WO2014043331A2 - Formes phosphorylées spécifiques de l'elk-1 utilisées comme biomarqueurs et cibles thérapeutiques contre le cancer - Google Patents

Formes phosphorylées spécifiques de l'elk-1 utilisées comme biomarqueurs et cibles thérapeutiques contre le cancer Download PDF

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WO2014043331A2
WO2014043331A2 PCT/US2013/059438 US2013059438W WO2014043331A2 WO 2014043331 A2 WO2014043331 A2 WO 2014043331A2 US 2013059438 W US2013059438 W US 2013059438W WO 2014043331 A2 WO2014043331 A2 WO 2014043331A2
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elk
protein
antibody
phosphorylated
cancer
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PCT/US2013/059438
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WO2014043331A3 (fr
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James Eberwine
Jai-Yoon Sul
Jacqueline MORRIS
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The Trustees Of The University Of Pennsylvania
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons

Definitions

  • Elk-1 is a member of the Ets oncogene family of transcription factors, which are characterized by a conserved DNA-binding domain, or Ets domain.
  • Phosphorylation of Elk- 1 at S383 serves as an integration point within cells for redundant activation of upstream MAPK signaling cascades by distinct external stimuli.
  • Growth factors and mitogens activate the Raf/MEK/ERK pathway, resulting in regulation of growth and differentiation, through transcriptional activation by Elk-1 pS383 (Rao et al., 1994, Oncogene, 9: 1855-1860; Li et al, 2005, J Biol Chem, 280: 6036-6046). Stress, inflammatory cytokines, and other growth factors activate the p38 MAPK and
  • SAPK/JNK pathways resulting in regulation of inflammation, apoptosis, growth, and differentiation, also through transcriptional activation by Elk-1 at pS383 (Rao et al., 1994, Oncogene, 9: 1855-1860; Li et al, 2005, J Biol Chem, 280: 6036-6046).
  • Elk-1 regulates transcription by interacting with serum response factor (SRF) at serum response elements (SRE) within the promoters of genes that subsequently execute the functions initiated by extracellular signals (Janknecht et al., 1994, Oncogene, 9: 1273-1278).
  • Elk-1 is most notably known for contributing to regulation of growth and proliferation through transcriptional activation of immediate early genes such as c-Fos and Zif-268 (Sgambato et al, 1998, J Neurosci, 18: 214-226; Hipskind et al, 1991, Nature, 354: 531-534).
  • Transcriptional activation of Elk- 1 through these signaling cascades results in regulation of a wide range of normal cellular functions, including cell-cell and cell-matrix adhesion, proliferation and apoptosis (York et al., 1998, Nature, 392: 622- 626; Shao et al, 1998, Oncogene, 17: 527-532).
  • Carcinogenesis involves dysregulation of these processes and Elk-1 activation has been implicated in cancers of many tissues including breast, pancreas, and colon.
  • the activation domain of Elk-1 contains many phosphorylation sites, which, excluding the S383 site, remain largely uncharacterized (Gille et al, 1995, The EMBO Journal, 14: 951-962). Mutation of these sites to create nonphosphorylatable mutants revealed that preventing phosphorylation at the T417 site, but not the S383 site, blocked the apoptotic response when the mutant mRNA were introduced into neuronal dendrites (Sharma et al, 2010, PLoS ONE, 5: e9002).
  • the present invention provides a method of diagnosing cancer in a subject in need thereof, comprising: determining the level of phosphorylated Elk-1 protein in a biological sample from the subject, comparing the level of phosphorylated Elk-1 protein in the biological sample with a comparator control, and diagnosing the subject with cancer when the level of phosphorylated Elk-1 protein in the biological sample is different than the level of phosphorylated Elk-1 protein of the comparator control.
  • the level of phosphorylated Elk-1 protein in the biological sample is elevated when compared with the comparator control.
  • the level of phosphorylated Elk-1 protein in the biological sample is reduced when compared with the comparator control.
  • the phosphorylated Elk-1 protein is Elk-1 pT417. In one embodiment, the phosphorylated Elk-1 protein is Elk-1 pT363. In one embodiment, the phosphorylated Elk-1 protein is Elk-1 pT363, pT417.
  • the level of phosphorylated Elk-1 protein in the biological sample is determined by measuring an activity of phosphorylated Elk-1 protein in the biological sample.
  • the comparator control is at least one of a positive comparator control, a negative comparator control, a historical control, a historical norm, or the level of a reference molecule in the biological sample.
  • the reference molecule is total Elk-1 protein.
  • the cancer diagnosed by the method of the invention is colorectal adenocarcinoma.
  • the cancer is stomach cancer or breast cancer.
  • the subject is human.
  • the method comprises administering an effective cancer treatment to the subject.
  • the present invention provides a composition comprising a phosphorylated Elk-1 protein inhibitor.
  • the phosphorylated Elk-1 protein inhibitor is an antibody that specifically binds to phosphorylated Elk-1 protein.
  • the antibody specifically binds to Elk-1 pT417. In one embodiment, the antibody specifically binds to Elk-1 pT363. In one embodiment, the antibody is at least one of a polyclonal antibody, a monoclonal antibody, an intracellular antibody, an antibody fragment, a single chain antibody (scFv), a heavy chain antibody, a synthetic antibody, a chimeric antibody, or humanized antibody.
  • a polyclonal antibody a monoclonal antibody, an intracellular antibody, an antibody fragment, a single chain antibody (scFv), a heavy chain antibody, a synthetic antibody, a chimeric antibody, or humanized antibody.
  • the phosphorylated Elk-1 protein inhibitor is at least one of a chemical compound, a protein, a peptide, a peptidomemetic, an aptamer, a ribozyme, an antisense nucleic acid or a small molecule chemical compound.
  • the present invention also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a phosphorylated Elk-1 protein inhibitor.
  • the phosphorylated Elk-1 protein inhibitor is an antibody that specifically binds to phosphorylated Elk-1 protein.
  • the antibody specifically binds to Elk-1 pT417.
  • the antibody specifically binds to Elk-1 pT363.
  • the antibody is at least one of a polyclonal antibody, a monoclonal antibody, an intracellular antibody, an antibody fragment, a single chain antibody (scFv), a heavy chain antibody, a synthetic antibody, a chimeric antibody, or humanized antibody.
  • the phosphorylated Elk-1 protein inhibitor is at least one of a chemical compound, a protein, a peptide, a peptidomemetic, an aptamer, a ribozyme, an antisense nucleic acid or a small molecule chemical compound.
  • the cancer is colorectal adenocarcinoma. In one embodiment, the subject the subject is human.
  • the present invention provides a method of identifying a test compound as a modulator of phosphorylated Elk-1 protein, comprising determining the level phosphorylated Elk-1 protein in the presence of a test compound, determining the level of phosphorylated Elk-1 protein in the absence of a test compound, comparing the level of phosphorylated Elk-1 protein in the presence of the test compound with the level of phosphorylated Elk-1 protein in the absence of the test compound, and identifying the test compound as a modulator of phosphorylated Elk-1 protein when the level of
  • phosphorylated Elk-1 protein in the presence of the test compound is different than the level of phosphorylated Elk-1 protein in the absence of the test compound.
  • the phosphorylated Elk-1 protein is Elk-1 pT417. In one embodiment, the phosphorylated Elk-1 protein is Elk-1 pT363. In one embodiment, the phosphorylated Elk-1 protein is Elk-1 pT363, pT417.
  • the test compound when the level of phosphorylated Elk-1 protein is higher in the presence of the test compound, the test compound is identified as an Elk-1 phosphorylation activator. In one embodiment, when the level of phosphorylated Elk-1 protein is lower in the presence of the test compound, the test compound is identified as an Elk-1 phosphorylation inhibitor.
  • the level of phosphorylated Elk-1 protein is determined by measuring the level of phosphorylated Elk-1 protein. In one embodiment, the level of phosphorylated Elk-1 protein is determined by measuring an activity of phosphorylated Elk-1 protein.
  • the test compound is at least one of a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, a nucleic acid, an antisense nucleic acid, an aptamer, a ribozyme, or a small molecule chemical compound.
  • the present invention provides an antibody that specifically binds to phosphorylated Elk- 1 protein. In one embodiment, the antibody specifically binds to Elk-1 pT417. In one embodiment, the antibody specifically binds to Elk-1 pT363.
  • the antibody is at least one of a polyclonal antibody, a monoclonal antibody, an intracellular antibodies, an antibody fragment, a single chain antibody (scFv), a heavy chain antibody, a synthetic antibody, a chimeric antibody, or humanized antibody.
  • the antibody is isolated from phage display.
  • Figure 1 is a set of images depicting the results of experiments detecting Elk-1 non-phosphoT417 and Elk-1 pT417 in normal tissue using immunohistochemistry.
  • a tissue microarray composed of different normal and cancer tissues was processed for Elk-1 non-phosphoT417 or Elk-1 pT417 immunohistochemistry and aligned to the corresponding H&E slide, depicted in Figure II through Figure 1L.
  • Elk-1 pT17 ( Figure 1A) and non-phosphoT417 ( Figure IE) are localized to distinct epithelial cell nuclei in skin.
