WO2006099539A1 - Reg iv, cible pour le diagnostic et le traitement du cancer - Google Patents

Reg iv, cible pour le diagnostic et le traitement du cancer Download PDF

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Publication number
WO2006099539A1
WO2006099539A1 PCT/US2006/009524 US2006009524W WO2006099539A1 WO 2006099539 A1 WO2006099539 A1 WO 2006099539A1 US 2006009524 W US2006009524 W US 2006009524W WO 2006099539 A1 WO2006099539 A1 WO 2006099539A1
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reg
cancer
protein
prostate
antibody
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PCT/US2006/009524
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English (en)
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Robert E. Reiter
Zhennan Gu
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The Regents Of The University Of California
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Publication of WO2006099539A1 publication Critical patent/WO2006099539A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Prostate cancer is the most common malignancy and the second leading cause of cancer-related death in American men. Prostate cancer is a biologically and clinically heterogeneous disease. A majority of men with this malignancy harbor slow-growing tumors that may not impact an individual's natural lifespan, while others are struck by rapidly progressive, metastatic tumors. PSA screening is limited by a lack of specificity and an inability to predict which patients are at risk to develop hormone refractory metastatic disease. Recent studies advocating a lower PSA threshold for diagnosis may increase the number of prostate cancer diagnoses and further complicate the identification of patients with indolent vs.
  • markers may also play a role in the biology of metastatic or hormone refractory prostate cancer progression.
  • Recent examples of genes present in primary tumors that correlate with outcome and play a role in the biology of prostate cancer progression include EZH2 and LIM kinase (Varambally et al., Nature, 419: 624-629 (2002); Yoshioka et al., Proc Natl Acad Sci USA, 100: 7247-7252 (2003)). However, neither of these two genes is secreted.
  • Reg IV a new member of the regenerating family of secreted C-lectin proteins
  • GI gastrointestinal
  • Their pleotropic functions include promoting tissue regeneration, proliferation and resistance to apoptosis (Macadam et al., Br J Cancer, 83: 188-195 (2000)).
  • Reg IV encodes a secreted protein, which is not expressed in the normal prostate.
  • Reg IV is expressed at low levels in a subset of primary tumors and is moderately or highly expressed in a majority of hormone refractory and metastatic tumors. These results demonstrate that Reg IV is a marker of prostate cancer metastasis or hormone refractory growth as well as a marker with respect to other urogenital cancers, including bladder cancer.
  • the present invention provides methods of diagnosis and providing a prognosis for individuals at risk for cancers that overexpress a Reg IV protein or mRNA transcript, particularly urogenital cancers including prostate and/or bladder cancer.
  • the methods generally comprise contacting a test tissue sample from an individual at risk of having a cancer that overexpresses a Reg IV protein or mRNA transcript with an antibody that specifically binds to a Reg IV protein; and determining the presence or absence of a Reg IV protein in the test tissue sample in comparison to a control tissue sample from an individual known to be negative for cancer.
  • the tissue sample is serum, but can also be biopsy tissue, particularly urogenital tissues including prostate tissue or bladder tissue.
  • the present invention further provides methods of inhibiting the growth of and promoting the regression of a cancerous tumor that overexpresses a Reg IV protein or mRNA transcript, the method comprising inhibiting the binding of Reg IV protein to a Reg IV receptor on a cell of the tumor tissue.
  • the methods find particular use in treating any cancer that overexpresses a Reg IV protein or mRNA transcript, particularly urogenital cancers including prostate and bladder cancers.
  • the present invention also provides methods of identifying compounds that inhibit the binding of a Reg IV protein to a Reg IV receptor, wherein said compounds find use in inhibiting the growth of and promoting the regression of a cancerous tumor that overexpresses Reg IV protein, for example, a tumor of a urogenital tissue including a prostate or bladder cancer tumor.
  • the screening methods can be carried out in vitro (i.e., by ELISA) and in vivo.
  • the invention provides methods of diagnosing a cancer in a subject by determining the level of Reg IV protein expression or activity in a biological sample or biopsy of the cancer or tumor from the subject wherein an increased level of Reg IV protein expression or activity in the sample or biopsy is indicative of cancer.
  • determining the Reg IV protein levels involves steps of (a) contacting a tissue sample or biopsy from the subject with an antibody that specifically binds to Reg IV protein; and (b) determining whether or not the Reg IV protein is overexpressed in the sample or biopsy; thereby diagnosing the cancer.
  • the cancer can be a prostate cancer, ovarian cancer, renal cancer, breast cancer, colon cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma, or hepatocarcinoma.
  • the tissue sample can be a needle biopsy, a surgical biopsy or a bone marrow biopsy.
  • a tissue sample can be fixed or embedded in paraffin.
  • a tissue sample can be, for instance, from prostate, ovary, bone, blood, lymph node, liver, or kidney.
  • the antibody in some embodiments is a monoclonal antibody.
  • An elevated level of Reg IV protein in a sample is indicative of cancer.
  • the cancer is a prostate or bladder cancer.
  • the diagnosis of cancer is made upon the basis of the Reg IV levels as well as optionally on other conventional indicators of cancer. For instance, the diagnosis can be based upon both the Reg IV protein findings and the histology or growth characteristics of the cancer cells. In the case of prostate cancer, for instance, the Reg IV protein findings can supplement the Gleason scoring system to provide a more accurate or reliable indicator of carcinogenicity and likelihood of disease progression. In some embodiments of the above, the diagnosis is also based upon serum PSA levels.
  • the method alternatively determines the Reg IV protein by measuring mRNA transcript levels by (a) contacting a tissue sample with a primer set of a first oligonucleotide and a second oligonucleotide that each specifically hybridize to Reg IV protein 3 nucleic acid; (b) amplifying Reg IV protein nucleic acid in the sample; and (c) determining whether or not Reg IV protein nucleic acid is overexpressed in the sample; thereby diagnosing the cancer.
  • the first oligonucleotide can comprise a nucleotide sequence of Reg IV and the second oligonucleotide can comprise a nucleotide sequence complementary to that of Reg IV 3 cDNA.
  • both nucleotides are less than 50 base pairs in length.
  • the cancer is a prostate or bladder cancer.
  • the diagnosis is also based upon serum PSA levels.
  • the invention provides a method of prognosis for a cancer that overexpresses Reg IV by assessing the likelihood that the cancer will be invasive, metastasize, recur or be resistant to therapy.
  • the invention provides a method of further diagnosing a cancer that overexpresses Reg IV or has increased Reg IV transcriptional activity and therefore has an increased liklihood of invasiveness, metastasizing, recurrence or resistance to therapy.
  • the method comprises the steps of (a) contacting a tissue sample with an antibody that specifically binds to Reg IV; and (b) determining whether or not the Reg IV is overexpressed in the sample; thereby diagnosing the cancer that overexpresses Reg IV.
  • the cancer may be diagnosed before or after obtaining and analyzing the sample for Reg IV expression or activity levels.
  • the cancer may have been initially identified on the basis of histological appearance (e.g., Gleason score in the case of prostate cancer) and not on the basis of the Reg IV level determination.
  • the cancer can have been diagnosed as such with or without, or despite, knowledge of Reg IV level.
  • the cancer can be a prostate cancer or bladder cancer, renal cancer, breast cancer, colon cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma, or hepatocarcinoma.
  • the tissue sample can be a needle biopsy, a surgical biopsy or a bone marrow biopsy.
  • a tissue sample can be fixed or embedded in paraffin.
  • a tissue sample can be, for instance, from prostate, ovary, bone, lymph node, liver, or kidney.
  • the antibody in some embodiments is a monoclonal antibody.
  • An elevated level of Reg IV in a sample is prognostic of, and associated with, an increased risk of recurrence or resistance to therapy for the cancer, hi a preferred embodiment, the cancer is a prostate cancer or bladder cancer. In some embodiments of the above, the diagnosis is also based upon PSA levels.
  • the method of diagnosing a cancer that overexpresses Reg IV comprises the steps of (a) contacting a tissue sample with a primer set of a first oligonucleotide and a second oligonucleotide that each specifically hybridize to Reg IV nucleic acid; (b) amplifying Reg IV nucleic acid in the sample; and (c) determining whether or not Reg IV nucleic acid is overexpressed in the sample; thereby diagnosing the cancer that overexpresses Reg IV.
  • the first oligonucleotide can comprise a nucleotide sequence of Reg IV cDNA and the second oligonucleotide can comprise a nucleotide sequence complementary to that of Reg IV cDNA.
  • both nucleotides are less than 50 base pairs in length, hi the above methods, a increased level of Reg IV in a sample is, prognostic for, and associated with, an increased risk of recurrence, metastasis, hormone independende or resistance to therapy for the cancer.
  • the cancer is a prostate or bladder cancer, hi some embodiments of the above, the diagnosis is also based upon serum PSA levels.
  • the invention provides a method of targeting patients for more aggressive or alternative cancer therapy or increased surveillance for a cancer recurrence based upon an elevated level of Reg IV in a tissue sample from the patient taken before, during, or after surgical removal of the cancerous tissue (e.g., prostectomy) or before, during, or after another cancer treatment.
  • the Reg IV activity or expression levels can be determined as described above, hi some embodiments of the above, the diagnosis is also based upon serum PSA levels.
  • the cancer that overexpresses Reg IV can be, for instance, a prostate cancer, ovarian cancer, renal cancer, lung cancer, breast cancer, colon cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma or hepatocarcinoma.
