WO2002020032A1 - Agonistes et antagonistes de stat3 et applications therapeutiques de ceux-ci - Google Patents

Agonistes et antagonistes de stat3 et applications therapeutiques de ceux-ci Download PDF

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WO2002020032A1
WO2002020032A1 PCT/US2001/028254 US0128254W WO0220032A1 WO 2002020032 A1 WO2002020032 A1 WO 2002020032A1 US 0128254 W US0128254 W US 0128254W WO 0220032 A1 WO0220032 A1 WO 0220032A1
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stat3
cells
compound
expression
activity
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PCT/US2001/028254
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WO2002020032A9 (fr
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Hua Yu
Drew Pardoll
Richard Jove
William Dalton
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John Hopkins University
University Of South Florida
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Priority to CA002421723A priority Critical patent/CA2421723A1/fr
Priority to US10/380,020 priority patent/US20040052762A1/en
Priority to EP01970740A priority patent/EP1324763A4/fr
Publication of WO2002020032A1 publication Critical patent/WO2002020032A1/fr
Publication of WO2002020032A9 publication Critical patent/WO2002020032A9/fr
Priority to US11/526,367 priority patent/US20070072822A1/en
Priority to US12/420,704 priority patent/US20090227489A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to methods for modulating, i.e., agonizing or antagonizing, Stat3 (signal transducer and activator of transcription3) signaling activity for use in gene therapy.
  • Inhibition and/or activation of Stat3 signaling is an effective therapeutic approach to modulate angiogenesis and the immune-response in various diseases.
  • STATs Signal transducers and activators of transcription
  • STAT proteins were originally defined in the context of normal cell signaling where STATs have been implicated in control of cell proliferation, differentiation, and apoptosis (Bromberg and Darnell, 2000, Oncogene, 19:2468-2473; Darnell et al, 1994, Science 264:1415-1421).
  • Stat3 ⁇ is a dominant-negative Stat3 variant, which is a truncated form of Stat3 that contains the dimerization and DNA binding domain but lacks the transactivation domain (Catlett-Falcone et al., 1999, Immunity, 10:105-115). As a consequence, Stat3 ⁇ can bind DNA but cannot transactivate gene expression, thus blocking Stat3 signaling in a trans- dominant negative fashion in most cases. Blocking Stat3 by Stat3 ⁇ in U266 cells down- regulated expression of the Stat3 -regulated Bcl-X L gene, resulting in a dramatic sensitization of cells to Fas-mediated apoptosis in vitro (Catlett-Falcone et al., 1999, supra). Effective gene therapy requires the killing of genetically untransduced cells
  • the present invention provides methods for use of Stat3 agonists and antagonists for treatment of disclose involving angiogenesis and immune disorders.
  • the invention is based, in part, on the Applicants' discovery that inhibition of Stat3 signaling results in the induction of a cascade of immunologic danger signals, which are normally produced only during inflammation and infection.
  • the cellular expression of a Stat3 antagonist results in the production of soluble factors which can induce the expression of pro- inflammatory cytokines and chemokines in neighboring cells.
  • the present invention takes advantage of this "bystander effect" of Stat3 activity modulators to provide methods and compositions for the treatment of a variety of conditions, diseases and disorders.
  • the invention further provides methods for identification of such soluble factors, herein termed "immunological danger signals".
  • the term "immunologic danger signals” refers to soluble factors produced as a result of inhibition of Stat3, which induce an immune response, such as a pro-inflammatory signal, e.g., a pro-inflammatory cytokine.
  • the present invention provides a method for modulating angiogenesis comprising administering to an individual in need of treatment an effective amount of a compound that agonizes or antagonizes the activity of Stat3.
  • the present invention further provides a method for the treatment or prevention of a hypoxic or ischemic condition or disorder, comprising administering to an individual in need of treatment an effective amount of a compound that increases the activity of Stat3, so that the hypoxic or ischemic condition or disorder is treated or prevented.
  • the compound is Stat3.
  • the compound is a constitutive active form of Stat3 (Stat3-C).
  • the compound is interleukin-6.
  • the condition or disorder is the result of ischemia, coronary-atherosclerosis, myocardial infarction, tissue ischemia in the lower extremities, infarction, inflammation, trauma, stroke, vascular occlusion, prenatal or postnatal oxygen deprivation, suffocation, choking, near drowning, carbon monoxide poisoning, smoke inhalation, trauma, including surgery and radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotensive shock, septic shock, anaphylactic shock, insulin shock, cardiac arrest, dysrhythmia, or nitrogen narcosis.
  • a method for the treatment or prevention of a proliferative angiopathy with neovascularization comprising administering to an individual in need of treatment an effective amount of a compound that decreases the activity of Stat3, so that the a proliferative angiopathy is treated or prevented.
  • the proliferative angiopathy is diabetic microangiopathy.
  • the compound is Stat3 ⁇ .
  • the compound is a negative regulatory protein.
  • the compound is a Stat3 antisense nucleic acid molecule.
  • the compound is a ribozyme specific to Stat3.
  • the compound is an inhibitor of a positive regulator of Stat3.
  • the compound is an antibody specific to Stat3.
  • the invention further provides a method for suppressing an immune response, comprising administering to an individual in need of treatment an effective amount of a compound that increases the activity of Stat3.
  • the compound is Stat3.
  • the compound is a constitutive active form of Stat3 (Stat3-C).
  • the compound is interleukin-6.
  • the treatment of the individual ameliorates a symptom of an autoimmune disease.
  • the autoimmune disease is insulin dependent diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten- sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia,
  • thrombocytopenia purpura rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, or dense deposit disease.
  • a method for activating an immune response comprising administering to an individual in need of treatment an effective amount of a compound that decreases the activity of Stat3, with the proviso that the treatment is not a cancer treatment.
  • the compound is Stat3 ⁇ .
  • the compound is a negative regulatory protein.
  • the compound is a Stat3 antisense nucleic acid molecule.
  • the compound is a ribozyme specific to Stat3.
  • the compound is an inhibitor of a positive regulator of Stat3.
  • the compound is an antibody specific to Stat3.
  • the therapeutic compound is delivered via gene therapy.
  • the compound is delivered to the individual with a pharmaceutically acceptable carrier.
  • the invention further provides a method for identifying an immunologic danger signal comprising: (a) inhibiting Stat3 signaling activity in cells in culture; (b) separating the supernatant from said cells; (c) adding said supernatant, or fractions thereof, to immune cells; and (d) assaying for activation of said immune cells; such that if immune cells are activated by a cell supernatant or a fraction thereof, then an immunological danger signal is identified.
  • the invention further provides a composition comprising the cell supernatant or a fraction thereof that is the product of this method.
  • the immune cells are macrophages.
  • said assaying for activation of said immune cells comprises assaying said macrophages for NO production.
  • said assaying for activation of said immune cells comprises assaying said macrophages for iNOS expression.
  • said assaying for activation of said immune cells comprises assaying said macrophages for RANTES expression.
  • the immune cells of the method are neutrophils.
  • said assaying for activation of said immune cells comprises assaying said neutrophils for TNF- ⁇ expression.
  • the immune cells are T cells.
  • said assaying for activation of said immune cells comprises assaying said T cells for for IFN- ⁇ expression.
  • said assaying for activation of said immune cells comprises assaying said T cells for IL-2 expression
  • the cells of the method are B16 cells.
  • the Stat3 is suppressed by a Stat3 signaling activity antagonist.
  • the antagonist is a dominant negative Stat3 mutant.
  • the antagonist is a negative regulatory protein.
  • the antagonist is a Stat3 antisense nucleic acid molecule.
  • the antagonist is a ribozyme specific to Stat3.
  • the antagonist is an inhibitor of a positive regulator of Stat3.
  • the antagonist is an antibody specific to Stat3.
  • Stat signal transducer and activator of transcription
  • Stat3, signal transducer and activator of transcription3 signal transducer and activator of transcription3
  • TRAIL TNF-related apoptosis-inducing ligand
  • EMSA electrophoretic mobility shift assay
  • hSIE high-affinity sis-inducible element
  • EGFP enhanced green fluorescence protein
  • FACS fluorescence-activated cell sorting
  • pIRES vector comprising an internal ribosome entry site
  • IL interleukin
  • Stat3 ⁇ or Stat3beta a dominant negative form of signal transducer and activator of transcription3
  • Stat3-C a constitutive active form of signal transducer and activator of transcription3
  • VEGF vascular endothelial growth factor
  • pIRE palindromic interferon response element.
  • FIG. 1 Inhibition of endogenous Stat3 (SEQ. ID. NO:l; SEQ. ID. NO:2) DNA-binding activity in B16 cells by overexpression of Stat3 ⁇ (SEQ. ID. NO:3; SEQ. LO. NO:4).
  • EMS A was performed with nuclear extracts prepared from B16 cells transfected with no DNA (lane 1), empty vector (lane 2) or Stat3 ⁇ expression vector (lane 3). Extracts from
  • ⁇ NTH3T3 fibroblasts stimulated with EGF were used as a positive control for Statl and Stat3 (lane 4).
  • Supershifts were performed using antibodies recognizing either Stat3 ( ⁇ -ST3) or Stat3 ⁇ ( ⁇ -ST3 ⁇ ) with extracts derived from B 16 cells transfected with no DNA (lanes 5-7), the empty vector (lanes 8-10), or Stat3 ⁇ vector (lanes 11-13).
  • ST3:3, and ST1:3, ST1:1 indicate migration of complexes containing Stat3 or Stat3 ⁇ homodimers, Statl /Stat3 ⁇ heterodimers and Statl/Statl homodimers, respectively.
  • the asterisk indicates the position of supershifted complexes.
  • FIG. 2A-C Soluble factors produced by Stat3 ⁇ -transfected B16 cells induce growth inhibition of non-transfected B16 cells.
  • A Growth inhibition analysis using supernatants 5 derived from either empty vector or Stat3 ⁇ transfected B16 cells collected at 0 h, 12 h, 24 h, 36 h, 48 h after transfection. Growth inhibition of B16 cells by supernatants from Stat3 ⁇ - B16 at various times was expressed as % inhibition based on the formula, (No. cells control - No. cells experimental)/No. cells control x 100.
  • 3 H-TdR incorporation assays 0.25 ⁇ Ci 3 H-TdR was added during the last 4 h of incubation.
  • MTT assays 5 ⁇ l MTT (10
  • B Cell cycle analysis. B16 cells were transfected in the lower chambers of Transwell units. Five hours later, non-transfected B 16 cells were added to upper chambers. Another 48 h later, B16 cells in the upper chambers were harvested for cell cycle analysis.
  • C Apoptosis assays. After incubating with transfected cells in the lower chambers for 48 h, cells in upper chambers were harvested and
  • AAD and FL3-H represents Annexin V-PE.
  • FIG. 3A-C Overexpression of Stat3 ⁇ induces cell cycle arrest and apoptosis in B16 30 cells.
  • FIG. 5A-B Blocking Stat3 signaling in B16 cells stimulates production of soluble factors capable of inducing iNOS-dependent nitric oxide production by macrophages.
  • A. Kinetics of availability of soluble factors after Stat3 ⁇ transfection. Supernatants from transfected B16 cells were taken out at the times indicated. The data shown represent one of two experiments with similar results and expressed as ⁇ M nitrite ⁇ SD, n 4.
  • Macrophages activated by the supernatant derived from Stat3 ⁇ -transfected B16 cells confers NO-mediated cytostatic activity against untransfected B 16 cells.