  • Elk-1 pT417 is localized to epithelial cell nuclei in breast (Figure IB), stomach (Figure 1C), and colon (Figure ID), while non- phosphoT417 is localized to the cytoplasm in these cells ( Figure IE through Figure 1H; respectively).
  • Samples were imaged at 200x magnification, while insets represent 400x magnification of the corresponding boxed area (boxes) in the larger image.
  • Specificity of the antibodies is shown in Figure 1M.
  • the Elk-1 pT417 and non-phosphoT417 antibodies recognize a peptide of approximately 48 kDa (asterisks), which is the published size of the Elk-1 protein, in normal human colon lysates. Pre-incubation of each antibody with the corresponding blocking peptide prevented the antibody from binding to this band ($). All samples shown were run on the same gel and blotted onto the same membrane.
  • Figure 2 comprising Figure 2A through Figure 21, is a set of images depicting the results of experiments detecting Elk-1 non-phosphoT417 and Elk-1 pT417 in cancer tissues using immunohistochemistry.
  • a tissue microarray composed of different normal and cancer tissues was stained for Elk-1 non-phosphoT417 or Elk-1 pT417 and aligned to the corresponding H&E slide, depicted in Figure 2G through Figure 21.
  • Elk-1 pT417 is found in the nucleus of an adenocarcinoma of the rectum ( Figure 2A), an infiltrating ductal carcinoma of the breast ( Figure 2B), and an adenocarcinoma of the stomach ( Figure 2C) while non-phosphoT417 is localized to the cytoplasm ( Figure 2D through Figure 2F; respectively). Samples were imaged at 200x magnification.
  • Figure 3 comprising Figure 3 A through Figure 31, is a set of images depicting the results of experiments demonstrating that Elk-1 pT417 levels are higher in adenocarcinoma compared to normal colon.
  • Archival tissue including adenocarcinoma of the colon and corresponding normal tissue, was processed for both Elk-1 non- phosphoT417 and Elk-1 pT417 immunohistochemistry and aligned to corresponding H&E images ( Figure 3G and Figure 3H).
  • Figure 4 is a set of images depicting the results of experiments demonstrating that Elk-1 pT417 levels correlate with differentiation grade of colonic adenocarcinoma.
  • Figure 4E is a graph illustrating the quantification of Elk-1 pT417 positive cells in the examined tissue.
  • Elk-1 pT417 immunofluorescence was quantified by calculating the number of pT417-positive nuclei as the percentage of DAPI -positive cells and expressed as means and SEMs.
  • a one-way analysis of variance revealed a significant main effect of differentiation grade on percentage of Elk-1 pT417 positive cells (p ⁇ 0.001).
  • Figure 5 is a set of images depicting the detection of Elk-1 pT417 in various forms of brain cancer, including oligodendroglioma, glioblastoma, and ganglioglioma.
  • Figure 6 is a set of images depicting the detection of phosphorylated Elk-1 in ganglioglioma and glioblastoma.
  • Figure 6 A is an image illustrating the detection of Elk-1 pT363 in ganglioglioma.
  • Figure 6B is an image illustrating the detection of Elk-1 pT363 and Elk-1 pT417 in
  • Figure 7 is a set of images depicting the detection of Elk-1 pT417 (top) and Elk-1 pT363 (bottom) in ganglioglioma.
  • Figure 8 is a set of images depicting the detection of Elk-1 pT417 in a neuroendocrine carcinoma of the lung.
  • Figure 9, comprising Figure 9A and Figure 9B, is a set of images depicting the detection of Elk-1 pT417 in different cancers.
  • Figure 9 A is an image illustrating the detection of Elk-1 pT417 in a ductal carcinoma of the breast.
  • Figure 9B is an image illustrating the detection of Elk-1 pT417 in an adenocarcinoma of the stomach.
  • Figure 10 is a set of images depicting the detection of Elk-1 pT417 in an adenocarcinoma of the stomach.
  • Figure 11 is a set of images depicting the detection of Elk-1 pT417 in an invasive ductal carcinoma of the breast.
  • Figure 12 is a set of images depicting the detection of Elk-1 pT363 in a glioblastoma.
  • Figure 13 is a set of images depicting the detection of Elk-1 pT363 in a high grade glial tumor, glioblastoma.
  • Figure 14 is an image depicting the detection of Elk-1 pT417 in a high grade glial tumor, glioblastoma.
  • Figure 15 is a set of images depicting the detection of Elk-1 pT363 in a ganglioglioma found in the frontal lobe of the brain.
  • Figure 16 is a set of images depicting the detection of Elk-1 pT417 in a ganglioglioma found in the frontal lobe of the brain.
  • Figure 17 is a set of images depicting the detection of Elk-1 pT363 in a neuroendocrine carcinoma found in the left lower lobe of the lung.
  • the present invention relates generally to compositions and methods for detecting cancer in a biological sample and diagnosing cancer, wherein a phosphorylated form of Elk-1 is detected in a biological sample of a subject.
  • the cancer is classified based on the level of phosphorylated Elk-1. Such classification is useful in selecting a treatment for cancer and in evaluating the effectiveness of a cancer treatment.
  • the invention is diagnostic method assessing the level of Elk-1 phosphorylation in a biological sample that is suspected to be cancerous or precancerous.
  • the level of Elk-1 T417-phosphorylation is measured in the sample.
  • the level of Elk- 1 T363 -phosphorylation is measured in the sample.
  • a biological sample of non-cancerous tissue is used as a negative comparator control.
  • a biological sample from known cancerous tissue is used as a positive comparator control.
  • the comparator control includes a historical norm or a demographic sampling of a population.
  • the biological sample is collected and the phosphorylated variant of Elk- 1 is compared to the total Elk-1 protein
  • the level of Elk-1 pT417 is compared with the total level of Elk-1 in a cell.
  • the level of Elk-1 pT363 is compared with the total level of Elk-1 in a cell.
  • the ratio of phosphorylated Elk-1 protein to total Elk-1 protein is useful for determining whether the biological sample contains cancer.
  • a higher level of phosphorylated Elk-1 relative to a comparator control is indicative of cancer.
  • the level of phosphorylated Elk-1 is indicative of the stage or grade of the cancer present in the biological sample.
  • the method comprises administering a treatment to a subject based upon the detected level of phosphorylated Elk-1 in a sample obtained from the subject.
  • the present invention also relates to compositions and methods of treating cancers related to elevated levels of phosphorylated forms of Elk-1.
  • the present invention provides methods of treating cancer in a subject by classifying cancer cells based on the levels Elk-1 pT417 or pT363 and administering an effective treatment to the subject.
  • the method comprises administering an effective dose of one or more inhibitors of the invention based on the classification.
  • an inhibitor of the invention includes a molecule that decreases the level of expression or activity of Elk-1 pT417.
  • an inhibitor of the invention includes a molecule that decreases the level of expression or activity of Elk-1 pT363.
  • amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
  • an element means one element or more than one element.
  • adenoma thus relates to a benign epithelial neoplasm. Adenomas are usually well circumscribed, they can be flat or polyploid and the neoplastic cells do not infiltrate or invade adjacent tissue.
  • adenoma is understood as equivalent to "non-progressed adenoma.”
  • progressed adenoma refers to an adenoma that harbors a focus of a cancer.
  • Colorectal adenomas are common in the elderly population, but only a small proportion of these pre-malignant tumors (estimated approximately 5%) progresses to malignant tumors (i.e., colorectal adenocarcinoma).
  • adenocarcinoma relates to a malignant neoplasm of epithelial cells.
  • adenocarcinoma is a cancer that originates in glandular tissue. This tissue is part of a more general type of tissue known as epithelial tissue.
  • Epithelial tissue includes skin, glands and a variety of other tissue lining or surrounding the cavities and organs of the body. Embryo logically, the epithelium is derived from ectoderm, endoderm and mesoderm. In order to be classified as
  • adenocarcinoma the cells do not necessarily need to be part of a gland, as long as they have secretory properties.
  • adenocarcinomas are also often referred to as
  • Glandular cancer or "glandular carcinoma.”
  • An adenocarcinoma can occur in some higher mammals, including humans. Highly differentiated adenocarcinomas tend to resemble the glandular tissue that they are derived from, while poorly differentiated may not.
  • a pathologist could verify whether a tumor is an adenocarcinoma or some other type of cancer by staining the cells from a biopsy. Such an independent examination may be used as additional means of diagnosis or diagnostic verification once a diagnosis has been obtained according to the method(s) of the present invention.
  • antibody refers to an immunoglobulin molecule that is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • the antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab and F(ab) 2 , as well as single chain antibodies (scFv), camelid antibodies and humanized antibodies (Harlow et al, 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al, 1988, Science 242:423-426).
  • the term “heavy chain antibody” or “heavy chain antibodies” comprises immunoglobulin molecules derived from camelid species, either by immunization with an antigen and subsequent isolation of sera, or by the cloning and expression of nucleic acid sequences encoding such antibodies.
  • the term “heavy chain antibody” or “heavy chain antibodies” further encompasses immunoglobulin molecules isolated from an animal with heavy chain disease, or prepared by the cloning and expression of V H (variable heavy chain immunoglobulin) genes from an animal.
