HK1216430B - Synthetic agonists of tlr9 - Google Patents

Description

Synthetic agonists of TLR9

The application is a divisional application of an invention patent divisional application with the application date of 2008-7-31 and the application number of 201210311690.3 and the name of 'a novel synthetic agonist of TLR 9'.

RELATED APPLICATIONS

The present application claims U.S. provisional patent application serial No. 60/953,251 filed on 8/1/2007; U.S. provisional patent application serial No. 60/983,601 filed on 30/10/2007; U.S. provisional patent application serial No. 60/987,151 filed on 12/11/2007; and U.S. provisional patent application serial No. 61/015,292 filed on 20.12.2007, the entire contents of which are hereby incorporated by reference.

Technical Field

The present invention relates to synthetic chemical compositions useful for modulating Toll-like receptor (TLR) -mediated immune responses. In particular, the invention relates to Toll-like receptor 9(TLR9) agonists that produce a unique cytokine and chemokine profile (profile).

Background

Toll-like receptors (TLRs) are present on many cells of the immune system and have been shown to be involved in the innate immune response (Hornung, V.et al (2002) J.Immunol.168: 4531-4537). In vertebrates, this family consists of 11 proteins called TLR 1-TLR 11 (Poltorak, a. et al (1998) Science 282: 2085. 2088; Underhill, D.M. et al (1999) Nature 401: 811. 815; Hayashi, F. et al (2001) Nature 410: 1099. 1103; Zhang, D. et al (2004) Science 303: 2. 1526; Meier, A. et al (2003) Microbiol.5: 561. 570; Campos, M.A. et al (2001) J.munol. 167: 748. 423; Hoebe, K. et al (2003) Nature 424: 743; Lund, J. 2003) J. 198. J. 152303: 748. 2004; Imebe, K. et al (2003) Nature 424: 19: 47. J. 134. 2004; Eur. 1529. J. 134. 2004; Meyer, J. 10. J. 10. J. 133. J. 133. 134. 2004).

TLRs are key means by which vertebrates recognize foreign molecules and mount immune responses to foreign molecules, and also provide a means to link innate and adaptive immune responses (Akira, S. et al (2001) Nature Immunol.2: 675-680; Medzhitov, R. (2001) Nature Rev. Immunol.1: 135-145). Some TLRs are located on the cell surface to detect and initiate a response to extracellular pathogens, while other TLRs are located intracellularly to detect and initiate a response to intracellular pathogens.

TLR9 is known to recognize unmethylated CpG motifs in bacterial DNA and in synthetic oligonucleotides (Hemmi, H., et al (2000) Nature 408: 740-. Other modifications of CpG-containing phosphorothioate oligonucleotides can also affect their ability to function as modulators of immune responses via TLR9 (see, e.g., Zhao et al (1996) Biochem. Pharmacol.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.9: 3453-3458; Zhao et al (2000) Bioorg. Med. Chem. Lett.10: 1051-1054; Yu, D. et al (2000) Bioorg Med. Chem. Lett.10: 2585-2588; Yu, D. et al (2001) Bioorg. Cheg. chem. 10: 22611-chem. 9; Cheimp. 9-9; Ka-chem. 9). Naturally occurring agonists of TLR9 have been shown to produce anti-tumor activity (e.g., tumor growth and angiogenesis) leading to potent anti-cancer responses (e.g., anti-leukemia) (Smith, J.B. and Wickstrom, E. (1998) J.Natl.cancer Inst.90: 1146-. In addition, TLR9 agonists have been shown to work synergistically with other known anti-tumor compounds (e.g., cetuximab (cetuximab), irinotecan (irinotecan)) (Vincenzo, D. et al (2006) Clin. cancer Res.12(2): 577-.