  • FIG. 7A-B Stat3 ⁇ expression in B16 cells upregulates the expression of pro- inflammatory chemokines and cytokines, which can stimulate peritoneal macrophages to produce NO. Elevated expression of pro-inflammatory cytokines and chemokines in B16 cells as a result of Stat3 ⁇ expression (A. TNF- ⁇ , IL-6, IFN- ⁇ ; B. IP-10).
  • Total RNAs were prepared form mock-transfected, GFP -transfected, Stat3 ⁇ -transfected and UV-irradiated B16 cells. Data shown have been confirmed with at least one more experiment, in which RNAs were prepared from independent transfectants and UV-irradiated B16 tumor cells.
  • FIG. 8A-B Factors secreted by Stat3 ⁇ -transfected B16 cells upregulate the expression of pro-inflammatory cytokines and chemokines by macrophages and neutrophils. Macrophages and neutrophils were incubated with supernatants derived from either empty vector-transfected, Stat3 ⁇ -transfected or UV-irradiated (macrophage only) B16 cells.
  • A RNAse protection assays using RNAs prepared from macrophages treated with various supernatants as indicated. Data shown represent RNAs pooled from two RNA preparations isolated from macrophages stimulated with supernatants derived from two independent transfections. B.
  • FIG. 9A-B Expression of Stat3 ⁇ in tumor cells leads to activation of macrophages and T lymphocytes in vivo.
  • Mice were injected with B16 cells (2 X lOVmouse) transfected with either GFP or Stat3 ⁇ expression vectors. Five days later, peritoneal macrophages and lymphocytes were tested for NO production and IFN- ⁇ production, respectively.
  • FIG. 10A-B Transfection of NIH3T3-Src cells with either: A. Stat3 ⁇ expression vector; or B. Stat3 anti-sense oligos, results in reduced levels of VEGF protein as shown by Western blot.
  • Src tyrosine kinase-mediated VEGF upregulation requires Stat3.
  • Src tyrosine activity is known to upregulate VEGF expression. In Src-transformed NIH3T3 cells, VEGF expression is high.
  • FIG. 11 Expression of constitutively-activated Stat3 (SEQ. ID. NO:5) increases the production of VEGF in NTH3 fibroblasts.
  • Left panel Stat3 DNA-binding activity in NIH3T3 stable clones transfected with Stat3C, a mutant forai of Stat3 that is constitutively activated.
  • FIG 12A-B Blocking Stat3 signaling in tumor cells inhibits VEGF promoter activity.
  • Both B 16 (A) and SCK (B) murine tumor cells were transfected with constructs containing the luciferase cDNA in the absence (pluc) or presence of the VEGF promoter (VEGF). While VEGF promoter activity was high in both tumor cells, co-transfection with anti-sense oligonucleotides (ASO) against Stat3 resulted in a dramatic reduction of VEGF promoter activity.
  • Figure 13 Inhibition of Stat3 signaling in tumor cells reduces the expression of the endogeneous VEGF gene.
  • B16 tumor cells were transfected with either: A. Stat3 ⁇ ; or B.
  • Stat3 anti-sense oligonucleotides at 100 nM, 200 nM, or 300 nM.
  • Western blot analyses indicated that a decrease in Stat3 protein is correlated with a reduction in VEGF protein, ⁇ -actin is used here to indicate the amount of protein loaded in each lane.
  • Stat3 is an essential regulator of several cellular and physiological processes, such as cell cycle, apoptosis, the immune response, and angiogenesis, as exemplified by the experiments in Section 6, 7 and 8.
  • Stat3 activity modulators can up-regulate or down-regulate cell cycle, apoptosis, immune-response, and angiogenesis respectively.
  • agonists and antagonists of Stat3 activity can be used to modulate cell cycle, apoptosis, immune-response, and angiogenesis to treat disorders involving dysfunctions of cell cycle, control of apoptosis, immune-response, and angiogenesis.
  • Such methods and compositions may be used to treat and/or prevent such diseases or disorders as, for example, ischemic diseases and proliferative angiopathies with neovascularization.
  • the methods and compositions described herein may be used to augment the immuneresponse to treat various diseases, such as cancer or inflammatory diseases, or to suppress the immune response to treat diseases and disorders such as autoimmune disorders.
  • target diseases and disorders are further described hereinbelow.
  • the invention provides methods of treatment and prophylaxis by administration to a subject of an effective amount of an agonist or antagonist of Stat3 activity, which are also referred to collectively herein as "Stat3 activity modulators” or “pharmaceuticals of the invention".
  • Stat3 activity modulators include, but are not limited to, peptides, polypeptides, nucleic acids, and small molecules.
  • polypeptide Stat3 activity modulators include, e.g., Stat3 ⁇ , a dominant negative form of the Stat3 gene constitutive active Stat3, the wild-type Stat3 gene, product and antibodies specific to Stat3.
  • Nucleotide sequences that can be used to inhibit Stat3 gene expression include, for example, antisense and ribozyme molecules, as well as gene or regulatory sequence replacement constructs designed to enhance the expression of Stat3, Stat3beta, or constitutive active Stat3 (e.g., expression constructs that place the Stat3 gene under the control of a strong promoter system).
  • Such Stat3 activity modulators are described in detail hereinbelow.
  • the invention further provides methods for the identification of "immunologic danger signals” and compositions comprising such immunologic danger signals, which may be used to stimulate an immune response.
  • the term "immunologic danger signal” refers to a signal which stimulates an immune response, such as a pro-inflammatory signal, e.g. pro-inflammatory cytokines and chemokines. Such methods for identification of immunological danger signals are further described hereinbelow.
  • methods are provided for stimulating angiogenesis using Stat3 agonists.
  • the therapeutic effect of activating Stat3 signaling in this embodiment of the invention lies in the promotion of a) de novo formation of blood vessels, and b) sprouting from pre-existing vessels.
  • both phenomenon will be jointly referred to as angiogenesis.
  • the use of Stat3 agonists to promote angiogenesis may be used, for example, in preventing or treating ischemic diseases.
  • Stat3 agonists may be administered to patients in need of such treatment to increase stimulated vessel growth, and consequentially increase tissue perfusion and blood flow, thereby overcoming the vascular insufficiency characteristic of ischemic diseases.
  • Angiogenic molecules can be administered by way of gene transfer.
  • the angiogenic protein such as Stat3, a constitutive active form of Stat (Stat3-C), and agonists of Stat3 signaling
  • the gene can be delivered in an expression vector via a variety of approaches, including direct injection, electroporation, by way of transfected cells, or commercially available liposome preparations.
  • the expression vector usually consisting of a replication-defiecient adenovirus, retrovisus, lentivirus, and/or an adeno-associated virus, is taken up by the host cells via receptor-mediated mechanisms and/or endocytosis (see 5.6.3).
  • the present invention relates to administering nucleotide sequences encoding constitutive active Stat3, agonists of Stat3, such as, but not limited to, interleukin-6 (IL-6), as well as the normal form of Stat3.
  • constitutive form of Stat3 is encoded by the Stat3-C mutant form of the Stat3 gene.
  • Stat3-C substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription, thus giving rise to a constitutive active molecule (Bromberg et al, 1999, Cell 98:295-303).
  • replacing those tyrosine residues in STAT3 that are being phosphorylated upon activation with aspartic acid residues may result in a constitutive active molecule.
  • acidic amino acids such as aspartic acid can mimic a phosphate.
  • Stat3 is activated upon phosphorylation at said tyrosine residues, mimicking such phosphates constitutively by incorporation of an aspartic acid can render the molecule to be constitutively active.
  • site directed mutagenesis approaches which are well known to the skilled artisan can be used.
  • the present invention also relates to the expression of proteins that activate Stat3, such as IL-6. Expression of said protein components via gene therapy and resulting activation of Stat3 can be used in order to promote angiogenesis in ischemic diseases.
  • Another embodiment of the invention relates to the expression of the normal form of the Stat3 protein component.
  • nucleotide sequence to be expressed in a gene therapy approach has to be operatively linked to a promoter sequence.
  • enhancer/promoter sequences are essential for the expression of a given gene. Enhancer/promoter sequences also confer temporal and spatial regulation onto the expression pattern of a given gene.
  • enhancer/promoter sequences should be chosen dependent on the indicated disorder.
  • tissue specific expression will be the preferred embodiment of the invention; in other cases systemic expression of the nucleotide sequence may be preferred. This decision will depend on the indicated disorder, and ultimately on the clinician. Expression specific to the tissue affected by vascular insufficiency or ischemia can be conferred by enhancer/promoter sequences that are active only in that tissue. Combining the right promoter sequences with the gene to be expressed will require some experimentation involving standard techniques known to the skilled artisan. In other disorders, inducible expression of the pro-angiogenic molecule, such as Stat3C, Stat3, or IL-6, may be indicated.
  • the pro-angiogenic molecule such as Stat3C, Stat3, or IL-6
  • enhancer/promoter sequences are active only in the absence and/or presence of a particular factor, which can be a metabolite, an anorganic molecule or a protein component.
  • a particular factor which can be a metabolite, an anorganic molecule or a protein component.
  • enhancer/promoter sequences that are induced upon hypoxia are the preferred embodiment of the invention.
  • Placing the expression of the nucleotide sequence of the invention under control of hypoxia constitutes a self-regulatory system.
  • the expression of the angiogenic gene i.e. Stat3, Stat3C or other constitutive forms of Stat3, IL-6, respectively.
  • the resulting newly formed vascular tissue provides an increased blood-flow in the affected tissue, thus increasing the oxygen concentration in said tissue. Consequently, the expression of the recombinant angiogenic gene will cease. This method ensures sufficient neovascularization but prevents vascular overgrowth that may be associated with too long exposure to or too high expression of angiogenic factors.
  • Angiogenic molecules can be delivered by administering the recombinant proteins.
  • Recombinant Stat3, Stat3-C, or IL-6 can be synthesized and purified as fusion proteins by recombinant DNA techniques. Fusing a "peptide tag" such as a polyhistidine tag, glutathione S-transferase (GST), or the E. coli maltose binding protein (MBP) to the angiogenic protein facilitates its purification.
  • a "peptide tag” such as a polyhistidine tag, glutathione S-transferase (GST), or the E. coli maltose binding protein (MBP)
  • GST glutathione S-transferase
  • MBP E. coli maltose binding protein
  • the fusion proteins can be synthesized in different host systems, such as, but not restricted to, bacteria, insects cells or mammalian
  • the proteins can be immuno-purified using antibodies specific to the respective protein.
  • An examplary approach comprises covalently linking antibodies specific to the protein which is to be purified to a solid matrix. Protein 0 extracts of the host cells expressing the desired protein are added to the matrix under conditions that allow binding of said protein to the matrix via non-covalent binding to the antibodies. After contaminants have been removed by washing under suitable conditions, the protein can be eluted.
  • the recombinant proteins can then be administered by the drug delivery system of 5 choice dependent on whether systemic or local administration of the protein is preferred for the treatment or prevention, respectively, of the indicated disorder.
  • Various delivery systems are known and are described in section 5.6.4.
  • agonists of Stat3 signaling such as but not limited too Stat3, Stat3-C, or IL-6 causes increased angiogenesis and subsequent increase in blood flow, thus
  • a plurality of disorders are caused by the overgrowth of blood vessels, herein referred to as proliferative angiopathies with neovascularization.
  • An examplary disorder of this kind is diabetic microangiopathy with neovascularization. This disease is characterized by swollen retinal vessels that leak fluid as well an excess of retinal vessels which is diagnosed as diabetic retinopathy and can lead to blindness in the affected patients.
  • the presented invention relates to inhibiting angiogenesis in proliferative angiopathies with neovascularization (other than cancer) by reducing the activity of Stat3 signaling.