  • synthetic antibody an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • aptamer refers to a small molecule that can bind specifically to another molecule. Aptamers are typically either polynucleotide- or peptide-based molecules.
  • a polynucleotide aptamer is a DNA or RNA molecule that adopts a highly specific three-dimensional conformation designed to have appropriate binding affinities and specificities towards specific target molecules, such as peptides, proteins, drugs, vitamins, among other organic and inorganic molecules.
  • polynucleotide aptamers can be selected from a vast population of random sequences through the use of systematic evolution of ligands by exponential enrichment.
  • a peptide aptamer is typically a loop of about 10 to about 20 amino acids attached to a protein scaffold that binds to specific ligands.
  • Peptide aptamers may be identified and isolated from combinatorial libraries, using methods such as the yeast two-hybrid system.
  • biomarker and “marker” are used herein interchangeably. They refer to a substance that is a distinctive indicator of a biological process, biological event and/or pathologic condition.
  • a sample may be of any biological tissue or fluid from which biomarkers of the present invention may be assayed. Examples of such samples include but are not limited to blood, lymph, urine, gynecological fluids, biopsies, amniotic fluid and smears. Samples that are liquid in nature are referred to herein as "bodily fluids.” Body samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to aspirate bodily fluids. Methods for collecting various body samples are well known in the art.
  • a sample will be a "clinical sample,” i.e., a sample derived from a patient.
  • samples include, but are not limited to, bodily fluids which may or may not contain cells, e.g., blood (e.g., whole blood, serum or plasma), urine, saliva, tissue or fine needle biopsy samples, and archival samples with known diagnosis, treatment and/or outcome history.
  • Biological or body samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • the sample also encompasses any material derived by processing a biological or body sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample, proteins or nucleic acid molecules extracted from the sample.
  • Processing of a biological or body sample may involve one or more of: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.
  • cancer as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
  • colon as used herein, relates to the colon and/or the rectum, i.e. the complete large intestine.
  • doctor control relates to a level of expression or activity which may be determined at the same time as the test sample by using a sample previously collected and stored from a subject whose disease state, e.g. cancerous, non-cancerous, is/are known.
  • “Differentially increased expression” or “up regulation” refers to biomarker product levels which are at least 10% or more, for example, 20%, 30%>, 40%>, or 50%, 60%, 70%, 80%, 90% higher or more, and/or 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold higher or more, as compared with a control.
  • “Differentially decreased expression” or “down regulation” refers to biomarker product levels which are at least 10%> or more, for example, 20%>, 30%>, 40%>, or 50%, 60%, 70%, 80%, 90% lower or less, and/or 0.9 fold, 0.8 fold, 0.6 fold, 0.4 fold, 0.2 fold, 0.1 fold or less, as compared with a control.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • a disease or disorder is "alleviated” if the severity of a sign or symptom of the disease, or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.
  • Signal transduction is any process by which a cell converts one signal or stimulus into another, most often involving ordered sequences of biochemical reactions carried out within the cell. The number of proteins and molecules participating in these events increases as the process emanates from the initial stimulus resulting in a “signal cascade.”
  • downstream effector refers to a protein or molecule acted upon during a signaling cascade, which in term acts upon another protein or molecule. The term “downstream” indicates the direction of the signaling cascade.
  • an effective amount and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • endogenous refers to any material from or produced inside the organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside the organism, cell, tissue or system.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein,
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and/or compound of the invention in a kit.
  • the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.
  • Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
  • the "level" of one or more biomarkers means the absolute or relative amount or concentration of the biomarker in the sample.
  • detection means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's clinical parameters.
  • abnormal and “healthy” are used herein interchangeably. They include an individual or group of individuals who have not undergone kidney
  • kidney injury transplantation and who have not shown any signs or symptoms of kidney injury, damage or dysfunction.
  • normal is also used herein to qualify a sample (e.g., a blood sample) obtained from a healthy individual.
  • non-cancerous relates in the context of the present invention to a condition in which neither benign nor malign proliferation can be detected. Suitable means for said detection are known in the art. Preferably, the term “non-cancerous” excludes a benign proliferation state as present in adenomas.
  • Naturally-occurring refers to the fact that the object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man is a naturally-occurring sequence.
  • nucleic acid is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases
  • nucleic acid typically refers to large polynucleotides.
  • the left-hand end of a single-stranded polynucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.
  • the direction of 5 ' to 3 ' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences.”
  • precancerous refers to cells or tissues that have characteristics relating to changes that may lead to malignancy or cancer, such as mutations controlling cell growth and proliferation.
  • Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • protein typically refers to large polypeptides.
  • peptide typically refers to short polypeptides.
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • a “polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid.
  • a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
  • the following abbreviations for the commonly occurring nucleic acid bases are used. "A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • transdominant negative mutant gene refers to a gene encoding a protein product that prevents other copies of the same gene or gene product, which have not been mutated (i.e., which have the wild-type sequence) from functioning properly (e.g., by inhibiting wild type protein function).
  • the product of a transdominant negative mutant gene is referred to herein as "dominant negative” or "DN” (e.g., a dominant negative protein, or a DN protein).
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear
  • vector includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • the term "subject” refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like) that can undergo kidney transplantation, but may or may not have undergone kidney transplantation.
  • the subject is a human being.
  • the subject is often referred to as an "individual” or a "patient.”
  • kidney transplant patient refers to an individual that has undergone kidney transplantation. The terms "individual” and "patient” do not denote a particular age.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description
  • the invention provides compositions and methods for assessing cancer by measuring a protein with a different phosphorylation state in cancerous cells compared with non-cancerous cells.
  • the cancer is colorectal
  • the protein marker for detecting cancer includes Elk-1 (Ets-like protein- 1) and is measured for its phosphorylated state to detect the presence or absence of cancer.
  • the phosphorylated form of Elk-1 is Elk-1 pT417, where the threonine residue at amino acid 417 is phosphorylated.
  • the phosphorylated form of Elk-1 is Elk-1 pT363, where the threonine residue at amino acid 363 is phosphorylated.
  • the phosphorylated form of Elk-1 is Elk-1 pT363, pT417, where the threonine residue at amino acid 417 and the threonine residue at position 363 are phosphorylated.
  • Elk-1 pT417, Elk-1 pT363, and Elk-1 pT363, pT417 refer to Elk-1 protein phosphorylated at residues analogous to T417 and T363, dependent upon species specific Elk-1 amino acid sequences.
  • the invention also provides compositions and methods for assessing the differentiation state of a cancer, by measuring a protein with a different phosphorylation state in one differentiation state of cancerous cells compared with cancerous cells in another differentiation state.
  • the cancer is colorectal
  • the protein marker for assessing the differentiation state of cancer includes Elk-1 (Ets-like protein- 1) and is measured for its phosphorylated state to detect the differentiation state of cancer.
  • the phosphorylated form of Elk- 1 is Elk-1 pT417.
  • the phosphorylated form of Elk-1 is Elk-1 pT363.
  • the phosphorylated form of Elk-1 is Elk-1 pT363, pT417.
  • the cancer detected by the methods of the invention is colorectal adenocarcinoma.
  • the present invention is partly based upon the finding that Elk-1 pT417 is increased in colorectal adenocarcinoma cells compared to normal colonic tissue. Therefore, in one embodiment, the present invention provides compositions and methods of detecting colorectal adenocarcinoma in a subject comprising measuring an increased level of Elk-1 pT417 in a sample from the subject compared to the level of Elk- 1 pT417 found in non-colorectal adenocarcinoma cells.
  • the present invention provides compositions and methods of detecting cancer in a subject comprising measuring a decreased level of Elk-1 pT417 in a sample from the subject compared to the level of Elk-1 pT417 found in noncancerous cells.
  • the present invention is partly based upon the finding that Elk-1 pT417 is decreased in carcinomas of certain tissue, compared to normal tissue. For example, it is shown elsewhere herein that Elk-1 pT417 is decreased in cancerous tissue of the breast (infiltrating duct carcinoma) and stomach (adenocarcinoma) compared to normal breast and stomach tissue, respectively.
  • Elk-1 (Ets-like protein- 1) is a transcription factor that is a member of the ternary complex factor (TCF) subfamily of Ets domain proteins. Elk-1 is involved in regulating cell proliferation, differentiation, and development through mitogen-activated protein kinase (MAPK) pathways. TCFs are able to form a ternary complex with the serum response factor (SRF) and the serum-response element (SRE), and are involved in SRF-driven gene expression. As a subgroup of TCFs, Ets protein family members, Elk-1, Sapl, and Sap2, possess an Ets domain and a winged helix-loop-helix (HLH) DNA binding domain recognizing specific DNA sequences.
  • TCF ternary complex factor
  • SRF serum response factor
  • SRE serum-response element
  • Elk-1 N- terminal Ets-DNA binding domain of Elk-1 is important for DNA recognition, a B domain containing 20 amino acids mediates protein-protein interaction, and C-terminal of Elk-1 possesses phosphorylation sites for ER , JNK, and p38 MAPKs.
  • Elk-1 is mostly activated by ERK proteins, and binds to the SRE modulating immediate early gene expression in signaling pathway. Accordingly, phosphorylated Elk-1, such as Elk-1 pT417 or Elk-1 pT363, can be used as a marker for diagnosis of cancer, as well as a target of a treatment for the cancer.
