • the invention concerns a composition
  • a composition comprising an antibody which binds a MLN64 (SEQ ID NO:6) or PNMT (SEQ ID NO:4) polypeptide in admixture with a pharmaceutically acceptable carrier.
  • the composition comprises a therapeutically effective amount of the antibody.
  • the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent.
  • the composition is sterile.
  • the invention further concerns antagonists and agonists of a MLN64 or PNMT polypeptide that inhibit one or more of the functions or activities of the MLN64 or PNMT polypeptide.
  • the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the MLN64 or PNMT polypeptides.
  • the nucleic acid preferably is DNA, and hybridization preferably occurs under stringent conditions.
  • Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the respective amplified genes, or as antisense primers in amplification reactions.
  • sequences can be used as part of ribozyme and/or triple helix sequence which, in turn, may be used in regulation of the amplified genes.
  • the invention concerns a method for determining the presence of a MLN64 or PNMT polypeptide comprising exposing a cell suspected of containing the MLN64 or PNMT polypeptide to an anti-MLN64 or PNMT antibody and determining binding of the antibody to the cell.
  • the present invention concerns a method of diagnosing tumour in a mammal, comprising detecting the level of expression of a gene encoding a MLN64 or PNMT polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample indicates the presence of tumour in the mammal from which the test tissue cells were obtained.
  • the present invention concerns a method of diagnosing tumour in a mammal, comprising (a) contacting an anti-MLN64 or anti-PNMT antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-MLN64 or anti-PNMT antibody and the MLN64 or PNMT polypeptide in the test sample.
  • the detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type. A larger quantity of complexes formed in the test sample indicates the presence of tumour in the mammal from which the test tissue cells were obtained.
  • the antibody preferably carries a detectable label.
  • the present invention concerns a cancer diagnostic kit, comprising an anti-MLN64 or anti-PNMT antibody and a carrier (e.g., a buffer) in suitable packaging.
  • the kit preferably contains instructions for using the antibody to detect the MLN64 or PNMT polypeptide.
  • the invention concerns a method for inhibiting the growth of tumour cells comprising exposing a cell which overexpresses a MLN64 (SEQ ID NO:6) or PNMT (SEQ ID NO:4) polypeptide to an effective amount of an agent inhibiting the expression and/or activity of the MLN64 or PNMT polypeptide.
  • the agent preferably is an anti-MLN64 or anti-PNMT antibody, a small organic and inorganic molecule, peptide, phosphopeptide, antisense or ribozyme molecule, or a triple helix molecule.
  • the agent is a prodrug that is relatively non- toxic but can be metabolized into an effective cytotoxic active drug form by the action of the enzyme phenylethanolamine N-methyltransferase. Since the tumour cells overexpress this gene product of the PNMT gene the production of the active drug would occur primarily in or very near the tumour cell and therefore would preferentially kill them.
  • the agent e.g., anti-MLN64 or anti- PNMT antibody induces cell death in the tumour cells while having little or no effect on other cells which do not overexpress a MLN64 or PNMT gene polypeptide expression product.
  • the tumour cells are further exposed to additional treatments including but not limited to, radiation treatment and/or a cytotoxic or chemotherapeutic agent.
  • the invention concerns a kit or an article of manufacture, comprising a container, a label on the container; and a composition comprising an active agent contained within the container; wherein the composition is effective for inhibiting the growth of tumour cells, the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a MLN64 or PNMT polypeptide, and the active agent in the composition is an agent inhibiting the expression and/or activity of the MLN64 or PNMT polypeptide.
  • the active agent is an anti-MLN64 or anti-PNMT antibody.
  • this invention provides a method for identifying a compound capable of inhibiting the expression and/or activity of a MLN64 or PNMT polypeptide, comprising contacting a candidate compound with a MLN64 or PNMT polypeptide under conditions and for a time sufficient to allow these two components to interact.
  • a candidate compound or the MLN64 or PNMT polypeptide is immobilized on a solid support.
  • the non-immobilized component carries a detectable label.
  • the present invention is based, in part, on the discovery that gene expression analysis can be combined with gene mapping techniques to produce a novel and powerful method (GEM) to identify small or large gene deletions or amplifications in cancer tissue.
  • GEM novel and powerful method
  • the gene expression profile can be compared for genes which map within the same or nearby chromosomal locations.
  • the overexpression or underexpression of a gene known to be deleted or amplified in the cell-proliferative disorder disease state can be correlated with the expression pattern of other genes at nearby chromosomal locations. If these other genes show a similar pattern then these other genes are likely to be co-amplified or co- deleted with the index gene.
  • this invention provides a simple and rapid method to determine which genes are amplified or deleted in cancer tissue and therefore provide means for diagnosis, prognosis and therapeutic intervention.
  • this invention provides two genes that are co-amplified along with c-erbB-2 (SEQ ID NO:1) in a significant percentage of one type of cell-proliferative disorder, i.e., breast cancer.
  • PNMT SEQ ID NO:3
  • SEQ ID NO5 steroidogenic acute regulatory protein related
  • SEQ ID NO5 the polypeptide expression product of which is a steroidogenic acute regulatory protein that binds and transports cholesterol and promotes steroidogenesis in placenta and brain.
  • TNM system determines the state or stage of the cancer, based on the tumour size, the degree of lymph node involvement and the presence of metastasis.
  • the stage of the cancer at the time of detection determines the outcome measured as percent free of recurrence at 10 years. This is the percentage of patients who have not experienced a recurrence of the original cancer in the 10 years after the original tumour is removed by mastectomy or lumpectomy.
  • the symptoms of breast cancer vary a great deal and depend on the location and size of the primary tumour, and the presence, location and extent of metastases.
  • the symptoms may include one or more of the following; unilateral or bilateral palpable breast mass, nipple discharge, breast skin changes, breast pain, which may or may not be cyclic in nature, i.e., with menses, bloody or watery nipple discharge, a palpable axillary mass, or other evidence of lymph node involvement.
  • CNS cerebrospinal sarcoma
  • spinal cord or other skeletal metastases and leptomeningeal metastases can cause local or diffuse pain, esp. back pain, and neurological symptoms or dysfunction including, parathesias, paraplegia, weakness or loss of sensation and hypercalcaemia. Seizures, headache, mental status changes or even paralysis or stroke are common with CNS involvement.
