IMMUNE REGULATORY OLIGONUCLEOTIDE (IRO) COMPOUNDS TO MODULATE TOLL-LIKE RECEPTOR BASED IMMUNE RESPONSE

Abstract

The invention provides novel immune regulatory oligonucleotides (IRO) as antagonist of TLRs and methods of use thereof. These IROs have unique sequences that inhibit or suppress TLR-mediated signaling in response to a TLR ligand or TLR agonist. The methods may have use in the prevention and treatment of cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, skin disorders, allergy, asthma or a disease caused by a pathogen.

Claims

1. An antagonist of TLR7 and/or TLR9 having the structure
5-N.sub.pN.sub.3N.sub.2N.sub.1C*G1N.sup.1N.sup.2N.sup.3N.sup.zN.sup.4N.sup.5-3, wherein C*G* is an oligonucleotide motif wherein C* is 5-Me-dC, and G1 is 7-deaza-dG; N.sub.1-N.sub.2, at each occurrence, is independently a 2-O-Me-ribonucleotide; N.sup.3, at each occurrence, is independently a nucleotide; N.sup.1-N.sup.3, at each occurrence, is independently a nucleotide; N.sub.m and N.sup.m, at each occurrence, is independently a nucleotide; N.sup.4-N.sup.5, at each occurrence, is independently a 2-O-Me-ribonucleotide; p is 5; and z is 3; provided that the compound contains less than 3 consecutive guanosines.

2. The antagonist according to claim 1 having the structure 5-CTATCTGUC*G1TTCTCTGU-3 or 5-CTATCTGAC*G1TTCTCTGU-3.

3. A pharmaceutical composition comprising an antagonist according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. An immune regulatory oligonucleotide (IRO) compound that is an antagonist of TLR7 and/or TLR9, selected from compound number 1 through compound number 67 and compound number 69 through compound number 111.

5. A pharmaceutical composition comprising an antagonist according to claim 4 and a pharmaceutically acceptable carrier.

6. A method for inhibiting a TLR7- and/or TLR9-mediated immune response in a mammal, the method comprising administering to the mammal an antagonist according to any one of claim 1, 2 or 4 or a composition thereof.

7. The method according to claim 6, 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.

8. The method according to claim 6, wherein the mammal is a human.

9. The method according to claim 6, wherein the antagonist 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.

10. A method for inhibiting the activity of a TLR7 and/or TLR9 agonist comprising administering an antagonist according to any one of claim 1, 2 or 4 or a composition thereof.

11. The method according to claim 10, comprising administering the antagonist prior to or at the same time as the TLR7 and/or TLR9 agonist.

12. A method for therapeutically treating a mammal having an autoimmune disease mediate by TLR7 and/or TLR9, wherein the autoimmune disease is selected from psoriasis, rheumatoid arthritis, 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-Barr syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious anaemia, polymyositis, primary biliary cirrhosis, schizophrenia, Sjgren's syndrome, temporal arteritis, vasculitis, vitiligo, vulvodynia or Wegener's granulomatosis, such method comprising administering to the mammal an antagonist according to any one of claim 1, 2 or 4 or a composition thereof.

13. The method according to claim 12, wherein the antagonist 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.

14. The method according to claim 12, 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.

15. The method according to claim 12, wherein the mammal is a human.

16. A method for preventing an autoimmune disease mediate by a TLR7 and/or TLR9, wherein the autoimmune disease is selected from psoriasis, rheumatoid arthritis, 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-Barr syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious anaemia, polymyositis, primary biliary cirrhosis, schizophrenia, Sjgren's syndrome, temporal arteritis, vasculitis, vitiligo, vulvodynia or Wegener's granulomatosis, such method comprising administering to a mammal an antagonist according to any one of claim 1, 2 or 4 or a composition thereof.

17. The method according to claim 16, wherein the antagonist 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 or co-stimulatory molecules.

18. The method according to claim 16, 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.

19. The method according to claim 16, wherein the mammal is a human.

20. The composition according to claim 3, further comprising one or more vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, TLR antagonists, peptides, proteins, gene therapy vectors, DNA vaccines, adjuvants or co-stimulatory molecules.

21. The composition according to claim 5, further comprising one or more vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, TLR antagonists, peptides, proteins, gene therapy vectors, DNA vaccines, adjuvants or co-stimulatory molecules.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a synthetic scheme for the linear synthesis of immune regulatory compounds of the invention. DMTr=4,4-dimethoxytrityl; CE=cyanoethyl.

[0023] FIG. 2 is a synthetic scheme for the parallel synthesis of immune regulatory oligonucleotides of the invention. DMTr=4,4-dimethoxytrityl; CE=cyanoethyl.

[0024] FIGS. 3A-3C depicts the ability of TLR7/9 antagonists according to the invention to inhibit TLR7-induced cytokines/chemokines by TLR7/9 antagonists in mouse splenocytes treated according to Example 3.

[0025] FIGS. 4A-4C depicts the ability of TLR7/9 antagonists according to the invention to inhibit TLR9-induced cytokines/chemokines by TLR7/9 antagonists in mouse splenocytes treated according to Example 3.

[0026] FIG. 5 depicts the ability of TLR7/9 antagonists according to the invention to inhibit TLR7-induced IL-12 in vivo in mice treated according to Example 4.

[0027] FIG. 6 depicts the ability of TLR7/9 antagonists according to the invention to inhibit TLR9-induced IL-12 in vivo in mice treated according to Example 4.

[0028] FIG. 7 depicts the ability of TLR7/9 antagonists according to the invention to inhibit TLR7-induced IL-12 in vivo over time in mice treated according to Example 4.

[0029] FIG. 8 depicts the ability of TLR7/9 antagonists according to the invention to inhibit TLR9-induced inhibition of IL-12 in vivo over time in mice treated according to Example 4.

[0030] FIGS. 9A-9B depicts the ability of TLR7/9 antagonists according to the invention to selectively inhibit mouse TLR7 & 9-induced cytokines in vivo in mice treated according to Example 4.

[0031] FIGS. 10A-10B demonstrates that TLR7/9 antagonists according to the invention do not inhibit the activity of TLR3 or TLR5 in vivo in mice treated according to Example 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention relates to the therapeutic use of novel oligonucleotide-based compounds as immune modulatory agents for immunotherapy applications. The invention provides novel oligonucleotide-based compounds that provide distinct immune inhibition profiles through their interaction with TLR7 and/or TLR9. Specifically, 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. These IROs have chemical modifications, and/or internucleotide linkages, and/or linkers between oligonucleotides that provide their inhibition or suppression of TLR7- and/or TLR9-mediated signaling in response to endogenous and/or exogenous TLR ligands or agonists. The references cited herein reflect the level of knowledge in the field and are hereby incorporated by reference in their entirety. Any conflicts between the teachings of the cited references and this specification shall be resolved in favor of the latter.

[0033] The invention further 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 bowel syndrome, sepsis, and bacteria, parasitic, and viral infections in adult and pediatric human and veterinary applications. Thus, the invention provides IRO compounds having optimal levels of immune modulatory effect for immunotherapy and methods for making and using such compounds. In addition, IRO compounds of the invention are useful in combination with, for example, vaccines, antigens, antibodies, allergens, chemotherapeutic agents (both chemotherapy and targeted therapies), and/or antisense oligonucleotides for prevention and treatment of diseases.