  • the present invention is based on the discovery that Elk-1 pT417 is present in epithelial cell nuclei of various normal and cancer tissues and that the number of pT417 positive cells correlates with differentiation grade of colonic adenocarcinomas.
  • the present invention is also based on the discovery that Elk-1 pT363 is present in epithelial cell nuclei of various normal and cancer tissues.
  • the invention provides a protein marker for detecting cancer present in a biological sample.
  • the invention further provides a protein marker for determining the differentiation grade of cancer present in a biological sample.
  • the cancer is colonic adenocarcinoma.
  • the protein marker is Elk-1 pT417. In other embodiments, the protein marker is Elk-1 pT363.
  • the present invention is not limited to detecting colonic adenocarcinoma.
  • Exemplary cancers that may be detected by the present methods include, but are not limited to, brain cancer, spinal cord cancer, breast cancer, stomach cancer, and carcinomas, such as transitional cell carcinoma, colonic adenocarcinoma,
  • adenocarcinoma of the rectum gastric carcinoma, colorectal carcinoma, infiltrating duct carcinoma, neuroendocrine carcinoma, hepatocellular carcinoma, squamous cell carcinoma, adenocarcinoma of the stomach, and small bowel malignant stromal tumor.
  • Exemplary cancers of the brain and spinal cord that may be detected by way of the present invention include, but are not limited to, oligodendroglioma, glioblastoma, and ganglioglioma.
  • the detection and comparison of the levels of phosphorylated Elk-1, in a biological sample can be both diagnostic and prognostic of cancer.
  • an elevated level of phosphorylated Elk-1 protein in a biological sample is indicative of cancer or of a greater risk or predisposition of the subject to develop cancer.
  • a decreased level of phosphorylated Elk-1 protein, in a biological sample is indicative of cancer or a greater risk or predisposition of the subject to develop cancer.
  • the level of phosphorylated Elk- l is assessed in a cell population. In another embodiment, the level of phosphorylated Elk-1 is assessed in a single cell. In another embodiment, the level of phosphorylated Elk-1 is assessed in a subcellular compartment, including, for example, the nucleus and cytoplasm. Therefore, in various embodiments of the invention, the detection and measurement of the level of expression of phosphorylated Elk-1 in a biological sample is used in a diagnostic assay, a prognostic assay, to monitor a clinical trial and in a screening assay.
  • the invention provides diagnostic and prognostic assays for detecting phosphorylated Elk-1. Also provided are diagnostic and prognostic assays for detecting interactions between phosphorylated Elk-1, and Elk-1 target molecules.
  • the invention relates to a method of detecting Elk-1, in a biological sample, or to determine the stage or severity of the cancer in the subject, or to determine the predisposition of a subject to develop cancer, or to monitor the effect of the therapy administered to a subject with cancer.
  • the method comprises: a) quantifying the level of phosphorylated Elk-1, in a biological sample from a subject, and; b) comparing the level to that of a comparator control ; wherein an increase in the level relative to that of the comparator control is indicative of cancer, i.e., it is an indication that the subject is suffering from cancer or has a predisposition to develop cancer.
  • the level of phosphorylated Elk-1, in a biological sample as compared to that of a comparator control also indicates the stage or severity of cancer in a subject, or can be useful for monitoring the effect of the therapy administered to a subject with cancer.
  • the method comprises administering a treatment to a subject based upon the detected level of phosphorylated Elk-1 in a sample obtained from the subject.
  • the biological sample is a colon tissue sample, which can be obtained by conventional methods, e.g., by biopsy, by using methods well known to those of ordinary skill in the related medical arts.
  • Methods for obtaining the biological sample from the biopsy include gross apportioning of a mass, or
  • Samples can be obtained from subjects previously diagnosed or not with cancer, or from subjects who are receiving or have previously received anti-cancer treatment.
  • the sample size required for analysis may range from 1, 10, 50, 100, 200, 300, 500, 1,000, 5,000, 10,000, to 50,000 or more cells.
  • the appropriate sample size may be determined based on the cellular composition and condition of the biopsy. The standard preparative steps for the determination are well known to one of ordinary skill in the art.
  • the method of the invention comprises (i) contacting the protein extracted from the sample with a composition comprising one or more antibodies that specifically bind to one or more epitopes of phosphorylated Elk-1 and (ii) quantifying the complexes of Elk-1 -antibodies that are formed.
  • immunological assays immunological assays
  • the amount of phosphorylated Elk-1 protein can be quantified by means of specific antibodies to phosphorylated Elk-1, i.e., antibodies which recognize (or bind) phosphorylated Elk-1, such as, phospho-specific antibodies, antibodies which recognize any epitope on phosphorylated Elk-1 protein, for example, antibodies that recognize the binding between the phosphorylation site (e.g., a serine, threonine or tyrosine residue) and a phosphorus-containing moiety, or any epitope, for example, a conformational epitope, generated in the phosphorylated Elk-1 protein as a result of the phosphorylation of Elk-1.
  • antibodies which recognize (or bind) phosphorylated Elk-1 such as, phospho-specific antibodies, antibodies which recognize any epitope on phosphorylated Elk-1 protein, for example, antibodies that recognize the binding between the phosphorylation site (e.g., a serine, threonine or tyrosine residue) and
  • the antibodies can be in the form of monoclonal antibodies, polyclonal antibodies, intact or recombinant fragments of antibodies, combibodies and Fab or scFv of antibody fragments. These antibodies can be human, humanized or non-human in origin.
  • the antibodies used in these assays can be labeled or unlabeled; the unlabeled antibodies can be used in agglutination assays; the labeled antibodies can be used in a wide range of assays.
  • Antibody labels include
  • radionucleotides include but are not limited to Western-blot or Western transfer, ELISA (Enzyme-linked immunosorbent assay), RIA
  • Radioimmunoassay Radioimmunoassay
  • Competitive EIA Competitive enzyme immunoassay
  • DAS- ELISA Double antibody sandwich-ELISA
  • immunocyto-chemical and immunohistochemical techniques techniques based on biochips or protein microarrays that use specific antibodies, and colloidal precipitation-based assays in formats such as dipsticks.
  • Other techniques to detect and quantify the phosphorylation of Elk- 1 protein are affinity chromatography, ligand binding assays and lectin binding assays.
  • the final step of the method of the invention involves comparing the level of phosphorylated Elk-1 protein quantified in a biological sample obtained from the subject to the level of phosphorylated Elk-1 protein in a comparator control sample (i.e., positive control, negative control, historical norm, baseline level or reference value).
  • the level of phosphorylated Elk-1 protein in comparator control samples can be determined by measuring the level of phosphorylated Elk-1 protein in a colon tissue sample from cancer-free subjects (i.e., negative control subjects with respect to cancer).
  • An increase in the level of phosphorylation of Elk-1 protein in a biological sample from the subject under study relative to the level of Elk-1 protein in a comparator control sample is indicative of cancer, i.e., it is an indication that said subject is suffering from cancer or has a predisposition to develop cancer.
  • the level of phosphorylated Elk-1 protein in a biological sample as compared to that of a comparator control sample can also indicate the stage or severity of cancer in a subject, or can be useful for monitoring the effect of the therapy administered to a subject with cancer.
  • the cancer is colonic or colorectal carcinoma.
  • the method comprises comparing the level of phosphorylated Elk-1 protein quantified in a biological sample from the subject under study to the level of phosphorylated Elk-1 protein in a tissue control sample (i.e., baseline level or reference value).
  • the levels of phosphorylated Elk-1 protein in tissue control samples can be determined by measuring levels of phosphorylated Elk-1 protein in a biological sample from cancer free subjects (i.e., control subjects with respect to cancer).
  • a decrease in the level of phosphorylation of Elk-1 protein in a biological sample from the subject under study relative to the level of Elk-1 protein in a tissue control sample is indicative of cancer, i.e., it is an indication that said subject is suffering from cancer or has a predisposition to develop cancer.
  • the level of phosphorylated Elk-1 protein in a biological sample as compared to that of a tissue control sample can also indicate the stage or severity of cancer in a subject, or can be useful for monitoring the effect of the therapy administered to a subject with said condition.
  • the method of the invention based on the measurement of the level (concentration) of phosphorylated Elk-1 protein in colon biological samples is highly sensitive and specific.
  • the invention refers to the use of a phosphorylated Elk-1 protein or a peptide sequence derived from a phosphorylated Elk-1 protein to detect or diagnose cancer in a subject, or to determine the stage or severity of the condition in a subject, or to determine the predisposition of a subject to develop cancer, or to monitor the effect of the therapy administered to a subject with the cancer.
  • agent of the invention characterized by its ability to detect Elk-1 and/or
  • Non- limiting examples of an agent capable to detect Elk-1 isoforms include an antibody, an aptamer, a molecular probe, peptide, peptidomimetic, small molecule, and conjugates thereof.
  • agent characterized by its ability to modulate the expression and/or activity and/or phosphorylation state of the Elk- 1 protein.