  • the cancer is a prostate or bladder cancer.
  • Patients identified as having raised Reg IV levels and accordingly being at high risk of metastasis, recurrence or a therapy resistant cancer can be treated with exogenous or endogenous hormone ablation, optionally supplemented with chemotherapy and/or radiation.
  • the hormone ablation is androgen ablation (e.g., treatment with finasteride and other anti- tesosterone or anti-DHT agents).
  • the invention provides a method of treating or inhibiting a cancer, a therapy resistant cancer, a metastasis of cancer, or recurrence of cancer, that overrexpresses Reg IV in a subject comprising administering to the subject a therapeutically effective amount of one or more inhibitors of Reg IV or Reg IV expression.
  • the cancer that overexpresses Reg IV can be, for instance, a prostate cancer, bladder cancer, ovarian cancer, renal cancer, lung cancer, breast cancer, colon cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma or hepatocarcinoma.
  • the cancer is a prostate or bladder cancer.
  • the compound can be a compound as identified in the following aspect.
  • the overexpression can be identified as described in the previous aspects.
  • the compound can be administered concurrently with another cancer therapy.
  • the invention also provides a method of identifying a compound that inhibits cancer, therapy resistant cancer, or metastasis, or a recurrence of cancer, the method comprising the steps of contacting a cell with a compound; and determining the effect of the compound on the expression or activity of the Reg IV polypeptide in the cell; wherein compounds which decrease the Reg IV expression or activity levels are identified as being able to inhibit cancer, its metastasis, or progression to a hormone-independent or treatment resistant state.
  • the compound decreases the expression of Reg IV in the cell.
  • the cell is a cancer cell and, more particularly, may be cancer cell of a particular tissue type or origin (e.g., prostate, ovary, kidney, lung, breast, colon, leukemia, non-Hodgkin's lymphoma, multiple myeloma or hepatocarcinoma) which has overexpression of Reg IV
  • the cancer that overexpresses Reg IV is prostate cancer, bladder cancer, ovarian cancer, renal cancer, lung cancer, breast cancer, colon cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma or hepatocarcinoma.
  • the cancer is a prostate cancer or bladder cancer.
  • the invention also provides a method of localizing a cancer that overexpresses Reg IV in vivo, and is therefore likely to be invasive, likely to metastasize, become hormone independent, or refractory to treatment, the method comprising the step of imaging in a subject a cell overexpressing Reg IV wherein the cancer that overexpresses Reg IV is selected from the group consisting of prostate cancer, bladder cancer, ovarian cancer, renal cancer, breast cancer, lung cancer, colon cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma and hepatocarcinoma.
  • Reg IV proteins and Reg IV -encoding nucleic acid molecules may be used in various immunotherapeutic methods to promote immune-mediated destruction of cancers (e.g., prostate or bladder tumors), particularly, when such tumors are invasive.
  • cancers e.g., prostate or bladder tumors
  • the invention provides methods of treating cancer, particularly an invasive cancer or a metastasis, or preventing the progression of a cancer to a treatment resistant, hormone-independent, or metastasizing state by administering antibodies that bind to Reg IV to reduce their respective activity in the patient.
  • the antibodies are conjugated to effector moieties which thereby are preferentially cytotoxic to cells overexpressing the Reg IV.
  • the antibodies are humanized monoclonal antibodies.
  • the invention provides RNA interference (RNAi)-based methods of treating cancer or preventing the progression of a cancer to a treatment resistant, hormone-independent, or metastasizing state by administration of short interfering RNA molecule (siRNA) or an antisense molecule specific for Reg IV and which accordingly are capable of inhibit the expression of Reg IV.
  • siRNA short interfering RNA molecule
  • the siRNA molecule is a short hairpin RNAi.
  • the tissue, cancer, subject, or patient to be treated or diagnosed can be human or mammalian.
  • FIG. 1 Figure 1: Reg IV expression in prostate cancer xenografts and in normal tissues and Reg IV secretion.
  • Northern analysis of Reg IV in (a) prostate xenografts, showing overexpression in two hormone refractory (AI) sublines of LAPC 9, and (b) multiple normal tissues, with notable expression in colon. Expression was also seen in pancreas and small bowel in a multiple tissue dot blot (not shown here), (c) A Myc-tagged Reg IV cDNA was transiently transfected into 293 T cells and recovered from the conditioned media with an anti-Myc antibody. A control antibody did not identify this band, indicating that it is Reg IV. Likewise, a Myc antibody did not pull down a specific protein from a vector-only transfectant control (not shown).
  • FIG. 2 In situ expression analysis of Reg IV expression.
  • A The antisense probe (right panel) shows Reg IV (brown) expression in an androgen- independent tumor but not in the adjacent normal tissue to the left of the tumor. The sense control, which is negative, is in the left panel.
  • B Progression of Reg IV expression.
  • In the upper left panel (a) is normal prostate, which is not staining.
  • To the right (b) is a negative Gleason 6 cancer.
  • On the bottom left (c) is a Gleason 9/10 primary tumor staining strongly for Reg IV (note intense purple color).
  • To the right (d) is a strongly staining lymph node metastasis.
  • FIG. 3 Reg IV expression in normal, primary and metastatic prostate cancer.
  • the mean expression score +/- standard deviation for Reg IV expression is shown for normal prostate, primary prostate cancer and metastatic prostate cancer.
  • the results summarize a prostate cancer tissue array representing the gamut of prostate tissue.
  • Figure 4 Association of Reg IV expression with Gleason score. The mean expression score +/- standard deviation for Reg IV expression is shown for Gleason 5-6 and 7-10 prostate tumors.
  • Figure 5 Reg IV mRNA and amino acid sequences from LAPC 9 hormone refractory xenograft.
  • the RegIV protein and/or mRNA transcripts are overexpressed in a number of prostate tumors after neoadjuvant hormone ablation therapy. As predicted by its sequence homology, it is secreted from transiently transfected cells. Message RNA transcripts are over-expressed in a majority of hormone refractory metastases represented on two high-density tissue microarrays. In comparison, it is not expressed by any normal prostate specimens and only at low levels in ⁇ 40% of primary tumors. These data support Reg IV as a candidate marker and cancer associated antigen for hormone refractory metastatic prostate cancer and urogenital cancers.
  • Reg IV refers to nucleic acids, e.g., gene, pre-mRNA, mRNA, and polypeptides, polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein, for example, as depicted in Figure 5; (2) specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising a referenced amino acid sequence as depicted in Figure 5, immunogenic fragments thereof, and conservatively modified variants thereof; (3) specifically hybridize
  • a polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal.
  • the nucleic acids and proteins of the invention include both naturally occurring or recombinant molecules.
  • Cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid tumors and lymphoid cancers, kidney, breast, lung, kidney, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer, lymphoma, including non-Hodgkins and Hodgkins lymphoma, leukemia, and multiple myeloma.
  • Urinital cancer refers to human cancers of urinary tract and genital tissues, including but not limited to kidney, bladder, urinary tract, urethra, prostrate, penis, testicle, vulva, vagina, cervical and ovary tissues.
  • the cancer will generally comprise Reg IV-expressing cells, such that the anti-Reg IV antibody herein is able to bind to the cancer. While the cancer may be characterized by overexpression of the Reg IV receptor, the present application further provides a method for treating cancer which is not considered to be an Reg TV overexpressing cancer.
  • the cancer to be treated herein may be one characterized by excessive activation of Reg IV.
  • a diagnostic or prognostic assay will be performed to determine whether the patient's cancer is characterized by overexpression of
  • Reg IV Various assays for determining such amplification/overexpress ion are contemplated and include the immunohistochemistry, FISH and shed antigen assays, southern blotting, or PCR techniques. Moreover, the Reg IV overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label (e.g. a radioactive isotope) and externally scanning the patient for localization of the label.
  • a detectable label e.g. a radioactive isotope
  • apoptosis-mediated cancers e.g., through death receptor cell signaling, for example, Fas ligand receptor, TRAIL receptors, TNF-Rl, chemotherapeutic drugs, radiation
  • non-apoptosis mediated cancer therapies including chemotherapy, hormonal therapy, radiotherapy, and immunotherapy.
  • “Overexpression” generally refers to RNA or protein expression of Reg IV in a test tissue sample that is significantly higher that RNA or protein expression of Reg IV in a control tissue sample from a normal individual.
  • the tissue sample is autologous.
  • Cancerous test tissue samples typically have at least one fold higher expression of Reg IV, mRNA or protein, often up to two-, three-, four-, five-, eight-, or ten-fold or more fold higher expression of Reg IV in comparison to normal tissue samples.
  • the terms “overexpress,” “overexpression” or “overexpressed” interchangeably refer to a gene that is transcribed or translated at a detectably greater level, usually in a cancer cell, in comparison to a normal cell.
  • Overexpression therefore refers to both overexpression of protein and RNA (due to increased transcription, post transcriptional processing, translation, post translational processing, altered stability, and altered protein degradation), as well as local overexpression due to altered protein traffic patterns (increased nuclear localization), and augmented functional activity, e.g., as in an increased enzyme hydrolysis of substrate.
  • Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell or comparison cell (e.g., a BPH cell).
  • cancer that overexpresses Reg IV and “cancer associated with the overexpression of Reg IV” interchangeably refer to cancer cells or tissues that overexpress Reg IV in accordance with the above definition.
