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WO2009154610A1 - Composés d'oligonucléotides de régulation immune (iro) permettant de moduler une réponse immune fondée sur un récepteur de type toll - Google Patents

Composés d'oligonucléotides de régulation immune (iro) permettant de moduler une réponse immune fondée sur un récepteur de type toll Download PDF

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Publication number
WO2009154610A1
WO2009154610A1 PCT/US2008/067184 US2008067184W WO2009154610A1 WO 2009154610 A1 WO2009154610 A1 WO 2009154610A1 US 2008067184 W US2008067184 W US 2008067184W WO 2009154610 A1 WO2009154610 A1 WO 2009154610A1
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Prior art keywords
iro
nucleotide
disease
tlr
linkage
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PCT/US2008/067184
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English (en)
Inventor
Ekambar R. Kandimalla
Daqing Wang
Yukui Li
Dong Yu
Fugang Zhu
Lakshmi Bhagat
Sudhir Agrawal
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Idera Pharmaceuticals, Inc.
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Priority to PCT/US2008/067184 priority Critical patent/WO2009154610A1/fr
Publication of WO2009154610A1 publication Critical patent/WO2009154610A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • IMMUNE REGULATORY OLIGONUCLEOTIDE COMPOUNDS TO MODULATE TOLL-LIKE RECEPTOR BASED IMMUNE RESPONSE
  • the invention generally relates to the field of immunology and immunotherapy, and more specifically to immune regulatory oligonucleotide (IRO) compositions and their use for inhibition and/or suppression of Toll-like Receptor- mediated immune responses.
  • IRO immune regulatory oligonucleotide
  • TLRs Toll-like receptors
  • TLRs are a key means by which mammals recognize and mount an immune response to foreign molecules and also provide a means by which the innate and adaptive immune responses are linked (Akira, S. et al. (2001) Nature Immunol. 2:675-680; Medzhitov, R. (2001) Nature Rev. Immunol. 1 :135-145). TLRs have also been shown to play a role in the pathogenesis of many diseases, including autoimmunity, infectious disease, and inflammation (Cook, D.N. et al. (2004) Nature Immunol. 5:975-979) and the regulation of TLR-mediated activation using appropriate agents may provide a means for disease intervention.
  • TLRs are located on the cell surface to detect and initiate a response to extracellular pathogens and other TLRs are located inside the cell to detect and initiate a response to intracellular pathogens.
  • Table 1 provides a representation of TLRs and the known agonists therefore (Diebold, S. S. et al. (2004) Science 303:1529-1531; Liew, F. et al. (2005) Nature 5:446-458; Hemmi H et al.
  • TLR9 recognizes unmethylated CpG motifs present in bacterial and synthetic DNA (Hemmi, H. et al. (2000) Nature 408:740-745).
  • Other modifications of CpG-containing phosphorothioate oligonucleotides can also affect their ability to act as modulators of immune response through TLR9 (see, e.g., Zhao et al., Biochem. Pharmacol. (1996) 51 :173-182; Zhao et al. (1996) Biochem Pharmacol. 52:1537-1544; Zhao et al. (1997) Antisense Nucleic Acid Drug Dev. 7:495-502; Zhao et al (1999) Bioorg. Med. Chem. Lett.
  • TLRs T helper cells involved in classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs) are ThI cells.
  • CTLs cytotoxic T lymphocytes
  • ThI cells T helper cells involved in classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs)
  • CTLs cytotoxic T lymphocytes
  • Th2 cells have been shown to be activated in response to bacteria and parasites and may mediate the body's adaptive immune response (e.g. IgE production and eosinophil activation) through the secretion of IL-4 and IL-5.
  • the type of immune response is influenced by the cytokines produced in response to antigen exposure and the differences in the cytokines secreted by ThI and Th2 cells may be the result of the different biological functions of these two subsets.
  • TLRs While activation of TLRs is involved in mounting an immune response, an uncontrolled stimulation of the immune system through TLRs may exacerbate certain diseases in immune compromised subjects.
  • ODNs oligodeoxyoligonucleotides
  • GGGG "GGGG" or "GC” sequences
  • GC GC sequences
  • GGGG containing ODNs will suppress systemic lupus (Patole, P. et al. (2005) J. Am. Soc. Nephrol. 16:3273-3280). Additionally, Gursel, L, et al., J. Immunol, 171 : 1393-1400 (2003), describe repetitive TTAGGG elements, which are present at high frequency in mammalian telomeres, down-regulate CpG-induced immune activation. Shirota, H., et al, J. Immunol, 173: 5002-5007 (2004), demonstrate that synthetic oligonucleotides containing the TTAGGG element mimic this activity and could be effective in the prevention/treatment of certain ThI -dependent autoimmune diseases.
  • G containing ODNs are acting as antagonists of TLRs.
  • US 6,426,334, Agrawal et al demonstrate that administering CpG oligonucleotides containing GGGG strings have potent antiviral and anticancer activity, and further that administration of these compounds will cause an increase in serum IL- 12 concentration.
  • CpG oligos containing polyG sequences are known to induce immune responses through TLR9 activation (Verthelyi D et al, J Immunol.
  • polyG oligonucleotides are also known to inhibit HIV and ReI A (McShan WM, et al, J Biol Chem., 267(8):5712-21, 1992; Rando, RF et al., J Biol Chem, 270(4): 1754-60, 1995; Benimetskaya L, et al., Nucleic Acids Res., 25(13):2648-56, 1997).
  • ODNs containing an immune stimulatory CpG motif and 4 consecutive G nucleotides (class A ODNs) induce interferon- ⁇ production and a ThI shift in the immune response.
  • Class A ODN have been shown to induce a TLR-mediated immune response.
  • oligonucleotides containing guanosine strings have been shown to form tetraplex structures, act as aptamers and inhibit thrombin activity (Bock LC et al., Nature, 355:564-6, 1992; Padmanabhan, K et al., J Biol Chem., 268(24): 17651-4, 1993).
  • thrombin activity Bock LC et al., Nature, 355:564-6, 1992; Padmanabhan, K et al., J Biol Chem., 268(24): 17651-4, 1993.
  • the invention provides novel immune regulatory oligonucleotides
  • IRO immunosorbentatives mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated renin receptor mediated peptide, oligonucleotide sequence, oligonucleotide motif that would be immune stimulatory but for the modification.