  • the agent which can be identified and evaluated according to the present invention, can be any agent selected from the following: a) a compound which modulates phosphorylation of Elk-1, b) a cytotoxic agent, such as a toxin, a molecule with radioactive atoms or chemotherapeutic agents, including but not limited to small organic and inorganic molecules, peptides, phosphopeptides, antisense molecules, ribozymes, triple-helix molecules, double stranded RNA etc., which modulates the expression and/or the activity and/or the phosphorylation of the Elk-1 protein, c) an antibody, or combination of antibodies, specific for one or more epitopes on a phosphorylated Elk-1 protein, preferably a human or humanized monoclonal antibody, including fragments thereof, single-chain antibodies and anti-idiotype antibodies
  • the agent of the invention inhibits the expression and/or activity and/or phosphorylation state of the Elk-1 protein. In another embodiment, the agent of the invention promotes or enhances the expression and/or activity and/or phosphorylation of the Elk-1 protein.
  • the present invention provides an agent having a therapeutic benefit, where the agent is targeted to Elk-1 or phosphorylated Elk-1.
  • the agent comprises a therapeutic domain and a targeting domain that binds to phosphorylated Elk.
  • the targeting domain is an antibody, or fragment thereof, that specifically binds to phosphorylated Elk-1.
  • the targeting domain is an aptamer.
  • the therapeutic domain is a composition able to induce the death of a tumor cell.
  • the therapeutic domain is a pro-apoptotic domain.
  • the agent of the invention is an antibody that specifically binds to phosphorylated Elk-1 protein, sometimes referred herein as an antibody of the invention.
  • the antibody specifically binds to Elk-1 pT417.
  • the antibody specifically binds to Elk-1 pT363.
  • Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.
  • Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals.
  • the choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost.
  • Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species.
  • Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity and are cleared rapidly from the blood.
  • the antibodies of the present invention may be polyclonal antibodies.
  • Such polyclonal antibodies can be produced in a mammal, for example, following one or more injections of an immunizing agent, and preferably, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected into the mammal by a series of administrations (e.g., subcutaneous injection, intraperitoneal injection, etc.).
  • the immunizing agent may include phosphorylated Elk-1 or a fragment thereof or a fusion protein thereof.
  • a crude protein preparation which has been enriched for a phosphorylated Elk-1 protein or a fragment thereof can be used to generate antibodies.
  • Such proteins, fragments or preparations are introduced into the non-human mammal in the presence of an appropriate adjuvant. If the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies are purified by immunoaffinity
  • the antibodies may be monoclonal antibodies.
  • Monoclonal antibodies may be produced by hybridomas, wherein a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent, e.g., Kohler and Milstein, 1975, Nature 256:495.
  • the immunizing agent will typically include the phosphorylated Elk-1 protein or a fragment thereof or a fusion protein thereof and optionally a carrier.
  • lymphocytes may be immunized in vitro.
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired, or peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired.
  • the lymphocytes are fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to produce a hybridoma cell.
  • a suitable fusing agent such as polyethylene glycol
  • immortalized cell lines are transformed mammalian cells, for example, myeloma cells of rat, mouse, bovine or human origin.
  • the hybridoma cells are cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells.
  • the culture medium (supernatant) in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against phosphorylated Elk-1 protein by conventional techniques, such as by immunoprecipitation or by an in vitro binding assay, such as RIA or ELISA.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be isolated from the phosphorylated Elk-1- specific hybridoma cells and sequenced, e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies.
  • the DNA may be inserted into an expression vector, which is then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for the murine heavy and light chain constant domains for the homologous human sequences, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • the non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies may also be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, in vitro methods are suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine technniques known in the art.
  • Antibodies and antibody fragments characteristic of hybridomas of the invention can also be produced by recombinant means by extracting messenger RNA, constructing a cDNA library, and selecting clones which encode segments of the antibody molecule.
  • the antibodies of the invention may further comprise humanized antibodies or human antibodies.
  • humanized antibody refers to humanized forms of non-human (e.g., murine) antibodies that are chimeric antibodies,
  • Humanized antibodies include human immunoglobulins in which residues from a complementary determining region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non-human species such as mouse, rat or rabbit having the desired binding specificity, affinity and capacity.
  • CDR complementary determining region
  • the humanized antibody will comprise substantially all of at least one, and generally 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 consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Heteroconjugate antibodies which comprise two covalently joined antibodies, are also within the scope of the present invention.
  • Heteroconjugate antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be prepared using a disulfide exchange reaction or by forming a thioether bond.
  • the antibodies of the invention are preferably specific for the phosphorylated Elk-1 protein and so, do not bind peptides derived from other proteins with high affinity.
  • the antibodies may be used as functional modulators, most commonly as antagonists.
  • antibody modulators of the invention are derived from monoclonal antibodies specific for phosphorylated Elk-1 protein.
  • Monoclonal antibodies capable of blocking or neutralizing phosphorylated Elk-1 protein are generally selected by their ability to inhibit a phosphorylated Elk-1 protein biological activity.
  • monoclonal antibodies, Fv fragments, Fab fragments, or other binding compositions derived from monoclonal antibodies of the invention have a high affinity to phosphorylated Elk-1 proteins.
  • the affinity of monoclonal antibodies and related molecules to phosphorylated Elk-1 proteins may be measured by conventional techniques.
  • the antibodies of the present invention include those cloned from a phage antibody library.
  • a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same.
  • Amplified cDNA is cloned into Ml 3 expression vectors creating a library of phage which express human Fab or scFv fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab or scFv immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.
  • the antibody of the invention comprises an anti- Elk- 1, anti-Elk- l-pT363, or anti-Elk- l-pT417 antibody isolated using phage display. In other various embodiments, the antibody of the invention comprises anti- Elk-1 scFv, anti-Elk- l-pT363 scFv, or anti-Elk- l-pT417 scFv mAbs isolated using phage display.
  • Phage display libraries allow for the in vitro identification of human antibody products directed against molecular targets.
  • the methods of the present invention provide that phage display libraries enable the in vitro identification of human antibody products directed against an isolated Elk-1 protein or fragment thereof.
  • Antibody phage display of the present invention provides the linkage between genotype and phenotype.
  • selection of phage clones of the present invention is based on binding affinity.
  • selection of phage clones of the present invention is based on binding specificity.
  • selection of phage clones of the present invention is based on functional activity of the displayed antibody (phenotype).
  • selection of phage clones of the present invention is based on binding affinity, specificity, and functional activity of the displayed antibody (phenotype).
  • Each phage carries the DNA for the antibody it displays on its surface, the phenotype is directly linked to the antibody genotype (cDNA sequence).
  • the present invention provides a high affinity human antibody which is specific for phosphorylated Elk-1.
  • the present invention provides that light chain shuffling while retaining the heavy chain variable region using phage display technology is able to modify the ultrafme specificity of the antigen-binding construct.
  • the present invention provides that heavy chain shuffling while retaining the light chain variable region using phage display technology is able to modify the ultrafme specificity of the antigen-binding construct.
  • the present invention provides that heavy chain promiscuity is limited in in vivo developed antibodies.
  • the present invention provides that light chain promiscuity is limited in in vivo developed antibodies. In another embodiment, the present invention allows such to form antigen- binding structures following random assortment of heavy and light chain variable regions. In another embodiment, the present invention provides that phage display technology selects against clones which are restricted with respect to light chain usage. In another embodiment, the present invention provides that phage display technology selects against clones which are restricted with respect to heavy chain usage. In another embodiment, the present invention provides that phage display technology represents a population which is restricted in this respect.
  • the antibodies of the present invention are useful for detecting phosphorylated Elk-1 proteins. Such detection methods are advantageously applied to diagnosis and/or prognosis of cancer.
  • the antibodies of the present invention are useful for modulating the expression and/or activity and/or phosphorylation state of the Elk-1 protein.
  • the antibodies of the invention can be used to isolate phosphorylated Elk-1 proteins by standard techniques, such as affinity chromatography or immunoprecipitation.
  • an antibody of the invention can facilitate the purification of natural phosphorylated Elk-1 proteins from cells and of recombinantly produced phosphorylated Elk-1 proteins expressed in host cells.
  • the antibodies of the invention can be used to isolate phosphorylated Elk-1 proteins in order to evaluate the level thereof in a specific tissue, for example, colon tissue.
  • antibodies of the invention can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a label group.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent of the invention, or a pharmaceutically acceptable salt, derivative or prodrug thereof together with a pharmaceutically acceptable carrier, adjuvant, or vehicle, for administration to a patient.
  • the agent of the invention is an antagonist of the invention.
  • the use of an agent of the invention in the manufacture of a pharmaceutical composition to prevent or treat cancer constitutes another aspect of the invention.
  • the agent of the present invention is an aptamer.
  • the agent is a protein aptamer.
  • the agent is a polynucleotidal aptamer.
  • the aptamer of the invention is used to detect phosphorylated Elk-1, including Elk-1 pT363 and Elk-1 pT417, in a biological sample.
  • the aptamer of the invention is used to modulate Elk-1 phosphorylation and/or activity.
  • an apatmer is a nucleic acid or oligonucleotide molecule that binds to a specific molecular target, such as phosphorylated Elk-1.