  • cancer-associated antigen or “tumor-specific marker” or “tumor marker” interchangeably refers to a molecule (typically protein, carbohydrate or lipid) that is preferentially expressed in a cancer cell in comparison to a normal cell, and which is useful for the preferential targeting of a pharmacological agent to the cancer cell.
  • a marker or antigen can be expressed on the cell surface or intracellularly.
  • a cancer-associated antigen is a molecule that is overexpressed or stabilized with minimal degradation in a cancer cell in comparison to a normal cell, for instance, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell.
  • a cancer-associated antigen is a molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell.
  • a cancer-associated antigen will be expressed exclusively in a cancer cell and not synthesized or expressed in a normal cell.
  • Exemplified cell surface tumor markers include the proteins c-erbB-2 and human epidermal growth factor receptor (HER) for breast cancer, PSMA for prostate cancer, and carbohydrate mucins in numerous cancers, including breast, ovarian and colorectal.
  • Exemplified intracellular tumor markers include, for example, mutated tumor suppressor or cell cycle proteins, including p53.
  • An "agonist” refers to an agent that binds to a polypeptide or polynucleotide of the invention, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide or polynucleotide of the invention.
  • an "antagonist” refers to an agent that inhibits expression of a polypeptide or polynucleotide of the invention or binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of a polypeptide or polynucleotide of the invention.
  • “Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonists, antagonists, and their homologs and mimetics.
  • modulator includes inhibitors and activators.
  • Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists.
  • Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists.
  • Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity.
  • Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%.
  • Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • test compound or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi, oligonucleotide, etc.
  • the test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity.
  • Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • a fusion partner e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • HTS high throughput screening
  • a "small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 Daltons and less than about 2500 Daltons, preferably less than about 2000 Daltons, preferably between about 100 to about 1000 Daltons, more preferably between about 200 to about 500 Daltons.
  • Cytotoxic agents include "cell-cycle-specific” or “antimitotic” or "cytoskeletal- interacting” drugs. These terms interchangeably refer to any pharmacological agent that blocks cells in mitosis. Such agents are useful in chemotherapy.
  • cell-cycle- specific-drugs bind to the cytoskeletal protein tubulin and block the ability of tubulin to polymerize into microtubules, resulting in the arrest of cell division at metaphase.
  • Exemplified cell-cycle-specific drags include vinca alkaloids, taxanes, colchicine, and podophyllotoxin.
  • Exemplified vinca alkaloids include vinblastine, vincristine, vindesine and vinorelbine.
  • Exemplifed taxanes include paclitaxel and docetaxel.
  • Another example of a cytoskeletal-interacting drug includes 2-methoxyestradiol.
  • siRNA refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA or RNAi molecule is expressed in the same cell as the gene or target gene.
  • siRNA or “RNAi” thus refers to the double stranded RNA formed by the complementary strands.
  • the complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity.
  • an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.
  • the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • siRNA molecules and vectors are well known to those of ordinary skill in the art.
  • an efficient process for designing a suitable siRNA is to start at the AUG start codon of the mRNA transcript (e.g., see, Figure 5) and scan for AA dinucleotide sequences (see, Elbashir et al. EMBO J 20: 6877-6888 (2001).
  • Each AA and the 3' adjacent nucleotides are potential siRNA target sites.
  • the length of the adjacent site sequence will determine the length of the siRNA. For instance, 19 adjacent sites would give a 21 Nucleotide long siRNA siRNAs with 3' overhanging UU dinucleotides are often the most effective.
  • RNA pol EI terminates transcription at 4-6 nucleotide poly(T) tracts to create RNA molecules having a short poly(U) tail.
  • siRNAs with other 3' terminal dinucleotide overhangs can also effectively induce RNAi and the sequence may be empirically selected.
  • target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences can be avoided by conducting a BLAST search ⁇ see, www.ncbi.nlm.nih.gov/BLAST).
  • the siRNA can be administered directly or an siRNA expression vectors can be used to induce RNAi can have different design criteria.
  • a vector can have inserted two inverted repeats separated by a short spacer sequence and ending with a string of T's which serve to terminate transcription.
  • the expressed RNA transcript is predicted to fold into a short hairpin siRNA .
  • the selection of siRNA target sequence, the length of the inverted repeats that encode the stem of a putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5'-overhangs, can vary.
  • a preferred order of the siRNA expression cassette is sense strand, short spacer, and antisense strand.
  • Hairp siRNAs with these various stem lengths can be suitable.
  • the length of the loops linking sense and antisense strands of the hairpin siRNA lean have varying lengths (e.g., 3 to 9 nucleotides, or longer).
  • the vectors may contain promoters and expression enhancers or other regulatory elements which are operably linked to the nucleotide sequence encoding the siRNA.
  • the expression "control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. These control elements may be designed to allow the clinician to turn off or on the expression of the gene by adding or controlling external factors to which the regulatory elements are responsive.
  • Construction of suitable vectors containing the desired therapeutic gene coding and control sequences employs standard ligation and restriction techniques, which are well understood in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Determining the functional effect refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a polynucleotide or polypeptide of the invention, e.g., measuring physical and chemical or phenotypic effects.
  • Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g.
  • RNA stability e.g., G-protein binding; GPCR phosphorylation or dephosphorylation; signal transduction, e.g., receptor-ligand interactions, second messenger concentrations (e.g., cAMP, IP3, or intracellular Ca2+); identification of downstream or reporter gene expression (CAT, luciferase, ⁇ -gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
  • CAT reporter gene expression
  • Samples or assays comprising a nucleic acid or protein disclosed herein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%.
  • Activation is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200- 500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
  • Biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc.
  • a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, Mouse; rabbit; or a bird; reptile; or fish.
  • a "biopsy” refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (i.e., prostate, lymph node, liver, bone marrow, blood cell), the size and type of the tumor (i.e., solid or suspended (i.e., blood or ascites)), among other factors. Representative biopsy techniques include excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An “excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it.
  • An “incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor.
  • a diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy” of the tumor mass, or a “fine-needle aspiration biopsy” which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al, eds., 16th ed., 2005, Chapter 70, and throughout Part V.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection ⁇ see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol.
  • a preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J. MoI Biol 215:403-410 (1990), respectively.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring,- and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et at., J.
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • a particular nucleic acid sequence also implicitly encompasses "splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition.
  • polypeptide any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • An example of potassium channel splice variants is discussed in Leicher, et ah, J. Biol. Chem. 273(52):35095-35101 (1998).
  • the terms "polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) ⁇ see, e.g., Creighton, Proteins (1984)).
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 0 C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 65 0 C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in IX SSC at 45 0 C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • telomere length a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48 0 C depending on primer length.
  • a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 5O 0 C to about 65°C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 9O 0 C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • a Reg IV antibody or anti-Reg IV antibody is one which specifically binds and recognizes a Reg IV protein or Reg IV antigen.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to VH-C H I by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region ⁇ see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990))
  • antibodies e.g., recombinant, monoclonal, or polyclonal antibodies
  • many technique known in the art can be used (see, e.g., Kohler & Milstein,
  • the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridorna and used to produce a recombinant monoclonal antibody.
  • Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3 rd ed. 1997)).
  • Techniques for the production of single chain antibodies or recombinant antibodies U.S. Patent 4,946,778, U.S. Patent No.
  • transgenic mice or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al,
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)).
  • Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al, EMBO J. 10:3655-3659 (1991); and Suresh et al, Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91/00360; WO 92/200373; and EP 03089).
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers ⁇ see, e.g., Jones et ah, Nature 321:522-525 (1986); Riechmann et ah, Nature 332:323-327 (1988); Verhoeyen et ah, Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • the antibody is conjugated to an "effector" moiety.
  • the effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety.
  • the antibody modulates the activity of the protein.
  • the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background.
  • Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins.
  • This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein ⁇ see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity) .
  • terapéuticaally effective dose or amount herein is meant a dose that produces effects for which it is administered.
  • the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)).
  • pharmaceutically acceptable salts or “pharmaceutically acceptable carrier” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et at, Journal of Pharmaceutical Science 66:1-19 (1977)).
  • Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for the present invention.
  • the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
  • the present invention provides compounds which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
  • Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
  • the present invention provides methods of diagnosis and providing a prognosis for individuals at risk for a cancer that overexpresses a Reg TV protein or mRNA transcript, particularly urogenital cancers including prostate and/or bladder cancer.
  • the methods generally comprise contacting a test tissue sample from an individual at risk of having a cancer that overexpresses a Reg IV protein or mRNA transcript with an antibody that specifically binds to a Reg IV protein; and determining the presence or absence of a Reg IV protein in the test tissue sample in comparison to a control tissue sample from an individual known to be negative for a cancer that overexpresses a Reg IV protein or mRNA transcript.
  • the tissue sample is serum, but can also be a tissue from a biopsy, particularly from a urogenital tissue including prostate tissue or bladder tissue.
  • the antibody is a monoclonal antibody.
  • a positive diagnosis for a cancer that overexpresses a Reg IV protein or mRNA transcript is indicated when a higher level of Reg IV protein is detected in a test tissue sample in comparison to a control tissue sample from an individual known not to have cancer, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3- fold, 4-fold, five-fold, eight-fold, ten-fold higher or more.