  • the invention further provides novel IRO compositions having the structure 5-N m - N 3 N 2 N 1 CGN 1 N 2 N 3 - N m -3', wherein CG is an oligonucleotide motif and C is cytosine or a pyrimidine nucleotide derivative or non-nucleotide linkage, and G is guanosine a purine nucleotide derivative or non-nucleotide linkage; N1-N3, at each occurrence, is independently a nucleotide, nucleotide derivative or non- nucleotide linkage; Nm, at each occurrence, is independently a nucleotide, nucleotide derivative or non-nucleotide linkage; provided that at least one Nl to N3 and/or C and/or G is a nucleotide derivative or non-nucleotide linkage; and further provided that compound contains less than 4 consecutive guanosine nucleotides wherein
  • the invention further provides for a pharmaceutical composition comprising any an IRO and a pharmaceutically acceptable carrier.
  • the invention provides for a method for modifying a TLR-stimulating oligonucleotide comprising an immune stimulatory oligonucleotide motif comprising incorporating chemical modifications into the immune stimulatory oligonucleotide motif and/or to the sequence flanking the immune stimulatory oligonucleotide motif, wherein the immune stimulatory activity of the immune stimulatory oligonucleotide motif is suppressed by the chemical modifications.
  • the invention further provides a method for inhibiting a TLR-mediated immune response in a vertebrate, the method comprising administering to the vertebrate an IRO compound in a pharmaceutically effective amount, wherein the route of administration is parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form.
  • inhibiting TLR stimulation comprising administering an IRO compound according to the invention, wherein the TLR is selected from TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9.
  • the invention further provides a method for inhibiting the activity of a
  • TLR agonist comprising administering an IRO compound, wherein the IRO is administered at the same time, prior to or after the TLR agonist.
  • the TLR agonist is selected from an agonist of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9.
  • the invention further provides a method for therapeutically treating a vertebrate having a disease mediated by a TLR, such method comprising administering to the vertebrate an IRO compound according to the invention in a pharmaceutically effective amount.
  • the disease is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, malaria, Lyme disease, ocular infections, conjunctivitis, skin disorders, psoriasis, scleroderma, cardiovascular disease, atherosclerosis, chronic fatigue syndrome, sarcoidosis, transplant rejection, allergy, asthma or a disease caused by a pathogen.
  • Preferred autoimmune disorders include without limitation lupus erythematosus, multiple sclerosis, type I diabetes mellitus, irritable bowl syndrome, Chron's disease, rheumatoid arthritis, septic shock, alopecia universalis, acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas disease, chronic obstructive pulmonary disease, coeliac disease, dermatomyositis, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
  • Preferred inflammatory disorders include without limitation airway inflammation, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, Behcet's disease,_hypersensitivities, inflammatory bowel disease, reperfusion injury, rheumatoid arthritis, transplant rejection, ulcerative colitis, uveitis, conjunctivitis and vasculitis.
  • the invention further provides a method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, malaria, Lyme disease, ocular infections, conjunctivitis, skin disorders, psoriasis, scleroderma, cardiovascular disease, atherosclerosis, chronic fatigue syndrome, sarcoidosis, transplant rejection, allergy, asthma or a disease caused by a pathogen in a vertebrate, such method comprising administering to the vertebrate an IRO compound according to the invention in a pharmaceutically effective amount.
  • Preferred autoimmune disorders include without limitation lupus erythematosus, multiple sclerosis, type I diabetes mellitus, irritable bowl syndrome, Chron's disease, rheumatoid arthritis, septic shock, alopecia universalis, acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas disease, chronic obstructive pulmonary disease, coeliac disease, dermatomyositis, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain- Barre syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
  • Preferred inflammatory disorders include without limitation airway inflammation, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, Behcet's disease, ⁇ hypersensitivities, inflammatory bowel disease, reperfusion injury, rheumatoid arthritis, transplant rejection, ulcerative colitis, uveitis, conjunctivitis and vasculitis.
  • the IRO compound is administered in combination with one or more vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, TLR antagonists, peptides, proteins, gene therapy vectors, DNA vaccines, adjuvants, kinase inhibitors or co-stimulatory molecules or combinations thereof.
  • the route of administration is parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form.
  • Figure 1 demonstrates IRO inhibition of the TLR9 agonist activity of an IMO.
  • Figure 2 demonstrates the specificity of one IRO compound as an antagonist of TLR9 vs TLR3.
  • Figure 3 demonstrates dose-dependent inhibition by an IRO.
  • Figures 4A - 4D demonstrate that pre-administration and simultaneous administration of IRO can inhibit an agonist of TLR9.
  • Figures 5A and 5B demonstrate that two CpG oligonucleotides linked at their 5' ends show TLR-inhibitory properties.
  • Figure 6 demonstrates that an IRO inhibited TLR9 agonist activity in human cell cultures.
  • Figure 7 demonstrates an IRO effect on OVA induced Th2 and ThI immune responses.
  • Figure 8 demonstrates that an IRO reversed Th2 inhibitory properties and inhibited ThI immune responses induced by an IMO.
  • Figure 9 demonstrates antibody responses to an IMO and an IRO.
  • Figure 10 demonstrates early inhibitory activity of selected IROs on
  • Figure 11 demonstrates early inhibitory activity of selected IROs on
  • Figure 12 demonstrates early inhibitory activity of selected IROs on
  • Figure 13 demonstrates long-term antagonist activity of selected IROs on TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.
  • Figure 14 demonstrates long-term antagonist activity of selected IROs on TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.
  • Figure 15 demonstrates long-term antagonist activity of selected IROs on TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.
  • Figure 16 demonstrates that an IRO inhibits proliferation of wild type
  • FIGS. 17A through 17C demonstrate that an IRO inhibited IL-6 and
  • Figures 18A through 18E demonstrate that MRL-lpr mice injected with an IRO reduced levels of anti-DNA IgGl and IgG2a antibodies in serum and protein in urine.
  • FIG. 19 demonstrates that an IRO inhibits serum anti-DNA IgG2a in
  • the present invention relates to the therapeutic use of novel oligonucleotides as immune modulatory agents for immunotherapy applications.
  • the invention provides Immune Regulatory Oligonucleotide (IRO) compounds as antagonists of toll-like receptors (TLRs) to inhibit and/or suppress a TLR-mediated immune response.
  • IROs Immune Regulatory Oligonucleotide
  • TLRs toll-like receptors
  • These IROs have unique sequences that inhibit or suppress TLR-mediated signaling in response to endogenous and/or exogenous TLR ligands or agonists.