  • aptamers are obtained from an in vitro evolutionary process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which selects target-specific aptamer sequences from combinatorial libraries of single stranded oligonucleotide templates comprising randomized sequences.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • nucleotide components of an aptamer include modified or non-natural nucleotides, for example nucleotides that have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide is replaced by 2'-F or 2'- -NH 2 ), which in some instances, improves a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • individual aptamers having the same nucleotide sequence differ in their secondary structure.
  • the aptamers of the invention are conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • aptamers are specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross- linker. (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • a method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules is known to those of skill in the art and is described in U.S. Pat. No. 5,270,163.
  • the method known as SELEX (Selective Evolution of Ligands by Exponential Enrichment) involves selection from a mixture of candidate
  • oligonucleotides from a library comprising a large sequence variations (e.g. about 10 15 ) and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX method includes the steps of contacting the mixture with the desired target, partitioning unbound nucleic acids from those nucleic acids which have bound to the target molecule, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
  • the SELEX method is modified to encompass the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics.
  • modifications include chemical substitutions at the ribose and/or phosphate and/or base positions.
  • SELEX-identified nucleic acid ligands containing modified nucleotides are described, for example, in U.S. Pat. No. 5,660,985. These include oligonucleotides containing nucleotide derivatives chemically modified at the 2' position of ribose, 5 position of pyrimidines, and 8 position of purines.
  • 5,756,703 describes oligonucleotides containing various 2'- modified pyrimidines.
  • U.S. Pat. No. 5,580,737 describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'— NH.sub.2), 2'- fluoro (2'-F), and/or 2'-0-methyl (2'-OMe) substituents.
  • nucleic acids contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
  • the modifications are performed pre-SELEX . In other instances, the modifications are performed post-SELEX. Pre-SELEX process
  • nucleic acid ligands with both specificity for their SELEX target and improved in vivo stability.
  • Post-SELEX process modifications made to 2'-OH nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand.
  • aptamers are obtained from the screening of an RNA library.
  • a combinatorial RNA library is made by transcribing DNA templates or a DNA library. Each template consists of 108 bases with a 40-base randomized segment. This segment is flanked by two constant regions for primer annealing.
  • the 5' constant sequence includes a promoter for T7 RNA polymerase. The two restriction sites are EcoRI located in the 5' constant region and Hindlll located in the 3' constant region, respectively.
  • an aptamer is obtained from the screening of a ssDNA library (Chen et al, 2009, PLoS ONE, 4(12): e8142).
  • any biological sample known to comprise at least one of Elk- 1, Elk-1 pT363, and Elk-1 p417 is used as the protein target in SELEX.
  • the invention provides methods and compositions for blocking phosphorylation of Elk-1.
  • the invention provides an inhibitor of Elk-1 phosphorylation.
  • the invention comprises an inhibitor of phosphorylated Elk-1.
  • the inhibitor of the invention comprises an antibody, siRNA, a ribozyme, an antisense, an aptamer, a peptidomimetic, a small molecule, or any combination thereof.
  • inhibiting Elk-1 phosphorylation comprises blocking the activity of a serine/threonine kinase. Agents which are capable of decreasing or inhibiting phosphorylation activity are referred to as antagonists or inhibitors of that activity.
  • antagonist as used in the art, is generally taken to refer to a compound which binds to an enzyme and inhibits the activity of the enzyme.
  • serine/threonine kinase for example an upstream kinase, or the expression of modulators of the activity of the serine/threonine kinase.
  • the antagonist may bind to and compete for one or more sites on the relevant molecule, for example, Elk-1 or upstream kinase, preferably, a catalytic site or a binding site of the serine/threonine kinase.
  • the antagonist of the kinase preferably interferes with, or prevents, the binding of Elk-1 to the upsteam kinase. Preferably, it competes for an Elk-1 binding region of the kinase.
  • binding blocks the interaction between the molecule and another entity (for example, the interaction between Elk-1 and its upstream kinase).
  • the antagonist need not necessarily bind directly to a catalytic or binding site, and may bind for example to an adjacent site, for example, an adjacent site in the kinase polypeptide, or even another protein (for example, a protein which is complexed with the enzyme) or other entity on or in the cell, so long as its binding reduces the activity of the enzyme or molecule or molecules in question.
  • an antagonist may include a substrate of the enzyme, or a fragment of this which is capable of binding to the enzyme.
  • whole or fragments of a substrate generated natively or by peptide synthesis may be used to compete with the substrate for binding sites on the enzyme.
  • the antagonist may also include a peptide or other small molecule which is capable of interfering with the binding interaction.
  • Blocking the phosphorylation of Elk- 1 may also be achieved by reducing the level of expression of Elk- 1 and/or one or more serine/threonine kinase in the cell.
  • the cell may be treated with antisense compounds, for example
  • oligonucleotides having sequences specific to Elk-1 mR A or the kinase mR A having sequences specific to Elk-1 mR A or the kinase mR A.
  • the term "antagonist” includes but is not limited to agents such as an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty acid and a carbohydrate.
  • An agent may be used herein,
  • antagonists are also intended to include, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanized, a peptide hormone, a receptor, a signaling molecule or other protein; a nucleic acid, including, but not limited to, an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e.g.
  • a yeast artificial chromosome or a part thereof, R A, including mR A, tRNA, rR A or a ribozyme, or a peptide nucleic acid (PNA); a virus or viruslike particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptide (e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate.
  • R A including mR A, tRNA, rR A or a ribozyme, or a peptide nucleic acid (PNA); a virus or viruslike particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptid
  • Small molecules including inorganic and organic chemicals, which bind to and occupy the active site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented, are also included.
  • Examples of small molecules include but are not limited to small peptides or peptide-like molecules.
  • the invention provides methods and compositions for enhancing phosphorylation of Elk- 1 such as by increasing the activity of a
  • phosphorylation activity are referred to as agonists or activators of that activity.
  • agonist as used in the art, is generally taken to refer to a compound which binds to an enzyme and promotes the activity of the enzyme.
  • the term as used here, however, is intended to refer broadly to any agent which enhances or promotes the activity of a molecule, not necessarily by binding to it. Accordingly, as used generally, it includes agents which affect phosphorylation of Elk- 1, for example the expression of a serine/threonine kinase, for example an upstream kinase, or the expression of modulators of the activity of the serine/threonine kinase.
  • the agonist or activator may promote the binding of the relevant molecule, for example, Elk-1 , to an upstream kinase, thereby inducing the relevant molecule
  • the agonist or activator may bind to both Elk-1 and a serine/threonine kinase, thereby forming a complex comprising both Elk-1 and the serine/threonine kinase.
  • the agonist or activator may also influence the cellular localization of Elk-1, such that Elk-1 translocates to a region where kinase activity may be preferentially be located.
  • Enhancing the phosphorylation of Elk- 1 may also be achieved by increasing the level of expression of one or more serine/threonine kinase in the cell.
  • the cell may be treated with nucleic acid sequences encoding the one or more serine/threonine kinase.
  • the term "agonist” includes but is not limited to agents such as an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty acid and a carbohydrate.
  • An agent may be
  • agonist and “agent” are also intended to include, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanized, a peptide hormone, a receptor, a signaling molecule or other protein; a nucleic acid, including, but not limited to, an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e.g., a yeast artificial chromosome) or a part thereof, RNA, including mR A, tRNA, rRNA or a ribozyme, or a peptide
  • small molecules include but are not limited to small peptides or peptide-like molecules.
  • compositions containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients.
  • the therapeutic agents of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.
  • the pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.
  • the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative.
  • the active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.
  • the pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art.
  • pharmaceutically acceptable carriers such as phosphate buffered saline solutions pH 7.0-8.0.
  • the expression vectors, transduced cells, polynucleotides and polypeptides (active ingredients) of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the organism. They can be administered by any conventional means available for use in conjunction with
  • pharmaceuticals either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's
  • the active ingredients of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans.
  • a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans.
  • Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs that are described in U.S. Patent Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890.
  • Other adjuvants, which are useful include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate and
  • DDA dimethyldioctadecylammonium bromide
  • IL-12 IL-12
  • Other components may include a polyoxypropylene-polyoxyethylene block polymer
  • control release preparations can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate.
  • concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release.
  • the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.
  • the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in a mammal body to achieve a particular effect (see, e.g., Rosenfeld et al, 1991; Rosenfeld et al, 1991a; Jaffe et al, supra; Berkner, supra).
  • Rosenfeld et al, 1991; Rosenfeld et al, 1991a; Jaffe et al, supra; Berkner, supra One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be
  • administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
  • each dosage unit e.g., a teaspoonful, tablet, solution, or
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate.
  • the specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition in the particular host.
  • compositions can be further approximated through analogy to compounds known to exert the desired effect.
  • the invention also provides a method (also referred to herein as a
  • screening assay for identifying candidate agents (e.g., small molecules, nucleic acids, peptides, antibodies, peptidomimetics or other drugs) which have a modulatory effect on, for example, the expression level of Elk- 1, the phosphorylation of Elk- 1 or on the biological activity of Elk- 1.