  • the detection methods can be carried out, for example, using standard ELISA techniques known in the art ⁇ reviewed in Gosling, Immunoassays: A Practical Approach, 2000, Oxford University Press). Detection is accomplished by labeling a primary antibody or a secondary antibody with, for example, a radioactive isotope, a fluorescent label, an enzyme or any other detectable label known in the art.
  • invention provides methods of diagnosis and providing a prognosis for individuals at risk for a cancer that overexpresses a Reg IV protein or mRNA transcript, particularly a urogenital cancer including prostate and/or bladder cancer, by contacting a test tissue sample from an individual at risk of having a cancer that overexpresses a Reg IV protein or mRNA transcript with a primer set of a first oligonucleotide and a second oligonucleotide that each specifically hybridize to a Reg IV nucleic acid; amplifying the Reg IV nucleic acid in the sample; and determining the presence or absence of the Reg IV nucleic acid in the test tissue sample in comparison to a control tissue sample from an individual known to be negative for a cancer that overexpresses a Reg IV protein or mRNA transcript.
  • the tissue sample is serum, but can also be a tissue from a biopsy, particularly a urogenital tissue including a prostate or bladder tissue.
  • a positive diagnosis for a cancer that overexpresses a Reg TV protein or mRNA transcript is indicated when a higher level of Reg IV transcribed RNA is detected in a test tissue sample in comparison to a control tissue sample from an individual known not to have cancer.
  • the invention also provides methods for improving the response to cancer therapy in a cancer that overexpresses a Reg IV protein or mRNA transcript by administering a therapeutically effective amount of a compound that inhibits the binding of Reg IV protein to a Reg IV receptor on a cell of the cancer tumor tissue.
  • the methods of inhibiting Reg IV binding to its receptor are carried out concurrently with another anticancer therapy, including, for example, known chemotherapeutics, immunotherapeutics, and radiotherapy for the reversal of resistance, tumor progression, and metastasis.
  • the present invention further provides methods of inhibiting the growth of and promoting the regression of a tumor that overexpresses Reg IV protein, the methods comprising inhibiting the binding of Reg IV protein to a Reg IV receptor on a cell of the tumor tissue.
  • the methods can be carried out by administering to an individual in need thereof a sufficient amount of a compound that inhibits the binding of a Reg IV protein to a Reg TV receptor.
  • the compound specifically binds to a Reg IV protein.
  • the compound specifically binds to a Reg IV receptor.
  • the compound prevents the transcription or the translation of a Reg IV protein.
  • the methods find particular use in treating urogenital cancers, including prostate and bladder cancers.
  • the compound comprises a polypeptide, including an antibody or an analog or fragment of a Reg IV polypeptide.
  • the methods find particular application in the diagnosis, prognosis and treatment of prostate and bladder cancers.
  • the methods are applied to hormone refractory or therapy resistant cancers.
  • the methods are applied to metastatic cancers. For example comparisons of differential expression of a Reg IV protein and/or mRNA can be used to determine the stage of cancer of an individual having a cancer that overexpresses a Reg IV protein or mRNA transcript.
  • Treatment will generally involve the repeated administration of the anti-ReglV antibodies, immunoconjugates, inhibitors, and siRNA preparations via an acceptable route of administration such as intravenous injection (TV), at an effective dose.
  • TV intravenous injection
  • Dosages will depend upon various factors generally appreciated by those of skill in the art, including without limitation the type of cancer and the severity, grade, or stage of the cancer, the binding affinity and half life of the agents used, the degree of Reg IV expression in the patient, the extent of circulating shed Reg IV antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic agents used in combination with the treatment method of the invention.
  • Typical daily doses may range from about 0.1 to 100 mg/kg.
  • Doses in the range of 10-500 mg of the mAb or immunoconjugates per week may be effective and well tolerated, although even higher weekly doses may be appropriate and/or well tolerated.
  • the principal determining factor in defining the appropriate dose is the amount of a particular agent necessary to be therapeutically effective in a particular context. Repeated administrations may be required in order to achieve tumor inhibition or regression. Initial loading doses may be higher. The initial loading dose may be administered as an infusion. Periodic maintenance doses may be administered similarly, provided the initial dose is well tolerated.
  • the agents may be injected directly into the bladder. Because agents administered directly to bladder will be cleared from the patient rapidly, it may be possible to use non-human or chimeric antibodies effectively without significant complications of antigenicity.
  • the invention further provides vaccines formulated to contain a Reg IV protein or fragment thereof.
  • a tumor antigen in a vaccine for generating humoral and cell- mediated immunity for use in anti-cancer therapy is well known in the art and, for example, has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159: 3113- 3117).
  • Such methods can be readily practiced by employing a Reg IV protein, or fragment thereof, or a Reg IV-encoding nucleic acid molecule and recombinant vectors capable of expressing and appropriately presenting the Reg IV immunogen.
  • viral gene delivery systems may be used to deliver a Reg IV -encoding nucleic acid molecule.
  • Various viral gene delivery systems which can be used in the practice of this aspect of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and Sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).
  • Non-viral delivery systems may also be employed by using naked DNA encoding a Reg IV protein or fragment thereof introduced into the patient (e.g., intramuscularly) to induce an anti-tumor response.
  • the full-length human Reg TV cDNA may be employed.
  • Reg IV nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes can be determined using specific algorithms (e.g., Epimer, Brown University) to identify peptides within a Reg IV protein which are capable of optimally binding to specified HLA alleles.
  • CTL epitopes can be determined using specific algorithms (e.g., Epimer, Brown University) to identify peptides within a Reg IV protein which are capable of optimally binding to specified HLA alleles.
  • CTL epitopes can be determined using specific algorithms (e.g., Epimer, Brown University) to identify peptides within a Reg IV protein which are capable of optimally binding to specified HLA alleles.
  • CTL epitopes can be determined using specific algorithms (e.g., Epimer, Brown University) to identify peptides within a Reg IV protein which are capable of optimally binding to specified HLA alleles
  • Dendritic cells can be used to present Reg IV peptides to T cells in the context of MHC class I and II molecules.
  • autologous dendritic cells are pulsed with Reg IV peptides capable of binding to MHC molecules.
  • dendritic cells are pulsed with the complete Reg IV protein.
  • Yet another embodiment involves engineering the overexpression of the Reg IV gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al., 1996, Cancer Res.
  • Anti-idiotypic anti-Reg IV antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a Reg IV protein.
  • generation of anti-idiotypic antibodies is well known in the art and can readily be adapted to generate anti-idiotypic anti-Reg IV antibodies that mimic an epitope on a Reg IV protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al., 1996, Cancer Immunol Immunother 43: 65-76).
  • Such an anti- idiotypic antibody can be used in anti-idiotypic therapy as presently practiced with other anti- idiotypic antibodies directed against tumor antigens.
  • the invention further provides methods for inhibiting cellular activity (e.g., cell proliferation, activation, or propagation) of a cell expressing multiple Reg IV antigens on its cell surface.
  • This method comprises reacting the immunoconjugates of the invention (e.g., a heterogeneous or homogenous mixture) with the cell so that the Reg IV antigens on the cell surface forms a complex with the immunoconjugates.
  • the greater the number of Reg IV antigens on the cell surface the greater the number of Reg IV -antibody complexes can be used.
  • the greater the number of Reg TV -antibody complexes the greater the cellular activity that is inhibited.
  • a heterogeneous mixture includes Reg IV antibodies that recognize different or the same epitope, each antibody being conjugated to the same or different therapeutic agent.
  • a homogenous mixture includes antibodies that recognize the same epitope, each antibody being conjugated to the same therapeutic agent.
  • the invention further provides methods for inhibiting the biological activity of Reg IV by blocking Reg IV from binding its receptor.
  • the methods comprises contacting an amount of Reg IV with an antibody or immunoconjugate of the invention under conditions that permit a Reg IV -immunoconjugate or Reg IV -antibody complex thereby blocking Reg TV from binding its ligand and inhibiting the activity of Reg IV.
  • the invention provides a method of treating cancer, particularly a cancer which overexpresses Reg IV, or of inhibiting the growth of a cancer cell overexpressing a Reg IV protein by treating a subject or contacting the cancer cell with an antibody or fragment thereof that recognizes and binds the Reg IV protein in an amount effective to inhibit the growth of the cancer cell.
  • the cancer cell is a prostate cancer cell or a bladder cancer cell.
  • the contacting antibody can be a monoclonal antibody and/or a chimeric antibody.
  • the chimeric antibody comprises a human immunoglobulin constant region.
  • the antibody is a human antibody or comprises a human immunoglobulin constant region.
  • the antibody fragment comprises an Fab, F(ab) 2 , or Fv.
  • the fragment comprises a recombinant protein having an antigen-binding region.
  • the invention provides methods for treating cancer, particularly, a cancer overexpressing Reg IV or selectively inhibiting a cell expressing or overexpressing a Reg IV antigen by reacting any one or a combination of the immunoconjugates of the invention with the cell in an amount sufficient to inhibit the cell.
  • Such amounts include an amount to kill the cell or an amount sufficient to inhibit cell growth or proliferation.
  • the dose and dosage regimen will depend on the nature of the disease or disorder to be treated associated with Reg IV, its population, the site to which the antibodies are to be directed, the characteristics of the particular immunotoxin, and the patient.
  • the amount of immunoconjugate can be in the range of 0.1 to 200 mg/kg of patient weight.
  • the immunoconjugate can comprise the anti-Reg IV antibody or the fragment linked to a therapeutic agent.
  • the therapeutic agent can be cytotoxic agent.