  • Liver metastases may cause liver failure with elevated liver function tests, jaundice and/or other evidence of liver dysfunction. Lung involvement can cause difficulty breathing, pneumonia, or other respiratory symptoms. While the above symptoms are common in breast cancer with or without metastases since the tumour cells can invade and proliferate in any tissue in the body it is possible for almost symptom complex to occur in patients with breast cancer. [36] Numerous prognostic factors have been identified in breast cancer patients, including the degree of invasion of the tumour locally, the number of involved axillary lymph nodes and tumour size, and these factors are incorporated in the staging system described above.
  • c-erb B2 avian erythoblastic leukaemia viral oncogene homolog 2 gene (c-erb B2) (also called HER-2/neu) This gene was first identified by transfection studies in which NIH 3T3 cells were transformed with DNA from chemically induced rat neuroglioblastomas. (See, Shih C, er a/. Nature 290:261 , 1981)
  • a well known mechanism of gene (e.g., oncogene) overexpression in cancer cells is gene amplification.
  • gene amplification In this process, multiple copies of a particular gene are produced in the chromosome of the ancestral cell. The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome, (see, Alitalo er a/., Adv. Cancer Res. 47, 235-281, 1986) It is believed that the overexpression of the gene parallels gene amplification, i.e., is proportionate to the number of copies made.
  • EGFR epidermal growth factor receptor
  • HER2 also known as c-erbB2 or ErbB2 or p185 ne "
  • HER3 ErbB3
  • HER4 ErbB4 or tyro2
  • EGFR vascular endothelial growth factor alpha
  • TGF- ⁇ transforming growth factor alpha
  • the second member of the ErbB family, p185" eu was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats.
  • the activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein.
  • c-erb B2 encodes a 185-kd transmembrane glycoprotein receptor (p 185 HER2 ; HER2) that has partial homology with the other members of the (EGFR) (epidermal growth factor receptor) family, see Hynes NE & Stern DF. Biochem. Biophys. Acta. 1198:165-184 (1994); Kraus MH. Proc. Natl. Acad. Sci. USA 90:9193-9197 (1989); and Plowman GD, et al. Proc. Natl. Acad. Sci. USA 90:1746- 1750 (1993).
  • EGFR epidermal growth factor receptor
  • the c-erbB2 gene (SEQ ID NO:1) is overexpressed in about 25% to 30% of human breast cancer and predicts a worse prognosis as measured by lower overall survival and disease-free survival, see Slamon DJ, et al. Science 244:707-712 (1989); Press MF, et al. J. Clin. Oncol. 15:2894-2904 (1997); and Seshadri R, et al. J. Clin. Oncol. 11:1936-1942 (1993). This overexpression is primarily due to gene amplification.
  • c-erbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (see, Slamon et al., (1987) and (1989), supra; Ravdin and Chamness, Gene 159:19- 27 (1995) and Hynes and Stern, Biochim Biophys Ada 1198:165-184 (1994]), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines (Baselga et al., Oncology 11 (3 Suppl l):43-48 [1997]).
  • CMF cyclophosphamide, methotrexate, and fluoruracil
  • anthracyclines Baselga et al., Oncology 11 (3 Suppl l):43-48 [1997]
  • Genomic amplification is a common feature of many types of cancer cells and can also be provoked in non-cancer cells by selecting for resistance to certain toxic agents. Amplification may involve large regions of the chromosomal DNA containing numerous genes. These gene may be involved in carcinogenesis directly or may increase the oncogenic properties of other genes by altered expression of downstream signalling molecules, see Stein D et al. EMBO J. 13: 1331-1334 (1994). Thus, genomic amplification is a critical process in the expression of oncogenic properties.
  • CGH comparative genomic hybridization
  • FISH fluorescence in-situ hybridization
  • tumour types contain amplified chromosomal sites, such as the site 17q22-q24, and the increase in gene number may significantly influence tumour behaviour.
  • types of tumours include; neurobastoma, mesotheliomas, rabdomyosarcomas and dermatofibrosarcomas, see, Barlund M, Genes, Chromosomes & Cancer 20: 372-376 (1997).
  • the available techniques to determine genomic amplification have shortcomings, including limited resolution and dynamic range. Therefore, there is a need for more rapid and more generally applicable techniques to determine the presence and extent of gene amplification in cancer tissues and specifically to determine which genes are both amplified in copy number and, in addition, overexpressed with the resulting increase in mRNA and polypeptide expression products.
  • HER2-positive patients responding clinically to treatment with taxanes are greater than three times those of HER2-negative patients.
  • a recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 (sold under the trademark HERCEPTINTM) has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol. 14:737-744 [1996]).
  • the murine MAb 4D5 directed against the extracellular domain of p185 HER2 , is a potent inhibitor of growth of human breast cancer cells that overexpress c-erb B2, see, Lewis GD, et al. Cancer Immunol. Immunother. 37:255-263 (1993).
  • this anti- HER2 antibody has been shown to also enhance the anti-tumour activity of paclitaxel and doxorubicin against c-erb B2 overexpressing human breast cancer xenografts and HERCEPTINTM is now a valuable treatment in human patients with breast cancer, see, Beselga J, et al. Cancer Res. 58:2825-2831 (1998), see, Vogel, et al.
  • genes which map in the same region of the chromosome as c-erb B2 may be included in the amplicon and may be co-amplified and co-expressed at high levels in patients whose tumours overexpress c-erb B2. This has been found to be the case with steriodogenic acute regulatory protein related (MLN64)
  • This gene has been mapped to the q12-q21 region of the long of chromosome 17 (see, Tomasetto C et al. Genomics, 28, 367-376, 1995). This is a region known to exhibit various sites of DNA mutation, deletion, or amplification in primary breast carcinoma. This region contains the c-erb B2 gene and it has been shown that MLN64 is overexpressed with c erb B2 in breast cancer cell lines, see Tomasetto C et al. Supra and MLN64 is also amplified and over expressed with c erb B2 in human breast carcinomas, see Moog-Lutz C et al. Int. J. Cancer 71: 183-191 , 1997.
  • MLN64 over-expression was correlated with that of the c-erb B2 gene at both mRNA and protein level, suggesting that these two genes are on the same amplicon on human chromosome 17 and that therefore MLN64 could constitute an additive target gene on this amplicon, see Moog-Lutz C et al. supra.