  • the invention relates to inhibiting Stat3 using negative regulators of Stat3, such as a dominant negative form of Stat3, Stat3beta.
  • the invention comprises inhibiting Stat3 using negative regulators of Stat3, such as SOCS and PIAS, inhibitors of Stat3 expression, such as antisense oligonucleotides and ribozymes, antibodies inhibitors of positive regulators of Stat3, such as inhibitors of the Src tyrosine kinase.
  • negative regulators of Stat3 such as SOCS and PIAS
  • inhibitors of Stat3 expression such as antisense oligonucleotides and ribozymes
  • antibodies inhibitors of positive regulators of Stat3 such as inhibitors of the Src tyrosine kinase.
  • the Stat3 activity modulator is Stat3beta, a dominant negative form of Stat3.
  • STAT3beta lacks the C-terminal transactivation domain.
  • STAT3beta fails to activate a pIRE containing promoter in transient transfection assays. Instead, co-expression of STAT3beta inhibits the transactivation potential of STAT3, thus effectively inhibiting Stat3 activity (Caldenhoven et al, 1996, Journal of Biological Chemistry 271 : 13221-13227).
  • the dominant negative form of Stat3, Stat3beta can be administered by a gene therapy approach as described 5.6.3.
  • Stat3beta is delivered to the targeted tissue in form of a nucleotide sequence encoding Stat3beta under conditions that allow Stat3beta expression.
  • the gene In order for the Stat3beta gene to be expressed, the gene must be operatively linked to an enhancer/promoter sequence.
  • tissue-specific and or inducible enhancer/promoter sequences can be used.
  • Alternative embodiments of the inventions comprise other inhibitors of Stat3 signaling, such as, but not limited to, the SOCS negative regulatory molecues and the PIAS family of negative regulatory proteins (Starr and Hilton 1999, Bioessays 21 :47-52). These factors can also be administered via gene therapy as described in 5.6.3.
  • the respective gene In order for these genes to be expressed, the respective gene must be operatively linked to an enhancer/promoter sequence. In order to target only certain organs or tissues, tissue- specific and/or inducible enhancer/promoter sequences can be employed. Additionally, the invention relates to suppressing the expression of endogenous
  • antisense Stat3 nucleotide sequence This can be achieved by administering nucleotide sequences that are in antisense orientation relative to the Stat3 encoding mRNA (hereinafter referred to as antisense Stat3 nucleotide sequence; see Example 3, Fig. 12). Those nucleotide sequences can vary in length from 20 basepairs up to the length of the entire Stat3 cDNA. Antisense nucleotide sequences of different length may differ in their efficacy as drugs, and it may take some experimentation to find the right length to treat the indicated disorder. Said antisense Stat3 nuleotide sequences can be delivered via gene transfer as described in 5.6.3.
  • anitsense Stat3 nucleotide sequence In order for these antisense nucleotide sequences to be expressed, the anitsense Stat3 nucleotide sequence must be operatively linked to an enhancer/promoter sequence. For targeting only certain organs or tissues, tissue-specific and/or inducible enhancer/promoter sequences can be employed.
  • small RNA therapeutics such as ribozymes.
  • Small RNA therapeutics can be delivered via gene therapy by linking the nucleotide sequences encoding said RNA therapeutics opatively to an enhancer/promoter sequence.
  • the invention encompasses the administration of a vector comprising the nucleotide sequence encoding the Stat3 specific ribozyme operatively linked to an enhancer/promoter to the patient by methods described in 5.6.3, thus resulting
  • Anti-angiogenic molecules can also be delivered by administering the recombinant 0 proteins.
  • Recombinant Stat3beta, SOCS, or PIAS respectively can be synthesized and purified as fusion proteins by recombinant DNA techniques. Fusing a "peptide tag" such as a polyhistidine tag, glutathione S-transferase (GST), or the E. coli maltose binding protein (MBP) to the angiogenic protein facilitates its purification.
  • the fusion proteins can be synthesized in different host systems, such as, but not restricted to, bacteria, insects cells or
  • proteins can be immuno-purified using antibodies specific to the respective protein.
  • the recombinant proteins can then be administered by the drug delivery system of choice dependent on whether systemic or local administration of the protein is preferred for the treatment or prevention, respectively, of the indicated disorder.
  • Various delivery ⁇ systems are known and are described in 5.8.
  • the invention relates to suppressing the expression of endogenous Stat3. This can be achieved by administering antisense Stat3 nucleotide sequences. Those nucleotide sequences can vary in length from 20 basepairs up to the length of the entire Stat3 cDNA. Antisense nucleotide sequences of different length may differ in their efficacy
  • Said antisense Stat3 nuleotide sequences can be delivered by administering directly in vitro synthesized antisense nucleotide sequences. Those antisense nucleotide sequences can be modified to increase their stability, thus lengthening their half-life, in a cell. Antisense Stat3 nucleic acids are described in detailed in Section 5.6.4.
  • RNA therapeutics such as ribozymes specific to Stat3 RNA.
  • the invention comprises the in vitro synthesis of small RNA therapeutics such as ribozymes specific to Stat3 RNA and administration of said small RNA therapeutics.
  • Those RNA therapeutics can be chemically modified in order to increase their sability and lengthen their half-life.
  • the invention relates to reducing neovascularization by antagonizing Stat3 signaling via inhibitors of positive regulators of Stat3 signaling such as the tyrosine kinase Src.
  • the invention encompasses the inhibition of Src by 0 administration of the drug SU6656 (Blake et al. 2000, Molecular Cellular Biology 20:9018- 9027).
  • the invention also comprises reducin neovascularization by antagonizing Stat3 signaling using antibodies specific to the Stat3 protein component.
  • the antibodies can be administered by the drug delivery system of choice dependent on whether systemic or local 5 administration of the protein is preferred for the treatment or prevention, respectively, of the indicated disorder.
  • Various delivery systems are known and are described in 5.8.
  • the invention provides methods for stimulating the immune response using antagonists of Stat3 signaling activity.
  • Immunologic danger signals are factors that attract cells of the immune-system to the site of 3 the infection or cancerous growth and activate an immune response.
  • immunologic danger signals include, but are not limited to: IFN-gamma inducible protein 10 (IP- 10), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-beta (IFN- beta).
  • the invention encompasses administration to a patient the ⁇ supernatant of cells in which Stat3 activity is suppressed by means comprising Stat3beta and Stat3 antisense nucleotide sequences.
  • the invention also encompasses inhibition of Stat3 signaling in the patient locally or systemically to augment the immune response in various diseases.
  • the various embodiments of the invention are described in more detail in the sections below. The goal of any of these embodiments is to increase the concentration 3 of immunologic danger signals either locally or systemically in the patient, thereby augmenting the immune response.
  • Such a strengthening of the patient's own defense system is desirable when the patients natural immune reaction is not sufficient to eliminate the pathogen or the malignant cells. More specifically, some tumors evade immune surveillance by suppressing the expression of said immunologic danger signals.
  • This embodiment of the invention relates to the inhibition of Stat3 signaling in cells such as B16 melanoma cells by such means as expression of Stat3beta, expression of negative regulators of Stat3 signaling as for example PIAS and SOCS, expression of Stat3 antisense nucleotide sequences, administration of in vitro synthesized Stat3 antisense nucleotide sequences, and antibodies specific to Stat3.
  • Stat3beta is expressed in B16 melanoma cells by means of transfection and supernatant is obtained from said cell culture.
  • the supernatant can then be administered to a patient in order to augment the immune response in various diseases.
  • diseases include infectious diseases and various malignancies.
  • the supernatant can be administered by any method known in the art. Some examples of which are described in section 5.6.4. Said supernatant can be converted into solid form by means such as to lyophilization
  • the pharmaceutical of the invention is Stat3beta, a dominant negative form of Stat3.
  • STAT3beta lacks the C-terminal transactivation domain.
  • STAT3beta fails to activate a pIRE containing promoter in transient transfection assays. Instead, co-expression of STAT3beta inhibits the transactivation potential of STAT3, thus effectively inhibiting Stat3 activity (Caldenhoven et al. 1996, Journal of Biological Chemistry 271:13221-13227).
  • the dominant negative form of Stat3, Stat3beta can be administered by a gene therapy approach as described 5.6.3.
  • Stat3beta is delivered to the targeted tissue in form of a nucleotide sequence encoding Stat3beta under conditions that allow Stat3beta expression.
  • the gene In order for the Stat3beta gene to be expressed, the gene must be operatively linked to an enhancer/promoter sequence.
  • tissue-specific and/or inducible enhancer/promoter sequences can be used. For a more detailed discussion of tissue-specific gene therapy see section 5.6.3.
  • Alternative embodiments of the inventions comprise other inhibitors of Stat3 signaling, such as, but not limited to, the SOCS negative regulatory molecules and the PIAS family of negative regulatory proteins (Starr and Hilton 1999, Bioessays 21:47-52). These factors can also be administered via gene therapy as described in 5.6.3.
  • the respective gene In order for these genes to be expressed, the respective gene must be operatively linked to an enhancer/promoter sequence. In order to target only certain organs or tissues, tissue-specific and/or inducible enhancer/promoter sequences can be employed.
  • the invention relates to suppressing the expression of endogenous Stat3.
  • This can be achieved by administering nucleotide sequences that are in antisense orientation relative to the Stat3 encoding mRNA (hereinafter referred to as antisense Stat3 nucleotide sequence; see Example 3, Fig. 12).
  • antisense Stat3 nucleotide sequence can vary in length from 20 basepairs up to the length of the entire Stat3 cDNA.
  • Antisense nucleotide sequences of different length may differ in their efficacy as drugs, and it may take some experimentation to find the right length to treat the indicated disorder.
  • Such antisense Stat3 nuleotide sequences can be delivered via gene transfer as described in 5.6.3.
  • the antisense Stat3 nucleotide sequence must be operatively linked to an enhancer/promoter sequence.
  • tissue-specific and/or inducible enhancer/promoter sequences can be employed.
  • RNA therapeutics such as ribozymes.
  • Small RNA therapeutics can be delivered via gene therapy by linking the nucleotide sequences encoding RNA therapeutics operatively to an enhancer/promoter sequence.
  • the invention encompasses the administration of a vector comprising the nucleotide sequence encoding the Stat3 specific ribozyme operatively linked to an enhancer/promoter to a patient by methods described in 5.6.3, thus enhancing the immune response of the patient.
  • Antagonists of Stat3 signaling activity can be delivered by administering the recombinant proteins.
  • Recombinant Stat3beta, SOCS, or PIAS respectively can be synthesized and purified as fusion proteins by recombinant DNA techniques. Fusing a "peptide tag" such as a polyhistidine tag, glutathione S-transferase (GST), or the E. coli maltose binding protein (MBP) to the protein of the invention facilitates its purification.
  • GST glutathione S-transferase
  • MBP E. coli maltose binding protein
  • the fusion proteins can be synthesized in different host systems, such as, but not restricted to, bacteria, insects cells or mammalian cells. Alternatively the proteins can be immune- purified using antibodies specific to the respective protein.
  • the recombinant proteins can then be administered by the drug delivery system of choice dependent on whether systemic or local administration of the protein is preferred for the treatment or prevention, respectively, of the indicated disorder.
  • Various delivery systems are known and are described in 5.8.
  • the invention relates to suppressing the expression of endogenous
  • nucleotide sequences can vary in length from 20 basepairs up to the length of the entire Stat3 cDNA. Antisense nucleotide sequences of different length may differ in their efficacy as drugs, and it may take some experimentation to find the right length to treat the indicated disorder. Said antisense Stat3 nuleotide sequences can be delivered by administering directly in vitro synthesized antisense nucleotide sequences. Those antisense nucleotide sequences can be modified to increase their stability, thus lengthening their half-
  • RNA therapeutics such as ribozymes specific to Stat3 RNA.