  • the cytotoxic agent can be selected from a group consisting of ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethiduim bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin A chain, alpha-sarcin, gelonin mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, maytansinoids, and glucocorticoidric
  • a chemotherapeutic drug and/or radiation therapy can be administered further.
  • the patient also receives hormone antagonist therapy.
  • the contacting of the patient with the antibody or antibody fragment can be by administering the antibody to the patient intravenously, intraperitoneally, intramuscularly, intratumorally, or intradermally.
  • the patient has a urogenital cancer (e.g., bladder cancer, prostate cancer).
  • the patient suffers from prostate cancer and optionally further receives patient hormone ablation therapy.
  • the contacting comprises administering the antibody directly into the cancer or a metastasis of the cancer.
  • the immunoconjugate has a cytotoxic agent which is a small molecule.
  • cytotoxic agents such as maytansin, maytansinoids, saporin, gelonin, ricin or calicheamicin and analogs or derivatives thereof are also suitable.
  • Other cytotoxic agents that can be conjugated to the anti-Reg IV antibodies include BCNU, streptozoicin, vincristine and 5- fluorouracil. Enzymatically active toxins and fragments thereof can also be used.
  • the radio- effector moieties may be incorporated in the conjugate in known ways (e.g., bifunctional linkers, fusion proteins).
  • the antibodies of the present invention may also be conjugated to an effector moiety which is an enzyme which converts a prodrug to an active chemotherapeutic agent. See, WO 88/07378; U. S. Patent No. 4,975, 278; and U.S. Patent No. 6,949,245.
  • the antibody or immunoconjugate may optionally be linked to nonprotein polymers (e. g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol).
  • Conjugates of the antibody and cytotoxic agent may be made using methods well known in the art.
  • the conjugates may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diis
  • SPDP
  • a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.
  • the anti-Reg IV antibodies or immunoconjugates are administered to a human patient in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • Intravenous or subcutaneous administration of the antibody is preferred.
  • the administration may be local or systemic.
  • compositions for administration will commonly comprise an agent as described herein (e.g., Reg IV inhibitors, Reg IV antibodies and immunoconjugates, Reg IV siRNA and vectors thereof) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a typical pharmaceutical composition for intravenous administration will vary according to the agent. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
  • compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges.
  • antibodies when administered orally, should be protected from digestion. This is typically accomplished either by complexing the molecules with a composition to render them resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in an appropriately resistant carrier, such as a liposome or a protection barrier. Means of protecting agents from digestion are well known in the art.
  • compositions particularly, of the antibodies and immunoconjugates and inhibitors for use with the present invention can be prepared by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers.
  • Such formulations can be lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used.
  • Acceptable carriers, excipients or stabilizers can be acetate, phosphate, citrate, and other organic acids; antioxidants (e.g., ascorbic acid) preservatives low molecular weight polypeptides; proteins, such as serum albumin or gelatin, or hydrophilic polymers such as polyvinylpyllolidone; and amino acids, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents; and ionic and non-ionic surfactants (e.g., polysorbate); salt-forming counter-ions such as sodium; metal complexes (e. g. Zn-protein complexes); and/or non-ionic surfactants.
  • the antibody can be formulated at a concentration of between 0.5 - 200 mg/ml, or between 10-50 mg/ml.
  • the formulation may also provide additional active compounds, including, chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitory agent, and anti- hormonal agent.
  • the active ingredients may also prepared as sustained-release preparations (e.g., semi-permeable matrices of solid hydrophobic polymers (e.g., polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol) ), polylactides.
  • the antibodies and immunocongugates may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions can be administered for therapeutic or prophylactic treatments.
  • compositions are administered to a patient suffering from a disease (e.g., cancer) in a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient.
  • a "patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications. Li the preferred embodiment the patient is a mammal, preferably a primate, and in the most preferred embodiment the patient is human. Other known cancer therapies can be used in combination with the methods of the invention.
  • the compositions for use according to the invention may also be used to target or sensitize a cell to other cancer therapeutic agents such as 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.
  • the methods of the invention with other cancer therapies (e.g, radical prostatectomy), radiation therapy (external beam or brachytherapy), hormone therapy (e.g., orchiectomy, LHRH-analog therapy to suppress testosterone production, anti-androgen therapy), or chemotherapy.
  • Radical prostatectomy involves removal of the entire prostate gland plus some surrounding tissue. This treatment is used commonly when the cancer is thought not to have spread beyond the tissue. Radiation therapy is commonly used to treat prostate cancer that is still confined to the prostate gland, or has spread to nearby tissue. If the disease is more advanced, radiation may be used to reduce the size of the tumor.
  • Hormone therapy is often used for patients whose prostate cancer has spread beyond the prostate or has recurred.
  • LHRH Luteinizing hormone-releasing hormone
  • Anti-androgens e.g., flutamide, bicalutamide, and nilutamide
  • Total androgen blockade refers to the use of anti-androgens in combination with orchiectomy or LHRH analogs, the s combination is called.
  • Chemotherapy is an option for patients whose prostate cancer has spread outside of the prostate gland and for whom hormone therapy has failed.
  • doxorubicin Adriamycin
  • estramustine etoposide
  • mitoxantrone vinblastine
  • paclitaxel Two or more drags are often given together to reduce the likelihood of the cancer cells becoming resistant to chemotherapy.
  • Small cell carcinoma is a rare type of prostate cancer that is more likely to respond to chemotherapy than to hormonal therapy.
  • a "cardioprotectant” is also administered with the antibody Reg IV binding inhibitor or siRNA molecule for use to according to the invention.
  • a cardioprotectant is a compound or composition which prevents or reduces myocardial dysfunction (i.e. cardiomyopathy and/or congestive heart failure) associated with administration of a drug, such as an anthracycline antibiotic to a patient.
  • the cardioprotectant may, for example, block or reduce a free-radical-mediated cardiotoxic effect and/or prevent or reduce oxidative-stress injury.
  • cardioprotectants encompassed by the present definition include the iron-chelating agent dexrazoxane (ICRF- 187) (Seifert et al.
  • lipid-lowering agent and/or anti-oxidant such as probucol (Singal et al. J. MoI. Cell Cardiol. 27:1055-1063 (1995)); amifostine (aminothiol 2- [(3-aminopropyl)aniino]ethanethiol ⁇ dihydrogen phosphate ester, also called WR-2721, and the dephosphorylated cellular uptake form thereof called WR-1065) and S-3-(3- methylaminopropylamino)propylphosphoro- thioic acid (WR- 151327), see Green et al.
  • vitamin E ascorbic acid (vitamin C); free radical scavengers such as oleanolic acid, ursolic acid and N-acetylcysteine (NAC); spin trapping compounds such as alpha-phenyl-tert-butyl nitrone (PBN); (Paracchini et al., Anticancer Res. 13:1607-1612 (1993)); selenoorganic compounds such as P251 (Elbesen); and the like.
  • vitamin C ascorbic acid
  • free radical scavengers such as oleanolic acid, ursolic acid and N-acetylcysteine (NAC)
  • spin trapping compounds such as alpha-phenyl-tert-butyl nitrone (PBN); (Paracchini et al., Anticancer Res. 13:1607-1612 (1993)); selenoorganic compounds such as P251 (Elbesen); and the like.
  • the combined administrations contemplates coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • Reg IV protein modulators can be administered alone or coadministered in combination with conventional chemotherapy, radiotherapy or immunotherapy as well as currently developed therapeutics.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid such as water, saline or PEG 400
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor e.g., sucrose
  • an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • Aerosol formulations i.e., they can be "nebulized" to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • the composition can, if desired, also contain other compatible therapeutic agents.
  • Preferred pharmaceutical preparations deliver one or more active Reg IV protein modulators, optionally in combination with one or more chemotherapeutic agents or immunotherapeutic agents, in a sustained release formulation.
  • the Reg IV modulator is administered therapeutically as a sensitizing agent that increases the susceptibility of tumor cells to other cytotoxic cancer therapies, including chemotherapy, radiation therapy, immunotherapy and hormonal therapy.
  • the Reg IV modulators or inhibitors utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily.
  • the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. For example, dosages can be empirically determined considering the type and stage of cancer diagnosed in a particular patient.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
  • the pharmaceutical preparations for use according to the invention are typically delivered to a mammal, including humans and non-human mammals.
  • Non-human mammals treated using the present methods include domesticated animals (i.e., canine, feline, murine, rodentia, and lagomorpha) and agricultural animals (bovine, equine, ovine, porcine).
  • Modulation of a Reg IV protein, and corresponding modulation of cellular, e.g., tumor cell, proliferation can be assessed using a variety of in vitro and in vivo assays, including cell-based models. Such assays can be used to test for inhibitors and activators of a Reg IV protein, and, consequently, inhibitors and activators of cellular proliferation, including modulators of chemotherapeutic sensitivity and toxicity. Such modulators of a Reg IV protein are useful for treating disorders related to pathological cell proliferation, e.g., cancer. Modulators of Reg IV protein are tested using either recombinant or naturally occurring Reg IV, preferably human Reg IV.
  • Measurement of cellular proliferation modulation with a Reg IV protein or a cell expressing a Reg IV protein can be performed using a variety of assays, in vitro, in vivo, and ex vivo, as described herein.
  • a suitable physical, chemical or phenotypic change that affects activity e.g., enzymatic activity such as kinase activity, cell proliferation, or ligand binding (e.g., a Reg IV protein receptor) can be used to assess the influence of a test compound on the polypeptide of this invention.