  • the experimental methods of this invention depend, in part, on measurements of cellular constituents.
  • the cellular constituents measured can be from any aspect of the biological state of a cell. They can be from the transcriptional state, in which RNA abundances are measured, the translation state, in which protein abundance's are measured, the activity state, in which protein activities are measured.
  • the cellular characteristics can also be from mixed aspects, for example, in which the activities of one or more proteins are measured along with the RNA abundances (gene expressions) of other cellular constituents.
  • This section describes exemplary methods for measuring the cellular constituents in drug or pathway responses. This invention is adaptable to other methods of such measurement.
  • the transcriptional state of the other cellular constituents is measured.
  • the transcriptional state can be measured by techniques of hybridization to arrays of nucleic acid or nucleic acid mimic probes, described in the next subsection, or by other gene expression technologies, described in the subsequent subsection.
  • the result is data including values representing mRNA abundance and/or ratios, which usually reflect DNA expression ratios (in the absence of differences in RNA degradation rates).
  • aspects of the biological state other than the transcriptional state such as the translational state, the activity state, or mixed aspects can be measured.
  • the presence, progression or prognosis of cancer in a subject can be monitored by measuring a level of expression of mRNA or encoded protein corresponding to at least one of the genes identified in TABLE 1 , below, in a sample of bodily fluid or breast tissue obtained in the subject over time, i.e., at various stages of the cell-proliferative disorder.
  • the level of expression of the mRNA or encoded protein corresponding to the gene(s) identified as relevant to overall prognosis can provide valuable information concerning the treatment or progression of the cancer.
  • the level of expression of mRNA and protein corresponding to the gene(s) can be detected by standard methods as described below.
  • Formation of a complex between the protein and its binding partner can be detected by using detectably labelled proteins such as radiolabelled, fluorescently labelled, or enzymatically labelled protein or its binding partner, by immunoassay or by chromatographic detection.
  • detectably labelled proteins such as radiolabelled, fluorescently labelled, or enzymatically labelled protein or its binding partner
  • the protein or its binding partner can be immobilized to facilitate separation of complexes from uncomplexed forms of the protein and its binding partner and automation of the assay. Complexation of the protein to its binding partner can be achieved in any type of vessel, e.g., microtitre plates, micro-centrifuge tubes and test tubes.
  • the protein can be fused to another protein, e.g., glutathione-S-transferase to form a fusion protein which can be absorbed onto a matrix, e.g., glutathione Sepharose beads (Sigma Chemical, St.
  • the labelled protein partner e.g., labelled with 35 S
  • test compound e.g., labelled with 35 S
  • the beads are washed to remove unbound label and the matrix is immobilized and the radiolabel is determined
  • Another method for immobilizing proteins on matrices involves utilizing biotin and streptavidin.
  • the protein can be biotinylated using biotin NHS (N-hydroxy- succinimide) using well-known techniques and immobilized in the well of streptavidin-coated plates.
  • the present invention also provides for both prophylactic and therapeutic methods of treating a subject having, or at risk of having, a cell-proliferative disorder, including but not limited to a breast disorder.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the cell-proliferative disorder , such that development of the cell-proliferative disorder is prevented or delayed in its progression.
  • the tumour cell e.g., cancer cell
  • suitable therapeutic agents include, but are not limited to, antibodies as described in detail below.
  • measurements of the cellular constituents should be made in a manner that is relatively independent of when the measurements are made.
  • Transcriptional State Measurement Preferably, measurement of the transcriptional state is made by hybridization of nucleic acids to oligonucleotide arrays, which are described in this subsection. Certain other methods of transcriptional state measurement are described later in this subsection.
  • Transcript Arrays Generally.
  • the present invention makes use of "oligonucleotide arrays” (also called herein “microarrays”). Microarrays can be employed for analyzing the transcriptional state in a cell, and especially for measuring the transcriptional states of cancer cells.
  • transcript arrays are produced by hybridizing detectably labelled polynucleotides representing the mRNA transcripts present in a cell (e.g., fluorescently labelled cDNA synthesized from total cell mRNA or labelled cRNA.) to a microarray.
  • a microarray is a surface with an ordered array of binding (e.g., hybridization) sites for products of many of the genes in the genome of a cell or organism, preferably most or almost all of the genes.
  • Microarrays can be made in a number of ways, of which several are described below. However produced, microarrays share certain characteristics: The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other.
  • the microarrays are small, usually smaller than 5 cm.sup.2, and they are made from materials that are stable under binding (e.g. nucleic acid hybridization) conditions.
  • a given binding site or unique set of binding sites in the microarray will specifically bind the product of a single gene in the cell.
  • site physical binding site
  • positionally addressable arrays containing affixed nucleic acids of known sequence at each location are used.
  • cDNA or cRNA complementary to the total cellular mRNA when detectably labelled (e.g., with a fluorophore) cDNA or cRNA complementary to the total cellular mRNA is hybridized to a microarray, the site on the array corresponding to a gene (i.e., capable of specifically binding the product of the gene) that is not transcribed in the cell will have little or no signal (e.g., fluorescent signal), and a gene for which the encoded mRNA is prevalent will have a relatively strong signal.
  • a gene i.e., capable of specifically binding the product of the gene
  • Microarrays are known in the art and consist of a surface to which probes that correspond in sequence to gene products (e.g., cDNAs, mRNAs, cRNAs, polypeptides, and fragments thereof), can be specifically hybridized or bound at a known position.
  • the microarray is an array (i.e., a matrix) in which each position represents a discrete binding site for a product encoded by a gene (e.g., a protein or RNA), and in which binding sites are present for products of most or almost all of the genes in the organism's genome.
  • the "binding site” is a nucleic acid or nucleic acid analogue to which a particular cognate cDNA or cRNA can specifically hybridize.
  • the nucleic acid or analogue of the binding site can be, e.g., a synthetic oligomer, a full-length cDNA, a less-than full-length cDNA, or a gene fragment.
  • the microarray contains binding sites for products of all or almost all genes in the target organism's genome, such comprehensiveness is not necessarily required.
  • the microarray may have binding sites for only a fraction of the genes in the target organism.
  • the microarray will have binding sites corresponding to at least about 50% of the genes in the genome, often at least about 75%, more often at least about 85%, even more often more than about 90%, and most often at least about 99%.
  • the microarray has binding sites for genes relevant to testing and confirming a biological network model of interest.