Definitions

[0034] The term 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. 90:543; Protocols for Oligonucleotides and Analogs Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993; and Hunziker, J. et al. (1995) Mod. Syn. Methods 7:331-417; and Crooke, S. et al. (1996) Ann. Rev. Pharm. Tox. 36:107-129. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such 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. The term oligonucleotide also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (R.sub.P)- or (S.sub.P)-phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, the terms 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. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof.

[0035] 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. In certain embodiments, 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, for example, 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.

[0036] 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.

[0037] 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.

[0038] The term about generally means that the exact number is not critical. Thus, the number of 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.

[0039] The term 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.

[0040] The term antagonist generally refers to a substance that attenuates, inhibits or suppresses the effects of an agonist or ligand.

[0041] The term 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.

[0042] The term airway inflammation generally includes, without limitation, asthma.

[0043] The term 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. Typically the 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.

[0044] The term allergy generally refers to an inappropriate immune response characterized by inflammation and includes, without limitation, food allergies and respiratory allergies.

[0045] The term 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.

[0046] The terms autoimmune disease and autoimmune disorder generally refer to diseases or disorders in which self components undergo attack by the immune system.

[0047] The term 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, autoimmune diseases or disorders, airway inflammation, inflammatory diseases or disorders, infectious diseases, skin disorders, allergy, asthma or diseases caused by a pathogen.

[0048] The term physiologically acceptable generally refers to a material that does not interfere with the effectiveness of an IRO compound or composition according to the invention and that is compatible with a biological system such as a cell, cell culture, tissue or organism. Preferably, the biological system is a living organism, such as a vertebrate, or mammal.

[0049] The term 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, for example, Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.

[0050] The term co-administration generally refers to the administration of at least two different substances sufficiently close in time to modulate, suppress or inhibit 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.

[0051] The term 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.

[0052] The term an effective amount or a sufficient amount generally refers to an amount sufficient to affect a desired biological effect, such as beneficial results. Thus, an effective amount or sufficient amount will depend upon the context in which it is being administered. In the context of administering a compound or composition that modulates an immune response to a co-administered antigen, an effective amount of an IRO compound or composition according to the invention and antigen is an amount sufficient to achieve the desired modulation, inhibition or suppression as compared to the immune response obtained when the antigen is administered alone. An effective amount may be administered in one or more administrations.

[0053] The term in combination with generally means in the course of treating a disease or disorder in a patient, administering an IRO compound or composition according to the invention and an agent useful for treating the disease or disorder that does not diminish the immune inhibitory effect of the IRO compound or composition according to the invention. Such combination treatment may also include more than a single administration of an IRO compound or composition according to the invention and/or independently an agent. The administration of the IRO compound or composition according to the invention and/or the agent may be by the same or different routes.

[0054] The term individual or subject or vertebrate generally refers to a mammal. Mammals generally include, but are not limited to, humans, non-human primates, rats, mice, cats, dogs, horses, cattle, cows, pigs, sheep, and rabbits.

[0055] The term 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, DASATINIB, ZACTIMA, TYKERB and STI571.

[0056] The term nucleoside generally refers to compounds consisting of a sugar, usually ribose or deoxyribose, and a purine or pyrimidine base.

[0057] The term nucleotide generally refers to a nucleoside comprising a phosphate group attached to the sugar.

[0058] As used herein, the term 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)). Similarly, the term purine nucleoside refers to a nucleoside wherein the base component of the nucleoside is a purine base (e.g., adenine (A) or guanine (G)).

[0059] The terms 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 2deoxyribose and can be used in the backbone for an oligonucleotide.

[0060] The term inhibiting or suppressing generally refers to a decrease in or a prevention of a response or qualitative difference in a response, which could otherwise arise from eliciting and/or stimulation of a response.

[0061] The term 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. Preferably such linker is from about 2 angstroms to about 200 angstroms in length.

[0062] The term 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.

[0063] The terms oligonucleotide motif means an oligonucleotide sequence, including a dinucleotide selected from CpG, C*pG, C*pG* or CpG*. The terms CpG, C*pG, C*pG* and CpG* refer to oligonucleotide motifs that are immune stimulatory wherein C is cytosine, C* is a cytosine analog or derivative, G is a guanine and G* is a guanine analog or derivative.

[0064] 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. In other words, an oligonucleotide motif that would be immune stimulatory, but for one or more modifications refers to a TLR9-inducing moiety that would have TLR9 agonistic activity but for that fact that it has been functionally blocked or inhibited from inducing TLR9 mediated immune response through modification(s) of the TLR9-inducing moiety itself and/or by one or more chemical modification within the oligonucleotide based compound. Modifications that inhibit the activity of a TLR9-inducing moiety include, but not limited to, 2-OMe-ribonucleosides, 3-OMe-ribonucleosides, 3-nitropyrrole, 5-nitroindole, dU, -L-deoxynucleosides, -deoxynucleosides, abasic nucleoside, propanediol linker, amino linker, isopropoxyl, glycerol linker, 2-5-DNA, 2-5 RNA, and P-Me DNA.

[0065] The term treatment generally refers to an approach intended to obtain a beneficial or desired results, which may include alleviation of symptoms and/or delaying and/or ameliorating the progression of a disease or disorder.

[0066] Certain IROs according to the invention are shown in Table 2. In this table, the IRO compounds have all phosphorothioate (PS) linkages, except where indicated with o. Except where indicated, all nucleotides are deoxyribonucleotides.