  • the invention provides methods for suppressing an immune response caused by TLRs and can be used for immunotherapy applications such as, but not limited to, treatment of cancer, autoimmune disorders, asthma, respiratory allergies, food allergies, skin allergies, systemic lupus erythematosus (SLE), arthritis, pleurisy, chronic infections, inflammatory diseases, inflammatory bowl syndrome, sepsis, and bacteria, parasitic, and viral infections in adult and pediatric human and veterinary applications.
  • the invention further provides IRO compounds having optimal levels of immune modulatory effect for immunotherapy and methods for making and using such compounds.
  • IRO compounds of the invention are useful in combination with, for example, DNA vaccines, antigens, antibodies, and allergens; and in combination with chemotherapeutic agents (both traditional chemotherapy and modern targeted therapies) and/or antisense oligonucleotides for prevention and treatment of diseases.
  • chemotherapeutic agents both traditional chemotherapy and modern targeted therapies
  • antisense oligonucleotides for prevention and treatment of diseases.
  • oligonucleotide generally refers to a polynucleoside comprising a plurality of linked nucleoside units. Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods. In preferred embodiments each nucleoside unit can encompass various chemical modifications and substitutions as compared to wild-type oligonucleotides, including but not limited to modified nucleoside base and/or modified sugar unit. Examples of chemical modifications are known to the person skilled in the art and are described, for example, in Uhlmann E et al. (1990) Chem. Rev.
  • nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages.
  • internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages.
  • oligonucleotide also encompasses polynucleosides having one or more stereospecif ⁇ c internucleoside linkage (e.g., (R P )- or (5 * p)-phosphorothioate, alkylphosphonate, or phosphotriester linkages).
  • R P stereospecif ⁇ c internucleoside linkage
  • 5 * p p-phosphorothioate
  • alkylphosphonate e.g., phosphotriester linkages
  • oligonucleotide and dinucleotide are expressly intended to include polynucleosides and dinucleosides having any such internucleoside linkage, whether or not the linkage comprises a phosphate group.
  • these internucleoside linkages may be phosphodiester, phosphorothioate, or phosphorodithioate linkages, or combinations thereof.
  • the term "2'-substituted ribonucleoside” or “2 ' -substituted arabinoside” generally includes ribonucleosides or arabinonucleosides in which the hydroxyl group at the 2' position of the pentose moiety is substituted to produce a 2'- substituted or 2'-O-substituted ribonucleoside.
  • such substitution is with a lower hydrocarbyl group containing 1-6 saturated or unsaturated carbon atoms, with a halogen atom, or with an aryl group having 6-10 carbon atoms, wherein such hydrocarbyl, or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups.
  • Examples of 2'-O-substituted ribonucleosides or 2'-O-substituted-arabinosides include, without limitation 2 '-amino, 2'-fluoro, 2'-allyl, 2'-O-alkyl and 2'-propargyl ribonucleosides or arabinosides, 2'-O- methylribonucleosides or 2'-O-methylarabinosides and 2'-O- methoxyethoxyribonucleosides or 2'-O-methoxyethoxyarabinosides.
  • the term " 3' " when used directionally, generally refers to a region or position in a polynucleotide or oligonucleotide 3' (downstream) from another region or position in the same polynucleotide or oligonucleotide.
  • the term “ 5' " when used directionally, generally refers to a region or position in a polynucleotide or oligonucleotide 5' (upstream) from another region or position in the same polynucleotide or oligonucleotide.
  • nucleoside residues in the oligonucleotides is not critical, and oligonucleotides having one or two fewer nucleoside residues, or from one to several additional nucleoside residues are contemplated as equivalents of each of the embodiments described above.
  • agonist generally refers to a substance that binds to a receptor of a cell and induces a response.
  • An agonist often mimics the action of a naturally occurring substance such as a ligand.
  • antagonist generally refers to a substance that attenuates the effects of an agonist.
  • adjuvant generally refers to a substance which, when added to an immunogenic agent such as vaccine or antigen, enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture.
  • airway inflammation generally includes, without limitation, asthma.
  • allergen generally refers to an antigen or antigenic portion of a molecule, usually a protein, which elicits an allergic response upon exposure to a subject.
  • a subject is allergic to the allergen as indicated, for instance, by the wheal and flare test or any method known in the art.
  • a molecule is said to be an allergen even if only a small subset of subjects exhibit an allergic immune response upon exposure to the molecule.
  • allergy generally refers to an inappropriate immune response characterized by inflammation and includes, without limitation, food allergies and respiratory allergies.
  • antigen generally refers to a substance that is recognized and selectively bound by an antibody or by a T cell antigen receptor, resulting in induction of an immune response.
  • Antigens may include but are not limited to peptides, proteins, nucleosides, nucleotides, and combinations thereof. Antigens may be natural or synthetic and generally induce an immune response that is specific for that antigen.
  • autoimmune disorder generally refers to disorders in which
  • TLR-mediated disease or TLR-mediated disorder generally means any pathological condition for which activation of one or more TLRs is a contributing factor. Such conditions include but are not limited, cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, skin disorders, allergy, asthma or a disease caused by a pathogen.
  • physiologically acceptable generally refers to a material that does not interfere with the effectiveness of an IRO compound and that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • a biological system such as a cell, cell culture, tissue, or organism.
  • the biological system is a living organism, such as a vertebrate.
  • carrier generally encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microspheres, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, e.g., Remington 's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990.
  • co-administration generally refers to the administration of at least two different substances sufficiently close in time to modulate an immune response. Co-administration refers to simultaneous administration, as well as temporally spaced order of up to several days apart, of at least two different substances in any order, either in a single dose or separate doses.
  • complementary generally means having the ability to hybridize to a nucleic acid. Such hybridization is ordinarily the result of hydrogen bonding between complementary strands, preferably to form Watson-Crick or Hoogsteen base pairs, although other modes of hydrogen bonding, as well as base stacking can also lead to hybridization.
  • an "effective amount” or a “sufficient amount” generally refers to an amount sufficient to affect a desired biological effect, such as beneficial results.
  • an "effective amount” or “sufficient amount” will depend upon the context in which it is being administered.
  • an effective amount of an IRO compound and antigen is an amount sufficient to achieve the desired modulation as compared to the immune response obtained when the antigen is administered alone.
  • An effective amount may be administered in one or more administrations.
  • combination with generally means in the course of treating a disease or disorder in a patient, administering an IRO compound and an agent useful for treating the disease or disorder that does not diminish the immune modulatory effect of the IRO compound.
  • Such combination treatment may also include more than a single administration of an IRO compound and/or independently an agent.