  • a “gene” is identified as an open reading frame (ORF) of preferably at least 50, 75, or 99 amino acids from which a messenger RNA is transcribed in the organism (e.g., if a single cell) or in some cell in a multicellular organism.
  • ORF open reading frame
  • the number of genes in a genome can be estimated from the number of mRNAs expressed by the organism, or by extrapolation from a well- characterized portion of the genome.
  • the number of ORFs can be determined and mRNA coding regions identified by analysis of the DNA sequence. For example, the Saccharomyces cerevisiae genome has been completely sequenced and is reported to have approximately 6275 open reading frames (ORFs) longer than 99 amino acids.
  • the "binding site" to which a particular cognate cDNA specifically hybridizes is usually a nucleic acid or nucleic acid analogue attached at that binding site.
  • the binding sites of the microarray are DNA polynucleotides corresponding to at least a portion of each gene in an organism's genome.
  • DNAs can be obtained by, e.g., polymerase chain reaction (PCR) amplification of gene segments from genomic DNA, cDNA (e.g., by RT-PCR), or cloned sequences or the sequences may be synthesized de novo on the surface of the chip, for example by use of photolithography techniques, e.g., Affymetrix uses such a different technology to synthesize their oligos directly on the chip).
  • PCR primers are chosen, based on the known sequence of the genes or cDNA, that result in amplification of unique fragments (i.e. fragments that do not share more than 10 bases of contiguous identical sequence with any other fragment on the microarray).
  • each gene fragment on the microarray will be between about 20 bp and about 2000 bp, more typically between about 100 bp and about 1000 bp, and usually between about 300 bp and about 800 bp in length.
  • PCR methods are well known and are described, for example, in Innis et al.
  • nucleic acid for the microarray is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (Froehler et al., Nucleic Acid Res 14:5399-5407 (1986); McBride et al., Tetrahedron Lett. 24:245-248 (1993)).
  • Synthetic sequences are between about 15 and about 500 bases in length, more typically between about 20 and about 50 bases.
  • synthetic nucleic acids include non-natural bases, e.g., inosine.
  • nucleic acid analogues may be used as binding sites for hybridization.
  • An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., Nature 365:566-568 (1993); see also U.S. Pat. No. 5,539,083).
  • the binding (hybridization) sites are made from plasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags), or inserts therefrom (Nguyen et al., Genomics 29:207-209 (1995)).
  • the polynucleotide of the binding sites is RNA.
  • nucleic acid or analogue are attached to a solid support, which may be made from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other materials.
  • a preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al., Science 270:467-470 (1995). This method is especially useful for preparing microarrays of cDNA. See, also, DeRisi et al., Nature Genetics 14:457-460 (1996); Shalon et al., Genome Res. 6:639-645 (1996); and Schena et al., Proc. Natl. Acad. Sci. USA 93:10539-11286 (1995). Each of the aforementioned articles is incorporated by reference in its entirety for all purposes.
  • a second preferred method for making microarrays is by making high-density oligonucleotide arrays.
  • Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ (see, Fodor et al., Science 251 :767- 773 (1991); Pease et al., Proc. Natl. Acad. Sci. USA 91:5022-5026 (1994); Lockhart et al., Nature Biotech 14:1675 (1996); U.S. Pat. Nos.
  • oligonucleotides e.g., 25- mers
  • oligonucleotide probes can be chosen to detect alternatively spliced mRNAs.
  • microarrays e.g., by masking
  • any type of array for example, dot blots on a nylon hybridization membrane (see Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Ed.), Vol. 1-3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), which is incorporated in its entirety for all purposes), could be used, although, as will be recognized by those of skill in the art, very small arrays will be preferred because hybridization volumes will be smaller.
  • RNA is extracted from cells of the various types of interest in this invention using guanidinium thiocyanate lysis followed by CsCI centrifugation (Chirgwin et al., Biochemistry 18:5294-5299 (1979)).
  • Poly(A) + RNA is selected by selection with oligo-dT cellulose (see Sambrook et al., supra).
  • Cells of interest include wild-type cells, drug-exposed wild-type cells, cells with modified/perturbed cellular constituent(s), and drug-exposed cells with modified/perturbed cellular constituent(s).
  • Labelled cDNA is prepared from mRNA or alternatively directly from RNA by oligo dT-primed or random-primed reverse transcription, both of which are well known in the art (see e.g., Klug & Berger, Methods Enzymol. 152:316-325 (1987)). Reverse transcription may be carried out in the presence of a dNTP conjugated to a detectable label, most preferably a fluorescently labelled dNTP.
  • isolated mRNA can be converted to labelled antisense RNA synthesized by in vitro transcription of double-stranded cDNA in the presence of labelled dNTPs (Lockhart et al., Nature Biotech.
  • the cDNA or RNA probe can be synthesized in the absence of detectable label and may be labelled subsequently, e.g., by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labelled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
  • biotinylated dNTPs or rNTP or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs)
  • streptavidin e.g., phycoerythrin-conjugated streptavidin
  • fluorophores include fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others (see, e.g., Kricka, Nonisotopic DNA Probe Techniques (Academic Press, San Diego, Calif., 1992). It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.
  • a label other than a fluorescent label is used.
  • a radioactive label or a pair of radioactive labels with distinct emission spectra, can be used (see Zhao et al., Gene 156:207 (1995); Pietu et al., Genome Res. 6:492 (1996)).
  • use of radioisotopes is a less-preferred embodiment.
  • labelled cDNA is synthesized by incubating a mixture containing 0.5 mM dGTP, dATP and dCTP plus 0.1 mM dTTP plus fluorescent deoxyribonucleotides (e.g., 0.1 mM Rhodamine 110 UTP (Perkin Elmer Cetus) or 0.1 mM Cy3 dUTP (Amersham)) with reverse transcriptase (e.g., SuperscriptTM II, LTI Inc.) at 42°C. for 60 min.
  • fluorescent deoxyribonucleotides e.g., 0.1 mM Rhodamine 110 UTP (Perkin Elmer Cetus) or 0.1 mM Cy3 dUTP (Amersham)
  • reverse transcriptase e.g., SuperscriptTM II, LTI Inc.
  • nucleic acid hybridization and wash conditions are chosen so that the probe "specifically binds" or “specifically hybridizes” to a specific array site, i.e., the probe hybridizes, duplexes or binds to a sequence array site with a complementary nucleic acid sequence but does not hybridize to a site with a non- complementary nucleic acid sequence.