Certain TLR9 agonists consist of 3 '-3' linked DNA structures containing a core CpR dinucleotide, where R is a modified guanosine (U.S. Pat. No. 7,276,489). In addition, specific chemical modifications allow the preparation of specific oligonucleotide analogs that exert distinct modulation of immune responses. In particular, the structural activity correlation studies allow the identification of synthetic motifs (motif) and novel DNA-based compounds which generate specific modulations of the immune response which are distinct from the modulations generated by unmethylated CpG dinucleotides (Kandimalla, E.et al (2005) Proc. Natl.Acad.Sci.U S A102: 6925-6930; Kandimalla, E.et al (2003) Proc. Nat.Acad.Sci.U S A100: 14303-14314308; Cong, Y.et al (2003) Biochem Biophys Res.Commu.310: 1133-1139; Kandimalla, E.et al (2003) Biochem Biophys.Commu.Res.Comp.306: 948-3; Kandimalla, E.et al (2003) Biophys.Comp.No.2002; Biophys.No.No.No. 4431: 4548-97; Biophys.J.Biophys.31: 97-D.J.2002; Biochem Biophys.31: 4411-4411: 31-97; Biophys.J.D.J.97-97-J.J.97; Biochem Biophys.31: 97-D.31; Biochem Biophys.31: 31-97; Biophys.J.J.31; Biochem Biophys.31: 97-97; Biochem, D. et al (2002) Nucleic acids sRs.30: 1613-1619; yu, D, et al (2001) bioorg.Med.chem.9: 2803-2808; yu, D, et al (2001) bioorg.Med.chem.Lett.11: 2263-; kandimalla, E.et al (2001) bioorg.Med.chem.9: 807-813; yu, D, et al (2000) bioorg.Med.chem.Lett.10: 2585-; putta, M.et al (2006) nucleic acids Res.34: 3231-3238).

The inventors have surprisingly found that uniquely modifying the flanking sequences of the core CpR dinucleotide, the linkage between the nucleotides, or the linker linking the oligonucleotides, results in novel TLR9 agonists that produce distinct cytokine and chemokine profiles in vitro and in vivo. This ability to "tailor-modulate" (custom-tune) cytokine and chemokine responses to CpR-containing oligonucleotides provides the ability to prevent and/or treat a variety of disease conditions in a disease-specific and even patient-specific manner. Thus, new oligonucleotide analog compounds are needed to provide such custom-modulated responses.

Summary of The Invention

The present invention provides novel oligonucleotide-based compounds that each interact as an agonist with TLR9 to provide a distinct (distinting) immune response profile (profile), respectively. TLR9 agonists according to the invention are characterized by specific (specific) and unique (unique) chemical modifications that provide their distinct (discotic) immune response activation profiles.

The TLR9 agonists according to the invention induce an immune response in a variety of cell types and in a variety of in vitro and in vivo experimental models, with each agonist providing a distinct immune response profile. The TLR9 agonists according to the invention are used in the prevention and/or treatment of various diseases, alone, or in combination or co-administration with other drugs, or as adjuvants for antigens used as vaccines. They are therefore useful as tools for studying the immune system and for comparing the immune system of various animal species, such as humans and mice.

Thus, in a first aspect, the invention provides oligonucleotide-based agonists ("compounds") of TLR 9.

In a second aspect, the invention provides a pharmaceutical formulation comprising an oligonucleotide-based TRL9 agonist according to the invention and a pharmaceutically acceptable carrier.

In a third aspect, the invention provides a vaccine. The vaccine according to this aspect comprises a pharmaceutical formulation according to the invention and further comprises an antigen.

In a fourth aspect, the invention provides a method for generating a TLR 9-mediated immune response in an individual, such method comprising administering to the individual a compound, pharmaceutical formulation or vaccine according to the invention.

In a fifth aspect, the invention provides a method for therapeutically treating a patient having a disease or disorder, such method comprising administering to the patient a compound, pharmaceutical formulation or vaccine according to the invention.

In a sixth aspect, the present invention provides a method for preventing a disease or disorder comprising administering to a patient a compound, pharmaceutical formulation or vaccine according to the present invention.

The invention can be described by the following paragraphs: 1. an immunomodulatory compound selected from compound nos. 1 to 169.

2. A composition comprising an immunomodulatory compound according to paragraph 1 and a physiologically acceptable carrier.