  • the invention comprises the in vitro synthesis of small RNA therapeutics such as ribozymes specific to Stat3 RNA and administration of said small RNA therapeutics.
  • Those RNA therapeutics can be chemically modified in order to increase their stability and lengthen their half-life.
  • the invention relates to enhancing the immune response by antagonizing Stat3 signaling via inhibitors of positive regulators of Stat3 signaling such as the tyrosine kinase Src.
  • the invention encompasses the inhibition of Src by administration of the drug SU6656 (Blake et al. 2000, Molecular Cellular Biology
  • the invention also comprises augmenting the immune response by antagonizing Stat3 signaling using antibodies specific to the Stat3 protein.
  • the antibodies can be administered by the drug delivery system of choice dependent on whether systemic or local administration of the protein is preferred for the treatment or prevention, respectively, of the indicated disorder.
  • Various delivery systems are known and are described in 5.8.
  • the invention provides methods for inhibiting the immune response using agonists of Stat3 signaling activity.
  • Immunologic danger signals are factors that attract cells of the immune-system to the site of the infection or transplants and activate an immune response.
  • immunologic- danger signals include, but are not limited to: IFN-gamma inducible protein 10 (IP- 10), interleukin- 35 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-beta (IFN-beta).
  • the invention encompasses administration to a patient of agonists of Stat3 signaling locally or systemically to suppress the immune response in various diseases.
  • the various embodiments of the invention are described in more detail in the sections below.
  • the goal of any of these embodiments is to decrease the concentration of immunologic danger signals either locally or systemically in the patient, thereby suppressing the immune response.
  • Such a suppression of the patient's own defense system is desirable when the patient is suffering from autoimmune diseases or to ameliorate adverse reactions to transplants.
  • Immuno suppressant molecules can be administered by way of gene transfer.
  • the immuno suppressant protein such as Stat3, a constitutive active fonn of Stat (Stat3-C), and agonists of Stat3 signaling
  • Stat3-C a constitutive active fonn of Stat
  • the gene can be delivered in an expression vector via a variety of approachs, including direct injection, electroporation, by way of transfected cells, or commercially available liposome preparations.
  • the expression vector usually consisting of a replication-defiecient adenovirus, retrovisus, lentivirus, and/or an adeno-associated viras, is taken up by the host cells via receptor-mediated mechanisms and/or endocytosis.
  • the present invention relates to administering nucleotide sequences encoding constitutive active Stat3, agonists of Stat3, such as, but not limited to, interleukin-6 (IL-6), as well as the normal form of Stat3.
  • constitutive form of Stat3 is encoded by the Stat3-C mutant form of the Stat3 gene.
  • Stat3-C substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription, thus giving rise to a constitutive active molecule (Bromberg et al, 1999, Cell 98:295-303).
  • replacing those tyrosine residues in STAT3 that are being phosphorylated upon activation with aspartic acid residues may result in a constitutive active molecule.
  • acidic amino acids such as aspartic acid can mimic a phosphate.
  • Stat3 is activated upon phosphorylation at said tyrosine residues, mimicking such phosphates constitutively by incorporation of an aspartic acid can render the molecule to be constitutively active.
  • basic side directed mutagenesis approaches which are well known to the skilled artisan can be used.
  • the present invention also relates to the expression of proteins that activate Stat3, such as IL-6. Expression of said protein components via gene therapy and resulting activation of Stat3 can be used in various diseases where a suppression of the immune response is desirable.
  • Another embodiment of the invention relates to the expression of the normal form of the Stat3 protein component. Despite the regulation of Stat3 signaling in a cell, elevating Stat3 protein levels in a cell can also increase its function thereby suppressing the immune response.
  • nucleotide sequence to be expressed in a gene therapy approach has to be operatively linked to a promoter sequence.
  • enhancer/promoter sequences are essential for the expression of a given gene. Enhancer/promoter sequences also confer temporal and spatial regulation onto the expression pattern of a given gene.
  • enhancer/promoter sequences should be chosen dependent on the indicated disorder.
  • tissue specific expression will be the preferred embodiment of the invention; in other cases systemic expression of the nucleotide sequence may be preferred. This decision will depend on the indicated disorder, and ultimately on the clinician. Expression specific to the tissue affected by the immunologic disorder can be conferred by enhancer/promoter sequences that are active only in that tissue. Combining the right promoter sequences with the gene to be expressed will require some experimentation involving standard techniques known to the skilled artisan.
  • inducible expression of the immuno-suppressant protein such as Stat3C, Stat3, or IL-6, may be indicated.
  • different enhancer/promoter sequences are active only in the absence and/or presence of a particular factor, which can be a metabolite, an anorganic molecule or a protein component.
  • ⁇ J Immuno suppressant molecules can be delivered by administering the recombinant proteins.
  • Recombinant Stat3, Stat3-C, or IL-6, respectively can be synthesized and purified as fusion proteins by recombinant DNA techniques. Fusing a "peptide tag", such as a polyhistidine tag, glutathione S-transferase (GST), or the E. coli maltose binding protein (MBP) to the angiogenic protein facilitates its purification.
  • GST glutathione S-transferase
  • MBP E. coli maltose binding protein
  • the fusion proteins can ⁇ be synthesized in different host systems, such as, but not restricted to, bacteria, insects cells or mammalian cells. Methods of expressing said proteins in different systems and purifying them are described below.
  • the proteins can be immuno-purified using antibodies specific to the respective protein.
  • An examplary approach comprises covalently 5 linking antibodies specific to the protein which is to be purified to a solid matrix. Protein extracts of the host cells expressing the desired protein are added to the matrix under conditions that allow binding of said protein to the matrix via non-covalent binding to the antibodies. After contaminants have been removed by washing under suitable conditions, the protein can be eluted.
  • the recombinant proteins can then be administered by the drug delivery system of choice dependent on whether systemic or local administration of the protein is preferred for the treatment or prevention, respectively, of the indicated disorder.
  • Various delivery systems are known and are described below.
  • the invention relates to a method of identifying immunologic danger signals. Once ⁇ those immunologic danger signals have been identified, they can be synthesized and administered to patients in order to augment the immune-response in various diseases.
  • the invention encompasses the identification of immunologic danger signals secreted by melanoma B16 cells that have been genetically engineered to express the dominant negative form of Stat3, Stat3beta.
  • Stat3beta is expressed in melanoma B16 cells using the pIRES vector system (Clontech; Palo Alto, CA; Catlett-Falcone et al. 1999, Immunity 10:105-115).
  • the nucleotide sequence encoding Stat3beta can be inserted into pIRES or any other vector system suitable for transfection of mammalian cells by standard molecular biology techniques. Likewise, the DNA can be transfected into the cells by standard
  • the supernatant of cells expressing Stat3beta comprises immunologic danger signals (see Example 2).
  • the components of the supernatant can be separated by standard biochemical techniques such as, but not limited to, 5 gel-filtration chromotography or ion-exchange chromotography. These techniques are well known to the skilled artisan, and a minimum of experimentation will be required to determine the optimal conditions under which to purify individual components of the supernatant. After separation of the constituent components of the supernatant in individual components or fractions, said fractions are tested for their immunologic signaling effects on 0 different immune cells, such as macrophages, T-cell and neutrophils.
  • the components of the fraction must be seperated from each other and individually tested for their immunologic signaling activity in the respective assay.
  • standard biochemical techniques such as, but not limited to, gel-filtration chromotography or ion-exchange chromotography can be used for the isolation of the component of interest.
  • the invention relates to the identification of immunologic danger signals released from cells other than melanoma B 16 cells, but similarly expressing Stat3beta.
  • the invention encompasses a method of identifying immunologic danger signals released from cells, such as but not limited to, melanoma B16 cells, in which Stat3beta signaling is inhibited by specific antagonists of Stat3beta acitivity.
  • Such antagonists comprise antisense nucleotide sequences specific to Stat3beta and ribozymes that act specifically on Stat3beta RNA.
  • Immunologic signaling activity can be tested either in cell culture on various types of cells of the immune system or in an animal model. Accordingly, the fractions, which are obtained from the supernatant as described above, are added either to cells in culture, such as cultures of macrophages, T-cells and neutrophils, or, alternatively, are injected into an animal, preferrably a mouse. After a sufficient time period said cells are tested for immunologic activity. This can be accomplished for example by measuring the expression levels of markers of activation. In the case of macrophages such markers include, but are not limited to, the nitric oxide synthase, iNOS, and the chemokine RANTES.
  • interferon-gamma IFN-gamma
  • IL-2 interleukin-2
  • TNF-alpha tumor necrosis factor alpha
  • the length of the time period between stimulation and assay of expression of said markers may be changed and depends on the precise experimental conditions. A minimum of experimentation is necessary to establish the assay system to which the invention relates in such a way that it functions optimally.
  • the levels of iNOS, RANTES, IFN-gamma, IL-2, and TNF-alpha can be determined by immunoblotting, Northern blotting, RNAse protection assays, immunocytochemistry or similar techniques well known to the skilled artisan.
  • probes specific to iNOS, RANTES, IFN-gamma, IL-2, and TNF-alpha, respectively have to be employed.
  • Such probes comprise antibodies and antisense RNA molecules. The detection of such probes is well established in the art.
  • macrophages, T-cells and neutrophils can be isolated from the animal and subsequently analyzed or, alternatively, expression levels of iNOS, RANTES, IFN-gamma, IL-2, and TNF-alpha can be tested in situ by immunohistochemistry or in situ hybridization.
  • probes specific to iNOS, RANTES, IFN-gamma, IL-2, and TNF-alpha, respectively have to be employed.
  • probes comprise antibodies and antisense RNA molecules. The detection of such probes is well established in the art. Quantification and statistical analysis of the data is done by standard methods.
  • the Stat3 activity modulator comprises a protein which is encoded by a specific nucleotide sequence.
  • the pharmaceutical comprises a nucleotide sequence which is transcribed to generate a biologically active RNA molecule.
  • the Stat3 activity modulator comprises a nucleotide sequence which is to be transcribed and translated. In either case, said nucleotide sequence is inserted into an expression vector for propagation and expression in recombinant cells or in cells of the host in the case of gene therapy.
  • An expression construct refers to a nucleotide sequence encoding the Stat3 activity modulator, which can be either an RNA molecule or a protein, operably linked to one or more regulatory regions or enhancer/promoter sequences which enables expression of the protein of the invention in an appropriate host cell.
  • “Operably-linked” refers to an association in which the regulatory regions and the nucleotide sequence encoding the Stat3 activity modulator to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation.
  • the regulatory regions necessary for transcription of the Stat3 activity modulator can be provided by the expression vector.
  • cellular transcriptional factors such as RNA polymerase
  • the precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell.
  • a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence.
  • Such regulatory regions may include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.
  • Both constitutive and inducible regulatory regions may be used for expression of the Stat3 activity modulator. It may be desirable to use inducible promoters when the conditions optimal for growth of the host cells and the conditions for high level expression of the Stat3 activity modulator are different. Examples of useful regulatory regions are provided below (section 5.6.3).
  • linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art (Wu et al., 1987, Methods in Enzymol 152:343-349). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alternatively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site.
  • An expression construct comprising a sequence encoding the Stat3 activity modulator operably linked to regulatory regions (enhancer/promoter sequences) can be directly introduced into appropriate host cells for expression and production of the Stat3 activity modulator without further cloning.
  • the expression constructs can also contain
  • expression vectors may be used in the present invention which include, but are not limited to, plasmids, cosmids, phage, phagemids, or modified viruses.