  • one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch.
  • the polynucleotides are perfectly complementary (no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see, e.g., Shalon et al., supra, and Chee et al., supra).
  • Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, DNA, PNA) of labelled probe and immobilized polynucleotide or oligonucleotide.
  • length e.g., oligomer versus polynucleotide greater than 200 bases
  • type e.g., RNA, DNA, PNA
  • hybridization conditions are hybridization in 5 X SSC plus 0.2% SDS at 65°C. for 4 hours followed by washes at 25°C. in low stringency wash buffer (1 X SSC plus 0.2% SDS) followed by 10 minutes at 25°C. in high stringency wash buffer (0.1 X SSC plus 0.2% SDS) (Shena et al., Proc. Natl. Acad. Sci. USA, 93:10614 (1996)).
  • Useful hybridization conditions are also provided in, e.g., Tijessen, Hybridization With Nucleic Acid Probes (Elsevier Science Publishers B. V., 1993) and Kricka, Nonisotopic DNA Probe Techniques (Academic Press San Diego, Calif., 1992).
  • the fluorescence emissions at each site of a transcript array can be, preferably, detected by scanning confocal laser microscopy. In one embodiment, a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used. Alternatively, a laser can be used that allows specimen illumination at wavelengths specific to the fluorophores used and emissions from the fluorophore can be analyzed. In a preferred embodiment, the arrays are scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope objective.
  • Fluorescence laser scanning devices are described in Schena et al., Genome Res. 6:639-645 (1996) and in other references cited herein.
  • the fibre-optic bundle described by Ferguson et al., Nature Biotech. 14:1681- 1684 (1996), may be used to monitor mRNA abundance levels at a large number of sites simultaneously.
  • Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 bit analogue to digital board.
  • the scanned image is despeckled using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site.
  • a graphics program e.g., Hijaak Graphics Suite
  • the Agilent Technologies GeneArray scanner is a bench-top, 488 nm argon-ion laser-based analysis instrument.
  • the laser can be focused to a spot size of less than 4 microns. This precision allows for the scanning of probe arrays with probe cells as small as 20 microns.
  • the laser beam focuses onto the probe array, exciting the fluorescent labelled nucleotides. It then scans using the selected filter for the dye used in the assay. Scanning in the orthogonal coordinate is achieved by moving the probe array.
  • the laser radiation is absorbed by the dye molecules incorporated into the hybridized sample and causes them to emit fluorescence radiation. This fluorescent light is collimated by a lens and passes through a filter for wavelength selection.
  • the light is then focused by a second lens onto an aperture for depth discrimination and then detected by a highly sensitive photo multiplier tube (PMT).
  • PMT highly sensitive photo multiplier tube
  • ADC an analogue to digital converter
  • the output current of the PMT is converted into a voltage read by an analogue to digital converter (ADC) and the processed data is passed back to the computer as the fluorescent intensity level of the sample point, or picture element (pixel) currently being scanned.
  • ADC analogue to digital converter
  • the computer displays the data as an image, as the scan progresses.
  • the fluorescent intensity level of all samples, representing the expression profile of the sample is recorded in computer readable format.
  • an experimentally determined correction for "cross talk" (or overlap) between the channels for the two fluors may be made.
  • a ratio of the emission of the two fluorophores may be calculated. The ratio is independent of the absolute expression level of the cognate gene, but may be useful for genes whose expression is significantly modulated by drug administration, gene deletion, or any other tested event.
  • the term "similar”, when used to compare two or more values, means that the two values are within 20%, or more preferably within 10% of each other in numerical value when using the same units.
  • the transcriptional state of a cell may be measured by other gene expression technologies known in the art.
  • Several such technologies produce pools of restriction fragments of limited complexity for electrophoretic analysis, such as methods combining double restriction enzyme digestion with phasing primers (see, e.g., European Patent 0534858 A1, filed Sep. 24, 1992, by Zabeau et al.), or methods selecting restriction fragments with sites closest to a defined mRNA end (see, e.g., Prashar et al., Proc. Natl. Acad. Sci. USA 93:659-663 (1996)).
  • cDNA pools statistically sample cDNA pools, such as by sequencing sufficient bases (e.g., 20-50 bases) in each of multiple cDNAs to identify each cDNA, or by sequencing short tags (e.g., 9-10 bases) which are generated at known positions relative to a defined mRNA end (see, e.g., Velculescu, Science 270:484-487 (1995)). pathway pattern.
  • sequencing sufficient bases e.g., 20-50 bases
  • sequencing short tags e.g., 9-10 bases
  • aspects of the biological state other than the transcriptional state can be measured in order to obtain drug and pathway responses. Details of these embodiments are described in this section.
  • Translational State Measurements Expression of the protein encoded by the gene(s) can be detected by a probe which is detectably labelled, or which can be subsequently labelled. Generally, the probe is an antibody that recognizes the expressed protein.
  • antibody includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, and biologically functional antibody fragments sufficient for binding of the antibody fragment to the protein.
  • various host animals may be immunized by injection with the polypeptide, or a portion thereof. Such host animals may include, but are not limited to, rabbits, mice, and rats, to name but a few.
  • adjuvants may be used to increase the immunological response, depending on the host species, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Camette-Guerin) and Corynebaderium parvum.
  • BCG Bacille Camette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof.
  • an antigen such as target gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, may be immunized by injection with the encoded protein, or a portion thereof, supplemented with adjuvants as also described above.
  • mAbs Monoclonal antibodies (mAbs), which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler & Milstein, Nature, 256:495-497 (1975); and U.S. Patent No.
  • chimeric antibodies Morrison et al., Proc. Natl. Acad. Sci USA, 81:6851-6855 (1984); Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature, 314:452-454 (1985), by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived form a murine mAb and a human immunoglobulin constant region.
  • such fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F (ab') 2 fragments.
  • Fab expression libraries may be constructed, Huse et al., Science, 246:1275-1281 (1989), to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • immunoassay methods that utilize the antibodies described above.
  • immunoassay methods include, but are not limited to, dot blotting, western blotting, competitive and noncompetitive protein binding assays, enzyme-linked immunosorbant assays (ELISA), immunohistochemistry, fluorescence activated cell sorting (FACS), and others commonly used and widely described in scientific and patent literature, and many employed commercially.