3. A method of generating an immune response in an individual comprising administering to the individual a pharmaceutically effective amount of a compound according to paragraph 1.

4. A method for therapeutically treating an individual having a disease or disorder where modulation of an immune response is beneficial comprising administering to the individual a pharmaceutically effective amount of a compound according to paragraph 1.

5. The method according to paragraph 4, wherein the disease or disorder is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, skin disorders, allergy, asthma, or a disease caused by a pathogen or allergen.

6. The method according to paragraph 4, further comprising administering one or more chemotherapeutic compounds, targeted therapeutics, vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, kinase inhibitors, peptides, proteins, DNA vaccines, adjuvants, co-stimulatory molecules, or combinations thereof.

7. A method for prophylactically treating an individual having a disease or disorder where modulation of an immune response would be beneficial comprising administering to the individual a pharmaceutically effective amount of a compound according to paragraph 1.

8. The method according to paragraph 7, wherein the disease or disorder is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, skin disorders, allergy, asthma or a disease caused by a pathogen or allergen in an individual.

9. The method according to paragraph 7, further comprising administering one or more chemotherapeutic compounds, targeted therapeutics, vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, kinase inhibitors, peptides, proteins, DNA vaccines, adjuvants, co-stimulatory molecules, or combinations thereof.

Brief Description of Drawings

FIG. 1 is a synthetic scheme for the linear synthesis of the immunomodulatory compounds of the invention. DMTr ═ 4, 4' -dimethoxytrityl; CE ═ cyanoethyl.

FIG. 2 is a synthetic scheme for the parallel synthesis of immunomodulatory compounds of the invention. DMTr ═ 4, 4' -dimethoxytrityl; CE ═ cyanoethyl.

Figures 3A-3C depict NF- κ B activity in TLR 9-expressing HEK293 cells cultured, processed and analyzed according to example 2 below. Briefly, HEK293 cells were stimulated with 10. mu.g/ml of an immunomodulatory oligonucleotide according to the invention for 18 hours and NF-. kappa.B levels were determined using the SEAP (secreted human embryo alkaline phosphatase) assay.

Figures 3D-3G depict NF- κ B activity in TLR 9-expressing HEK293 cells cultured, processed and analyzed according to example 2 below. HEK293 cells were stimulated with 0 (PBS/medium), 0.1, 0.3, 1.0, 3.0, or 10.0. mu.g/ml of an immunomodulatory oligonucleotide according to the invention for 18 hours and NF-. kappa.B levels were determined using the SEAP (secreted human embryo alkaline phosphatase) assay. Figures 3A-3G more generally demonstrate that administration of an immune modulatory oligonucleotide according to the invention containing novel bases, linkers, and/or unique modifications results in a distinct TLR9 activation profile.

Figures 4A and 4B depict cytokine and chemokine concentrations of human PBMCs isolated, cultured, processed and analyzed according to example 3 below. Briefly, PBMCs were isolated from freshly obtained blood of healthy human volunteers and cultured with a dose of 10 μ g/ml of the immunomodulatory oligonucleotides according to the invention for 24 hours. Supernatants were collected and analyzed for cytokine and chemokine levels by Luminex multiplex assay. FIGS. 4A and 4B more generally demonstrate that administration of an immunomodulatory oligonucleotide comprising novel bases, linkers, and/or unique modifications according to the invention produces distinct cytokine and chemokine profiles.

Figures 4C-4H depict cytokine and chemokine concentrations of human PBMCs isolated, cultured, processed and analyzed according to example 3 below. Briefly, PBMCs were isolated from freshly obtained blood of healthy human volunteers and cultured with an immunomodulatory oligonucleotide according to the invention at a dose of 0(PBS), 0.1, 0.3, 1.0, 3.0, or 10.0 μ g/ml for 24 hours. Supernatants were collected and analyzed for cytokine and chemokine levels by Luminex multiplex assay. FIGS. 4C-4H more generally demonstrate that administration of an immunomodulatory oligonucleotide containing novel bases, linkers, and/or unique modifications according to the invention produces distinct cytokine and chemokine profiles.