  • the administration of the IRO compound and/or the agent may be by the same or different routes.
  • mammals generally refers to a mammal, such as a human. Mammals generally include, but are not limited to, humans, non-human primates, rats, mice, cats, dogs, horses, cattle, cows, pigs, sheep, and rabbits.
  • kinase inhibitor generally refers to molecules that antagonize or inhibit phosphorylation-dependent cell signaling and/or growth pathways in a cell.
  • Kinase inhibitors may be naturally occurring or synthetic and include small molecules that have the potential to be administered as oral therapeutics.
  • Kinase inhibitors have the ability to rapidly and specifically inhibit the activation of the target kinase molecules.
  • Protein kinases are attractive drug targets, in part because they regulate a wide variety of signaling and growth pathways and include many different proteins. As such, they have great potential in the treatment of diseases involving kinase signaling, including cancer, cardiovascular disease, inflammatory disorders, diabetes, macular degeneration and neurological disorders.
  • Examples of kinase inhibitors include sorafenib (Nexavar®), Sutent®, dasatinib, DasatinibTM, ZactimaTM, TykerbTM and STI571.
  • nucleoside generally refers to compounds consisting of a sugar, usually ribose or deoxyribose, and a purine or pyrimidine base.
  • nucleotide generally refers to a nucleoside comprising a phosphate group attached to the sugar.
  • pyrimidine nucleoside refers to a nucleoside wherein the base component of the nucleoside is a pyrimidine base (e.g., cytosine (C) or thymine (T) or Uracil (U)).
  • purine nucleoside refers to a nucleoside wherein the base component of the nucleoside is a purine base (e.g., adenine (A) or guanine (G)).
  • analog or “derivative” can be used interchangeable to generally refer to any purine and/or pyrimidine nucleotide or nucleoside that has a modified base and/or sugar.
  • a modified base is a base that is not guanine, cytosine, adenine, thymine or uracil.
  • a modified sugar is any sugar that is not ribose or 2'deoxyribose and can be used in the backbone for an oligonucleotide.
  • inhibiting generally refers to a decrease in a response or qualitative difference in a response, which could otherwise arise from eliciting and/or stimulation of a response.
  • non-nucleotide linker generally refers to any linkage or moiety that can link or be linked to the oligonucleotides other than through a phosphorous-containing linkage.
  • linker is from about 2 angstroms to about 200 angstroms in length.
  • nucleotide linkage generally refers to a direct 3 '-5' linkage that directly connects the 3' and 5' hydroxyl groups of two nucleosides through a phosphorous-containing linkage.
  • oligonucleotide motif means an oligonucleotide sequence, including a dinucleotide.
  • An "oligonucleotide motif that would be immune stimulatory, but for one or more modifications” means an oligonucleotide motif which is immune stimulatory in a parent oligonucleotide, but not in a derivative oligonucleotide, wherein the derivative oligonucleotide is based upon the parent oligonucleotide, but has one or more modifications.
  • CpG, C*pG, C*pG* and CpG* refer to oligonucleotide motifs that are immune stimulatory and comprise cytosine or a cytosine analog and a guanine or a guanine analog.
  • treatment generally refers to an approach intended to obtain a beneficial or desired results, which may include alleviation of symptoms, or delaying or ameliorating a disease progression.
  • the invention provides an immune regulatory oligonucleotide (IRO) compound.
  • IRO refers to an immune regulatory oligonucleotide compound that is an antagonist for one or more TLR, wherein the compound comprises an oligonucleotide motif and at least one modification, wherein the oligonucleotide motif would be immune stimulatory (e.g., unmethylated CpG), but for the one or more modifications that suppress the activity of the oligonucleotide motif, provided that compound contains less than 4 consecutive guanosine nucleotides and preferably less than 3 consecutive guanosine nucleotides.
  • Such modifications may be in the oligonucleotide 5' terminus, in a sequence flanking the oligonucleotide motif, and/or within the oligonucleotide motif. These modifications result in an IRO compound that suppresses TLR-modulated immune stimulation.
  • Such modifications can be to the bases, sugar residues and/or the phosphate backbone of the nucleotides/nucleosides flanking the oligonucleotide motif or within the oligonucleotide motif.
  • the modification when the modification is a 2' alkylation or alkoxylation then the modification is not 5' adjacent to the oligonucleotide motif; when the modification is a non-charged internucleoside linkage then the modification is not 5' adjacent to the oligonucleotide motif; and when the modification is a 3' alkylation or alkoxylation then the modification is not 5' or 3' adjacent to the oligonucleotide motif.
  • the IRO compound is not an antisense oligonucleotide.
  • N m - N 3 N 2 N 1 CGN 1 N 2 N 3 - N m -3' wherein CG is an immune stimulatory motif and C is cytosine or a pyrimidine nucleotide derivative or non-nucleotide linker, and G is guanosine, a purine nucleotide derivative or non-nucleotide linker; N1-N3, at each occurrence, is independently a nucleotide, nucleotide derivative or non-nucleotide linker; Nm, at each occurrence, is independently a nucleotide, nucleotide derivative or non-nucleotide linker; provided that at least one Nl to N3 and/or C and/or G is a nucleotide derivative or non-nucleotide linker; and further provided that compound contains less than 4 consecutive guanosine nucleotides and preferably less than 3 consecutive guanosines, wherein the immune stimulatory activity of the
  • IRO compounds may comprise at least two oligonucleotides covalently linked by a nucleotide linkage, or a non-nucleotide linker, at their 5'-, 3'- or 2'-ends or by functionalized sugar or by functionalized nucleobase via a non-nucleotide linker or a nucleotide linkage.
  • Such IRO compounds may be linear or branched.
  • the linker may be attached to the 3'-hydroxyl.
  • the linker comprises a functional group, which is attached to the 3'-hydroxyl by means of a phosphate -based linkage like, for example, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, or by non-phosphate-based linkages.
  • a phosphate -based linkage like, for example, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, or by non-phosphate-based linkages.
  • Possible sites of conjugation for the ribonucleotide are indicated in Formula I, below, wherein B represents a heterocyclic base and wherein the arrow pointing to P indicates any attachment to phosphorous.
  • the non-nucleotide linker is a small molecule, macromolecule or biomolecule, including, without limitation, polypeptides, antibodies, lipids, antigens, allergens, and oligosaccharides.
  • the non-nucleotidic linker is a small molecule.
  • a small molecule is an organic moiety having a molecular weight of less than 1,000 Da. In some embodiments, the small molecule has a molecular weight of less than 750 Da.