  • sandwich ELISA of which a number of variations exist, all of which are intended to be encompassed by the present invention.
  • unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule after a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex.
  • a second antibody labelled with a reporter molecule capable of inducing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen-labelled antibody.
  • any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.
  • Variations on the forward assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and added to the unlabeled surface bound antibody.
  • reporter molecules in this type of assay are either enzymes, fluorophore- or radionuclide-containing molecules.
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate an enzyme conjugated to the second antibody, usually by means of glutaraldehyde or periodate.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.
  • p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1 ,2-phenylenediamine or toluidine are commonly used.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • a solution containing the appropriate substrate is then added to the tertiary complex.
  • the substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an evaluation of the amount of protein which is present in the serum sample.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic colour visually detectable with a light microscope.
  • Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.
  • Measurement of the translational state may also be performed according to several additional methods.
  • whole genome monitoring of protein i.e., the "proteome,” Goffeau et al., supra
  • whole genome monitoring of protein i.e., the "proteome,” Goffeau et al., supra
  • binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome.
  • antibodies are present for a substantial fraction of the encoded proteins, or at least for those proteins relevant to testing or confirming a biological network model of interest.
  • Methods for making monoclonal antibodies are well known (see, e.g., Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor, N.Y., 1988), which is incorporated in its entirety for all purposes).
  • monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell.
  • proteins from the cell are contacted to the array, and their binding is assayed with assays known in the art.
  • proteins can be separated by two-dimensional gel electrophoresis systems
  • Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. See, e.g., Hames et al., Gel Electrophoresis of Proteins: A Practical Approach (IRL Press, New York, 1990); Shevchenko et al., Proc. Nat'IAcad. Sci. USA 93:1440-1445 (1996); Sagliocco et al., Yeast 12:1519-1533 (1996); Lander, Science 274:536-539 (1996).
  • the resulting electropherograms can be analyzed by numerous techniques, including mass spectrometric techniques, western blotting and immunoblot analysis using polyclonal and monoclonal antibodies, and internal and N-terminal micro- sequencing. Using these techniques, it is possible to identify a substantial fraction of all the proteins produced under given physiological conditions, including in cells (e.g., in yeast) exposed to a drug, or in cells modified by, e.g., deletion or over-expression of a specific gene.
  • response data may be formed of mixed aspects of the biological state of a cell. Response data can be constructed from, e.g., changes in certain mRNA abundances, changes in certain protein abundances, and changes in certain protein activities.
  • Antibody types include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to a target protein.
  • various host animals can be immunized by injection with the target protein, such host animals include, but are not limited to, rabbit, mice, rats, etc.
  • adjuvants can be used to increase the immunological response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as bacillus Calmette-Guerin (BCG) and Corynebaderium parvum.
  • BCG Bacillus Calmette-Guerin
  • Monoclonal Antibodies For preparation of monoclonal antibodies directed towards a target protein, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. Such techniques include, but are not restricted to, the hybridoma technique originally developed by Kohler & Milstein, Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983)), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, pp. 77-96 (Alan R. Liss, Inc., 1985)).
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545).
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., Proc. Natl. Acad. Sci. USA 80: 2026-2030 (1983)), or by transforming human B cells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies and Cancer Therapy, pp. 77-96 (Alan R. Liss, Inc., 1985)).
  • techniques developed for the production of "chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci.
  • monoclonal antibodies are advantageous, they can be alternatively selected from large antibody libraries using the techniques of phage display (Marks et al., 1992, J. Biol. C/?em._267:16007-16010). Using this technique, libraries of up to 10.sup.12 different antibodies have been expressed on the surface of fd filamentous phage, creating a "single pot" in vitro immune system of antibodies available for the selection of monoclonal antibodies (Griffiths et al., 1994, EMBO J. 13:3245-3260).
  • Selection of antibodies from such libraries can be done by techniques known in the art, including contacting the phage to immobilized target protein, selecting and cloning phage bound to the target, and subcloning the sequences encoding the antibody variable regions into an appropriate vector expressing a desired antibody format.
  • Antibody fragments that contain the idiotypes of the target protein can be generated by techniques known in the art.
  • such fragments include, but are not limited to: the F(ab').sub.2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments that can be generated by reducing the disulfide bridges of the F (ab').sub.2 fragment, the Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA (enzyme-linked immunosorbent assay). To select antibodies specific to a target protein, one may assay generated hybridomas or a phage display antibody library for an antibody that binds to the target protein.
  • compositions may be administered by an number of routes including, but not limited to, oral, intravenous, intramuscular, intra-articular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients re carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilising agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated m aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycatonic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder that may contain any or all of the following: 1-50 mM histidine, 0. 1 %-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • such labelling would include amount, frequency, and method of administration.
  • Those skilled in the art will employ different formulations for antisense nucleotides than for antagonists, e.g., antibodies or inhibitors.
  • Pharmaceutical formulations suitable for oral administration of proteins are described, e.g., in U.S. Patents 5,008,114; 5,505,962; 5,641 ,515; 5,681 ,811; 5,700,486; 5,766,633; 5,792,451 ; 5,853,748; 5,972,387; 5,976,569; and 6,051,561.
  • the level of mRNA corresponding to the marker can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art.
  • biological sample is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA.
  • RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from breast cells (see, e.g., Ausubel, et al., Ed., Current Protocols in Molecular Biology (John Wiley & Sons, New York, 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well-known to those of skill in the art, such as, for example, the single-step RNA isolation process of U.S. Patent No. 4,843,155.
  • the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • One preferred diagnostic method for the detection of mRNA levels involve contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected.
  • the nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present invention.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
  • the mRNA is immobilized on a solid surface and contacted with a probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array.
  • a skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.
  • An alternative method for determining the level of mRNA corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, U.S. Patent No. 4,683,202 (1987); ligase chain reaction, Barany, Proc. Natl. Acad. Sci. USA, 88:189-193 (1991); self- sustained sequence replication, Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874-1878 (1990); transcriptional amplification system, Kwoh et al., Proc. Natl. Ac. Sci.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice- versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • mRNA does not need to be isolated form the breast cells prior to detection.
  • a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.
  • determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-breast cancer sample, or between samples from different sources.
  • the expression level can be provided as a relatively expression level.