TABLE-US-00002 TABLE2 IRO compound# Sequence/Structure/SEQIDNO 1 5-UGUCG1TTCT-X1-TCTTG1CUGU-5; 5-SEQIDNO1-3-X1-3-SEDIDNO1-5 2 5-UGUCG1TTC-X1-CTTG1CUGU-5; 5-SEQIDNO2-3-X1-3-SEDIDNO2-5 3 5-UGUCG1TT-X1-TTG1CUGU-5; 5-SEQIDNO3-3-X1-3-SEDIDNO3-5 4 5-UGUCoG1TTCTo-Z-oTCTTG1oCUGU-5; 5-SEQIDNO4-3-Z-3-SEDIDNO4-5 5 5-GUCG1TTCTT-Z-TTCTTG1CUG-5; 5-SEQIDNO5-3-Z-3-SEDIDNO5-5 6 5-UGUCG2TTCT-Z-TCTTG2CUGU-5; 5-SEQIDNO6-3-Z-3-SEDIDNO6-5 7 5-UGUCG1TTCT-X4-TCTTG1CUGU-5; 5-SEQIDNO7-3-X4-3-SEDIDNO7-5 8 5-UGUCG1TTC-X4-CTTG1CUGU-5; 5-SEQIDNO8-3-X4-3-SEDIDNO8-5 9 5-UGUCoG1TTCTo-X4-oTCTTG1oCUGU-5; 5-SEQIDNO9-3-X4-3-SEDIDNO9-5 10 5-GUCG1TTCTT-X4-TTCTTG1CUG-5; 5-SEQIDNO10-3-X4-3-SEDIDNO10-5 11 5-UGUCG1TT-X4-TTG1CUGU-5; 5-SEQIDNO11-3-X4-3-SEDIDNO11-5 12 5-UGUCG1TTC-X5-CTTG1CUGU-5; 5-SEQIDNO12-3-X5-3-SEDIDNO12-5 13 5-UGUCG2TTC-X5-CTTG2CUGU-5; 5-SEQIDNO13-3-X5-3-SEDIDNO13-5 14 5-UGUCG1TTC-X6-CTTG1CUGU-5; 5-SEQIDNO14-3-X6-3-SEDIDNO14-5 15 5-UGUCG2TTC-X6-CTTG2CUGU-5; 5-SEQIDNO15-3-X6-3-SEDIDNO15-5 16 5-UGUCG1TTCT-X7-TCTTG1CUGU-5; 5-SEQIDNO16-3-X7-3-SEDIDNO16-5 17 5-UGUCG2TTCT-X7-TCTTG2CUGU-5; 5-SEQIDNO17-3-X7-3-SEDIDNO17-5 18 5-UGUCG1TTC-X7-CTTG1CUGU-5; 5-SEQIDNO18-3-X7-3-SEDIDNO18-5 19 5-TGUCG1TTCT-X-TCTTG1CUGT-5; 5-SEQIDNO19-3-X-3-SEDIDNO19-5 20 5-CTTGUCG1TTCT-X-TCTTG1CUGTTC-5; 5-SEQIDNO20-3-X-3-SEDIDNO20-5 21 5-TTGUCG1TTC-X-CTTG1CUGTT-5; 5-SEQIDNO21-3-X-3-SEDIDNO21-5 22 5-CTTTGUCG1TTC-X-CTTG1CUGTTTC-5; 5-SEQIDNO22-3-X-3-SEDIDNO22-5 23 5-TGUCG1TTCT-X7-TCTTG1CUGT-5; 5-SEQIDNO23-3-X7-3-SEDIDNO23-5 24 5-TTGUCG1TTC-X7-CTTG1CUGTT-5; 5-SEQIDNO24-3-X7-3-SEDIDNO24-5 25 5-GUCG1TTCTT-Z-TTCTTG1CUG-5; 5-SEQIDNO25-3-Z-3-SEDIDNO25-5 26 5-TGUCG1TTCA-X-ACTTG1CUGT-5; 5-SEQIDNO26-3-X-3-SEDIDNO26-5 27 5-TCTGACG1TTCT-X-TCTTG1CAGTCT-5; 5-SEQIDNO27-3-X1-3-SEDIDNO27-5 28 5-TCTGACG2TTCT-X-TCTTG2CAGTCT-5; 5-SEQIDNO28-3-X-3-SEDIDNO28-5 29 5-TTGUCG1TTA-X-ATTG1CUGTT-5; 5-SEQIDNO29-3-X-3-SEDIDNO29-5 30 5-CTCTGUCG1TTA-X-ATTG1CUGTCTC-5; 5-SEQIDNO30-3-X-3-SEDIDNO30-5 31 5-TGTC*GTTCT-X-TCTTGC*TGT-5; 5-SEQIDNO31-3-X-3-SEDIDNO31-5 32 5-TGTCGTTCT-X-TCTTGCTGT-5; 5-SEQIDNO32-3-X-3-SEDIDNO32-5 33 5-TGTC*GTTCT-X-TCTTGC*TGT-5; 5-SEQIDNO33-3-X-3-SEDIDNO33-5 34 5-TGTCGTTCT-X-TCTTGCTGT-5; 5-SEQIDNO34-3-X-3-SEDIDNO34-5 35 5-UGUCG1ACAT-X-TACAG1CUGU-5; 5-SEQIDNO35-3-X-3-SEDIDNO35-5 36 5-UGUCG1TTC-X-CTTG1CUGU-5; 5-SEQIDNO36-3-X-3-SEDIDNO36-5 37 5-UGUCG1TT-X-TTG1CUGU-5; 5-SEQIDNO37-3-X-3-SEDIDNO37-5 38 5-UoGUCG1TToCTo-X-oTCoTTG1CUGoU-5; 5-SEQIDNO38-3-X-3-SEDIDNO38-5 39 5-UoGoUCG1TTCTo-X-oTCTTG1CUoGoU-5; 5-SEQIDNO39-3-X-3-SEDIDNO39-5 40 5-UGACG1TTCT-X-TCTTG1CAGU-5; 5-SEQIDNO40-3-X-3-SEDIDNO40-5 41 5-UGUCG1ACAT-Z-TACAG1CUGU-5; 5-SEQIDNO41-3-Z-3-SEDIDNO41-5 42 5-UGUCG1TTCT-Z-TCTTG1CUGU-5; 5-SEQIDNO42-3-Z-3-SEDIDNO42-5 43 5-UGUCG1TTC-Z-CTTG1CUGU-5; 5-SEQIDNO43-3-Z-3-SEDIDNO43-5 44 5-UGUCG1TT-Z-TTG1CUGU-5; 5-SEQIDNO44-3-Z-3-SEDIDNO44-5 45 5-UGUC*GTTCT-X-TCTTGC*UGU-5; 5-SEQIDNO45-3-X-3-SEDIDNO45-5 46 5-T{circumflex over ()}GTC*GTTCT-X-TCTTGC*TGT-5; 5-SEQIDNO46-3-X-3-SEDIDNO46-5 47 5-UGUC*GTTCT-X-TCTTGC*UGU-5; 5-SEQIDNO47-3-X-3-SEDIDNO47-5 48 5-T{circumflex over ()}G{circumflex over ()}T{circumflex over ()}C*GTTCT-X-TCTTGC*T{circumflex over ()}G{circumflex over ()}T{circumflex over ()}-5; 5-SEQIDNO48-3-X-3-SEDIDNO48-5 49 5-UGUCG1ACAT-X1-TACAG1CUGU-5; 5-SEQIDNO49-3-X1-3-SEDIDNO49-5 50 5-UGACG2TTCT-X-TCTTG2CAGU-5; 5-SEQIDNO50-3-X-3-SEDIDNO50-5 51 5-TCTGUCG1TTCT-X-TCTTG1CUGTCT-5; 5-SEQIDNO51-3-X-3-SEDIDNO51-5 52 5-TCTGUCG2TTCT-X-TCTTG2CUGTCT-5; 5-SEQIDNO52-3-X-3-SEDIDNO52-5 53 5-UGUCG2TTCT-X-TCTTG2CUGU-5; 5-SEQIDNO53-3-X-3-SEDIDNO53-5 54 5-UGUCG2TT-Z-TTG2CUGU-5; 5-SEQIDNO54-3-Z-3-SEDIDNO54-5 55 5-TUGUCG1TTC-Z-CTTG1CUGUT-5; 5-SEQIDNO55-3-Z-3-SEDIDNO55-5 56 5-CTUGUCG1TT-Z-TTG1CUGUTC-5; 5-SEQIDNO56-3-Z-3-SEDIDNO56-5 57 5-UCG1TTCTTC-Z-CTTCTTG1CU-5; 5-SEQIDNO57-3-Z-3-SEDIDNO57-5 58 5-CTATCTGAC*GTTCTCTGT-3; 5-SEQIDNO58-3 59 