  • the small molecule is an aliphatic or aromatic hydrocarbon, either of which optionally can include, either in the linear chain connecting the oligoribonucleotides or appended to it, one or more functional groups including, but not limited to, hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, or thiourea.
  • the small molecule can be cyclic or acyclic.
  • Examples of small molecule linkers include, but are not limited to, amino acids, carbohydrates, cyclodextrins, adamantane, cholesterol, haptens and antibiotics.
  • the term "small molecule" is not intended to include a nucleoside.
  • the non-nucleotidic linker is an alkyl linker or amino linker.
  • the alkyl linker may be branched or unbranched, cyclic or acyclic, substituted or unsubstituted, saturated or unsaturated, chiral, achiral or racemic mixture.
  • the alkyl linkers can have from about 2 to about 18 carbon atoms. In some embodiments such alkyl linkers have from about 3 to about 9 carbon atoms.
  • Some alkyl linkers include one or more functional groups including, but not limited to, hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thioether.
  • such alkyl linkers may include peptides or amino acids.
  • the non-nucleotide linker may include, but are not limited to, those listed in Table 2.
  • the small molecule linker is glycerol or a glycerol homolog of the formula HO-(CH 2 ) O -CH(OH)-(CH 2 ) ? -OH, wherein o and/? independently are integers from 1 to about 6, from 1 to about 4, or from 1 to about 3.
  • the small molecule linker is a derivative of 1,3-diamino- 2-hydroxypropane.
  • Some such derivatives have the formula HO-(CH 2 ) m -C(O)NH-CH 2 -CH(OH)-CH 2 -NHC(O)-(CH 2 ) m -OH, wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 to about 6, or from 2 to about 4 [0083]
  • Some non-nucleotide linkers according to the invention permit attachment of more than two oligonucleotides.
  • the small molecule linker glycerol has three hydroxyl groups to which oligonucleotides may be covalently attached.
  • Some IROs according to the invention therefore, comprise two or more oligonucleotides linked to a nucleotide or a non-nucleotide linker. Such IROs are referred to as being "branched".
  • IRO compounds may comprise at least two oligonucleotides non- covalently linked, such as by electrostatic interactions, hydrophobic interactions, ⁇ -stacking interactions, hydrogen bonding and combinations thereof.
  • Non-limiting examples of such non-covalent linkage includes Watson-Crick base pairing, Hoogsteen base pairing and base stacking.
  • pyrimidine nucleosides in the immune regulatory oligonucleotides used in the compositions and methods according to the invention have the structure (II):
  • D is a hydrogen bond donor
  • D' is selected from the group consisting of hydrogen, hydrogen bond donor, hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;
  • A is a hydrogen bond acceptor or a hydrophilic group
  • A' is selected from the group consisting of hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;
  • X is carbon or nitrogen
  • S' is a pentose or hexose sugar ring, or a sugar analog.
  • the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog.
  • hydrogen bond donors include, without limitation, -NH-, -NH 2 , -SH and -OH.
  • (II) is a pyrimidine nucleoside derivative.
  • pyrimidine nucleoside derivatives include, without limitation, 5- hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, or N4-ethylcytosine, araC, 5-OH-dC, N3-Me-dC, and 4-thiouracil.
  • Chemical modified derivatives also include, but are not limited to, thymine or uracil analogues.
  • the sugar moiety S' in (II) is a sugar derivative. Suitable sugar derivatives include, but are not limited to, trehalose or trehalose derivatives, hexose or hexose derivatives, arabinose or arabinose derivatives.
  • the purine nucleosides in immune regulatory oligonucleotides used in the compositions and methods according to the invention have the structure (III):
  • D is a hydrogen bond donor
  • D' is selected from the group consisting of hydrogen, hydrogen bond donor, and hydrophilic group
  • A is a hydrogen bond acceptor or a hydrophilic group
  • X is carbon or nitrogen
  • each L is independently selected from the group consisting of C, O, N and S;
  • S' is a pentose or hexose sugar ring, or a sugar analog.
  • the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog.
  • hydrogen bond donors include, without limitation, -NH-, -NH 2 , -SH and -OH.
  • (III) is a purine nucleoside derivative.
  • purine nucleoside derivatives include, without limitation, guanine analogues such as 7-deaza-G, 7-deaza-dG, ara-G, 6-thio-G, Inosine, Iso-G, loxoribine, TOG(7-thio-8-oxo)-G, 8-bromo-G, 8-hydroxy-G, 5-aminoformycin B, Oxoformycin, 7-methyl-G, 9-p-chlorophenyl-8-aza-G, 9-phenyl-G, 9-hexyl-guanine, 7-deaza-9- benzyl-G, 6-Chloro-7-deazaguanine, 6-methoxy-7-deazaguanine, 8-Aza-7-deaza- G(PPG), 2-(Dimethylamino)guanosine, 7-Methyl-6-thioguanosine, 8- Benzyloxyguanosine, 9-Deazaguanosine, 1 -(gu
  • Chemically modified derivatives also include, but are not limited to, adenine analogues such as 9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine, 2-Amino-N2-O- , methyladenosine, 8-Aza-7-deaza-A, 7-deaza-A, Vidarabine, 2-Aminoadenosine, Nl- Methyladenosine, 8-Azaadenosine, 5-Iodotubercidin, and Nl-Me-dG.
  • the sugar moiety S' in (III) is a sugar derivative as defined for Formula II.
  • the immune regulatory nucleic acid comprises a nucleic acid sequence containing at least one B-L-deoxy nucleoside or 3'-deoxy nucleoside.
  • the immune regulatory oligonucleotide comprises a nucleic acid sequence containing at least one dinucleotide selected from CpG, C*pG, C*pG* and CpG*, wherein C is cytosine or T- deoxycytidine, G is guanosine or 2'-deoxyguanosine, C* is 2'-deoxythymidine, l-(2'- deoxy- ⁇ -D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, 2'-dideoxy-5- halocytosine, 2'-dideoxy-5-nitrocytosine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, T- deoxy-N4-alkyl-cytidine, 2'-de
  • the oligonucleotides each have from about 6 to about 35 nucleoside residues, preferably from about 9 to about 30 nucleoside residues, more preferably from about 11 to about 23 nucleoside residues. In some embodiments, the oligonucleotides have from about 6 to about 18.
  • the invention provides pharmaceutical formulations comprising an IRO compound according to the invention and a physiologically acceptable carrier.
  • the invention provides methods for inhibiting or suppressing TLR-mediated induction of an immune response in a vertebrate, such methods comprising administering to the vertebrate a IRO compound according to the invention.