  • the level of expression of the marker is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question.
  • the mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker.
  • the expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level.
  • the samples used in the baseline determination will be from breast cancer or from non-cancer cells of similar tissue type.
  • the choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is tumour specific (versus normal cells).
  • the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from normal cells provides a means for grading the severity of the cancer state.
  • a polypeptide corresponding to a marker is detected.
  • a preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide corresponding to a marker of the invention, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F (ab') 2) can be used.
  • labelled is intended to encompass direct labelling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labelling of the probe or antibody by reactivity with another reagent that is directly labelled.
  • indirect labelling include detection of a primary antibody using a fluorescently labelled secondary antibody and end labelling of a DNA probe with biotin such that it can be detected with fluorescently labelled streptavidin.
  • Proteins from tumour cells can be isolated using techniques that are well known to those of skill in the art.
  • the protein isolation methods employed can, for example, be such as those described in Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988).
  • a variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA); radioimmunoasay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA).
  • EIA enzyme immunoassay
  • RIA radioimmunoasay
  • ELISA enzyme linked immunoabsorbant assay
  • a skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether breast cells express a marker of the present invention.
  • antibodies, or antibody fragments can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins.
  • Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody.
  • Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • kits for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample e.g., a breast- associated body fluid, serum, plasma, lymph, cystic fluid, urine, stool, csf, acitic fluid, or blood.
  • a biological sample e.g., a breast- associated body fluid, serum, plasma, lymph, cystic fluid, urine, stool, csf, acitic fluid, or blood.
  • the kit can comprise a labelled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide).
  • Kits can also include instructions for interpreting the results obtained using the kit.
  • the kit can comprise, for example: 1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker or the invention; and, optionally, 2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • the kit can comprise, for example: 1 ) an oligonucleotide, e.g., a detectably labelled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention; or 2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein- stabilizing agent.
  • the kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the computer system may be a single hardware platform comprising internal components and being linked to external components.
  • the internal components of this computer system include processor element interconnected with a main memory.
  • computer system can be an Intel Pentium based processor of 200 Mhz or greater clock rate and with 32 MB or more of main memory.
  • the external components include mass data storage. This mass storage can be one or more hard disks (which are typically packaged together with the processor and memory). Typically, such hard disks provide for at least 1 GB of storage.
  • Other external components include user interface device, which can be a monitor and keyboards, together with pointing device, which can be a "mouse", or other graphic input devices.
  • the computer system is also linked to other local computer systems, remote computer systems, or wide area communication networks, such as the Internet. This network link allows the computer system to share data and processing tasks with other computer systems.
  • This software components Loaded into memory during operation of this system are several software components, which are both standard in the art and special to the instant invention. These software components collectively cause the computer system to function according to the methods of this invention. These software components are typically stored on mass storage. Alternatively, the software components may be stored on removable media such as floppy disks or CD-ROM (not illustrated).
  • the software component represents the operating system, which is responsible for managing the computer system and its network interconnections.
  • This operating system can be, e.g., of the Microsoft Windows family, such as Windows 95, Windows 98, or Windows NT, or a Unix operating system, such as Sun. Solaris.
  • Software includes common languages and functions conveniently present on this system to assist programs implementing the methods specific to this invention. Languages that can be used to program the analytic methods of this invention include C, C++, or, less preferably, JAVA.
  • the methods of this invention are programmed in mathematical software packages, which allow symbolic entry of equations and high-level specification of processing, including algorithms to be used, and thereby freeing a user of the need to procedurally program individual equations or algorithms.
  • Such packages include, e.g., Matlab from Mathworks (Natick, Mass.), Mathematica from Wolfram Research (Champaign, III.), and MathCAD from Mathsoft (Cambridge, Mass.)..
  • the analytic software component actually comprises separate software components that interact with each other.
  • Analytic software represents a database containing all data necessary for the operation of the system. Such data will generally include, but is not necessarily limited to, results of prior experiments, genome data, experimental procedures and cost, and other information, which will be apparent to those skilled in the art.
  • Analytic software includes a data reduction and computation component comprising one or more programs which execute the analytic methods of the invention.
  • Analytic software also includes a user interface (Ul) which provides a user of the computer system with control and input of test network models, and, optionally, experimental data.
  • the user interface may comprise a drag-and-drop interface for specifying hypotheses to the system.
  • the user interface may also comprise means for loading experimental data from the mass storage component (e.g., the hard drive), from removable media (e.g., floppy disks or CD-ROM), or from a different computer system communicating with the instant system over a network (e.g., a local area network, or a wide area communication network such as the internet).
  • a network e.g., a local area network, or a wide area communication network such as the internet.
  • the term "Antibodies” shall include polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Isolated means altered by the human hands from its natural state, i.e. if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein.
  • RNA polyribonucleotide
  • DNA polydeoxribonucleotide
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • nucleotide bases includes, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polypeptide refers to any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, - i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.
  • Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • allele refers to one of two or more alternative forms of a gene occuring at a given locus in the genome.
  • the term "homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined. Falling within this generic term are the terms “ortholog”, and “paralog”. "Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species. "Paralog” refers to a polynucleotide or polypeptide that within the same species which is functionally similar.
  • amino acid and nucleotide abbreviations which occur in the various amino acid sequences appearing herein are identified according to their well-known, three-letter or one-letter abbreviations.
  • the nucleotides, which occur in the various DNA fragments are designated with the standard single-letter designations used routinely in the art.
  • RNA refers to a nucleic acid molecule whose nucleotide sequence encodes an RNA or polypeptide.
  • a gene can be either RNA or DNA. Genes may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • isolated should be understood to mean a preparation free from naturally occurring materials with which such DNA or protein is normally associated in nature.
  • the term "essentially pure” should be understood to mean a “highly” purified preparation that contains at least 95% of the DNA or protein of interest.
  • cell extract is intended to include culture media, especially spent culture media from which the cells have been removed.
  • a cell extract that is said to contain the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation containing the DNA or protein of interest and obtained from cells that express the protein or contain the DNA of interest.
  • basal activity means the level of gene expression observed in the absence of a specific stimulus
  • the terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like which would be to a degree that would prohibit administration of the composition.
  • the term "cell-proliferative disorder” denotes malignant as well as non-malignant disorders in which cell populations morphologically appear to differ from the surrounding tissue.