Figures 4I-4N depict cytokine and chemokine concentrations of human PBMCs isolated, cultured, processed and analyzed according to example 3 below. Briefly, PBMCs are isolated from freshly obtained blood of healthy human volunteers and cultured with an immunomodulatory oligonucleotide according to the invention at a dose of 0(PBS), 1.0, or 10.0 μ g/ml for 24 hours. Supernatants were collected and analyzed for cytokine and chemokine levels by Luminex multiplex assay. FIGS. 4I-4N more generally demonstrate that administration of an immunomodulatory oligonucleotide containing novel bases, linkers, and/or unique modifications according to the invention produces distinct cytokine and chemokine profiles.

Figures 4O-4FF depict cytokine and chemokine concentrations of human PBMCs isolated, cultured, processed and analyzed according to example 3 below. Briefly, PBMCs were isolated from freshly obtained blood of healthy human volunteers and cultured with an immunomodulatory oligonucleotide according to the invention at a dose of 0(PBS), 0.1, 0.3, 1.0, 3.0, or 10.0 μ g/ml for 24 hours. Supernatants were collected and analyzed for cytokine and chemokine levels by Luminex multiplex assay. FIGS. 4O-4FF more generally demonstrate that administration of an immunomodulatory oligonucleotide containing novel bases, linkers, and/or unique modifications according to the invention produces distinct cytokine and chemokine profiles.

Figures 5A and 5B depict cytokine and chemokine concentrations of human plasmacytoid dendritic cells (pdcs) isolated, cultured, treated and analyzed according to example 3 below. Briefly, pdcs were isolated from freshly obtained blood PBMCs of healthy human volunteers and cultured with a dose of 10 μ g/ml of the immunomodulatory oligonucleotides according to the invention for 24 hours. Supernatants were collected and analyzed for cytokine and chemokine levels by Luminex multiplex assay. FIGS. 5A and 5B more generally demonstrate that administration of an immunomodulatory oligonucleotide comprising novel bases, linkers, and/or unique modifications according to the invention produces distinct cytokine and chemokine profiles.

FIGS. 6A-6F depict human B-cell proliferation induced by an immunomodulatory oligonucleotide according to the invention. Human B cells were isolated, cultured, processed and analyzed according to example 4 below. Briefly, human B cells isolated from freshly obtained blood PBMCs of healthy human volunteers were cultured with different doses of the immunomodulatory oligonucleotides according to the invention for 68 hours and used³H-thymidine is pulsed for 6-8 hours. Measurement using liquid scintillation counter³H-thymidine uptake. FIGS. 6A-6F more generally demonstrate that administration of an immunomodulatory oligonucleotide according to the invention containing novel bases, linkers, and/or unique modifications produces distinct cell proliferation profiles that vary by the base composition, unique modifications, and amount of the oligonucleotide administered.

Figure 7A depicts serum cytokine and chemokine induction in C57BL/6 mice treated according to example 5 below. Briefly, mice were injected subcutaneously with a 1mg/kg dose of the immunomodulatory oligonucleotides according to the invention, and 2 hours later sera were collected and analyzed for cytokine and chemokine levels by Luminex multiplex assay.

Figure 7B depicts serum cytokine induction in BALB/c mice treated according to example 5 below. Briefly, mice were injected subcutaneously with a 1mg/kg dose of the immunomodulatory oligonucleotide according to the invention, and 2 hours later sera were collected and analyzed for IL-12 levels by ELISA.

FIGS. 7C-7F depict serum cytokine induction in BALB/C mice treated in accordance with example 5 below. Briefly, mice were injected subcutaneously with a 0.25 or 1mg/kg dose of an immunomodulatory oligonucleotide according to the invention, and sera were collected after 2 hours and analyzed for IL-12 levels by ELISA. Figures 7A-7F more generally demonstrate that in vivo administration of immune modulatory oligonucleotides containing novel bases, linkers, and/or unique modifications according to the invention results in a distinct TLR9 activation profile, which may find application in a variety of diseases.