  • the vertebrate is a mammal.
  • IRO compound is administered to a vertebrate in need of immune suppression.
  • an IRO compound is capable of suppressing a TLR-based immune response to a further TLR ligand or TLR agonist.
  • a TLR agonist or TLR ligand e.g.
  • an immune modulatory oligonucleotide can be suppressed/inhibited by the simultaneous, pre- or post- administration of an IRO compound, and such suppression/inhibition may be maintained for an extended period of time (e.g. days) after administration.
  • This beneficial property of the current invention has a unique advantage for the prevention and/or treatment of a disease or disorder.
  • application of certain TLR- agonists in the course of treating the disease may cause unwanted immune stimulation that an IRO compound could suppress/inhibit.
  • Administration of the IRO simultaneously, pre and/or post administration of the TLR-agonist may allow therapeutic benefits from the TLR-agonist while suppressing/inhibiting the unwanted side effect(s).
  • pre-administration of an IRO could prevent an immune response (e.g., allergic reaction) to a subsequent or later challenge by a TLR-agonist.
  • administration of IRO compound can be by any suitable route, including, without limitation, parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form.
  • Administration of the therapeutic compositions of IRO compound can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease.
  • the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of IRO compound from about 0.0001 micromolar to about 10 micromolar.
  • a total dosage of IRO compound ranges from about 0.001 mg per patient per day to about 200 mg per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode.
  • the IRO compound may optionally be linked to one or more allergens and/or antigens (self or foreign), an immunogenic protein, such as keyhole limpet hemocyanin (KLH), cholera toxin B subunit, or any other immunogenic carrier protein.
  • IRO can also be used in combination with other compounds (e.g. adjuvants) including, without limitation, TLR agonists (e.g. TLR2 agonists and TLR9 agonists), Freund's incomplete adjuvant, KLH, monophosphoryl lipid A (MPL), alum, and saponins, including QS-21 and imiquimod, or combinations thereof.
  • TLR agonists e.g. TLR2 agonists and TLR9 agonists
  • MPL monophosphoryl lipid A
  • alum alum
  • saponins including QS-21 and imiquimod, or combinations thereof.
  • the invention provides methods for therapeutically treating a patient having a disease or disorder, such methods comprising administering to the patient a IRO compound according to the invention.
  • the disease or disorder to be treated is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, malaria, Lyme disease, ocular infections, conjunctivitis, skin disorders, psoriasis, scleroderma, cardiovascular disease, atherosclerosis, chronic fatigue syndrome, sarcoidosis, transplant rejection, allergy, asthma or a disease caused by a pathogen.
  • Preferred autoimmune disorders include without limitation lupus erythematosus, multiple sclerosis, type I diabetes mellitus, irritable bowl syndrome, Chron's disease, rheumatoid arthritis, septic shock, alopecia universalis, acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas disease, chronic obstructive pulmonary disease, coeliac disease, dermatomyositis, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
  • Preferred inflammatory disorders include without limitation airway inflammation, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, Behcet's disease,_hypersensitivities, inflammatory bowel disease, reperfusion injury, rheumatoid arthritis, transplant rejection, ulcerative colitis, uveitis, conjunctivitis and vasculitis.
  • Pathogens include bacteria, parasites, fungi, viruses, viroids, and prions. Administration is carried out as described for the third aspect of the invention.
  • the invention provides methods for preventing a disease or disorder, such methods comprising administering to the patient IRO compound according to the invention.
  • the disease or disorder to be prevented is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, malaria, Lyme disease, ocular infections, conjunctivitis, skin disorders, psoriasis, scleroderma, cardiovascular disease, atherosclerosis, chronic fatigue syndrome, sarcoidosis, transplant rejection, allergy, asthma or a disease caused by a pathogen.
  • Preferred autoimmune disorders include without limitation lupus erythematosus, multiple sclerosis, type I diabetes mellitus, irritable bowl syndrome, Chron's disease, rheumatoid arthritis, septic shock, alopecia universalis, acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas disease, chronic obstructive pulmonary disease, coeliac disease, dermatomyositis, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
  • Preferred inflammatory disorders include without limitation airway inflammation, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, Behcet's disease,Jiypersensitivities, inflammatory bowel disease, reperfusion injury, rheumatoid arthritis, transplant rejection, ulcerative colitis, uveitis, conjunctivitis and vasculitis.
  • Pathogens include bacteria, parasites, fungi, viruses, viroids, and prions. Administration is carried out as described for the third aspect of the invention.
  • the IRO compound can be administered in combination with any other agent useful for treating the disease or condition that does not diminish the immune modulatory effect of the IRO compound.
  • the agent useful for treating the disease or condition includes, but is not limited to, one or more vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonist, TLR antagonist, peptides, proteins, gene therapy vectors, DNA vaccines, adjuvants or kinase inhibitors to enhance the specificity or magnitude of the immune response, or co-stimulatory molecules such as cytokines, chemokines, protein ligands, trans-activating factors, peptides and peptides comprising modified amino acids.
  • the IRO compound may be administered in combination with one or more chemotherapeutic compound, targeted therapeutic agent and/or monoclonal antibody.
  • the agent can include DNA vectors encoding for antigen or allergen.
  • the IRO compounds of the invention can variously act as adjuvants and/or produce direct immune modulatory effects.
  • TLR-ligands are shown in the following examples, but do not limit the scope of ligands to which the IROs of the invention act as antagonists.
  • Oligonucleotides were synthesized on a 1 ⁇ M scale using an automated DNA synthesizer (Expedite 8909; PerSeptive Biosystems, Framingham, Mass.), following standard linear synthesis or parallel synthesis procedures (see e.g. FIGS. 5 and 6 of U.S. Patent Publication No. 20040097719).
  • r,2'-dideoxyribose phosphoramidite, propyl- 1- phosphoramidite, 2-deoxyuridine phosphoramidite, l,3-bis-[5-(4,4'- dimethoxytrityl)pentylamidyl]-2-propanol phosphoramidite and methyl phosponamidite were obtained from Glen Research (Sterling, Va.).
  • .beta.-L-2'- deoxyribonucleoside phosphoramidite, .alpha.-2'-deoxyribonucleoside phosphoramidite, mono-DMT-glycerol phosphoramidite and di-DMT-glycerol phosphoramidite were obtained from ChemGenes (Willmington, Mass.). (4- Aminobutyl)- 1,3 -propanediol phosphoramidite was obtained from Clontech (Palo Alto, Calif.). Arabinocytidine phosphoramidite, arabinoguanosine, arabinothymidine and arabinouridine were obtained from Reliable Pharmaceutical (St. Louis, Mo.).