  • the cell-proliferative disorder may be a transcriptional disorder that results in an increase or a decrease in gene expression level.
  • the cause of the disorder may be of cellular origin or viral origin.
  • Treatment can be prophylactic in order to make a cell resistant to a virus, or therapeutic, in order to ameliorate an established infection in a cell, by preventing production of viral products.
  • the "administration" of a therapeutic composition can be effected by any means, and includes, but is not limited to, subcutaneous, intravenous, intramuscular, intrastemal, infusion techniques, intraperitoneally administration and parenteral administration.
  • the phrases “gene amplification” and “gene duplication” are used interchangeably and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line.
  • the duplicated region (a stretch of amplified DNA) is often referred to as "amplicon”.
  • amplicon a stretch of amplified DNA
  • the amount of the messenger RNA (mRNA) produced i.e., the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed.
  • tumor refers to any and all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukaemia.
  • cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endomenial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • complementary nucleotide sequences refer to nucleotide sequences that are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • purines will base-pair with pyrimidine to form combinations of guanine:cytosine and adenine:thymine in the case of DNA, or adenine:uracil in the case of RNA.
  • Other less common bases e.g., inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others may be included in the hybridizing sequences and will not interfere with pairing.
  • treatment means an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a therapeutic agent may directly decrease the pathology of tumour cells, or render the tumour cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • pathology includes all phenomena that compromise the well-being of the patient.
  • the term "mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In preferred embodiments the mammal is a human.
  • carriers shall include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or non ionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum album
  • administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., I 131 , 1 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • chemotherapeutic agent means any chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teni
  • a “growth inhibitory agent” refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo.
  • the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and Ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.
  • doxorubicin means an athracycline antibiotic.
  • the full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy- ⁇ -L-lyxo- hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11 -trihydroxy-8-(hydroxyacetyl)-1 -methoxy-5, 12- naphthacenedione.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumour necrosis factors- alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature.
  • stringent conditions may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran
  • the term "moderately stringent conditions” may be identified as - described by Sambrook et al., Molecular Cloning: A Laboratory Manual, (New York: Cold Spring Harbor Press, 1989), and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above.
  • moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • biological activity in the context of an antibody or another molecule that can be identified by the screening assays disclosed herein (e.g., an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins.
  • a preferred biological activity is growth inhibition of a target tumour cell.
  • Another preferred biological activity is cytotoxic activity resulting in the death of the target tumour cell.
  • immunological cross-reactivity means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of another polypeptide having this activity with polyclonal antisera raised against the known active polypeptide.
  • antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds.
  • the immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (e.g., antibody) identified, to the corresponding polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 8-times, most preferably at least about 8-times higher) than the binding affinity of that molecule to any other known native polypeptide.
  • the immunologically cross-reactive molecule e.g., antibody
  • the term "antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein.
  • agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc.
  • small molecule is defined herein to have a molecular weight below about 500 daltons.
  • antibodies are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibodylike molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • antibody is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • the terms “native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a 13-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the 13-sheet structure.
  • antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region, of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called herein, "Fab” fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • the term "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen- binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen- binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • immunoglobulins can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, 1gE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, IgA, and lgA2.
  • the heavy- chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 [1991] and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No, 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 [1984]).
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequence
  • humanized antibodies means forms of non-human (e.g., murine) antibodies which are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the humanized antibody includes a PRIMATIZEDTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • PRIMATIZEDTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V domains which enables the sFv to form the desired structure for antigen binding.
  • a polypeptide linker between the V H and V domains which enables the sFv to form the desired structure for antigen binding.
  • VH heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • the term "autocrine" stimulatory pathway means a pathway in which self stimulation occurs by virtue of the cancer cell producing both an ErbB ligand and its cognate ErbB receptor.
  • the cancer may express or overexpress EGFR and also express or overexpress an EGFR ligand (e.g., EGF, TGF- ⁇ , or HB-EGF).
  • the cancer may express or overexpress ErbB2 and also express or overexpress a heregulin (e.g., ⁇ -HRG).
  • a cancer which "overexpresses" an ErbB receptor is one which has significantly higher levels of an ErbB receptor, such as ErbB2, at the cell surface thereof, compared to a noncancerous cell of the same tissue type.
  • Such overexpression may be caused by gene amplification or by increased transcription or translation.
  • ErbB receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the ErbB protein present on the surface of a cell (e.g., via an immunohistochemistry assay; IHC).
  • FISH fluorescent in situ hybridization
  • PCR polymerase chain reaction
  • RT-PCR real time quantitative PCR
  • a cancer which is "not characterized by overexpression of the ErbB2 receptor” is one which, in a diagnostic assay, does not express higher than normal levels of ErbB2 receptor compared to a noncancerous cell of the same tissue type.
  • a cancer which "overexpresses" an ErbB ligand is one which produces significantly higher levels of that ligand compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation.
  • Overexpression of the ErbB ligand may be determined diagnostically by evaluating levels of the ligand (or nucleic acid encoding it) in the patient, e.g., in a tumour biopsy or by various diagnostic assays such as the IHC, FISH, southern blotting, PCR or in vivo assays described above.
  • hormone independent cancer is one in which proliferation thereof is not dependent on the presence of a hormone which binds to a receptor expressed by cells in the cancer. Such cancers do not undergo clinical regression upon administration of pharmacological or surgical strategies that reduce the hormone concentration in or near the tumour.
  • hormone independent cancers include androgen independent prostate cancer, estrogen independent breast cancer, endometrial cancer and ovarian cancer.

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Abstract

L'invention concerne une amplification et/ou une délétion génomiques dans des tissus cancéreux et des procédés et compositions de diagnostic et de traitement du cancer. Le procédé selon l'invention consiste à mettre en oeuvre la combinaison de profilage d'expression génique et la cartographie génétique et l'identification de gènes spécifiques s'avérant être co-amplifiés avec le gène c-erb B2 dans le cancer du sein.
PCT/EP2004/012876 2003-11-13 2004-11-12 Detection d'amplification et de deletion genomiques dans le cancer WO2005047536A2 (fr)

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US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
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US9050297B2 (en) 2012-04-02 2015-06-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator
US9089604B2 (en) 2012-04-02 2015-07-28 Moderna Therapeutics, Inc. Modified polynucleotides for treating galactosylceramidase protein deficiency
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