5-CTATCTGACGTTCTCTGT-3; 5-SEQIDNO59-3 60 5-CTATCTGAC*GTTCTCTGT-3; 5-SEQIDNO60-3 61 5-CTATCTGACGTTCTCTGT-3; 5-SEQIDNO61-3 62 5-CTATCTG{circumflex over ()}A{circumflex over ()}CGTTCTCTGT-3; 5-SEQIDNO62-3 63 5-CTATCTGUC*GTTCTCTGT-3; 5-SEQIDNO63-3 64 5-CTATCTGUCGTTCTCTGT-3; 5-SEQIDNO64-3 65 5-CTATCTGUC*GTTCTCTGT-3; 5-SEQIDNO65-3 66 5-CTATCTGUCGTTCTCTGT-3; 5-SEQIDNO66-3 67 5-CTTGUC*G1TTCT-X-TCTTG1C*UGTTC-5 5-SEQIDNO67-3-X-3-SEDIDNO67-5 68 5-CTATCTGUC*G1TTCTCTGU-3 5-SEQIDNO68-3 69 5-UGUCG1TTCT-X-TCTTG1CUGU-5 5-SEQIDNO69-3-X-3-SEDIDNO69-5 70 5-TGUC*G1TTCT-X-TCTTG1C*UGT-5 5-SEQIDNO70-3-X-3-SEDIDNO70-5 71 5-CTTTGUC*G1TTC-X-CTTG1C*UGTTTC-5 5-SEQIDNO71-3-X-3-SEDIDNO71-5 72 5-GUC*G1TTCTT-X-TTCTTG1C*UG-5 5-SEQIDNO72-3-X-3-SEDIDNO72-5 73 5-TGUC*G1TTCA-X-ACTTG1C*UGT-5 5-SEQIDNO73-3-X-3-SEDIDNO73-5 74 5-CTTGUC*G1TTCT-X1-TCTTG1C*UGTTC-5 5-SEQIDNO74-3-X1-3-SEDIDNO74-5 75 5-CTTGUC*G2TTCT-X-TCTTG2C*UGTTC-5 5-SEQIDNO75-3-X-3-SEDIDNO75-5 76 5-CTTGUC*G1TTC-X5-CTTG1C*UGTTC-5 5-SEQIDNO76-3-X5-3-SEDIDNO76-5 77 5-CTTGUC*G1TTCT-X7-TCTTG1C*UGTTC-5 5-SEQIDNO77-3-X7-3-SEDIDNO77-5 78 5-CTTTGUC*oG1TTC-X-CTTG1oC*UGTTTC-5 5-SEQIDNO78-3-X-3-SEDIDNO78-5 79 5-CTTTGoUC*oG1TTC-X-CTTG1oC*UoGTTTC-5 5-SEQIDNO79-3-X-3-SEDIDNO79-5 80 5-CTTGUC*oG1TTCT-X-TCTTG1oC*UGTTC-5 5-SEQIDNO80-3-X-3-SEDIDNO80-5 81 5-CTTGoUC*oG1TTCT-X-TCTTG1oC*UoGTTC-5 5-SEQIDNO81-3-X-3-SEDIDNO81-5 82 5-CTGUC*oG1TTCTT-X-TTCTTG1oC*UGTC-5 5-SEQIDNO82-3-X-3-SEDIDNO82-5 83 5-CTGoUC*oG1TTCTT-X-TTCTTG1oC*UoGTC-5 5-SEQIDNO83-3-X-3-SEDIDNO83-5 84 5-UGUC*G1TTCT-X1-TCTTG1C*UGU-5 5-SEQIDNO84-3-X1-3-SEDIDNO84-5 85 5-UGUC*G2TTCT-X-TCTTG2C*UGU-5 5-SEQIDNO85-3-X-3-SEDIDNO85-5 86 5-UGUC*G1TTC-X5-CTTG1C*UGU-5 5-SEQIDNO86-3-X5-3-SEDIDNO86-5 87 5-UGUC*G1TTCT-X7-TCTTG1C*UGU-5 5-SEQIDNO87-3-X7-3-SEDIDNO87-5 88 5-CTATCTGUC*G1TTCTCTGT-3 5-SEQIDNO88-3 89 5-CTATCTGUC*G1TTCTCTGT-3 5-SEQIDNO89-3 90 5-TGAC*G1TTCT-X-TCTTG1C*AGT-5 5-SEQIDNO90-3-X-3-SEQIDNO90-5 91 5-CTTGAC*G1TTCT-X-TCTTG1C*AGTTC-5 5-SEQIDNO91-3-X-3-SEDIDNO91-5 92 5-CTTTGAC*G1TTC-X-CTTG1C*AGTTTC-5 5-SEQIDNO92-3-X-3-SEDIDNO92-5 93 5-GAC*G1TTCTT-X-TTCTTG1C*AG-5 5-SEQIDNO93-3-X-3-SEDIDNO93-5 94 5-TGAC*G1TTCA-X-ACTTG1C*AGT-5 5-SEQIDNO94-3-X-3-SEDIDNO94-5 95 5-CTTGAC*G1TTCT-X1-TCTTG1C*AGTTC-5 5-SEQIDNO95-3-X1-3-SEDIDNO95-5 96 5-CTTGAC*G2TTCT-X-TCTTG2C*AGTTC-5 5-SEQIDNO96-3-X-3-SEDIDNO96-5 97 5-CTTGAC*G1TTC-X5-CTTG1C*AGTTC-5 5-SEQIDNO97-3-X5-3-SEDIDNO97-5 98 5-CTTGAC*G1TTCT-X7-TCTTG1C*AGTTC-5 5-SEQIDNO98-3-X7-3-SEDIDNO98-5 99 5-CTTTGAC*oG1TTC-X-CTTG1oC*AGTTTC-5 5-SEQIDNO99-3-X-3-SEDIDNO99-5 100 5-CTTTGoAC*oG1TTC-X-CTTG1oC*AoGTTTC-5 5-SEQIDNO100-3-X-3-SEDIDNO100-5 101 5-CTTGAC*oG1TTCT-X-TCTTG1oC*AGTTC-5 5-SEQIDNO101-3-X-3-SEDIDNO101-5 102 5-CTTGoAC*oG1TTCT-X-TCTTG1oC*AoGTTC-5 5-SEQIDNO102-3-X-3-SEDIDNO102-5 103 5-CTGAC*oG1TTCTT-X-TTCTTG1oC*AGTC-5 5-SEQIDNO103-3-X-3-SEDIDNO103-5 104 5-CTGoAC*oG1TTCTT-X-TTCTTG1oC*AoGTC-5 5-SEQIDNO104-3-X-3-SEDIDNO104-5 105 5-UGAC*G1TTCT-X-TCTTG1C*AGU-5 5-SEQIDNO105-3-X-3-SEDIDNO105-5 106 5-UGAC*G1TTCT-X1-TCTTG1C*AGU-5 5-SEQIDNO106-3-X1-3-SEDIDNO106-5 107 5-UGAC*G2TTCT-X-TCTTG2C*AGU-5 5-SEQIDNO107-3-X-3-SEDIDNO107-5 108 5-UGAC*G1TTC-X5-CTTG1C*AGU-5 5-SEQIDNO108-3-X5-3-SEDIDNO108-5 109 5-UGAC*G1TTCT-X7-TCTTG1C*AGU-5 5-SEQIDNO109-3-X7-3-SEDIDNO109-5 110 5-CTATCTGAC*G1TTCTCTGT-3 5-SEQIDNO110-3 111 5-CTATCTGAC*G1TTCTCTGT-3 5-SEQIDNO111-3 112 5-CTATCTGAC*G1TTCTCTGU-3 5-SEQIDNO112-3
G1=7-deaza-dG; G2=AraG; C*=5-Me-dC; C*=2-O-Me-5-Me-C; AA/GA/TA/GA=2-O-(2-methoxyethyl)-ribonucelotides; X=Glycerol Linker (also known as 1,2,3-Propanetriol Linker); X1=1,2,4-Butanetriol Linker; Z=1,3,5-Pentanetriol Linker; X4=3-Trimethylamino-1, 2-propanediol Linker; X5=Bis-1,5-O-(3-thymidyl)-1,3,5-pentanetriol Linker; X6=Bis-1,5-O-[3-(1,2-dideoxy-D-ribosyl)]-1,3-5-pentanetriol Linker; X7=3-(2-Hydroxyethyl)-1,5-pentanediol Linker; G/U/A/C=2-O-Me-ribonucleotides; o=Phosphodiester linkage.