  • Arabinoguanosine phosphoramidite, arabinothymidine phosphoramidite and arabinouridine phosphoramidite were synthesized at Idera Pharmaceuticals, Inc. (Cambridge, Mass.) (Noronha et al. (2000) Biochem., 39:7050-7062). [00111] All nucleoside phosphoramidites were characterized by 31 P and 1 H
  • NMR spectra Modified nucleosides were incorporated at specific sites using normal coupling cycles. After synthesis, oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reverse phase HPLC, followed by dialysis. Purified oligonucleotides as sodium salt form were lyophilized prior to use. Purity was tested by CGE and MALDI-TOF MS.
  • HEK293 cells stably expressing TLR9 were transiently transfected with reporter gene, Seap, (Invivogen) for 6 hr. Cells were treated with 0.5 ⁇ g/ml 5 '-CTATCTGACGTTCTCTGT-S ' (mouse CpG sequence; IMO/SEQ ID NO 1; 0 dose) alone and various concentrations of IRO 5 or 6 for 18 hr. TLR9-dependent reporter gene expression was determined according to the manufacturer's protocol (Invivogen) and the results are expressed as % activity of TLR9 stimulating oligonucleotide (100%). The results are shown in Figure 1. These results demonstrate that IRO 5 inhibited TLR9 agonistic activity of IMO.
  • HEK293 cells stably expressing TLR9 or TLR3 were transiently transfected with reporter gene, Seap, (Invivogen) for 6 hr.
  • Cells were treated with 0.5 mg/ml IMOl (0.5 ⁇ g/ml), IRO 5 (2.0 ⁇ g/ml), R848 (5.0 ⁇ g/ml), or poly (I).poly(C) (0.5 ⁇ g/ml) and combinations of IMO+IRO, R848+IRO, or poly(I).poly(C)+IRO for 18 hr.
  • TLR9- or TLR3 -dependent reporter gene expression was determined according to the manufacturer's protocol (Invivogen) and the results are expressed as fold change in NF-kB activity. The results are shown in Figure 2. These results demonstrate that IRO 5 inhibits the activity of the TLR9 agonist but not agonist of TLR3, and more generally that IRO 's can selectively inhibit TLR activation.
  • C57BL/6 mice were injected s.c. at left underarm with 0.25 mg/kg stimulating IMO 3 and 1 mg/kg IRO 5 or 5 '-CTATCTCACCTTCTCTGT-S ' (non- CpG non-stimulatory control; oligo/SEQ ID NO 4) at right under arm either one hour before (-Ih) or at the same time as stimulating IMO (Oh).
  • Serum samples were taken at 2 hours after stimulating IMO injection and determined IL- 12 levels by ELISA.
  • the results in Figure 4 A demonstrate a decrease in serum IL- 12 levels after administration of IRO 5 or (oligo 4) either one hour before (-Ih) or at the same time as stimulating IMO (Oh).
  • C57BL/6 mice were injected s.c. at left underarm with 0.25 mg/kg stimulating IMO 3 and intranasal administration of 10 mg/kg IRO 102 at the same time as stimulating IMO (Oh).
  • Serum samples were taken at 2 hours after stimulating IMO injection and determined IL- 12 levels by ELISA.
  • the results in Figure 4B demonstrate a decrease in serum IL- 12 levels after intranasal administration of IRO 102 at the same time as s.c. of IMO.
  • C57BL/6 mice were injected s.c at left underarm with 0.25 mg/kg stimulating IMO 3 and 2 mg/kg or 10 mg/kg IRO 17, 99, 102 s.c. at right under arm either one hour before (-Ih), twenty- four hours before (-24) or seventy-two hours before (-72) as stimulating IMO (Oh). Serum samples were taken at 2 hours after stimulating IMO injection and determined IL- 12 levels by ELISA. The results are shown in Figure 4C-D. These results demonstrate pre-administration and simultaneous administration of IRO was able to inhibit agonist of TLR9, and more generally that IRO 's can inhibit TLR activation.
  • C57BL/6 mice were injected s.c. at left underarm with 0.25 mg/kg stimulating IMO 3 and 1 mg/kg IRO 21 or control oligo 4 at right under arm either one hour before (-Ih) or at the same time as stimulating IMO (Oh). Serum samples were taken at 2 hours after stimulating IMO injection and determined IL- 12 levels by ELISA. The results are shown in Figure 5A and 5B. These results demonstrate that a CpG oligonucleotide linked at its 5' ends show inhibitory properties, and more generally that immune stimulatory CpG oligonucleotides linked at their 5 ' ends can inhibit TLR activation.
  • CTATCTGTCGTTCTCTGT-3' human CpG sequence; IMO/SEQ ID NO 2
  • IROlO 40 ug IROlO for 24 hr.
  • IRO does not have an effect on Ovalbumin ("OVA") induced Th2 immune responses, whereas IMO compounds reduce OVA induced Th2 response and cause the production of ThI cytokines.
  • OVA Ovalbumin
  • IRO can reverse Th2 inhibitory properties and can inhibit ThI immune responses induced by IMO.
  • mice were immunized with HBsAg in the presence and absence of
  • HEK293 cells stably expressing TLR9 were transiently transfected with reporter gene, Seap, (Invivogen) for 6 hr. Cells were treated with 0.25 ⁇ g/ml IMO alone (IMOl; 0 dose) and various concentrations of IROs for 18 hr. TLR9-dependent reporter gene expression was determined according to the manufacturer's protocol (Invivogen) and the results are expressed as % inhibition of immune stimulating oligonucleotide activity. The results are shown in Tables 5 and 6 below. These results demonstrate that IROs inhibited activity of IMO.
  • IIMOl concentration was 0.25 ⁇ g/ml and IRO concentration was 2 ⁇ g/ml
  • IROs containing various modifications inhibit NF -KB activation of IMO in HEK293 cells expressing TLR9, and more generally IROs containing various modifications can inhibit NF- ⁇ B activation of IMO.
  • IROs containing various modifications inhibit NF- ⁇ B activation of IMO in HEK293 cells expressing TLR9, and more generally IROs containing various modifications can inhibit NF -KB activation of IMO.