Detailed Description

The present invention provides novel oligonucleotide-based compounds that each interact as an agonist with TLR9 to provide distinct immune response profiles, respectively. The TLR9 agonists according to the invention are characterized by unique chemical modifications that provide their distinct immune response activation profiles. All publications cited herein are indicative of the level of skill in the art and are incorporated herein by reference in their entirety. Any conflict between the teachings of these references and the present specification shall be resolved in favor of the latter.

The TLR9 agonists according to the invention induce an immune response in a variety of cell types and in a variety of in vivo and in vitro experimental models, with each agonist providing a distinct immune response profile. They are therefore useful as tools for studying the immune system and for comparing the immune system of various animal species, such as humans and mice. The TLR9 agonists according to the invention are used in the prevention and/or treatment of various diseases, alone, or in combination or co-administration with other drugs, or as adjuvants for antigens used as vaccines. Definition of

The term "2 ' -substituted nucleoside" or "2 ' -substituted arabinoside (arabinoside)" generally includes nucleosides or arabinonucleosides in which the hydroxyl group at the 2 ' position of the pentose or arabinose module is substituted to produce a 2 ' -substituted or 2 ' -O-substituted ribonucleoside. In certain embodiments, the substitution is with a lower alkyl group containing 1 to 6 saturated or unsaturated carbon atoms, with a halogen atom, or with an aryl group having 6 to 10 carbon atoms, wherein the alkyl or aryl group may be unsubstituted or may be substituted, for example, with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carboalkoxy (carboalkxy), or amino. Examples of 2 ' -O-substituted ribonucleosides or 2 ' -O-substituted cytarabine include, but are not limited to, 2 ' -amino, 2 ' -fluoro, 2 ' -allyl, 2 ' -O-hydrocarbyl and 2 ' -propargyl ribonucleosides or arabinosides, 2 ' -O-methyl ribonucleosides or 2 ' -O-methyl arabinosides and 2 ' -O-methoxyethoxy ribonucleosides or 2 ' -O-methoxyethoxy arabinosides.

The term "3 '" when used in an orientation generally refers to a region or position in a polynucleotide or oligonucleotide that is 3 ' (to the 3 ' position of the oligonucleotide) to another region or position in the same polynucleotide or oligonucleotide.

The term "5 '" when used in an orientation generally refers to a region or position in a polynucleotide or oligonucleotide that is 5 ' (toward the 5 ' position of the oligonucleotide) to another region or position in the same polynucleotide or oligonucleotide.

The term "about" generally means that the exact number is not critical. Thus, the number of nucleoside residues in the oligonucleotide is not critical, and oligonucleotides having one or two nucleoside residues less or one to several nucleoside residues more are contemplated as equivalents for each of the embodiments described above.

The term "airway inflammation" generally includes, but is not limited to, inflammation of the respiratory tract caused by allergens, including asthma.

The term "allergen" generally refers to an antigen or antigenic portion of a molecule (usually a protein) that elicits an allergic response upon exposure to a subject. Typically, the subject is allergic to the allergen, as indicated by, for example, the wheal (rheal) and flush (flare) test or any method known in the art. The molecule is said to be an allergen even if only a small fraction of subjects exhibit an allergic (e.g. IgE) immune response upon exposure to the molecule.

The term "allergy" generally includes, but is not limited to, food allergy, respiratory allergy, and skin allergy.

The term "antigen" generally refers to a substance that is recognized and selectively bound by an antibody or a T cell antigen receptor. Antigens may include, but are not limited to, peptides, proteins, nucleosides, nucleotides, and combinations thereof. An antigen may be natural or synthetic and generally induces an immune response specific for the antigen.