  • candidate agents e.g., small molecules, nucleic acids, peptides, antibodies, peptidomimetics or other drugs
  • the invention relates to a method for identifying and/or evaluating the efficacy of a potentially therapeutic agent against cancer, which comprises: a) contacting a culture of cells with a test compound under the appropriate conditions and for the required period of time for them to interact; b) determining the level of phosphorylated Elk-1 protein, preferably Elk-1 pT417; c) comparing the level obtained in step b) to that of a control culture of cells lacking the test compound; and d) selecting a test compound which causes the level of step b) to be altered.
  • a compound is selected that makes the level of step b) decrease.
  • a compound is selected that makes the level of step b) increase.
  • the selected compounds can be used, for example, for further testing as a potential agent for the prophylactic and/or therapeutic treatment of cancer.
  • the determination (detection and quantification) of the levels of phosphorylated Elk-1 protein can be performed in a similar manner to that described in connection with the method of the invention.
  • phosphorylated Elk-1 activates the expression of some genes, and, in some instances, increased levels of phosphorylated Elk-1 protein are found in biological samples from subjects suffering from cancer or having a greater risk or predisposition to develop cancer
  • Agents that are found by using the screening assays provided by the instant invention to be capable of decreasing Elk-1 phosphorylation by at least 5%, more preferably by at least 10%, still more preferably by at least 30%, still more preferably by at least 50%, still more preferably by at least 70%>, even more preferably by at least 90%>, may be selected for further testing as a prophylactic and/or therapeutic anti-cancer agent.
  • Elk-1 and phosphorylated Elk-1 can be determined by conventional techniques.
  • decreased levels of phosphorylated Elk-1 protein are found in biological samples from subjects suffering from cancer or having a greater risk or predisposition to develop cancer.
  • Agents that are found by using the screening assays provided by the instant invention to be capable of increasing Elk-1 phosphorylation by at least 5%, more preferably by at least 10%), still more preferably by at least 30%>, still more preferably by at least 50%>, still more preferably by at least 70%, even more preferably by at least 90%, may be selected for further testing as a prophylactic and/or therapeutic anti-cancer agent.
  • Elk-1 and phosphorylated Elk-1 can be determined by conventional techniques.
  • Agents that are found to be capable of modulating Elk-1 phosphorylation may be used, for example, to reduce the symptoms of cancer alone or in combination with other appropriate agents or treatments.
  • the invention relates to the use of a phosphorylated Elk-
  • peptides comprising
  • phosphorylation sites of phosphorylated Elk-1 protein can be used as antigens in order to obtain antibodies against phosphorylated Elk-1 protein.
  • the present invention envisions treating a disease, for example, cancer and the like, in a subject by the administration of an effective treatment to the subject.
  • the method comprises administering an effective treatment based upon the detected level of phosphorylated Elk-1 in a sample obtained from the subject.
  • the administered treatment may be any effective anti-tumor or anti-cancer treatment known in the art, including for example chemotherapy, radiation, surgery, adoptive T-cell therapy, and the like.
  • the method comprises administering to the subject an effective amount of a therapeutic agent of the invention.
  • the method comprises administering a therapeutic agent that inhibits the phosphorylation of Elk-1, such as Elk-1 pT417 or Elk- 1-363.
  • the method comprises administering a therapeutic agent that enhances the phosphorylation of Elk-1, such as Elk-1 pT417 or Elk- 1-363.
  • the subject is a mammal. In certain embodiments, the subject is a primate, cow, horse, pig, sheep, cat, dog, rat, mouse, or the like. In one embodiment, the subject is a human.
  • the present invention may be used to prevent or treat any cancer associated with Elk-1 phosphorylation.
  • Exemplary cancers that may be treated by the present methods include, but are not limited to, brain cancer, spinal cord cancer, breast cancer, stomach cancer, and carcinomas, such as transitional cell carcinoma, colonic adenocarcinoma, adenocarcinoma of the rectum, gastric carcinoma, colorectal carcinoma, infiltrating duct carcinoma, neuroendocrine carcinoma, hepatocellular carcinoma, squamous cell carcinoma, adenocarcinoma of the stomach, and small bowel malignant stromal tumor.
  • Exemplary cancers of the brain and spinal cord that may be treated by way of the present invention include, but are not limited to, oligodendroglioma, glioblastoma, and ganglioglioma.
  • Administration of the therapeutic agent or modified cell in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the agents or modified cell of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • the amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art
  • One or more suitable unit dosage forms having the therapeutic agent(s) of the invention which, as discussed below, may optionally be formulated for sustained release (for example using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of which are incorporated by reference herein), can be administered by a variety of routes including parenteral, including by intravenous and intramuscular routes, as well as by direct injection into the diseased tissue.
  • the therapeutic agent or modified cell may be directly injected into the tumor.
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the therapeutic agents of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • a pharmaceutically acceptable carrier diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • the total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation.
  • a "pharmaceutically acceptable” is a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • the active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.
  • the therapeutic agent of the invention is administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
  • immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies
  • cytoxin fludaribine
  • cyclosporin FK506, rapamycin
  • mycophenolic acid steroids
  • Example 1 Elk-1 phosphorylated at threonine-417 is present in diverse cancers and correlates with differentiation grade of colonic adenocarcinoma
  • Elk-1 is a member of the Ets family of transcription factors, which are identified by a conserved Ets DNA-binding domain that mediates transcriptional regulation at Ets sequence containing promoters.
  • the activation domain of Elk-1 is important for executing its physiological functions and contains many phosphorylation sites targeted by various MAP kinases following exposure to cell stressors or mitogenic stimuli. The different combinations of phosphorylated sites allow specificity of cellular responses mediated through redundant signaling pathways activated by distinct stimuli. Through phosphorylation of S383, MAP kinase activating stimuli have been shown to regulate various processes important in carcinogenesis through transcriptional regulation in various cell lines, including proliferation.
  • Phosphorylation at the T417 site (pT417), but not the S383 site, is involved in neuronal apoptosis induced through dendritic signaling mechanisms and associates with neuronal lesions in many Lewy Body diseases. This points to distinct roles for these different phosphorylation sites in pathophysiologic pathways.
  • the S383 site remains the best characterized in the context of normal function and carcinogenesis in cell lines and less is known about the biochemistry of other phosphorylation sites, particularly in more biochemically relevant models.
  • Elk-1 pT417 is present in epithelial cell nuclei of various normal and cancer tissues and that the number of pT417 positive cells correlates with differentiation grade of colonic adenocarcinomas. This nuclear localization and correlation with tumor differentiation in adenocarcinoma demonstrates an important transcriptional and biochemical role of this phosphorylation site in carcinogenesis of this tumor type. Further description of the data presented herein may be found in Morris et al., 2012, Human Pathology, 44(5): 766-776, the content of which is incorporated by reference in its entirety herein.
  • Tissue microarrays were made-to-order from Imgenex (IMH-1)
  • Two-mm diameter tissue cores were punched from various normal and tumor tissues and arrayed in a 4x6 grid in a paraffin block.
  • the TMA's were then cut in 4 ⁇ thick serial sections and mounted on glass slides (Yukawa et al, 2004, J Nephrol, 17: 26- 33). Thin serial sections allow detection of multiple antigens within the same tumor cells by performing IHC for each antigen on distinct slides.
  • the microarrays were supplied with information such as age and sex of patient and type of tumor. Some tumors were also supplied with patient survival data.
  • Paraffin-embedded tissue sections were de-paraffmized in four changes of fresh xylene for 5 minutes each. Sections were then rehydrated in graded alcohol washes, followed by two changes in Millipore water for 2.5 minutes each. Antigen retrieval was performed by immersing slides in 800 mL of 0.01 M Citric acid, pH 6.0 in a one-liter beaker and microwaving in a commercial microwave for 4 minutes at 100% power followed by 1.5 minutes at 10% power. The beaker was then removed and placed at room temperature for 20 minutes. Sections were quickly rinsed with Millipore water and washed with 0.01 M TBS, pH 8.4 2 times for 5 minutes each. The following steps, excluding washes, were performed in a humidified chamber.
  • Paraffin-embedded slides were de-paraffinized in 4 changes of fresh xylene, 5 minutes each.
  • slides were first incubated vertically in a dry oven at 62°C for 1 hour to remove the protective layer of paraffin. Slides were hydrated in graded ethanol solutions followed by a 5-minute incubation in 0.15 M PB, pH 7.4.
  • slides were laid flat on the bottom of a Tupperware container and covered with 0.01 M citrate buffer, pH 6.0 and placed in a commercial microwave oven for 5 minutes each at 100%, 50%, and 10%> power followed by a 20 minute incubation at room temperature. Slides were then rinsed in 0.15 M PB.
  • Slides were washed again 3 times 5 minutes in 5/5 block and incubated in biotinylated goat anti-rabbit IgG in 5/5 block for 1 hour at room temperature. Slides were washed 3 times 5 minutes in 0.15 M PB before incubation in ABC complex for 1 hour at room temperature. Slides were washed again 3 times 5 minutes in 0.15 M PB before incubating in SG substrate for 1.5 minutes. Slides were then dehydrated in sequential ethanol washes and cleared in xylenes 4 times 5 minutes each. Slides were mounted with Permount.