  • mice were injected subcutaneously (s.c.) at left underarm with 0.25 mg/kg to 10 mg/kg TLR agonist and 1 mg/kg to 20 mg/kg IRO 5, 17 or 37 or 5 '-TCCTGGCGGGGAAGT-S ' (poly dG control; oligo/SEQ ID NO 49) at right under arm at one hour (-Ih) or up to forty-eight hours (-48) before or at the same time as TLR agonist (Oh). Serum samples were taken at 2 hours after stimulating IMO injection and determined IL- 12 levels by ELISA. The results are shown in Tables 7- 22 below.
  • IRO 5 inhibited IMO induced IL-12 production when injected up to 6 hr after IRO administration. More generally, these results demonstrate that an IRO can inhibit TLR activation and IMO induced IL-12 production when IMO is administered or initially becomes present hours after IRO administration.
  • IRO 5 potently inhibited IMO induced IL- 12 production when injected up to 6 hr after IRO administration. More generally, these results demonstrate that an IRO can substantially inhibit TLR activation and IMO induced IL- 12 production when IMO is administered or initially becomes present hours after IRO administration.
  • IRO 5 potently inhibited IMO induced IL-12 production when injected up to 48 hr after IRO administration. More generally, these results demonstrate that an IRO can substantially inhibit TLR activation and IMO induced IL-12 production when IMO is administered or initially becomes present hours after IRO administration.
  • IRO 17 inhibited IMO induced IL- 12 production when injected up to 6 hr or more after IRO administration. More generally, these results demonstrate that an IRO can inhibit TLR activation and IMO induced IL- 12 production when IMO is administered or initially becomes present hours after IRO administration.
  • IRO 37 inhibited IMO induced IL-12 production when injected up to 3 hr after IRO administration. More generally, these results demonstrate that an IRO can inhibit TLR activation and IMO induced IL-12 production when IMO is administered or initially becomes present hours after IRO administration.
  • a poly dG compound known to show TLR9 antagonist activity inhibited IMO induced IL-12 production when injected up to 6 hr after IRO administration.
  • control poly dG oligo antagonistic effects are short-term and transient.
  • a poly dG compound known to show TLR9 antagonist activity inhibited IMO induced IL- 12 production when injected up to 6 hr after IRO administration.
  • control poly dG oligo antagonistic effects are short and transient.
  • IRO 5 shows a low transient inhibition of R848 induced IL-12 production when injected up to 1 hr after IRO administration. More generally, these data demonstrate that an IRO can inhibit activity of intracellular TLRs.
  • IRO 5 shows potent inhibition of IMO induced MCP-I production when injected up to 1 hr after IRO administration. More generally, these data demonstrate that an IRO can inhibit TLR activation and IMO induced MCP-I production.
  • IRO 5 shows a low transient inhibition of R848 induced MCP-I production when injected up to 1 hr after IRO administration. More generally, these data demonstrate that an IRO can inhibit TLR activation and MCP-I production through intracellular TLRs.
  • IRO 5 shows a low transient inhibition of Polyl.PolyC induced MCP-I production when injected up to 1 hr after IRO administration. More generally, these data demonstrate that an IRO can inhibit TLR activation and MCP-I production of a Polyl.PolyC
  • IRO 5 shows potent inhibition of IMO induced IL- 12 production when injected up to 7 days after IRO administration. More generally, these data demonstrate that an IRO can inhibit TLR activation and IMO induced IL- 12 production in mammals.
  • IRO 5 shows potent inhibition of IMO induced IL-12 production when injected up to 72 hr after IRO administration. More generally, these data demonstrate that an IRO can inhibit TLR activation and IMO induced IL-12 production in mammals hours after the IRO is administered.
  • IRO 5 shows inhibition of R848 induced IL-12 production when injected up to 72 hr after IRO administration. More generally, these data demonstrate that an IRO can inhibit the activity of an agonist of intracellular TLR' s and TLR agonist induced IL- 12 production in mammals hours after the IRO is administered.
  • IRO 5 shows no inhibition of Polyl.PolyC induced IL-12 production when injected 72 hr after IRO administration.
  • mice were subcutaneously injected with 2 mg/kg IRO in their right flank one hour (- Ih) before subcutaneous administration of a TLR agonist to the left flank. Serum samples were taken at 2 hours after administration of the TLR agonist and were analyzed using multiple cytokine/chemokine detecting Luminex kits obtained from Biosource (Camarillo, CA). Manufacture recommended protocols were followed. Cytokine/chemokine values were determined from mean values falling on the standard curve determined on a Luminex 100 instrument. Luminex analysis was performed using STarStation software (Applied Cytometry Systems, Sacramento, CA).
  • TLR3, TLR7, TLR8, and TLR9 intracellular TLRs
  • extracellular TLRs e.g. TLR2, TLR4, and TLR5
  • mice were subcutaneously injected with 10 mg/kg IRO in their right flank seventy- two hours (-72h) before subcutaneous administration of a TLR agonist (as described above) to the left flank. Serum samples were taken at 2 hours after administration of the TLR agonist and were analyzed as described above. The results are shown in Figures 13-15. These results demonstrate pre-administration administration of an IRO was able to inhibit TLR agonist, and that the inhibitory activities of IRO were effective even when administered 72 hours prior to the administration of the agonist.
  • mice were injected once a week s.c. with 100 ⁇ g doses of IRO-5 from wk 9 to 18, and 21 to 23 or IRO-17 starting from wk 10 to 15, 100 ⁇ g three times week in weeks 18-21 and 40 mg three times a week in weeks 22 to 24. Blood and urine were collected every week before IRO injection. Mice were sacrificed Wk 24. Serum anti-DNA IgGl levels were determined by ELISA. The results are shown in Figures 18A through 18E. These results demonstrate that IRO 5 and IRO 17 can inhibit IgGl and IgG2A production and urine protein in Lupus prone mice.

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Abstract

L'invention concerne de nouveaux oligonucléotides de régulation immune (IRO) comme antagonistes de TLR et des procédés d'utilisation de ceux-ci. Ces IRO possèdent des séquences uniques inhibant ou supprimant une signalisation induite par TLR, en réponse à un ligand TLR ou un agoniste TLR. Les procédés peuvent être utilisés dans la prévention et le traitement du cancer, d'un trouble auto-immun, d'une inflammation des voies aériennes, de troubles inflammatoires, d'une maladie infectieuse, de troubles de la peau, d'allergie, d'asthme ou d'une maladie causée par un pathogène.
PCT/US2008/067184 2008-06-17 2008-06-17 Composés d'oligonucléotides de régulation immune (iro) permettant de moduler une réponse immune fondée sur un récepteur de type toll WO2009154610A1 (fr)

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