The term "autoimmune disorder" generally refers to a disorder in which the "self antigen is subject to attack by the immune system. The term includes, but is not limited to, lupus erythematosus (lupus erythematosis), multiple sclerosis (multiple sclerosis), type I diabetes mellitus (type I diabetes mellitis), irritable bowel syndrome (irritable bowel syndrome), Crohn's disease, rheumatoid arthritis (rhematoid arthritis), septic shock (septic shock), alopecia universalis (alpropetia univisalis), acute disseminated encephalomyelitis (acutised acquired infectious encephalomyelitis), Addison's disease, ankylosing spondylitis (analysing spinal syndrome), antiphospholipid syndrome (antiphospholipid syndrome), autoimmune hepatitis (autoimmune meningitis), autoimmune meningitis (Bullous autoimmune disease), Bullous autoimmune disease (inflammatory bowel disease), cervical spondylosis (cervical syndrome), cervical spondylosis (cervical syndrome, cervical syndrome, Graves 'disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis suppurativa (hid)radionitis suppurativa), idiopathic thrombocytopenic purpura (idiophatic thrombocytopathic purpura), interstitial cystitis (interstitial cystitis), maculopathy (morphea), myasthenia gravis (myasthenia gravis), narcolepsy (narcolepsy), neuromyotonia nervosa (neuromyotonia), pemphigus (pemphigus), pernicious anemia (pernicious anaemia), polymyositis (polymyositis), primary biliary cirrhosis (primary biliary cirrhosis), schizophrenia (schizophrenia), sjogren's syndrome (MRT (R)syndrome), temporal arteritis (giant cell arteritis), vasculitis (vasculitis), vitiligo (vitigo), vulvodynia (vulvodynia), and Wegener's granulomatosis, autoimmune asthma (autoimmune asthma), septic shock (septic shock), psoriasis (psoriasis), and malaria (malacia).

The term "cancer" generally includes, but is not limited to, any malignant growth or tumor caused by abnormal or uncontrolled cellular proliferation and/or division. Cancer can occur in humans and/or animals, and can occur in any and all tissues. Treatment of a patient with cancer with the present invention may comprise administration of a compound, pharmaceutical formulation or vaccine according to the present invention such that abnormal or uncontrolled cell proliferation and/or division is affected.

The term "carrier" generally encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid-containing vesicle, microsphere, liposome encapsulation, or other substance known in the art for use in pharmaceutical formulations. It will be appreciated 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 substances is described, for example, in Remington's Pharmaceutical Sciences 18 th edition (A. Gennaro eds., Mack Publishing Co., Easton, PA, 1990).

The term "pharmaceutically acceptable" or "physiologically acceptable" generally refers to a substance that does not interfere with the efficacy of a compound according to the present invention and 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.

The term "co-administration" generally refers to the administration of at least two different substances in close enough time proximity to modulate an immune response. Preferably, co-administration refers to the simultaneous administration of at least two different substances.

The term "pharmaceutically effective amount" generally refers to an amount sufficient to achieve a desired biological effect, such as a beneficial result. Thus, a "pharmaceutically effective amount" will depend on the context in which it is administered. A pharmaceutically effective amount may be administered in one or more prophylactic or therapeutic administrations.

The term "in combination with …" generally refers to the administration of a compound according to the invention and/or other agents useful for treating a disease or condition and that do not abrogate the TLR9 antagonist effects of the compounds of the invention in the course of treating a patient. Such administration can be in any order, including simultaneous administration, and orders spaced apart over a period of several seconds up to several days. Such combined treatment may also include more than one administration of a compound according to the invention and/or of a separate further agent. Administration of the compounds according to the invention and other agents may be by the same or different routes.

The terms "individual" and "subject" generally refer to a mammal, such as a human. Mammals generally include, but are not limited to, humans, non-human primates, rats, mice, cats, dogs, horses, cows (cats), cows (cows), pigs, sheep, and rabbits.

The term "kinase inhibitor" generally refers to a molecule that antagonizes or inhibits a phosphorylation-dependent cellular signaling and/or growth pathway in a cell. Kinase inhibitors may be naturally occurring or synthetic, and include small molecules with the potential to be administered as oral therapeutics. Kinase inhibitors have the ability to rapidly and specifically inhibit the activation of a target kinase molecule. Protein kinaseEnzymes are attractive drug targets, in part because they regulate a wide variety of signaling and growth pathways and include many different proteins. They therefore have great potential in the treatment of diseases involving kinase signaling, including cancer, cardiovascular diseases, inflammatory disorders, diabetes, macular degeneration and neurological disorders. Examples of kinase inhibitors include sorafenibdasatinib、Dasatinib^TM、Zactima^TM、Tykerb^TMAnd STI 571.