  • the functional effects are determined using intact cells or animals, one can also measure a variety of effects, such as, ligand binding, kinase activity, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism, changes related to cellular proliferation, cell surface marker expression, DNA synthesis, marker and dye dilution assays (e.g., GFP and cell tracker assays), contact inhibition, tumor growth in nude mice, etc.
  • ligand binding kinase activity
  • transcriptional changes to both known and uncharacterized genetic markers e.g., northern blots
  • changes in cell metabolism e.g., changes in cell metabolism
  • changes related to cellular proliferation e.g., cell surface marker expression
  • DNA synthesis e.g., DNA synthesis
  • marker and dye dilution assays e.g., GFP and cell tracker assays
  • contact inhibition e.g., tumor growth in nude mice, etc.
  • Assays to identify compounds with Reg IV modulating activity can be performed in vitro. Such assays can use a full length Reg IV protein or a variant thereof (see, e.g., Figure 5), or a mutant thereof, or a fragment of a Reg IV protein. Purified recombinant or naturally occurring Reg IV protein can be used in the in vitro methods of the invention. In addition to purified Reg IV protein, the recombinant or naturally occurring Reg IV protein can be part of a cellular lysate or a cell membrane. As described below, the binding assay can be either solid state or soluble. Preferably, the protein or membrane is bound to a solid support, either covalently or non-covalently.
  • the in vitro assays of the invention are substrate or ligand binding or affinity assays, either non-competitive or competitive.
  • Other in vitro assays include measuring changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein.
  • Other in vitro assays include enzymatic activity assays, such as phosphorylation or autophosphorylation assays).
  • a high throughput binding assay is performed in which the Reg IV protein or a fragment thereof is contacted with a potential modulator and incubated for a suitable amount of time.
  • the potential modulator is bound to a solid support, and the Reg IV protein is added.
  • the Reg IV protein is bound to a solid support.
  • modulators can be used, as described below, including small organic molecules, peptides, antibodies, and Reg IV ligand analogs.
  • assays can be used to identify Reg IV-modulator binding, including labeled protein-protein binding assays, electrophoretic mobility shifts, immunoassays, enzymatic assays such as kinase assays, and the like.
  • the binding of the candidate modulator is determined through the use of competitive binding assays, where interference with binding of a known ligand or substrate is measured in the presence of a potential modulator.
  • microtiter plates are first coated with either a Reg IV protein or a Reg IV protein receptor, and then exposed to one or more test compounds potentially capable of inhibiting the binding of a Reg IV protein to a Reg IV protein receptor.
  • a labeled (i.e., fluorescent, enzymatic, radioactive isotope) binding partner of the coated protein, either a Reg IV protein receptor or a Reg IV protein is then exposed to the coated protein and test compounds. Unbound protein is washed away as necessary in between exposures to a Reg IV protein, a Reg IV protein receptor, or a test compound. An absence of detectable signal indicates that the test compound inhibited the binding interaction between a Reg IV protein and a Reg IV protein receptor.
  • detectable signal i.e., fluorescence, colorimetric, radioactivity
  • the presence or absence of detectable signal is compared to a control sample that was not exposed to a test compound, which exhibits uninhibited signal.
  • the binding partner is unlabeled, but exposed to a labeled antibody that specifically binds the binding partner.
  • Reg IV protein is expressed in a cell, and functional, e.g., physical and chemical or phenotypic, changes are assayed to identify Reg IV and modulators of cellular proliferation, e.g., tumor cell proliferation.
  • Cells expressing Reg IV proteins can also be used in binding assays and enzymatic assays. Any suitable functional effect can be measured, as described herein.
  • cellular morphology e.g., cell volume, nuclear volume, cell perimeter, and nuclear perimeter
  • ligand binding e.g., kinase activity, apoptosis
  • cell surface marker expression e.g., cell proliferation, GFP positivity and dye dilution assays (e.g., cell tracker assays with dyes that bind to cell membranes)
  • DNA synthesis assays e.g., 3 H- thymidine and fluorescent DNA-binding dyes such as BrdU or Hoechst dye with FACS analysis
  • Suitable cells for such cell based assays include both primary cancer or tumor cells and cell lines, as described herein, e.g., A549 (lung), MCF7 (breast, p53 wild-type), H1299 (lung, p53 null), HeIa (cervical), PC3 (prostate, p53 mutant), MDA-MB-231 (breast, ⁇ 53 wild-type). Cancer cell lines can be p53 mutant, p53 null, or express wild type p53.
  • the Reg IV protein can be naturally occurring or recombinant. Also, fragments of Reg IV or chimeric Reg IV proteins can be used in cell based assays.
  • Cellular Reg IV polypeptide levels can be determined by measuring the level of protein or mRNA.
  • the level of Reg IV protein or proteins related to Reg TV are measured using immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the Reg IV polypeptide or a fragment thereof.
  • immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the Reg IV polypeptide or a fragment thereof.
  • amplification e.g., using PCR, LCR, or hybridization assays, e.g., northern hybridization, RNAse protection, dot blotting, are preferred.
  • the level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
  • Reg IV expression can be measured using a reporter gene system.
  • a reporter gene system can be devised using an Reg IV protein promoter operably linked to a reporter gene such as chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, ⁇ -galactosidase and alkaline phosphatase.
  • the protein of interest can be used as an indirect reporter via attachment to a second reporter such as red or green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).
  • the reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art.
  • Animal models of cellular proliferation also find use in screening for modulators of cellular proliferation.
  • transgenic animal technology including gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, or gene overexpression, will result in the absence or increased expression of the Reg TV protein.
  • the same technology can also be applied to make knock-out cells.
  • tissue-specific expression or knockout of the Reg IV protein may be necessary.
  • Transgenic animals generated by such methods find use as animal models of cellular proliferation and are additionally useful in screening for modulators of cellular proliferation.
  • Knock-out cells and transgenic mice can be made by insertion of a marker gene or other heterologous gene into an endogenous Reg IV gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting an endogenous Reg IV with a mutated version of the Reg IV gene, or by mutating an endogenous Reg TV, e.g., by exposure to carcinogens.
  • a DNA construct is introduced into the nuclei of embryonic stem cells.
  • Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion ⁇ see, e.g., Capecchi et at, Science 244:1288 (1989)). Chimeric targeted mice can be derived according to Hogan et al.
  • Normal cells require a solid substrate to attach and grow. When the cells are transformed, they lose this phenotype and grow detached from the substrate.
  • transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, regenerate normal phenotype and require a solid substrate to attach and grow.
  • Soft agar growth or colony formation in suspension assays can be used to identify Reg IV modulators.
  • transformed host cells e.g., cells that grow on soft agar
  • RKO or HCTl 16 cell lines can be used.
  • Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique, 3 rd ed., Wiley-Liss, New York (1994), herein incorporated by reference. See also, the methods section of Garkavtsev et al. (1996), supra, herein incorporated by reference.
  • Normal cells typically grow in a flat and organized pattern in a petri dish until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. When cells are transformed, however, the cells are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, the transformed cells grow to a higher saturation density than normal cells. This can be detected morphologically by the formation of a disoriented monolayer of cells or rounded cells in foci within the regular pattern of normal surrounding cells. Alternatively, labeling index with [ 3 H] -thymidine at saturation density can be used to measure density limitation of growth. See Freshney (1994), supra. The transformed cells, when contacted with cellular proliferation modulators, regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
  • Reg IV modulators which are capable of inhibiting abnormal proliferation and transformation in host cells.
  • transformed host cells e.g., cells that are not contact inhibited
  • RKO or HCTl 16 cell lines can be used.
  • labeling index with [ 3 H] -thymidine at saturation density is a preferred method of measuring density limitation of growth.
  • Transformed host cells are contacted with a potential Reg IV modulator and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with [ 3 H] -thymidine is determined autoradiographically. See, Freshney (1994), supra.
  • the host cells contacted with a Reg IV modulator would give arise to a lower labeling index compared to control (e.g., transformed host cells transfected with a vector lacking an insert).
  • Growth factor or serum dependence can be used as an assay to identify Reg IV modulators.
  • Transformed cells have a lower serum dependence than their normal counterparts ⁇ see, e.g., Temin, J. Natl. Cancer lnsti 37:167-175 (1966); Eagle et al, J. Exp. Med. 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells.
  • the cells When transformed cells are contacted with a Reg TV modulator, the cells would reacquire serum dependence and would release growth factors at a lower level.
  • Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts.
  • plasminogen activator PA
  • PA plasminogen activator
  • Tumor specific markers For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells ⁇ see, e.g., Gullino, Angio genesis, tumor vascularization, and potential interference with tumor growth. In Mihich (ed.): "Biological Responses in Cancer.” New York, Academic Press, pp. 178-184 (1985)).
  • tumor angiogenesis factor TAF
  • TAF tumor angiogenesis factor
  • Tumor specific markers can be assayed to identify Reg IV modulators which decrease the level of release of these markers from host cells. Typically, transformed or tumorigenic host cells are used. Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al. , /. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305-312 (1980); Gulino, Angiogenesis, tumor vascularization, and potential interference with tumor growth. In Mihich, E. (ed): "Biological Responses in Cancer.” New York, Plenum (1985); Freshney Anticancer Res. 5:111-130 (1985).