[0067] In a first aspect, the invention provides immune regulatory oligonucleotide (IRO) compounds. The term IRO refers to an immune regulatory oligonucleotide-based compound that is an antagonist for TLR7- and/or TLR9, wherein the compound comprises an oligonucleotide motif and at least one modification, wherein the oligonucleotide motif would be immune stimulatory but for the one or more modifications that functionally block or inhibit the activity of the oligonucleotide motif, provided that the 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 the 5 sequence flanking the oligonucleotide motif, and/or within the immune stimulatory oligonucleotide motif. These modifications result in an IRO compound that antagonize, inhibit, suppresses or prevent TLR7- and/or TLR9-mediated immune stimulation. Such modifications can be to the bases, sugar residues and/or the phosphate backbone of the nucleotides/nucleosides flanking the immune stimulatory oligonucleotide motif or within such oligonucleotide motif.

[0068] The general structure of the IRO compound has the structure 5-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3 (SEQ ID NO: 70) wherein CG is an oligonucleotide motif selected from CpG, C*pG, C*pG* or CpG* wherein C is cytosine, C* is a cytosine analog or derivative, G is a guanine and G* is a guanine analog or derivative; N.sub.1-N.sub.3, at each occurrence, is independently a nucleotide or nucleotide derivative; N.sup.1-N.sup.3, at each occurrence, is independently a nucleotide or nucleotide derivative; N.sub.m and N.sup.m, at each occurrence, is independently a nucleotide, nucleotide derivative or non-nucleotide linker; provided that at least one of N.sub.1, N.sub.2, and N.sub.3 and/or C and/or G of the oligonucleotide motif is a nucleotide derivative that functionally blocks or inhibits the activity of the oligonucleotide motif; and further provided that the compound contains less than 4 consecutive guanosine nucleotides and preferably less than 3 consecutive guanosines, wherein the immune stimulatory activity of the oligonucleotide motif is antagonized, inhibited, suppressed or prevented by the nucleotide derivative; and wherein m is a number from 0 to about 30.

[0069] In preferred embodiments, N.sub.1 is a nucleotide derivative that functionally blocks or inhibits the activity of the oligonucleotide motif. In preferred embodiments N.sub.1 and N.sub.2, or N.sub.1 and N.sub.3, or N.sub.2 and N.sub.3, or N.sub.1, N.sub.2 and N.sub.3 are nucleotide derivatives that functionally blocks or inhibits the activity of the oligonucleotide motif.

[0070] In preferred embodiments the IRO compound is not an antisense oligonucleotide.

[0071] In certain embodiments of the invention, the IRO compound may comprise at least two oligonucleotides (for example 2, 3, 4, 5 or 6 oligonucleotides), wherein at least two oligonucleotides are covalently linked via a direct nucleotide to nucleotide linkage at their 3 ends through the 3 positions of the sugars or through a modified sugar or modified nucleobase or via a non-nucleotide linker at their 3 ends through the 3 positions of the sugars or through a modified sugar or modified nucleobase. In preferred aspects of this embodiment, at least one of oligonucleotides of the IRO compound has the structure 5-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3 (SEQ ID NO: 70), wherein N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G, N.sup.1, N.sup.2, N.sup.3 and N.sub.m are as described above for the general structure of the IRO compound. In more preferred aspects of this embodiment, at least two of the oligonucleotides of the IRO compound have the structure 5-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3 (SEQ ID NO: 70), wherein N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G, N.sup.1, N.sup.2, N.sup.3 and N.sup.m are as described above for the general structure of the IRO compound. Such an IRO compound may have the structure 5-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3-X-3-N.sup.m-N.sup.3N.sup.2N.sup.1GCN.sub.1N.sub.2N.sub.3-N.sub.m-5 (5-SEQ ID NO: 70-3-X-3-SEQ ID NO: 70-5), wherein X is a nucleotide linkage or a non-nucleotide linker and N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G, N.sup.1, N.sup.2, N.sup.3 and N.sub.m are as described above for the general structure of the IRO compound.

[0072] In certain embodiments of the invention, the IRO compound that is an antagonist of TLR7 and/or TLR9 has the structure 5-N.sub.pN.sub.3N.sub.2N.sub.1C*G*N.sup.1N.sup.2N.sup.3N.sup.zN.sup.4N.sup.5-3 (SEQ ID NO: 71), wherein C*G* is an oligonucleotide motif wherein C* is 5-Me-dC, and G* is 7-deaza-dG; N.sub.1-N.sub.2, at each occurrence, is independently a 2-O-Me-ribonucleotide; N.sub.3, at each occurrence, is independently a nucleotide or nucleotide derivative; N.sup.1-N.sup.3, at each occurrence, is independently a nucleotide or nucleotide derivative; N.sub.p and N.sup.z, at each occurrence, is independently a nucleotide or nucleotide derivative; N.sup.4-N.sup.5, at each occurrence, is independently a 2-O-Me-ribonucleotide; p is a number from 0 to about 30 and z is a number from 0 to about 30; provided that the compound contains less than 3 consecutive guanosines. In certain embodiments p and z are independently a number from 1 to about 20. In certain embodiments p and z are independently a number from 2 to about 15. In certain embodiments p and z are independently a number from 3 to about 10.

[0073] In certain embodiments of the invention, the IRO compound that is an antagonist of TLR7 and/or TLR9 has the structure 5-N.sub.pN.sub.3N.sub.2N.sub.1C*G*N.sup.1N.sup.2N.sup.3N.sup.zN.sup.4N.sup.5-3 (SEQ ID NO: 72), wherein C*G* is an oligonucleotide motif wherein C* is 5-Me-dC, and G* is 7-deaza-dG; N.sub.1-N.sub.2, at each occurrence, is independently a 2-O-Me-ribonucleotide; N.sub.3, at each occurrence, is independently a nucleotide; N.sup.1-N.sup.3, at each occurrence, is independently a nucleotide; N.sub.p and N.sup.z, at each occurrence, is independently a nucleotide; N.sup.4-N.sup.5 at each occurrence, is independently a 2-O-Me-ribonucleotide; p is 5 and z is 3; provided that the compound contains less than 3 consecutive guanosines. Alternatively, SEQ ID NO: 72 can be written as 5-NNNNNN.sub.3N.sub.2N.sub.1C*G*N.sup.1N.sup.2N.sup.3NNNN.sup.4N.sup.5-3.

[0074] In preferred embodiments, two oligonucleotides having the structure 5-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3 (SEQ ID NO: 70) are covalently linked via a direct nucleotide to nucleotide linkage at their 3 ends through the 3 positions of the sugars or through a modified sugar or modified nucleobase or via a non-nucleotide linker at their 3 ends through the 3 positions of the sugars or through a modified sugar or modified nucleobase. In preferred aspects of this embodiment, the IRO compound has the structure 5-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3-X-3-N.sup.m-N.sup.3N.sup.2N.sup.1GCN.sub.1N.sub.2N.sub.3-N.sub.m-5 (5-SEQ ID NO: 70-3-X-3-SEQ ID NO: 70-5), wherein X is a nucleotide linkage or a non-nucleotide linker and N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G, N.sup.1, N.sup.2, N.sup.3 and N.sup.m are as described above for the general structure of the IRO compound. In preferred embodiments, the two oligonucleotides are covalently linked directly via a nucleotide linkage. In more preferred embodiments, the two oligonucleotides are covalently linked via a non-nucleotide linker.