  • Elk-1 pT417 or non-phosphoT417 antibody was incubated with 5,000 times molar excess of the corresponding competing peptide for 1 hour at room temperature prior to membrane incubation.
  • the membrane was washed in 0.1% TBST before incubation in horseradish peroxidase conjugated anti-rabbit antibody diluted 1 : 10,000 in 5% BSA, 5% NGS, 0.01 % Triton X- 100 in TBS.
  • the membrane was washed again in 0.1% TBST before development of the blot with Thermo Scientific SuperSignal West Femtomax Chemiluminescent Substrate.
  • Regions from each histologically unique area of the tumor or from normal tissue were acquired with a Zeiss confocal LSM 710.
  • the corresponding H&E slide was used to align the tumor areas with the DAPI fluorescence on the immunofluorescence slide.
  • a threshold was applied to all images to remove the lowest 10%> of signal intensities.
  • the total number of Elk-1 pT417 positive epithelial cells in each region were counted and expressed as a percentage of the total number of epithelial cells derived from the DAPI image. Percentages from at least three regions were averaged for each tumor.
  • Elk-1 pT417 is present in normal epithelial cell nuclei
  • Tissue microarrays were processed for Elk-1 pT417 (pT417) or Elk-1 non- phosphoT417 (non-phosphoT417) immunohistochemistry, aligned to corresponding H&E slides for analysis, and qualitatively assessed based on relative staining
  • the pT417 antibody specifically recognizes Elk-1 phosphorylated at the T417 site, while the non-phosphoT417 antibody recognizes the T417 site when it is not phosphorylated.
  • Tissue samples were organized into categories discussed below based on the intensity of staining and proportion of cells positive for pT417 and non-phosphoT417.
  • Elk-1 pT417 moderately labeled a subset of epithelial cell nuclei within normal lung epithelium, while non-phosphoT417 diffusely and moderately labeled the cytoplasm of these cells. Within the liver, pT417 moderately stained the nuclei of a subset of hepatocytes, while the nonphosphorylated form weakly stained the cytoplasm. The specificity of the Elk-1 pT417 and the non- phospho-T417 antibodies was confirmed by performing peptide competition experiments with detection by Western blot (Figure 1M).
  • Elk-1 pT417 is present in diverse cancer types from various tissues
  • pT417 moderately labeled a subset of hepatocyte nuclei in a hepatocellular carcinoma (Table 1).
  • non-phosphoT417 strongly stained the cytoplasm, suggesting an increase in total Elk-1 compared to normal liver.
  • Elk-1 nonphosphoT417 and Elk-1 pT417 immunohistochemistry of each sample on the tissue microarray was assigned a qualitative score of negative, weak, moderate, or strong based on the intensity of staining in the most and least intensely stained samples.
  • the proportion of cells stained by Elk-1 pT417 is given with designations of low, medium, or high compared with samples with the lowest and highest proportions of positive nuclei.
  • the localization of each form of Elk-1 is also given and defined as nuclear or cytoplasmic Elk-1 pT417 is present at higher levels in colonic adenocarcinoma compared to normal colon
  • the intensity of staining was measured qualitatively by a pathologist and reported as intensity scores on a scale of 0 to 3.
  • Elk-1 pT417 levels correlate with differentiation grade of colonic adenocarcinoma
  • Samples were categorized as the following: normal, tubulovillous adenoma (adenocarcinoma precursor), or adenocarcinoma. Adenocarcinomas were further divided into well, moderately, or poorly differentiated groups. Tumor cells in DAPI images were identified by aligning fluorescence images with H&E images. As in the various normal and cancer tissues screened above, Elk-1 pT417 was exclusively nuclear in all tissues ( Figure 4A through Figure 4D).
  • Elk-1 staining was quantified by calculating the number of Elk-1 pT417 positive nuclei as a percentage of the total number of nuclei, as determined by DAPI. Analysis of percentages by differentiation grade revealed a significant main effect (One way ANOVA, p ⁇ 0.001).
  • Epithelia are self-renewing in that a population of stem cells constantly proliferate and differentiate to repopulate older cells of the epithelium that are discarded through anoikis.
  • stem cells within the base of the colonic crypts divide such that at any given time a portion of the stem cell pool remains and a separate pool of progenitors continues to proliferate.
  • the progenitor cells proliferate as they migrate up the crypt through the transit-amplifying region towards the surface, where they differentiate and are eventually replaced by newly produced progenitors.
  • the proliferation of stem cells is in part controlled by a gradient of WNT/beta-catenin signaling that is highest at the base of the crypt and decreases toward the top.
  • EphB-ephrinB the relative concentrations of which contribute to migration control of progenitor cells through regulation of cell-cell contacts
  • an inhibitory protein complex that includes APC binds beta- catenin and targets it for degradation (Verzi et al, 2008, Organogenesis, 4: 87-91). Upon Wnt binding to its receptor, Frizzled, inhibition is released and beta-catenin translocates to the nucleus to activate transcription of target genes (Verzi et al, 2008, Organogenesis, 4: 87-91).
  • a genetic mutation in the APC gene causes loss of ability to interact with beta-catenin, preventing its degradation and allowing constitutive transcription of target genes and consequently uncontrolled proliferation (Segditsas et al, 2006, Oncogene, 25: 7531-7537).
  • JNK Jun N-terminal kinase
  • JNK activates Elk-1 in response to cellular stress such as DNA damage (Whitmarsh et al, 1997, Mol Cell Biol, 17: 2360- 2371; Zhang et al, 2007, Mol Cell Biol, 27: 2861-2869) or growth inhibitory signals (Tyagi et al, 2003, Oncogene, 22: 1302-1316) through phosphorylation at the S383 site, resulting in transcription of c-FOS.
  • JNK also phosphorylates and activates c-JUN, which complexes with c-FOS to form the transcription factor AP-1.
  • the active phosphorylated form of c-JUN in epithelial cells of colon adenocarcinoma is significantly higher in more well-differentiated tumors (i.e. low in poorly differentiated cases), while c-FOS expression does not correlate with tumor differentiation (Konstantinopoulos et al., 2007, Int J Colorectal Dis, 22: 57-68).
  • the lack of similarity in staining of Elk-1 pT417 and c-FOS suggests that this
  • INK/SAPK has been shown to produce a phosphorylation pattern of Elk- 1 distinct from that of ERKl/2 following stressful stimuli in NIH 3T3 fibroblasts (Gille et al, 1995, Curr Biol, 5: 1191-1200).
  • JNK SAPK and p38 retain the ability to translocate into the nucleus in non-adherent cells exposed to stressful cellular stimuli, such as DNA-damaging agents (Aplin et al, 2002, J Cell Sci, 115: 2781-2790). Combined with the loss of ERKl/2 nuclear translocation, this would result in phosphorylation of a combination of sites on Elk-1 different from that in the adherent state, potentially contributing to activation of a distinct cellular outcome. Thus, as a tumor progresses and more cells lose cellular contacts, more cells may demonstrate this altered balance of phosphorylation of Elk-1. The T417 site could potentially be a part of that pattern in that increasing levels of Elk-1 pT417 across decreasingly differentiated cancers may indicate this signaling imbalance.
  • Elk-1 pT417 is located in the nuclei of normal epithelial cells in various glandular tissues, suggesting a role for Elk-1 phosphorylated at this site in transcriptional regulation.
  • Elk-1 pT417 was shown to label nuclei of epithelial cells in tumors from various tissues. The overall trend of Elk-1 pT417 staining was different for different cancers compared to normal tissue and this could be the result of differences in the balance of signaling components in different tissues as well as the differentiated state of the tumors.
  • Elk-1 pT417 was shown to be elevated in colonic adenocarcinoma by qualitative assessment of intensity and quantitative assessment of positive cell numbers.
  • Example 2 Expression of Elk-1 pT417 and Elk-1 pT363 in human cancer Experiments were conducted using phosphospecific antibodies for Elk-1 at amino acid residues 417 and 363 to demonstrate that Elk-1 pT417 and Elk-1 pT363 are strongly expressed in various cancers including brain tumors, lung cancer, and gastric cancer.
  • Elk-1 pT417 was strongly expressed in the nucleus of atypical ganglion cells and in various forms of brain cancer, including

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention concerne des compositions et des méthodes permettant d'évaluer le cancer en dosant une protéine ayant un état de phosphorylation différent dans les cellules cancéreuses par rapport aux cellules non cancéreuses.
PCT/US2013/059438 2012-09-12 2013-09-12 Formes phosphorylées spécifiques de l'elk-1 utilisées comme biomarqueurs et cibles thérapeutiques contre le cancer WO2014043331A2 (fr)

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US201261700047P 2012-09-12 2012-09-12
US61/700,047 2012-09-12

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WO2014043331A2 true WO2014043331A2 (fr) 2014-03-20
WO2014043331A3 WO2014043331A3 (fr) 2015-07-23

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PCT/US2013/059438 WO2014043331A2 (fr) 2012-09-12 2013-09-12 Formes phosphorylées spécifiques de l'elk-1 utilisées comme biomarqueurs et cibles thérapeutiques contre le cancer

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US7598028B2 (en) * 2006-11-28 2009-10-06 The Regents Of The University Of Michigan Compositions and methods for detecting and treating prostate disorders

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