  • expression vectors comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the sequence encoding the Stat3 activity modulator, and one or more selection markers.
  • the expression vector must be used with a compatible host cell which may be derived from a prokaryotic or an eukaryotic organism including but not limited to bacteria, yeasts, insects, mammals, and humans.
  • Non-limiting examples of regulatory regions that can be used for expression in E. coli may include but not limited to lac, trp, lpp, phoA, recA, tac, T3, T7 and ⁇ P L (Makrides, 1996, Microbiol Rev, 60:512-538).
  • Non-limiting examples of prokaryotic expression vectors may include the ⁇ gt vector series such as ⁇ gtl 1 (Huynh et al., 1984 in "DNA Cloning Techniques", Vol. I: A Practical Approach (D. Glover, ed.), pp.
  • a potential drawback of a prokaryotic host- vector system is the inability to perform many of the post-translational processing of mammalian cells.
  • an eukaryotic host- vector system is preferred, a mammalian host-vector system is more preferred, and a human host- vector system is the most preferred.
  • the Stat3 activity modulator in mammalian host cells, a variety of regulatory regions can be used, for example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.
  • Inducible promoters that may be useful in mammalian cells include but are not limited to those associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), ⁇ -interferon gene, and hsp70 gene (Williams et al., 1989, Cancer Res. 49:2735-42 ; Taylor et al., 1990, Mol. Cell Biol., 10: 165-75). It may be advantageous to use heat shock promoters or stress promoters to drive expression of the Stat3 activity modulator in recombinant host cells.
  • the expression vector may contain selectable or screenable marker genes for initially isolating, identifying or tracking host cells that contain DNA encoding the elected Stat3 activity modulator.
  • selectable or screenable marker genes for initially isolating, identifying or tracking host cells that contain DNA encoding the elected Stat3 activity modulator.
  • stable expression in mammalian cells is preferred.
  • a number of selection systems may be used for mammalian cells, including but not limited to the Herpes simplex viras thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc. Natl. Acad. Sci. USA
  • adenine phosphoribosyltransferase genes can be employed in tk “ , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci.
  • the Stat3 activity modulator is a protein (hereinafter: the protein of the invention)
  • generating a fusion protein comprising a peptide tag can aid its purification.
  • a fusion protein can be made by ligating the nucleotide sequence encoding the protein of the invention to the sequence encoding the peptide tag in the proper reading frame. If genomic sequences are used, care should be taken to ensure that the modified gene remains within the same translational reading frame, uninterrupted by translational stop signals and/or spurious messenger RNA splicing signals.
  • the peptide tag is fused at its amino terminal to the carboxyl terminal of the protein of the invention.
  • the precise site at which the fusion is made is not critical. The optimal site can be determined by routine experimentation.
  • peptide tags known in the art may be used in the modification of the protein of the invention, such as but not limited to the immunoglobulin constant regions, polyhistidine sequence (Petty, 1996, Metal-chelate affinity chromatography, in Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience), glutathione S-transferase (GST; Smith, 1993, Methods Mol. Cell Bio. 4:220- 229), the E. coli maltose binding protein (Guan et al., 1987, Gene 67:21-30), and various cellulose binding domains (U.S.
  • Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, (see, for example, Kriegler M. in “Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990), such as monkey kidney cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 36:59, 1977; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin. Proc. Natl. Acad. Sci.
  • monkey kidney cell line transformed by SV40 COS-7, ATCC CRL 1651
  • human embryonic kidney line (293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 36:59,
  • mice sertoli cells (Mather, Biol. Reprod. 23:243-251, 1980); mouse fibroblast cells (NTH-3T3), monkey kidney cells (CVI ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC CCL51).
  • a number of viral-based expression systems may also be utilized with mammalian cells to produce the Stat3 activity modulator.
  • Vectors using DNA virus backbones have been derived from simian viras 40 (SV40) (Hamer et al., 1979, Cell 17:725), adenovirus (Van Doren et al., 1984, Mol Cell Biol 4:1653), adeno-associated viras (McLaughlin et al., 1988, J Virol 62: 1963), and bovine papillomas viras (Zinn et al., 1982, Proc Natl Acad Sci 79:4897).
  • SV40 simian viras 40
  • adenovirus Van Doren et al., 1984, Mol Cell Biol 4:1653
  • adeno-associated viras McLaughlin et al., 1988, J Virol 62: 1963
  • bovine papillomas viras Za
  • the donor DNA sequence may be ligated to an adenoviras transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenoviras genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing heterologous products in infected hosts.
  • a non-essential region of the viral genome e.g., region El or E3
  • yeast a number of vectors containing constitutive or inducible promoters may be used with Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts).
  • Saccharomyces cerevisiae bakeer's yeast
  • Schizosaccharomyces pombe Schizosaccharomyces pombe
  • Pichia pastoris Pichia pastoris
  • Hansenula polymorpha methylotropic yeasts
  • Autographa califomica nuclear polyhidrosis virus (AcNPV) a baculovirus
  • AcNPV Autographa califomica nuclear polyhidrosis virus
  • the sequences encoding the protein of the invention may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). These recombinant virases are then used to infect host cells in which the inserted DNA is expressed.
  • AcNPV promoter for example the polyhedrin promoter
  • any of the cloning and expression vectors described herein may be synthesized and assembled from known DNA sequences by well known techniques in the art.
  • the regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic.
  • Some vectors and host cells may be obtained commercially. Non-limiting examples of useful vectors are described in Appendix 5 of Current Protocols in Molecular Biology, 1988, ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, which is incorporated herein by reference; and the catalogs of commercial suppliers such as Clontech Laboratories, Stratagene Inc., and Invitrogen, Inc.
  • Expression constructs containing cloned nucleotide sequence encoding the protein of the invention can be introduced into the host cell by a variety of techniques known in the art, including but not limited to, for prokaryotic cells, bacterial transformation (Hanahan, 1985, in DNA Cloning, A Practical Approach, 1:109-136), and for eukaryotic cells, calcium phosphate mediated transfection (Wigler et al., 1977, Cell 11:223-232), liposome-mediated transfection (Schaefer-Ridder et al, 1982, Science 215:166-168), electroporation (Wolff et al., 1987, Proc Natl Acad Sci 84:3344), and microinjection (Cappechi, 1980, Cell 22:479- 488).
  • Cell lines that stably express protein of the invention may be engineered by using a vector that contains a selectable marker.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while the protein of the invention is expressed continuously.
  • the protein of the invention can be recovered and purified from recombinant cell cultures by known methods, including ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, immunoaffmity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • ammonium sulfate precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, immunoaffmity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • total protein has to be prepared from the cell culture. This procedure comprises collection, washing and lysis of said cells and is well known to the skilled artisan.
  • the invention provides methods for purification of the protein of the invention which are based on the properties of the peptide tag present on the protein of the invention.
  • One approach is based on specific molecular interactions between a tag and its binding partner.
  • the other approach relies on the immunospecific binding of an antibody to an epitope present on the tag or on the protein which is to be purified.
  • affinity chromatography well known in the art is generally applicable to both of these approaches.
  • a method that is generally applicable to purifying protein of the invention that are fused to the constant regions of immunoglobulin is protein A affinity chromatography, a technique that is well known in the art.
  • Staphylococcus protein A is a 42 kD polypeptide that binds specifically to a region located between the second and third constant regions of heavy chain immunoglobulins. Because of the Fc domains of different classes, subclasses and species of immunoglobulins, affinity of protein A for human Fc regions is strong, but may vary with other species. Subclasses that are less preferred include human IgG-3, and most rat subclasses. For certain subclasses, protein G (of Streptococci) may be used in place of protein A in the purification.
  • Protein- A sepharose (Pharmacia or Biorad) is a commonly used solid phase for affinity purification of antibodies, and can be used essentially in the same manner for the purification of the protein of the invention fused to an immunoglobulin Fc fragment.
  • Bound protein of the invention can be eluted by various buffer systems known in the art, including a succession of citrate, acetate and glycine-HCl buffers which gradually lowers the pH. This method is less preferred if the recombinant cells also produce antibodies which will be copurified with the protein of the invention. See, for example, Langone, 1982, J. Immunol, meth. 51:3; Wilchek et al., 1982, Biochem. Intl.
  • a polyhistidine tag may be used, in which case, the protein of the invention can be purified by metal chelate chromatography.
  • the polyhistidine tag usually a sequence of six histidines, has a high affinity for divalent metal ions, such as nickel ions (Ni 2+ ), which can be immobilized on a solid phase, such as nitrilotriacetic acid-matrices.
  • Polyhistidine has a well characterized affinity for Ni 2+ -NTA-agarose, and can be eluted with either of two mild treatments: imidazole (0.1-0.2 M) will effectively compete with the resin for binding sites; or lowering the pH just below 6.0 will protonate the histidine sidechains and disrupt the binding.
  • the purification method comprises loading the cell culture lysate onto the Ni 2+ -NTA-agarose column, washing the contaminants through, and eluting the protein of the invention with imidazole or weak acid.
  • Ni 2+ -NTA-agarose can be obtained from commercial suppliers such as Sigma (St. Louis) and Qiagen.
  • Antibodies that recognize the polyhistidine tag are also available which can be used to detect and quantitate the protein of the invention.
  • GST glutathione-S-transferase
  • a protein of the invention-GST fusion expressed in a prokaryotic host cell such as E. coli, can be purified from the cell culture lysate by absorption with glutathione agarose beads, followed by elution in the presence of free reduced glutathione at neutral pH. Since GST is known to form dimers under certain conditions, dimeric protein of the invention may be obtained. See, Smith, 1993, Methods Mol. Cell Bio. 4:220-229.
  • Another useful peptide tag that can be used is the maltose binding protein (MBP) of E. coli, which is encoded by the malE gene.
  • MBP maltose binding protein
  • the protein of the invention binds to amylose resin while contaminants are washed away.
  • the bound protein of the invention-MBP fusion is eluted from the amylose resin by maltose. See, for example, Guan et al., 1987, Gene 67:21-30.
  • the second approach for purifying the protein of the invention is applicable to peptide tags that contain an epitope for which polyclonal or monoclonal antibodies are available. It is also applicable if polyclonal or monoclonal antibodies specific to the protein of the invention are available.
  • nucleotide sequences encoding Stat3, Stat3beta, Stat3-C, IL-6 or nucleotide sequences encoding therapeutic RNA molecules, such as antisense RNA and ribozymes specific to Stat3, are administered to treat, or prevent various diseases.
  • These nucleotide sequences are collectively referred to as nucleotide sequences of the invention.
  • Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleotide sequence.
  • the nucleotide sequences produce their encoded protein or RNA molecule that mediates a therapeutic effect.
  • nucleic acid molecules are used in which the nucleotide sequence of the invention is flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the nucleotide sequence of the invention (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al, 1989, Nature 342:435-438).
  • the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, for example by constracting them as part of an appropriate nucleic acid expression vector and administering the vector so that the nucleic acid sequences become intracellular.
  • Gene therapy vectors can be administered by infection using defective or attenuated retrovirals or other viral vectors (see, e.g., U.S. Patent No.
  • nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06 180; WO 92/22635; W092/20316; W093/14188, and WO 93/20221).
  • the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al, 1989, Nature 342:435-438).
  • viral vectors that contain the nucleotide sequence of the invention are used.
  • a retroviral vector can be used (see Miller et al, 1993, Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA.