[0075] As a non-limiting example, the non-nucleotide linker covalently linking the two oligonucleotides may be attached to the 3-hydroxyl of the sugar. In such embodiments, 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 a non-phosphate-based linkage. Possible sites of conjugation for the linker to the 3 end of the oligonucleotide are indicated in Formula I, below, wherein B represents a heterocyclic base and wherein the arrow pointing to P indicates any attachment to phosphorous.

##STR00001##

[0076] In certain embodiments according to this aspect of the invention, the non-nucleotide linker is a small molecule, macromolecule or biomolecule, including, without limitation, polypeptides, antibodies, lipids, antigens, allergens, and oligosaccharides. In certain other embodiments, the non-nucleotide linker is a small molecule. For purposes of the invention, 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.

[0077] In some embodiments, the small molecule is an aliphatic or aromatic hydrocarbon, either of which optionally can include, either in the linear chain connecting the oligonucleotides 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. However, for purposes of describing the non-nucleotide linker, the term small molecule is not intended to include a nucleoside.

[0078] In some embodiments, the non-nucleotide 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 2 to about 9 carbon atoms. In other embodiments, the alkyl linker has less than 3 carbon atoms. In further embodiments, the alkyl linker has at least 3 carbon atoms and preferentially more than three 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 can include, but are not limited to, 1,2 propanediol, 1,2,3 propanetriol, 1,3 propanediol, 1,2,4-Butanetriol, 1,3,5-Pentanetriol, 3-trimethylamino-1,2-propanediol, Bis-1,5-O-(3thymidyl(-1,3,5-pentanetriol, Bis-1,5-O-[3-(1,2-dideoxy-D-robosyl)]-1,3,5-pentanetriol, 3-(2-Hydroxyethyl)-1,5-pentanediol, triethylene glycol hexaethylene glycol, polyethylene glycol linkers (e.g. [OCH2-CH2-].sub.n (n=1-9)), methyl linkers, ethyl linkers, propyl linkers, butyl linkers or hexyl linkers. In some embodiments, such alkyl linkers may include peptides or amino acids.

[0079] In some embodiments, the non-nucleotide linker may include, but are not limited to, those listed in Table 3.

TABLE-US-00003 TABLE 3 Representative Non-Nucleotidic Linkers [00002] [00003] [00004] [00005] [00006] [00007] [00008]

[0080] In some embodiments, the small molecule linker is glycerol or a glycerol homolog of the formula HO(CH.sub.2).sub.oCH(OH)(CH.sub.2).sub.pOH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4, or from 1 to about 3. In some other embodiments, the small molecule linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO(CH.sub.2).sub.mC(O)NHCH.sub.2CH(OH)CH.sub.2NHC(O)(CH.sub.2).sub.mOH, 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.

[0081] Some non-nucleotide linkers according to the invention permit attachment of more than two oligonucleotides. For example, 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.

[0082] IRO compounds also 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.

[0083] In preferred embodiments one of the oligonucleotides of the IRO compound is not an antisense oligonucleotide. In more preferred embodiments neither of the oligonucleotides of the IRO compound is an antisense oligonucleotide.

[0084] In certain embodiments, pyrimidine nucleosides in the immune regulatory oligonucleotides used in the compositions and methods according to the invention have the structure (II):

##STR00009##

wherein:

[0085] D is a hydrogen bond donor;

[0086] 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;

[0087] A is a hydrogen bond acceptor or a hydrophilic group;

[0088] A is selected from the group consisting of hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;

[0089] X is carbon or nitrogen; and

[0090] S is a pentose or hexose sugar ring, or a sugar analog.

[0091] In certain embodiments, 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.

[0092] In some embodiments hydrogen bond donors include, without limitation, NH, NH.sub.2, SH and OH. Preferred hydrogen bond acceptors include, without limitation, CO, CS, and the ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of cytosine.

[0093] In some embodiments, structure (II) is a pyrimidine nucleoside derivative. Examples of pyrimidine nucleoside derivatives include, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, or N4-ethylcytosine, araC, 5-OH-dC, N3-Me-dC, 2-O-Me-C, 2-O-Me-U, 2-O-Me-T, and 4-thiouracil. Chemical modified derivatives also include, but are not limited to, thymine or uracil analogues. In some embodiments, 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.

[0094] In some embodiments, the purine nucleosides in immune regulatory oligonucleotides used in the compositions and methods according to the invention have the structure (III):

##STR00010##

[0095] wherein:

[0096] D is a hydrogen bond donor;

[0097] D is selected from the group consisting of hydrogen, hydrogen bond donor, and hydrophilic group;

[0098] A is a hydrogen bond acceptor or a hydrophilic group;

[0099] X is carbon or nitrogen;

[0100] each L is independently selected from the group consisting of C, O, N and S; and

[0101] S is a pentose or hexose sugar ring, or a sugar analog.

[0102] In certain embodiments, 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.

[0103] In certain embodiments hydrogen bond donors include, without limitation, NH, NH.sub.2, SH and OH. In certain embodiments hydrogen bond acceptors include, without limitation, CO, CS, NO.sub.2 and the ring nitrogen atoms of an aromatic heterocycle, e.g., N1 of guanine.

[0104] In some embodiments, structure (III) is a purine nucleoside derivative. Examples of 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-(B-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, 1-(2-deoxy--D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, 2-O-methyl-G, and N1-Me-dG. 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, N1-Methyladenosine, 8-Azaadenosine, 5-Iodotubercidin, and 2-O-Me-A. In some embodiments, the sugar moiety S in (III) is a sugar derivative as defined for Formula II.

[0105] In certain embodiments of the invention, the immune regulatory nucleic acid comprises a nucleic acid sequence containing at least one B-L-deoxy nucleoside or 3-deoxy nucleoside.

[0106] In certain embodiments of the invention, 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 2-deoxycytidine, G is guanosine or 2-deoxyguanosine, C* is 2-deoxythymidine, 1-(2-deoxy-B-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, 5-Me-dC, 2-dideoxy-5-halocytosine, 2-dideoxy-5-nitrocytosine, arabinocytidine, 2-deoxy-2-substituted arabinocytidine, 2-O-substituted arabinocytidine, 2-deoxy-5-hydroxycytidine, 2-deoxy-N4-alkyl-cytidine, 2-deoxy-4-thiouridine, 2-O-substituted ribonucleotides (including, but not limited to, 2-O-Me-5-Me-C, 2-O-(2-methoxyethyl)-ribonucelotides or 2-O-Me-ribonucleotides) or other pyrimidine nucleoside analogs or derivative, G* is 2-deoxy-7-deazaguanosine, 2-deoxy-6-thioguanosine, arabinoguanosine, 2-deoxy-2substituted-arabinoguanosine, 2-O-substituted-arabinoguanosine, 2-deoxyinosine, 2-0-substituted ribonucleotides (including, but not limited to, 2-O-(2-methoxyethyl)-ribonucelotides; or 2-O-Me-ribonucleotides) or other purine nucleoside analogs or derivative, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate, and wherein the activity of the at least one dinucleotide is regulated by the flanking sequence.

[0107] In some embodiments, the oligonucleotides of the IRO compound 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 nucleotide residues.

[0108] In some embodiments, the IRO compounds can be combined with 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.

[0109] In a second aspect, the invention provides a pharmaceutical composition comprising an IRO compound according to the invention and a physiologically acceptable carrier.

[0110] In embodiments of this aspect of the invention, the composition can further comprise 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.

[0111] In a third aspect, the invention provides methods for inhibiting or suppressing TLR-mediated induction of an immune response in a mammal, such methods comprising administering to the mammal an IRO compound according to the invention. In some embodiments, the mammal is a human. In preferred embodiments, the IRO compound is administered to a mammal in need of immune suppression.