The term "linear synthesis" generally refers to synthesis that begins at one end of an oligonucleotide and proceeds linearly to the other end. Linear synthesis allows incorporation of identical or different (in terms of incorporated length, base composition and/or chemical modification) monomeric units into the oligonucleotide.

The term "mammal" is expressly intended to include warm-blooded vertebrates, including but not limited to humans.

The term "modified nucleoside" is generally a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or any combination thereof. In some embodiments, the modified nucleoside is a non-natural pyrimidine or purine nucleoside, as described herein. For purposes of the present invention, a modified nucleoside, pyrimidine or purine analog or non-naturally occurring pyrimidine or purine is used interchangeably to refer to a nucleoside that includes a non-naturally occurring base and/or a non-naturally occurring sugar moiety. For the purposes of the present invention, a base is considered to be non-natural if it is not guanine, cytosine, adenine, thymine or uracil.

The term "modulation" or "modulatory" generally refers to changes, such as an increase in response or qualitative differences in TLR 9-mediated responses.

The term "linker" generally refers to any moiety that can be attached to an oligonucleotide via a sugar, base, or backbone by means of covalent or non-covalent bonding. Linkers can be used to attach two or more nucleosides, or can be attached to the 5 'and/or 3' terminal nucleotides of an oligonucleotide. In certain embodiments of the invention, the linker may be a non-nucleotide linker.

The term "non-nucleotide linker" generally refers to a chemical moiety other than a nucleotide linkage that can be attached to an oligonucleotide by covalent or non-covalent bonding. Preferably, the non-nucleotide linker is about 2 angstroms to about 200 angstroms in length and may be in either a cis or trans orientation.

The term "nucleotide linkage" generally refers to a chemical linkage joining two nucleosides via their sugars (e.g., 3 '-3', 2 '-5', 3 '-5'), which consists of a phosphorus atom and a charged or neutral group (e.g., phosphodiester, phosphorothioate, or phosphorodithioate) between adjacent nucleosides.

The term "oligonucleotide-based compound" refers to a polynucleotide formed from a plurality of linked nucleoside units. The nucleoside unit may be or may be constituted by a portion of a virus, a bacterium, a cellular debris, an siRNA or a microrna. The oligonucleotides may also be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably synthetically produced. In a preferred embodiment, each nucleoside unit comprises a heterocyclic base and a pentofuranosyl, trehalose, arabinose, 2 ' -deoxy-2 ' -substituted nucleoside, 2 ' -deoxy-2 ' -substituted arabinose, 2 ' -O-substituted arabinose or hexose group. The nucleoside residues can be coupled to each other by each of a variety of known internucleoside linkages. The internucleoside linkage includes, but is not limited to, phosphodiester (phosphorodiester), phosphorothioate (phosphorothionate), phosphorodithioate (phosphorodithioate), alkylphosphonate (alkylphosphonate), alkylphosphonothioate (alkylphosphorothionate), phosphotriester (phosphotriester), iminophosphate (phosphoramidite), siloxane (siloxane), carbonate (carbonate), carboalkoxy (carboalkoxy), aminoacetate (aminoacetate), carbamate (carbamate), morpholino (morpholino), borono (borano), thioether (thio), and the likeether), bridged phosphoramidates (bridged phosphoramidates), bridged methylene phosphonates (bridged methylene phosphonates), bridged phosphorothioates (bridged phosphorothioates), and sulfone (sulfone) internucleoside linkages. The term "oligonucleotide-based" also encompasses having one or more stereospecific (e.g., (R) internucleoside linkages_P) -or (S)_P) Phosphorothioate, hydrocarbyl phosphonate, or phosphotriester linkages). As used herein, the terms "oligonucleotide" and "dinucleotide" are expressly intended to include polynucleotides and dinucleosides having any of the internucleoside linkages, whether or not the linkages comprise a phosphate group. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphorothioate or phosphorodithioate linkages, or combinations thereof.