  • the nucleotide sequences of the invention to be used in gene therapy are cloned into one or more vectors, thereby facilitating delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al, 1994, Biotherapy 6:29 1-302, which describes the use of a retroviral vector to deliver the mdr 1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, 1994, J. Clin. Invest. 93:644-651; Klein et al, 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
  • mouse mammary tumor viras control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639- 1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338- 340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
  • antisense oligopeptide, ribozyme, and triple helix molecules.
  • Techniques for the production and use of such molecules are well known to those of skill in the art.
  • antisense targeting Stat3 mRNA inhibits Stat3 signaling, as described in Section 8 (see Figures 12 and 13).
  • Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • oligonucleotides complementary to non-coding regions of the Stat3 gene could be used in an antisense approach to inhibit translation of endogenous Stat3 mRNA.
  • Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • Stat3 antisense molecules complementary to coding or non-coding regions may be used, members of both are well known in the art.
  • Representative, non-limiting examples of Stat3 antisense molecules include the following: 5'- ACTCAAACTGCCCTCCTGCT-3'; 5'- TCTGAAGAAACTGCTTGATT-3'; 5'-GCCACAATCCGGGCAATCT-3'; 5'- TGGCTGCAGTCTGTAGAAGG-3'; 5'-TTTCTGTTCTAGATCCTGCA-3'; 5'- TAGTTGAAATCAAAGTCATC-3'; 5'-TTCCATTCAGATCTTGCATG-3'; 5'- TCTGTTCCAGCTGCTGCATC-3'; 5'-TCACTCACGATGCTTCTCCG-3'; 5'- GAGTTTTCTGCACGTACTCC-3' (see, e.g., U.S. Patent No. 6,159,694, issued December 12, 2000, which is incorporated herein in its entirety).
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcyto
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate (S-
  • ODNs a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an -anomeric oligonucleotide.
  • An -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual -units, the strands run parallel to each other (Gautier, et al., 1987, Nucl. Acids Res. 15, 6625-6641).
  • the oligonucleotide is a
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl. Acids Res.
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451), etc.
  • antisense nucleotides complementary to the target gene coding region sequen ccee ccoouulldd be used, those complementary to the transcribed, untranslated region are most preferred.
  • gene expression downregulation is achieved because specific target mRNAs are digested by RNAse H after they have hybridized with the antisense phosphorothioate oligonucleotides (S-ODNs). Since no rales exist to predict which antisense S-ODNs will be more successful, the best strategy is completely empirical and consists of trying several antisense S-ODNs.
  • Antisense phosphorothioate oligonucleotides will be designed to target specific regions of mRNAs of interest. Control S-ODNs consisting of scrambled sequences of the antisense S- ODNs will also be designed to assure identical nucleotide content and minimize differences potentially attributable to nucleic acid content. All S-ODNs will be synthesized by Oligos Etc. (Wilsonville, OR).
  • cells will be grown to 60-80% confluence on 100 mm tissue culture plates, rinsed with PBS and overlaid with lipofection mix consisting of 8 ml Opti-MEM, 52.8 1 Lipofectin, and a final concentration of 200 nM S-ODNs. Lipofections will be carried out using Lipofectin Reagent and Opti-MEM (Gibco BRL). Cells will be incubated in the presence of the lipofection mix for 5 hours. Following incubation the medium will be replaced with complete DMEM. Cells will be harvested at different time points post- lipofection and protein levels will be analyzed by Western blot.
  • Antisense molecules should be targeted to cells that express the target gene, either directly to the subject in vivo or to cells in culture, such as in ex vivo gene therapy protocols.
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.
  • the use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA.
  • a vector can be introduced e.g., such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive.
  • Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290, 304-310), the promoter contained in the 3 long terminal repeat of Rous sarcoma viras (Yamamoto, et al., 1980, Cell 22, 787-797), the herpes thymidine kinase promoter (Wagner, et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., 1982, Nature 296, 39-42), etc.
  • plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site.
  • viral vectors can be used that selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
  • Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and, therefore, expression of target gene product (see, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver, et al, 1990, Science 247, 1222-1225).
  • oligonucleotides which hybridize to the Stat3 gene are designed to be complementary to the nucleic acids encoding the Stat3 protein (SEQ LO. NO: 2).
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4, 469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event.
  • composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Patent No.
  • ribozymes that cleave mRNA at site specific recognition sequences can be __ used to destroy target gene mRNAs
  • the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224, 574-578; Zaug and Cech, 1986, Science, 231, 470-475; Zaug, et al., 1986, Nature, 324, 429-433; published International patent application No.
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in the target gene.
  • the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Endogenous target gene expression can also be reduced by inactivating or "knocking out” the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al., 1985, Nature 317, 230-234; Thomas & Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety).
  • targeted homologous recombination e.g., see Smithies, et al., 1985, Nature 317, 230-234; Thomas & Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989, Cell 5, 313-321; each of which is incorporated by reference herein in its entirety).
  • a mutant, non-functional target gene flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene.
  • ES embryonic stem
  • Such approaches are particularly suited modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas & Capecchi, 1987 and Thompson, 1989, supra).
  • this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e., the target gene promoter and/or enhancers) to form triple helical stractures that prevent transcription of the target gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the target gene i.e., the target gene promoter and/or enhancers
  • triple helical stractures that prevent transcription of the target gene in target cells in the body.
  • Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rales, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC+ triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called “switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the technique may so efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles that the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype.
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity may, be introduced into cells via gene therapy methods such as those described, below, in Section 5.7.2 that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized.
  • the target gene encodes an extracellular protein, it may be preferable to co-administer normal target gene protein in order to maintain the requisite level of target gene activity.
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • Stat3 its fragments or other derivatives, or analogs thereof, may be used as an im unogen to generate antibodies which immunospecifically bind such an immunogen.
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, lgM, IgD, IgA and IgY), class (e.g., IgG 1? IgG 2 , IgG 3 , IgG 4 , IgA j and IgA 2 ) or subclass of immunoglobulin molecule.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin or papain.
  • antibodies to a human Stat3 protein are produced.
  • antibodies to a domain of Stat3 are produced.
  • polyclonal antibodies to Stat3 or derivative or analog may be obtained.
  • rabbit polyclonal antibodies to an epitope of Stat3 encoded by a sequence or fragment of SEQ ID NO: 2, or a subsequence thereof can be obtained.
  • various host animals can be immunized by injection with the native Stat3, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • corynebacterium parvum any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).
  • human hybridomas Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030
  • EBV virus Cold-d Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96
  • techniques developed for the production of "chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • a specific domain of a STAT e.g., the transcriptional activation domain, DNA binding domain, dimerization domain, SH2 domain, or SH3 domain
  • an antibody that specifically binds a first Stat3 homolog but which does not specifically bind a different Stat3 homolog one can select on the basis of positive binding to the first Stat3 homolog and a lack of binding to the second Stat3 homolog.
  • Antibodies specific to a domain of Stat3 are also provided, such as to a transcriptional activation domain, DNA binding domain, a dimerization domain, SH2 domain, SH3 domain.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the Stat3 sequences of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
  • anti-Stat3 antibodies and fragments thereof containing the binding domain are used as therapeutics.
  • Anti-Stat3 antibodies can be obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Research Diagnostics, Inc. (Flanders, NJ) or Zymed Laboratories (South San Francisco, CA). Alternatively, anti-Stat3 antibodies antibodies can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • Stat3 agonists may be used to stimulate angiogenesis for the treatment or prevention of ischemic diseases.
  • Ischemia is caused by an impaired blood supply resulting from narrowed or blocked arteries that starve tissues of needed nutrients and oxygen.
  • any condition which reduces the availability of nutrients or oxygen to a tissue, resulting in stress, damage, and finally, cell death may be treated by the methods of the present invention.
  • Ischemic disorders that may be treated by the methods described herein include, but are not limited to, coronary-atherosclerosis induced myocardial infarction and tissue ischemia in the lower extremities.
  • Stat3 agonists may be used to protect cardiac tissue from injury sustained during ischemia, infarction, inflammation, or trauma.
  • These conditions arise from or include, but are not limited to stroke, vascular occlusion, prenatal or postnatal oxygen deprivation, suffocation, choking, near drowning, carbon monoxide poisoning, smoke inhalation, trauma, including surgery and radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotensive shock, septic shock, anaphylactic shock, insulin shock, sickle cell crisis, cardiac arrest, dysrhythmia, and nitrogen narcosis.
  • IDDM insulin dependent diabetes
  • the diseases set forth above, as referred to herein, include those exhibited by animal models for such diseases, such as, for example non-obese diabetic (NOD) mice for IDDM and experimental autoimmune encephalomyelitis (EAE) mice for multiple sclerosis.
  • NOD non-obese diabetic
  • EAE experimental autoimmune encephalomyelitis
  • the methods of the present invention can be used to treat such autoimmune diseases by reducing or eliminating the immune response to the patient's own (self) tissue, or, alternatively, by reducing or eliminating a pre-existing autoimmune response directed at tissues or organs transplanted to replace self tissues or organs damaged by the autoimmune response.
  • infectious diseases include those caused by intracellular pathogens such as virases, bacteria, protozoans, and intracellular parasites.
  • Virases include, but are not limited to viral diseases such as those caused by hepatitis type B virus, parvovirases, such as adeno-associated virus and cytomegalovirus, papovaviruses such as papilloma virus, polyoma virases, and SV40, adenovirases, herpes viruses such as herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), and Epstein-Barr virus, poxvirases, such as variola (smallpox) and vaccinia virus, RNA virases, including but not limited to human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), human T-cell lymphotropic
  • bacterial infections can be treated or prevented such as, but not limited to disorders caused by pathogenic bacteria including, but not limited to, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus , Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enter
  • the methods can be used to treat or prevent infections caused by pathogenic protozoans such as, but not limited to, Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, and Plasmodium malaria.
  • pathogenic protozoans such as, but not limited to, Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania
  • the diseases that can be treated or prevented by the methods of the present invention include, but are not limited to: human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medu
  • Diseases and disorders involving a deficiency in cell proliferation or in which cell proliferation is desired for treatment or prevention, and that can be treated or prevented by antagonizing Stat3, include but are not limited to degenerative disorders, growth deficiencies, hypoproliferative disorders, physical trauma, lesions, and wounds; for example, to promote wound healing, or to promote regeneration in degenerated, lesioned or injured tissues, etc. 5.8 PHARMACEUTICAL FORMULATIONS AND MODES OF ADMINISTRATION
  • a pharmaceutical of the invention comprises a substantially purified protein, nucleic acid, or chemical (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • Various delivery systems are known and can be used to administer the pharmaceutical of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • Nucleic acids and proteins of the invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents such as chemotherapeutic agents. Administration can be systemic or local.
  • nucleic acid or protein of the invention may be desirable to administer by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
  • a protein including an antibody, of the invention
  • care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al, 1989, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365; Lopez-Berestein, ibid., pp. 317-327; see generally, ibid.)
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321:574).
  • polymeric materials can be used (see Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida; Controlled Drag Bioavailability, Drag Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983, Macromol.
  • nucleic acid of the invention can be administered in vivo to promote expression of its encoded protein or RNA molecule, by constracting it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
  • a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • compositions pharmaceutical compositions
  • Such compositions comprise a therapeutically effective amount of a nucleic acid, chemical or protein of the invention, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as phannaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the nucleic acid or protein of the invention, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the pharmaceutical of the invention is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical of the invention may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical of the invention is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the amount of the nucleic acid or protein of the invention which will be effective in the treatment or prevention of the indicated disease can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the stage of indicated disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the present invention may be better understood by reference to the following non- limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
  • Effective cancer gene therapies require the killing of genetically untransduced tumor cells ("bystander" cells) concomitant with genetically transduced tumor cells. Because ⁇ transfection efficiency is a rate-limiting step for gene therapy, the efficacy of cancer gene therapy is enhanced by bystander effects.