  • the degree of invasiveness into Matrigel or some other extracellular matrix constituent can be used as an assay to identify Reg IV modulators which are capable of inhibiting abnormal cell proliferation and tumor growth.
  • Tumor cells exhibit a good correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent.
  • Li this assay tumorigenic cells are typically used as host cells. Therefore, Reg IV modulators can be identified by measuring changes in the level of invasiveness between the host cells before and after the introduction of potential modulators. If a compound modulates Reg IV, its expression in tumorigenic host cells would affect invasiveness.
  • the level of invasion of host cells can be measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125 I and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
  • GolG ⁇ cell cycle arrest can be used as an assay to identify Reg IV modulators.
  • cell lines such as RKO or HCTl 16 can be used to screen Reg IV modulators.
  • the cells can be co-transfected with a construct comprising a marker gene, such as a gene that encodes green fluorescent protein, or a cell tracker dye.
  • Methods known in the art can be used to measure the degree of G 1 cell cycle arrest.
  • a propidium iodide signal can be used as a measure for DNA content to determine cell cycle profiles on a flow cytometer.
  • the percent of the cells in each cell cycle can be calculated.
  • Cells contacted with a Reg IV modulator would exhibit, e.g., a higher number of cells that are arrested in GoZG 1 phase compared to control.
  • Knock-out cells and transgenic mice can be made by insertion of a marker gene or other heterologous gene into the endogenous Reg IV gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous Reg IV with a mutated version of Reg IV, or by mutating the endogenous Reg TV, e.g., by exposure to carcinogens.
  • a DNA construct is introduced into the nuclei of embryonic stem cells.
  • Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al, Science 244:1288 (1989)).
  • Chimeric targeted mice can be derived according to Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987). These knock-out mice can be used as hosts to test the effects of various Reg IV modulators on cell growth.
  • various immune-suppressed or immune-deficient host animals can be used.
  • genetically athymic "nude" mouse see, e.g., Giovanella et al, J. Natl. Cancer Inst. 52:921 (1974)
  • SCID mouse see, e.g., Giovanella et al, J. Natl. Cancer Inst. 52:921 (1974)
  • SCID mouse see, e.g., a SCID mouse, a thymectomized mouse, or an irradiated mouse
  • irradiated mouse see, e.g., Bradley et al, Br. J. Cancer 38:263 (1978); Selby et al, Br. J. Cancer 41:52 (1980)
  • Transplantable tumor cells (typically about 10 6 cells) injected into isogenic hosts will produce invasive tumors in a high proportions of cases, while normal cells of similar origin will not.
  • Hosts are treated with Reg IV modulators, e.g., by injection.
  • tumor growth is measured (e.g., by volume or by its two largest dimensions) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
  • Reg IV modulators which are capable, e.g., of inhibiting abnormal cell proliferation can be identified.
  • the present invention also provides methods of identifying compounds that inhibit the binding of a Reg IV protein to a Reg IV receptor, wherein said compounds find use in inhibiting the growth of and promoting the regression of a tumor that overexpresses Reg IV protein, for example a urogenital cancer tumor, including a prostate or bladder cancer tumor.
  • Screening assays can be carried out in vitro or in vivo. Typically, initial screening assays are carried out in vitro, and can be confirmed in vivo using cell based assays or animal models. For instance, proteins of the regenerating gene family are involved with cell proliferation. Therefore, compounds that inhibit the binding of a Reg IV protein to a Reg IV receptor can inhibit cell proliferation resulting from this binding interaction in comparison to cells unexposed to a test compound. Also, the binding of a Reg IV protein to a Reg IV receptor is involved with tissue injury responses, inflammation, and dysplasia.
  • compounds that inhibit the binding of a Reg IV protein to a Reg IV receptor can, for example, inhibit wound healing or the progression of dysplasia in comparison to an animal unexposed to a test compound. See, for example, Zhang, et al., World J Gastroenter (2003) 9:2635-41.
  • a compound that inhibits the binding of Reg IV to a Reg IV receptor is synthetic.
  • the screening methods are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel ⁇ e.g., in microtiter formats on microtiter plates in robotic assays).
  • the invention provides in vitro assays for inhibiting Reg IV binding to its receptor in a high throughput format.
  • "no modulator" control reactions which do not include a modulator provide a background level of Reg IV binding interaction to its receptor or receptors.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (96) modulators.
  • 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many different plates per day; assay screens for up to about 6,000-20,000, and even up to about 100,000-1,000,000 different compounds is possible using the integrated systems of the invention.
  • the steps of labeling, addition of reagents, fluid changes, and detection are compatible with full automation, for instance using programmable robotic systems or "integrated systems" commercially available, for example, through BioTX Automation, Conroe, TX; Qiagen, Valencia, CA; Beckman Coulter, Fullerton, CA; and Caliper Life Sciences, Hopkinton, MA.
  • any chemical compound can be tested as a potential inhibitor of Reg IV binding to its receptor for use in the methods of the invention. Most preferred are generally compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland), as well as providers of small organic molecule and peptide libraries ready for screening, including Chembridge Corp.
  • inhibitors of Reg IV-Reg IV receptor binding interaction are identified by screening a combinatorial library containing a large number of potential therapeutic compounds (potential modulator compounds).
  • potential modulator compounds potential modulator compounds
  • Such "combinatorial chemical or peptide libraries” can be screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity.
  • the compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)).
  • Other chemistries for generating chemical diversity libraries can also be used.
  • Such chemistries include, but are not limited to: peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication No.
  • WO 92/00091 benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with ⁇ -D-glucose scaffolding (Hirschmann etal., J. Amer. Chem. Soc.
  • Patent 5,539,083) antibody libraries ⁇ see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries ⁇ see, e.g., Liang etal, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries ⁇ see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
  • antibody libraries see, e.g., Vaughn et al, Nature Biotechnology, 14
  • RNA samples from a matched pair of androgen dependent and independent LAPC 9 xenografts were grown and prepared as described previously (Craft et al., Cancer Research, In Press (1999)). Total RNA was isolated by using Ultraspec RNA isolation systems (Biotecx). mRNA was purified using Oligotex mRNA Midi Kit (Qiagen). 2 micrograms of mRNA was reverse transcribed, and cDNA then labeled with Cy-5.
  • Labeled tumor cDNA was combined with a Cy-3 labeled common reference RNA derived from 11 different cell lines and hybridized to cDNA microarrays containing 22,648 elements representing 17,083 genes, as reported previously (Eisen, M. B. and Brown, P. O., Methods Enzymol, 303: 179-205 (1999)).
  • the slides were scanned with a GenePix microarray scanner (Axon Instruments) and were analyzed with Genepix software. Spots of insufficient quality by visual inspection were excluded from analysis. Data files were entered into the Stanford Microarray Database where spot intensity was correlated with gene identification 4 .
  • RNA Probes and in situ Hybridization A 399 by DNA fragment from the 3 'untranslated region of Reg IV (GeneBank AI 732541) was inserted into the pCR2.1 vector (Invitrogen) in both sense and antisense orientations under the control of the T7 promoter. Plasmids were linearized and digoxigenin labeled riboprobes were generated using the DIG RNA Labeling Kit (Roche Applied Science). Automated ISH was performed on the Discovery System (Ventana Medical Systems, Arlington, AZ, USA).
  • Sense and anti-sense riboprobes were diluted at 1:100 (1 ⁇ g of probe/ml) in hybridization solution (50% deionized formamide, 10% polyethylenglycol, 0.3M NaCl, 1OmM Tris pH 8.0, ImM EDTA, Denhardt's solution IX, yeast tRNA 500 ⁇ g/ml, 5OmM DTT).
  • hybridization was performed for 6 hours at 65°C with Dark blue cytoplasmic staining was scored as positive 100:1 of hybridization solution. After hybridization, slides were washed twice at 70 0 C for 6 minutes in l.OX SSC. The hybridization was followed by a 30 minute incubation with a biotinylated anti digoxigenin (Sigma Bio Sciences, St Louis, MO, USA), followed by alkalinephosphatase conjugated streptavidin for 16 minutes. Visualization was in NBT/BCIP (Ventana mapBlue) for 5 hours, and hematoxylin counterstain.
  • Cell lysates and conditioned medium were incubated with 3 micrograms of anti-myc monoclonal antibody (9E10, ATCC) and 20 microliters of protein G-Sepharose CL-4B (Amersham) for 2 hours at 4 0 C. Samples were washed and boiled in SDS sample buffer for 5 min and separated on 12.5% SDS-PAGE. The gel was treated with Amplify (Amersham) before drying and autoradiographed.
  • RNAs were extracted as described above. 10 micrograms of RNA was separated on a 1.2% agarose denaturing gel, transferred to nitrocellulose filters, and hybridized with RT-PCR-prepared DNA fragments of Reg IV (GeneBank AI732541). Probes were labeled with alpha 32 PdCTP by random priming using the Random Primer Labeling System (Amersham) and hybridization was carried out at 62°C in 6xSSC overnight, followed by washing with 2xSSC-0.1 % SDS and 0.2xSSC-0.1 % SDS at 62°C. For multiple tissue northern analysis, the hybridization was performed as described by the manufacture (Clontech).