[0112] According to this aspect of the invention, an IRO compound is capable of suppressing a TLR-based immune response to a further TLR ligand or TLR agonist. As discussed further in the Examples below, the activation of a TLR-based immune response by a TLR agonist or TLR ligand (for example, an immune stimulatory oligonucleotide) can be antagonized, inhibited, suppressed or prevented by the simultaneous, pre- or post-administration of an IRO compound, and such antagonism, inhibition, suppression or prevention may be maintained for an extended period of time (for example, 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. For example, application of certain TLR-agonists in the course of treating the disease may cause unwanted immune stimulation that an IRO compound could antagonize, suppress, inhibit or prevent. Administration of the IRO simultaneously, pre and/or post administration of the TLR-agonist may allow therapeutic benefits from the TLR-agonist while antagonizing, suppressing, inhibiting or preventing the unwanted side effect(s). Additionally, pre-administration of an IRO compound according to the invention could antagonize, suppress, inhibit or prevent an immune response (for example, an allergic reaction) to a subsequent or later challenge by a TLR-agonist. Preferably a TLR7 and/or TLR9 agonist.

[0113] In the methods according to this aspect of the invention, administration of IRO compound according to the invention can be by any suitable route, including, without limitation, parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intragastric, 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. When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood concentration of IRO compound from about 0.0001 micromolar to about 100 micromolar. More preferably, systemic administration would be at a sufficient dosage to attain a blood concentration of the IRO compound from about 0.001 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, 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 the IRO compound according to the invention daily, every second day, every third day, every fourth day, every fifth day, every sixth day or weekly. It may be desirable to administer simultaneously, or sequentially, a therapeutically effective amount of one or more of the IRO containing therapeutic compositions of the invention to an individual as a single treatment episode.

[0114] 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 (for example, adjuvants) including, without limitation, TLR agonists (e.g. TLR2 agonists, TLR4 agonists, and TLR9 agonists), Freund's incomplete adjuvant, KLH, monophosphoryl lipid A (MPL), alum, Merck alum adjuvant (MAA), and saponins, including QS-21 and imiquimod, or combinations thereof.

[0115] The methods according to this aspect of the invention are useful for model studies of the immune system. The methods are also useful for the prophylactic or therapeutic treatment of human or animal disease. For example, the methods are useful for pediatric, adult, and veterinary vaccine applications.

[0116] In a fourth aspect, 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. In various embodiments, 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 bowel 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-Barr syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious anaemia, polymyositis, primary biliary cirrhosis, schizophrenia, Sjgren's syndrome, temporal arteritis (giant cell arteritis), vasculitis, vitiligo, vulvodynia and Wegener's granulomatosis. 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.

[0117] In a fifth aspect, the invention provides methods for preventing a disease or disorder, such methods comprising administering to the patient IRO compound according to the invention. In various embodiments, 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 bowel 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-Barr syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious anaemia, polymyositis, primary biliary cirrhosis, schizophrenia, Sjgren's syndrome, temporal arteritis (giant cell arteritis), vasculitis, vitiligo, vulvodynia and Wegener's granulomatosis. 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.

[0118] In any of the methods according to the third, fourth or fifth aspect of the invention, the IRO compound can be administered in combination with any other agent useful for treating or preventing the disease or condition that does not abolish the immune antagonist, inhibitory, suppression or prevention effect or activity of the IRO compound. In any of the methods according to the invention, the agent useful for treating or preventing 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. For example, in the treatment of cancer, it is contemplated that the IRO compound may be administered in combination with one or more chemotherapeutic compound, targeted therapeutic agent and/or monoclonal antibody; And in preventing a disease, it is contemplated that the IRO compound may be administered in combination with one or more vaccine. Alternatively, the agent can include DNA vectors encoding for antigen or allergen. In these embodiments, the IRO compounds of the invention can variously act as adjuvants and/or produce direct immune modulatory effects.

[0119] The following examples are intended to further illustrate certain exemplary embodiments of the invention and are not intended to limit the scope of the invention. For example, representative 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.

Example 1

Synthesis of Oligonucleotides Containing Immune Regulatory Moieties

[0120] All IRO compounds of the invention were synthesized according to standard procedures (see e.g. U.S. Patent Publication No. 20040097719).

[0121] 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).

[0122] Deoxyribonucleoside phosphoramidites were obtained from (Aldrich-Sigma, St Louis, Mo.). 1,2-dideoxyribose phosphoramidite, propyl-1-phosphoramidite, 2-deoxyuridine phosphoramidite, 1,3-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.). Arabinoguanosine, was obtained from Reliable Pharmaceutical (St. Louis, Mo.). Arabinoguanosine phosphoramidite was synthesized at Idera Pharmaceuticals, Inc. (Cambridge, Mass.) (Noronha et al. (2000) Biochem., 39:7050-7062).

[0123] All nucleoside phosphoramidites were characterized by .sup.31P and .sup.1H 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.

Example 2

Inhibition of TLR7 and TLR9 Stimulation

[0124] C57BL/6 mice were injected s.c. at left underarm with 5 mg/kg of an IRO compound at 0 hours and 0.25 mg/kg TLR9 agonist or 10 mg/kg TLR7 agonist at 24 hours. Serum samples were taken at 2 hours after injection of the TLR9 or TLR7 agonist and IL-12 concentration was determined by ELISA. For IRO number 40, the TLR7 and TLR9 agonists were administered 72 hours after administration of the IRO. The results for all IROs are shown in Tables 4-11. These results demonstrate that an IRO compounds according to the invention can inhibit TLR7 and/or TLR9 activity in vivo, and more generally that IRO compounds according to the invention can inhibit TLR activation.

TABLE-US-00004 TABLE4 AntagonistActivityinvivoinmice %Inhibitionof %Inhibitionof Oligo TLR9agonist TLR7agonist No. SequencesandModification inducedIL-12 inducedIL-12 1 5-UGUCG1TTCT-X1-TCTTG1CUGU-5 44.8 94.0 2 5-UGUCG1TTC-X1-CTTG1CUGU-5 69.8 91.6 3 5-UGUCG1TT-X1-TTG1CUGU-5 63.7 89.8 4 5-UGUCoG1TTCTo-Z-oTCTTG1oCUGU-5 27.6 53.7 5 5-GUCG1TTCTT-Z-TTCTTG1CUG-5 75.9 97.1 6 5-UGUCG2TTCT-Z-TCTTG2CUGU-5 70.9 99.0 7 5-UGUCG1TTCT-X4-TCTTG1CUGU-5 83.2 92.6 8 5-UGUCG1TTC-X4-CTTG1CUGU-5 68.7 78.5 9 5-UGUCoG1TTCTo-X4-oTCTTG1oCUGU- 76.5 18.6 5 10 5-GUCG1TTCTT-X4-TTCTTG1CUG-5 87.8 100 11 5-UGUCG1TT-X4-TTG1CUGU-5 31.1 56.8 12 5-UGUCG1TTC-X5-CTTG1CUGU-5 7.3 80.4 13 5-UGUCG2TTC-X5-CTTG2CUGU-5 48.6 98.1 14 5-UGUCG1TTC-X6-CTTG1CUGU-5 64.6 92.2 15 5-UGUCG2TTC-X6-CTTG2CUGU-5 57.1 99.9 16 5-UGUCG1TTCT-X7-TCTTG1CUGU-5 96.5 98.5 17 5-UGUCG2TTCT-X7-TCTTG2CUGU-5 86.2 97.3 18 5-UGUCG1TTC-X7-CTTG1CUGU-5 94.0 98.1