  • Plasmids The bicistronic green fluorescent protein vector, pIRES-EGFP, was obtained from Clontech (Palo Alto, CA). Insertion of Stat3 ⁇ cDNA into the pIRES-EGFP plasmid to construct pIRES-Stat3 ⁇ was as described previously Catlett-Falcone et al, 1999, supra.
  • B 16 murine melanoma cells were grown in RPMI
  • Nuclear extracts and EMS A Nuclear extract preparation and EMSA analysis of STAT DNA-binding activity were performed as previously described Catlett-Falcone et al., 1999, supra.
  • B16 cells transfected with pIRES-EGFP or pIRES-Stat3 ⁇ were washed with CellScrubTM buffer (Gene Therapy Systems, San Diego, CA) 24 h after
  • Apoptosis of transiently-transfected B16 cells was analyzed after staining with Annexin V-PE by two-color flow cytometry.
  • Apoptosis of non-transfected tumor cells in the upper chambers of Transwell units was analyzed after staining with Annexin V-PE and VIA-PROBETM 7-AAD (Pharmingen, San Diego, CA) by two-color flow cytometry.
  • Cell cycle analysis Cell cycle analysis based on DNA content was performed.
  • 3 H-thymidine ( 3 H-TdR) incorporation assay 0.25 ⁇ ci 3 H-TdR was added to each well during the last 4 h of incubation, transferred to glassfiber filters by an automated cell harvester (Tomtec, Hamden, CT) and 3 H-TdR incorporation was determined with a liquid scintillation ⁇ -counter (Pharmacia Wallac, Finland).
  • 3 H-thymidine ( 3 H-TdR) incorporation assay 0.25 ⁇ ci 3 H-TdR was added to each well during the last 4 h of incubation, transferred to glassfiber filters by an automated cell harvester (Tomtec, Hamden, CT) and 3 H-TdR incorporation was determined with a liquid scintillation ⁇ -counter (Pharmacia Wallac, Finland).
  • MTT assays 5 ⁇ l MTT (10 mg/ml) was added to each well during the last 4 h of incubation. Cells were lysed in 100 ⁇ l
  • RNA isolation and RNase protection assay Total RNA was isolated from 5.0 x 10 6 cells by TRIzol reagent (Gibco BRL, Grand Island, NY). RNase protection assays (RPA) were carried out using the PharMingen Riboquant mAPO- 3 (TRAIL, FasL, CD95, and other death receptor associated genes) and mAPO- 2 (Bel- 2 family members) multi- probe templates according to the manufacturer's protocol (PharMingen, San Diego, CA). Briefly, the multi-probe template was synthesized by in vitro transcription with incorporation of [ 32 P]- ⁇ UTP and purified on a G50 Sephadex column (5- Prime to 3- Prime, Boulder, CO).
  • Stat3 ⁇ overexpression in B16 cells disrupts Stat3 DNA-binding activity.
  • FIG. 1 shows specific DNA- binding activities of endogenous Stat3 (lanes 1, 2) and ectopic Stat3 ⁇ (lane 3). EGF- induced Stat3 binding activity in NTH3T3 (lane 4) was used as a positive control.
  • EGF- induced Stat3 binding activity in NTH3T3 (lane 4) was used as a positive control.
  • By supershift analysis with antibody that recognizes Stat3 but not Stat3 ⁇ or antibody that recognizes Stat3 ⁇ but not Stat3 it was shown that there were Stat3-Stat3 homodimers in mock-transfected B16 cells and empty vector-transfected B16 cells.
  • Overexpression of Stat3 ⁇ in Stat3 ⁇ -transfected B 16 cells results in mostly Stat3 ⁇ -Stat3 ⁇ homodimer formation.
  • Stat3 ⁇ -mediated B16 cell growth inhibition involves both cell cycle arrest and apoptosis.
  • pIRES-EGFP or pIRES-Stat3 ⁇ vectors were transfected into B16 cells, respectively. While their transfection efficiencies were similar within each experiment as determined by the percentage of cells that exhibit green fluorescence at 24 hours post transfection (by FACS analysis), the number of live B16 cells decreases dramatically 48 h
  • FIG. 2A ⁇ later in the Stat3 ⁇ -transfected population.
  • FIG. 2B The cell cycle distributions of empty vector and Stat3 ⁇ -transfected cells were shown in Fig. 2B.
  • the Stat3 ⁇ transfected B16 cells show progressive accumulation in G 0 /G, phase, with concomitant decrease of the population
  • Stat3 ⁇ overexpression leads to production of soluble factors capable of inducing both cell cycle arrest and apoptosis.
  • 3 ⁇ - Stat3 ⁇ -dependent bystander effects were not mediated by cell-cell contact, but were mediated via soluble factors, supernatants were collected 24 h, 36 h, 48 h after Stat3 ⁇ vector transfection and subsequently used as conditioned medium for non-transfected B16 cells.
  • Different assays for growth inhibition were performed to show that the supernatants from Stat3 ⁇ -transfected B16 cells inhibit the growth of non-transfected B16 cells.
  • Fig. 3A shows that the conditioned media obtained from Stat3 ⁇ -transfected B16 cells inhibit B 16 cell growth, while that obtained from wild-type B 16 cells or empty vector-transfected B16 cells does not.
  • Stat3 ⁇ -transfected B 16 cells undergo apoptosis as demonstrated by Annexin V-PE and 7- AAD staining followed by FACS analysis (Fig. 3C).
  • These Stat3 ⁇ -induced soluble factors produced by transfected-B16 cells were also capable of inducing apoptosis of non- transfected Meth A cells (Fig. 3C).
  • TRAIL induced in Stat3 ⁇ -transfected B 16 cells.
  • RPAs RNase protection assays
  • RNA was _ isolated from various cell cultures and RPAs were carried out using probes specific for key physiologic regulators of apoptosis.
  • Stat3 ⁇ gene therapy as an effective cancer therapeutic approach is supported by the finding that in vivo the number of dying tumor cells greatly exceeds the number of tumor cells transfected with Stat3 ⁇ .
  • in vitro results presented herein demonstrate that overexpression of Stat3 ⁇ leads to apoptosis and cell cycle arrest of murine melanoma _ - B16 cells.
  • disruption of Stat3 signaling in B16 cells also results in the production of soluble factors.
  • the soluble factors were capable of inducing apoptosis and cell cycle arrest of non-transfected B16 tumor cells, showing that killing of bystander B 16 tumor cells in vivo is mediated by one or more soluble factors.
  • Constitutively-activated Stat3 correlates with elevated levels of members of the Bcl- 2 family of anti-apoptotic regulatory proteins, Bcl-X L and Mcl-1 in human malignancies. Inhibition of Stat3 activity by Stat3 ⁇ down regulates the expression of these anti-apoptotic proteins, resulting in apoptosis. In addition to inducing anti-apoptotic proteins, constitutive activation of Stat3 promotes the expression of proteins that were important for cell proliferation. In particular, cyclin Dl, which controls progression from Gl to S phase, is elevated in cells expressing the constitutively-activated mutant form of Stat3, Stat3C, or endogenous Stat3 activated by the Src oncoprotein. Down-regulation of these and other anti-apoptotic and pro-proliferation proteins by Stat3 ⁇ could, without being limited by
  • B16 tumors treated in vivo with a Stat3 dominant-negative variant, Stat3 ⁇ become infiltrated with iNOS-positive macrophages and T cells.
  • Inhibition of Stat3 signaling in B16 tumor cells results in secretion of soluble factors, which activate macrophages to produce additional inflammatory and tumoricidal mediators, including nitric oxide.
  • transfection of B16 cells with the Stat3 ⁇ gene upregulates
  • iNOS deficient macrophages abrogates soluble factor-induced, macrophage-mediated anti- B16 activity. Furthermore, our results demonstrate that inhibition of Stat3 signaling in B16 tumor cells results in elevated expression of IP- 10, IL-6, TNF- ⁇ and IFN- ⁇ . Production of the soluble factors, including these pro-inflammatory cytokines and chemokines, in turn upregulates the expression of RANTES in macrophages and TNF- ⁇ in neutrophils.
  • B16 melanoma cells were cultured in RPMI medium 25 with 10 % FBS. Transfections were performed using GenePORTERTM Transfection Reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. To determine transfection efficiency, fluorescence intensities of B16 cells transfected with either pIRES-EGFP or pIRES-Stat3 were measured by FACS (Becton Dickinson Immunocytometry, CA) 24 h after transfection. Supernatants were collected at 0 various time points as indicated in figures and figure legends. Supernatants were also collected from B 16 cells treated UV-irradiation at various time points. Mice.
  • mice Six- to eight-week old female C57/B6 mice were obtained from the National Cancer Institute (Frederick, MD). Cohorts of 3-5 mice per group were used for these experiments. To induce tumor, mice were shaved on the left flank and injected s.c. with 5 x 10 5 of B16 35 cells in 100 ⁇ l of PBS. Gene therapy with Stat3 ⁇ of established B16 tumors was described previously [Niu, 1999 #72]. Antibody staining of iNOS in B16 tumors. 3 ⁇ m paraffin sections were deparaffinized and endogenous peroxidase was blocked with 3% aqueous hydrogen peroxide.
  • the sections were incubated for 20 min in normal goat serum in PBS, followed by overnight incubation at 4° C with rabbit anti-iNOS polyclonal antibody (Transduction Laboratories). After washing with PBS, sections were incubated for 2 min with diaminobenzidine tetrahydrochloride, rinsed with tap water and counterstained with modified Mayer's hematoxylin. Sections were dehydrated, cleared and mounted. Peritoneal macrophages and neutrophils. Peritoneal macrophages were obtained and enriched. Neutrophils were obtained from peritoneal cavity 4 h after i.p. injection of 1 ml of 3% thioglycollate.
  • Macrophages were incubated for 48 h in conditioned medium containing 50 % supernatants from either non-transfected, or pIRES-Stat3 ⁇ or pIRES-EGFP transfected, or UV-irradiated B 16 cells. Macrophage supernatants (0.1 ml) were collected and examined for nitric oxide accumulation using Griess reagent. Neutrophil supernatants were tested for TNF- ⁇ production using ELIS A (R & D Systems, MN).
  • MTT assay was performed to ensure that the viability of macrophages and neutrophils cultivated in different supernatants was not affected. Macrophage-mediated antitumor cytotoxicity. Antitumor cytotoxic activity of macrophages against B 16 cells was determined by inhibition of DNA synthesis. Briefly, peritoneal macrophages (1 x 10 5 ) were incubated in 50 % supernatants derived from either wild type, pIRES-Stat3 ⁇ or pIRES-GFP transfected, or UV-irradiated B16 cells for 6 h.
  • B16 cells (1.0 x 10 4 /well) were added and co-cultured for 48 h with and without peritoneal macrophages.
  • NOS inhibitor N-monomethyl-L-arginine (NMA) (0.5 mM, Sigma) or H 2 O 2 quencher, catalase (500 U/ml, Boehringer Mannheim, Indianapolis, IN) were added to macrophages before adding supernatant from Stat3 ⁇ -transfected B16 cells.
  • the co-cultured cells were pulsed with 3 H-thymidine ( 3 H-TdR) (0.25 ⁇ Ci/well) during the last 6 h of incubation to estimate DNA synthesis.
  • 3 H-TdR 3 H-thymidine
  • RNA encoded by GAPDH housekeeping gene was used to normalize the amounts of RNAs loaded in each lane.