  • TMA prostate cancer progression
  • This TMA is composed of benign prostate tissue, localized prostate cancer, and hormone refractory metastatic prostate cancer. These cases came from well-fixed radical prostatectomy specimens from the University of Michigan (Ann Arbor, Michigan), the University Hospital UIm (UIm, Germany), and the rapid autopsy program from the University of Michigan Specialized Program of Research Excellence in Prostate Cancer (Rubin et al., Clin Cancer Res, 6: 1038-1045 (2000)). All samples were collected with prior Institutional Review Board approval at each respective institution.
  • This TMA was composed of classic acinar prostate cancers and areas demonstrating foamy gland features from the same cases. Benign tissue samples were also placed in the TMA to serve as a negative control. A second array containing predominately metastatic cases was also stained and scored.
  • Reg IV expression was determined using a validated scoring method (Varambally et al., Nature, 419: 624-629 (2002); Rubin et al., Jama, 287: 1662-1670 (2002); Dhanasekaran et al., Nature, All: 822-826 (2001); Kuefer et al., Am J Pathol, 161: 841-848 (2002)) where staining was evaluated for intensity. Benign epithelial glands and prostate cancer cells were scored for Reg IV staining intensity on a 4 tiered system ranging from negative to strong expression. A score of 1 was negative. A score of 2 was considered low expression. A score of 3 indicated moderate expression, and a score of 4 correlated with strong expression. Slides were read independently by two pathologists (MAR and ML) with >90% interobserver agreement.
  • MAR and ML pathologists
  • Myc.His tagged Reg IV construct was PCR amplified from a pcDNA Reg IV-Myc.His vector and inserted into the lenti viral vector CCR through restriction sites of EcoRI and Nhel (Barry et al., Hum Gene Therapy, 12: 1103-1108 (2001)).
  • Lentivirus stocks were generated by calcium phosphate mediated transfection of 293T cells. The titer of the virus was checked with 293T cells using CCR EGFP as a positive control and indicator.
  • LNCaP and LAPC-9 Prostate Xenograft models 5 xlO 6 LNCaP or LNCaP-Reg rV.Myc.His cells were mixed with equal volume of Matrigel and inoculated into SCID mice subcutaneously.
  • explanted tumor is digested with pronase and cultured in 10% FBS-RPMI 1640 for 16 hours.
  • Cells are then transduced by CCR lentivirus vector alone or lentivirus Reg. IV.Myc.His at M.O.I, of 5 for 2 hours. Cells are then washed with culture media, mixed with Matrigel, and inoculated back to SCID mice (IxIO 6 cells/mice).
  • LAPC 9 xenograft was established from metastatic prostate tumors and progresses in vivo from androgen dependence to independence.
  • RNAs from paired androgen dependent (AD) and androgen independent (AI) LAPC 9 tumors were labeled and hybridized to 24,000 spot cDNA arrays with common reference RNA.
  • 204 clones representing 101 named genes and 59 ESTs showed expression variation of at least 4-fold between the AD and AI samples.
  • GDF1(7.9) RGS1*(6.8) EDNRB(6.3) MME*(5.8) FGF12(5.0) ABCA5*(4.7) RGS5(5.9) RAB32(5.8) ANXAl *(4.8)
  • a full length cDNA was obtained by 5 1 and 3' RACE PCR, sequenced and found to be identical to Reg IV, a newly described member of the Reg gene family.
  • Reg IV has an open reading frame of 474 bp, predicting a peptide of 158 amino acids with an N terminal signal sequence of 22 amino acids. It is 39% similar to Reg I and Reg III, the other two members of this gene family in humans.
  • Reg IV Encodes a Secreted Protein of ⁇ 20 kd and is Detectable in Serum of Tumor- Bearing Animals
  • Reg IV is predicted to be a secreted protein based on the presence of a putative signal sequence and on its homology to Reg I and in. To confirm this prediction, we transiently expressed a Myc-tagged Reg IV cDNA construct in 293T cells and harvested the cell pellets and conditioned media. As shown in Figure Ic, the majority of Reg IV protein was found in the culture medium, consistent with the conclusion that Reg IV is a secreted protein. A single band of -20 kd was identified, again consistent with the predicted molecular weight of Reg IV.
  • Reg IV was identified in hormone refractory LAPC-9 sublines, suggesting that Reg IV is involved in hormone refractory prostate cancer progression.
  • sense and antisense Reg IV probes were generated and hybridized to four radical prostatectomy specimens obtained from patients with high-risk (high grade, locally advanced) tumors treated with neoadjuvant hormone ablation therapy for three to eight months.
  • AU four cases had residual disease, which stained specifically with the antisense Reg FV probe, but not the control sense probe. No staining was seen in residual adjacent normal tissue (Figure 2A).
  • Reg IV is secreted, it is also useful as a serum marker to identify patients with metastasis or at risk to develop metastases. This possibility is supported by the ability to detect Reg IV in the serum of tumor-bearing animals. Antibodies against Reg IV are currently being generated to assess Reg IV protein expression in tissue samples and to measure circulating Reg IV levels in normal and cancer patients. An important issue will be to determine if Reg IV expression in the gastrointestinal tract interferes with the detection of Reg IV from tumor tissue. [0183] Reg IV was cloned from a hormone refractory xenograft and is expressed by both androgen resistant local tumors and metastases.
  • Reg IV expression is related specifically to androgen independence and/or metastasis, since all of the metastases were obtained from hormone refractory patients. Reg IV expression does not appear to be regulated by androgen, since androgen starvation of both LAPC-4 and LNCaP prostate cancer cell lines in tissue culture did not result in Reg IV expression. Nor did androgen independent sublines of LNCaP or LAPC-4 express Reg IV in vivo.
  • Reg proteins have been associated with proliferation and regeneration, cell survival, resistance to apoptosis and cell adhesion. Hartupee and associates reported that Reg IV is highly expressed in ulcerative colitis and hypothesized that it might be related to the high rate of colon cancer in individuals with this disease (Hartupee et al., Biochim Biophys Acta, 1518: 287-293 ( 2001)). Violette et al. found a consistent relationship between Reg IV expression and chemotherapy resistance in colon cancer cell lines (Violette et al., hit J Cancer, 103: 185- 193 (2003)). They found that Reg IV is expressed by 5/7 chemoresistant lines, but is absent from all chemosensitive lines.
  • Reg IV is expressed by LS513, a cell line that survives but does not proliferate in the presence of chemotherapy, suggesting that Reg IV may be a survival factor rather than a mitogen.
  • Reg I alpha is a signaling intermediate in a survival pathway in motoneurons (Nishimune et al., Nat Cell Biol, 2: 906-914 (2000)).
  • the hypothesis that Reg IV plays a role in cell survival is consistent with its expression in hormone refractory prostate cancer.
  • the association of Reg IV expression with chemotherapy resistance is also consistent with the fact that a majority of patients with lethal prostate cancer metastases on our tissue array received chemotherapy during their clinical course.
  • Reg IV is the second gastrointestinal secreted protein that we have identified in prostate cancer.
  • Intestinal trefoil factor (ITF/TFF3) was initially identified in prostate cancer arrays and has since been reported to be expressed by -40% of localized prostate cancers and a higher percentage of metastases (Luo et al., Cancer Res, 61: 4683-4688 (2001); Garraway et al., Prostate. 61: 209-214 (2004); Faith et al., Prostate, 61: 215-227 (2004)).
  • Trefoil factors are known to play an important role in intestinal protection and restitution, a process in which mucosal continuity is reestablished following tissue injury, whereas Reg proteins are believed to play a role in tissue regeneration (Taupin et al., Nat Rev MoI Cell Biol, 4: 721- 732 (2003)). Both Reg and trefoil proteins are overexpressed in inflammatory bowel disease. Both are also overexpressed in malignancy. TFF3, for example, is an adverse prognostic factor in gastric cancer (Taupin et al., Nat Rev MoI Cell Biol 4: 721-732 (2003)).

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Abstract

La présente invention concerne des procédés de diagnostic qui utilisent une cible de traitement et de diagnostic pour le traitement des cancers qui surexprime Reg IV, y compris les cancers de la prostate et de la vessie. L'invention concerne aussi des procédés de découverte de médicaments destinés à identifier des agents pharmaceutiques qui inhibent ou empêchent la liaison de Reg IV à son récepteur, qui sont utiles lorsqu'ils sont utilisés isolément ou en combinaison avec des produits chimiothérapeutiques, immunothérapeutiques ou radiothérapeutiques connus à des fins d'inversion de la résistance, de la progression tumorale et des métastases du cancer associées à la surexpression de Reg IV.
PCT/US2006/009524 2005-03-15 2006-03-15 Reg iv, cible pour le diagnostic et le traitement du cancer WO2006099539A1 (fr)

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WO2008020596A2 (fr) * 2006-08-18 2008-02-21 Oncotherapy Science, Inc. Traitement ou prévention de cancers surexprimant le reg4 ou le kiaa0101

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US20040254101A1 (en) * 1995-06-06 2004-12-16 Human Genome Sciences, Inc. Colon specific gene and protein and cancer

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* Cited by examiner, † Cited by third party
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US20040254101A1 (en) * 1995-06-06 2004-12-16 Human Genome Sciences, Inc. Colon specific gene and protein and cancer

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008020596A2 (fr) * 2006-08-18 2008-02-21 Oncotherapy Science, Inc. Traitement ou prévention de cancers surexprimant le reg4 ou le kiaa0101
WO2008020596A3 (fr) * 2006-08-18 2008-06-12 Oncotherapy Science Inc Traitement ou prévention de cancers surexprimant le reg4 ou le kiaa0101

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