TABLE-US-00005 TABLE5 AntagonistActivityinvivoinmice %Inhibitionof %Inhibitionof Oligo TLR9agonist TLR7agonist No. SequencesandModification inducedIL-12 inducedIL12 19 5-TGUCG1TTCT-X-TCTTG1CUGT-5 45.2 89.8 20 5-CTTGUCG1TTCT-X-TCTTG1CUGTTC-5 74.5 77.9 21 5-TTGUCG1TTC-X-CTTG1CUGTT-5 66.5 86.8 22 5-CTTTGUCG1TTC-X-CTTG1CUGTTTC-5 47.5 88.9 23 5-TGUCG1TTCT-X7-TCTTG1CUGT-5 45.4 83.6 24 5-TTGUCG1TTC-X7-CTTG1CUGTT-5 42.5 88.3 25 5-GUCG1TTCTT-Z-TTCTTG1CUG-5 80.4 92.3 26 5-TGUCG1TTCA-X-ACTTG1CUGT-5 65.8 93.2

TABLE-US-00006 TABLE6 AntagonistActivityinvivoinmice Oligo %InhibitionofTLR9 No. SequencesandModification agonistinducedIL-12 27 5-TCTGACG1TTCT-X-TCTTG1CAGTCT-5 95.8 28 5-TCTGACG2TTCT-X-TCTTG2CAGTCT-5 97.4

TABLE-US-00007 TABLE7 AntagonistActivityinvivoinmice Oligo %InhibitionofTLR9 %InhibitionofTLR7 No. SequencesandModification agonistinducedIL-12 agonistinducedIL12 29 5-TTGUCG1TTA-X-ATTG1CUGTT-5 36.6 95.3 30 5-CTCTGUCG1TTA-X-ATTG1CUGTCTC- 22.6 91.6 5 31 5-TGTC*GTTCT-X-TCTTGC*TGT-5 78.9 32 5-TGTCGTTCT-X-TCTTGCTGT-5 73.4 33 5-TGTC*GTTCT-X-TCTTGC*TGT-5 75.5 34 5-TGTCGTTCT-X-TCTTGCTGT-5 85.8

TABLE-US-00008 TABLE8 AntagonistActivityinvivoinmice Oligo %InhibitionofTLR9 No. SequencesandModification agonistinducedIL-12 35 5-UGUCG1ACAT-X-TACAG1CUGU-5 65.1 36 5-UGUCG1TTC-X-CTTG1CUGU-5 35.7 37 5-UGUCG1TT-X-TTG1CUGU-5 26.5 38 5-UoGUCG1TToCTo-X-oTCoTTG1CUGoU-5 6.9 39 5-UoGoUCG1TTCTo-X-oTCTTG1CUoGoU-5 16.8

TABLE-US-00009 Table9 AntagonistActivityinvivoinmice Oligo %InhibitionofTLR9 No. SequencesandModification agonistinducedIL-12 40 5-UGACG1TTCT-X-TCTTG1CAGU-5 54.9 Table10 AntagonistActivityinvivoinmice Oligo %InhibitionofTLR9 No. SequencesandModification agonistinducedIL-12 41 5-UGUCG1ACAT-Z-TACAG1CUGU-5 86.9 42 5-UGUCG1TTCT-Z-TCTTG1CUGU-5 69.6 43 5-UGUCG1TTC-Z-CTTG1CUGU-5 60.8 44 5-UGUCG1TT-Z-TTG1CUGU-5 43.8

TABLE-US-00010 TABLE11 AntagonistActivityinvivoinmice oligo %InhibitionofTLR9 %InhibitionofTLR7 No. SequencesandModification agonistinducedIL-12 agonistinducedIL12 58 5-CTATCTGAC*GTTCTCTGT-3 72.6 79.8 62 5-CTATCTG{circumflex over ()}A{circumflex over ()}CGTTCTCTGT-3 68.1 30.9

TABLE-US-00011 TABLE12 AntagonistActivityinvivoinmice oligo %InhibitionofIL-12 No. Sequence TLR9 TLR7 71 5-CTTTGUC*G1TTC-X-CTTG1C*UGTTTC-5 55.5 94.9 77 5-CTTGUC*G1TTCT-X-TCTTG1C*UGTTC-5 71.5 92.8 78 5-CTTTGUC*oG1TTC-X-CTTG1oC*UGTTTC-5 25.6 83.4 80 5-CTTGUC*oG1TTCT-X-TCTTG1oC*UGTTC-5 6.7 68.2 82 5-CTGUC*oG1TTCTT-X-TTCTTG1oC*UGTC-5 9.0 78.4 88 5-CTATCTGUC*G1TTCTCTGT-3 63.2 77.7 89 5-CTATCTGUC*G1TTCTCTGT-3 36.4 40.5

Example 3

TLR7/TLR9 In Vitro Antagonist Study

[0125] C57BL/6 mice were used in this study. Mouse splenocytes were cultured for 24 hrs (at 37 C., 5% CO.sub.2) with TLR7/TLR9 antagonists over a dose range 0.3, 1, 5, 10 mg/ml or at a single dose, 10 mg/ml in the presence of a TLR7 agonist (200 mg/ml) or in the presence of a TLR9 agonist (1 mg/ml) or in the presence of PBS. Supernatants were collected and cytokine/chemokine responses were then evaluated in supernatants by multiplex assays using the Luminex xMAP system. Samples were assayed in duplicate (SD). Results are shown in FIGS. 3 and 4.

Example 4

TLR7/TLR9 In Vivo Antagonist Study

[0126] Female C57BL/6 mice (2/group) were s.c injected with 1, 5 or 15 mg/kg antagonist compound at 0 hr in the right flank. The mice were then injected with TLR7 agonist (10 mg/kg) or with TLR9 agonist (0.25 mg/kg) at 24 hrs in the left flank. Blood was collected by orbital bleeding 2 hrs post the agonist administration. The serum samples were then analyzed by IL-12 ELISA. Results are shown in FIGS. 5 and 6.

[0127] Additionally, female C57BL/6 mice (2/group) were s.c injected with 5 mg/kg antagonist compound at day 0 in the right flank. The mice were then injected with TLR7 (10 mg/kg) agonist at days 1, 2, 4, 9 and 11 or with TLR9 (0.25 mg/kg) agonist at days 1, 2, 4, 7, 9 and 11 in the left flank. Blood was collected by orbital bleeding 2 hrs post the agonist administration. The serum samples were then analyzed by IL-12 ELISA. Results are shown in FIGS. 7 and 8.

[0128] Female C57BL/6 mice (2/group) were also s.c injected with 5 mg/kg antagonist compound at 0 hr in the right flank. The mice were then injected with TLR9 (0.25 mg/kg) or TLR7 (10 mg/kg) agonists at 24 hrs in the left flank. Blood was collected by orbital bleeding 2 hrs post the agonist administration. Cytokine/chemokine responses were then evaluated in serum samples by multiplex assays using the Luminex xMAP system. Results are shown in FIG. 9.

[0129] Finally, female C57BL/6 mice (2/group) were s.c injected with 5 mg/kg antagonist compound at 0 hr in the right flank. The mice were then injected with TLR3 (10 mg/kg) or TLR5 (0.25 mg/kg) agonists at 24 hrs in the left flank. Blood was collected by orbital bleeding 2 hrs post the agonist administration. Cytokine/chemokine responses were then evaluated in serum samples by multiplex assays using the Luminex xMAP system. Results are shown in FIG. 10.

[0130] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.