  • RNA expression profiles of macrophages treated with supernatants derived from B16 cells transfected with either Stat3 ⁇ or GFP vector Similar protocols were used to determine RNA expression profiles of macrophages treated with supernatants derived from B16 cells transfected with either Stat3 ⁇ or GFP vector.
  • mCK-5 Top Panel
  • mCK-3 RPAs RNAs prepared from mock transfected B16 cells and UV-irradiated, apoptotic B16 cells were also included to serve as negative controls.
  • macrophage RNA analysis RNAs prepared from macrophages treated with supernatants from mock transfected and UV-irradiated B 16 cells were also included.
  • B16 tumors treated with Stat3b gene transfer were infiltrated with immune cells, including iNOS-positive macrophages
  • Nitric oxide-dependent cytotoxic activity against B16 tumor cells by soluble factor- activated macrophages Nitric oxide was the key mediator of the tumoricidal activity of macrophages. This example demonstrates that soluble factor-induced macrophage NO production leads to cytotoxic activity against B 16 tumor cells. 3 H-thymidine incorporation to estimate DNA synthesis and cell proliferation was performed. In the results summarized in Fig. 6, supernatants derived from various B16 cells were removed from macrophages after 6 hours of incubation.
  • B16 cells have little effect on the proliferation of non-transfected B16 cells, pre-incubation of macrophages in supernatant collected from pIRES-Stat3 ⁇ -transfected, but not control vector-transfected B 16 cells, induce strong cytostasis of non-transfected B 16 cells (Fig. 6).
  • Macrophage-mediated cytostasis of B16 cells was significantly blocked by a specific inhibitor of iNOS, NMA (Fig. 6).
  • addition of catalase which inhibits H 2 O 2 production, does not influence macrophage cytotoxic effects against B16 cells.
  • soluble factor-induced macrophage cytostasis against B16 cells was abrogated when macrophages derived from iNOS knockout mice were used instead of those from wild-type mice (Fig. 6).
  • Blocking Stat3 signaling in B16 cells elevates the expression of pro-inflammatory chemokines and cytokines. which in turn activates inflammatory cells to produce additional danger signals.
  • RNA expression profiles of a number of cytokines and chemokines in pIRES-Stat3 ⁇ transfected B 16 cells were determined.
  • results from these RNase protection assays using multi-template RNA probes indicated that the expression levels of IFN- ⁇ , TNF- ⁇ , IL-6 and IP-10 mRNAs, but not IL-4 and IL-10, were elevated in Stat3 ⁇ -transfected B16 cells in comparison with mock-transfected, control vector-transfected and UV-irradiated B 16 cells (Fig. 7). . g .
  • macrophages were activated to produce NO by these cytokines in vitro. Peritoneal macrophages were able to synthesize NO when stimulated by IFN- ⁇ and TNF- ⁇ simultaneously.
  • supernatant derived from Stat3 ⁇ -transfected B16 cells was capable of stimulating enhanced expression of RANTES by macrophages (Fig. 8 A) and
  • this example supports a mechanistic basis for the heavy infiltration of immune cells in tumors treated with Stat3 ⁇ gene transfer and indicate a critical immune component to the potent bystander effect of gene therapy targeting Stat3 signaling in tumor cells.
  • tumor site is not limited to direct cytotoxic activity against tumor cells.
  • iNOS and NO There is a critical role of iNOS and NO in mediating T cell-dependent antitumor responses: GM-CSF vaccine-induced antitumor T cell immune response requires NO/iNOS, and IL-12-induces antitumor T-cell responses that were also iNOS/NO dependent .
  • TNF- ⁇ causes necrosis of tumor cells.
  • Death of tumor cells in vivo itself promotes immunogenicity, because the release of tumor antigens under inflammatory conditions allows cross-priming of antigen-presenting cells, from which a tumor-specific T cell immunity can be elicited [Huang et al., 1994, Science 264:961-965; Huang et al, 1994, Ciba Found. Symp. 187:229-240; Dranoff et al., 1993, Proc. Natl. Acad. Sci.
  • Targeting Stat3 signaling may generate tumor-specific T cell immunity.
  • Stat3 constitutive activation of Stat3 contributes to oncogenesis by helping tumor cells evade immune surveillance.
  • the ability of normal cells to produce immunologic danger signals during infection and tissue destruction is well known, and Stat3 in hematopoietic cells is the regulator of macrophage activation.
  • Stat3 mediates immune suppression by IL- 10 signaling, which antagonizes the production of inflammatory cytokines such as TNF- ⁇ , IL-1 and IL-6, and suppresses iNOS activity.
  • mice with Stat3-/- macrophage and neutrophils were highly susceptible to endotoxin shock, with increased production of TNF- ⁇ , IL-1, IL-6 and IFN- ⁇ , and showed an enhanced T-helper 1 cell activity.
  • Stat3-/- macrophages display increased expression of MHC class II and B7-1 molecules, thus Stat3 signaling
  • Stat3 signaling is required for cell transformation by v-Src.
  • Activity of Src tyrosine kinase has been shown to regulate the expression of VEGF, a potent stimulator of angiogenesis, which is crucial for tumor growth and metastasis formation.
  • blocking Stat3 signaling inhibits v-Src-mediated VEGF upregulation, and expression of constitutively-activated Stat3 increases the production of VEGF in fibroblasts.
  • blocking Stat3 signaling inhibits transcriptional activity of the VEGF promoter and downregulates expression of the endogeneous VEGF gene.
  • constitutive Stat3 signaling upregulates VEGF expression, which in turn induces angiogenesis. Therefore, in the present invention, inhibition of Stat3 signaling inhibits angiogenesis mediated by downregulation of VEGF expression. And activation of Stat3 signaling promotes angiogenesis mediated by upregulation of VEGF expression.
  • Angiogenesis plays a critical role in a wide variety of disorders, such as ischemic diseases and proliferative angiopathies with neovascularization.
  • Vascular endothelial growth factor (VEGF) has been shown to be a potent endothelial cell-specific mitogen that stimulates angiogenesis.
  • An essential role of VEGF in tumorigenesis has been shown when systemic treatment of tumor-bearing animals with a neutralizing antibody to VEGF inhibits tumor growth, which correlates with reduced tumor vascularity.
  • Stat3 constitutive activation of Stat3 in numerous human solid tumors was caused by deregulated activities of c-Src tyrosine kinase.
  • Stat3 was constitutively activated by IL-6 mediated signaling. Because Src tyrosine kinase activity and IL-6 mediated signaling can lead to both Stat3 activation and VEGF upregulation in tumor cells, it is demonstrated herein that Stat3 regulates VEGF expression in tumor cells.
  • Stat3 signaling was required for v-Src-induced VEGF upregulation.
  • Blocking Stat3 signaling in tumor cells inhibits VEGF promoter activity.
  • B16 murine melanoma and SCK murine tumor cells were transiently transfected with Stat3 ⁇ and a reporter construct containing luciferase cDNA under the control of the VEGF promoter.
  • a plasmid construct containing the luciferase cDNA in the absence of the VEGF promoter was also transfected into 3T3 fibroblasts. Both of B16 and SCK tumor cells harbor constitutively activated Stat3.
  • VEGF promoter activity was readily detectable in both tumor cells as indicated by the high expression levels of luciferase protein.
  • cotransfection with Stat3 ⁇ but not the control vector, greatly inhibited the transcriptional activity of the VEGF promoter.
  • the inhibitory effect of Stat3 ⁇ on the transcriptional activity of the VEGF -. _ promoter was also observed in the tumor cells transfected with Stat3 anti-sense oligonucleotides (Fig. 12A-B). It was also shown that blocking Stat3 signaling in tumor cells inhibits the expression of the endogeneous VEGF gene.
  • B16 tumor cells were transiently transfected with Stat3 ⁇ and the expression of endogeneous VEGF gene was determined at the RNA and protein levels. As shown in Fig. 13, inhibition of constitutive activation of Stat3 in tumor cells downregulates expression of the endogeneous VEGF gene.
  • Blocking Stat3 signaling either by a Stat3 dominant-negative variant or antisense oligos, leads to antiangiogensis via down regulation of VEGF, thus adding a new dimension to the therapeutic effect of anti-Stat3 signaling.
  • VEGF In addition to antiangiogenesis, downregulation of VEGF may also contribute to increased immune responses associated with inhibition of Stat3 signaling in tumor cells.
  • VEGF produced by tumor cells have been shown to inhibit the functional maturation of dendritic cells, the most potent antigen presenting cells. Dendritic cells incubated with tumor cells in the presence of Stat3 antisense oligos, but not control oligos, undergo normal functional maturation.

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Abstract

L'invention concerne des procédés de modulation, c.-à-d. ayant une action agoniste ou antagoniste sur l'activité de signalisation de Stat3 (transducteur de signal et activateur de transcription3), utiles en thérapie génique. L'inhibition et/ou l'activation de la signalisation de Stat3 constitue une méthode efficace de modulation de l'angiogenèse et de la réaction immunitaire pour traiter et/ou prévenir l'inflammation, les infections, les affections immunitaires et l'ischémie.
PCT/US2001/028254 2000-09-08 2001-09-10 Agonistes et antagonistes de stat3 et applications therapeutiques de ceux-ci WO2002020032A1 (fr)

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CA002421723A CA2421723A1 (fr) 2000-09-08 2001-09-10 Agonistes et antagonistes de stat3 et applications therapeutiques de ceux-ci
US10/380,020 US20040052762A1 (en) 2001-09-10 2001-09-10 Stat3 agonists and antagonists and therapeutic uses thereof
EP01970740A EP1324763A4 (fr) 2000-09-08 2001-09-10 Agonistes et antagonistes de stat3 et applications therapeutiques de ceux-ci
US11/526,367 US20070072822A1 (en) 2000-09-08 2006-09-25 STAT3 agonists and antagonists and therapeutic uses thereof
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WO2004080394A2 (fr) * 2003-03-07 2004-09-23 Johns Hopkins University Antagonistes de stat3 et leur utilisation en tant que vaccins contre le cancer
US7951374B2 (en) 2004-12-14 2011-05-31 University Of South Florida Methods for inhibiting STAT3 signaling in immune cells
US8841257B2 (en) 2009-04-10 2014-09-23 Board Of Regents, The University Of Texas System Inhibitors of STAT3 and uses thereof
US9345682B2 (en) 1999-01-27 2016-05-24 University Of South Florida Inhibition of STAT3 signal transduction for human cancer therapy
US20220133825A1 (en) * 2016-07-19 2022-05-05 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Oncolytic viruses targeting stat3

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9345682B2 (en) 1999-01-27 2016-05-24 University Of South Florida Inhibition of STAT3 signal transduction for human cancer therapy
WO2004080394A2 (fr) * 2003-03-07 2004-09-23 Johns Hopkins University Antagonistes de stat3 et leur utilisation en tant que vaccins contre le cancer
WO2004080394A3 (fr) * 2003-03-07 2009-04-02 Univ Johns Hopkins Antagonistes de stat3 et leur utilisation en tant que vaccins contre le cancer
US7638122B2 (en) * 2003-03-07 2009-12-29 University Of South Florida Stat3 antagonists and their use as vaccines against cancer
US7951374B2 (en) 2004-12-14 2011-05-31 University Of South Florida Methods for inhibiting STAT3 signaling in immune cells
US8841257B2 (en) 2009-04-10 2014-09-23 Board Of Regents, The University Of Texas System Inhibitors of STAT3 and uses thereof
US20220133825A1 (en) * 2016-07-19 2022-05-05 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Oncolytic viruses targeting stat3

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WO2002020032A9 (fr) 2003-03-20
CA2421723A1 (fr) 2002-03-14
EP1324763A4 (fr) 2007-10-31

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