The term "peptide" generally refers to a polypeptide of sufficient length and composition to affect a biological response (e.g., antibody production or cytokine activity, whether or not the peptide is a hapten). The term "peptide" may include modified amino acids (whether naturally occurring or non-naturally occurring), wherein the modifications include, but are not limited to, phosphorylation, glycosylation, pegylation, lipidation (lipidation), and methylation.

The term "TLR 9 agonist" generally refers to an oligonucleotide-based compound capable of enhancing, inducing, or modulating immune stimulation mediated by TLR 9.

The term "treatment" or "treating" generally refers to an approach intended to achieve a beneficial or desired result (which may include alleviation of symptoms or delay or amelioration of disease progression).

Table 1 shows certain TLR9 agonists according to the invention. In this table, oligonucleotide-based TLR9 agonists are Phosphorothioate (PS) linkages unless indicated. However, one skilled in the art will recognize that Phosphodiester (PO) linkages or a mixture of PS linkages and PO linkages may be used. Unless indicated, all nucleotides are deoxyribonucleotides.

TABLE 1

G₁7-deaza-dG; g₂＝AraG；G₃7-deaza-araG;A/G/C/U2' -O-methyl ribonucleotide; dU is equal to U₁2' -deoxy-U; o ═ phosphodiester linkage; po ═ 5' -monophosphate; ps ═ 5' phosphorothioate linkage; pm ═ methylphosphonate (nonionic linkage); l ═ 1, 5-pentanediol linker;L1, 2-dideoxyribose; l is₁A triethylene glycol linker; l is₂A tetraethyleneglycol linker; l is₃A hexaethylene glycol linker; an M ═ cis, cis-1, 3, 5-cyclohexanetriol linker; an m ═ cis, trans-1, 3, 5-cyclohexanetriol linker; x ═ glycerol linker; x₁A 1,2, 4-butanetriol linker; x₂A 1,3, 5-tris (2-hydroxyethyl) cyanuric acid linker; x₃An iso-butanetriol linker; y ═ 1, 3-propanediol linker; y is₁A 1, 2-ethylene glycol linker; y is₂A 1, 4-butanediol linker; y is₃A 1, 5-pentanediol linker; and Z is a 1,3, 5-pentanetriol linker.

As described in example 2, the TLR9 agonists exemplified in table 1 were tested for immunostimulatory activity in TLR9 expressing HEK293 cells. The results shown in figures 3A, 3B, 3C, 3D, and 3E demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides alter their profile of NF-kB activation mediated by TLR9 after 18 hours of administration. More generally, these data demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides can be used to increase or decrease NF-kB activation.

As described in example 3, in IL-12, IL-10, IL-8, IL-6, IFN- α, IP-10, MIP-1 α, MIP-1 α, IL-1R α, IL-2R, and MCP-1 in human PBMC assays against TLR9 agonists exemplified in Table 1 immunostimulating activity the results shown in FIGS. 4A-4FF demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides alter their TLR9 mediated IL-12, IL-10, IL-8, IL-6, IFN- α, IP-10, MIP-1 α, MIP-1 β, IL-1R α, IL-2R, and/or MCP-1 activation profiles in human PBMCs more generally these data demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides can be used to increase or decrease IL-12, IL-10, IL-638, IL-6, IL- α, IL-1R, MIP-1R 6851, MIP-6851, IL-2R, and MIP-1 activation profiles.

As described in example 3, immunostimulatory activity was tested in human pDC assays for IL-12, IL-6, IFN- α, IP-10, MIP-1 α, MIP-1 β, and TNF α against the TLR9 agonists exemplified in Table 1 the results shown in FIGS. 5A and 5B demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides will alter their TLR 9-mediated immune activation profile in human pDC more generally, these data demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides can be used to increase or decrease IL-12, IL-6, IFN- α, IP-10, MIP-1 α, MIP-1 β, and TNF α activation.

Immunostimulatory activity was tested in human B cell proliferation assays against the TLR9 agonists exemplified in table 1 as described in example 4. The results shown in fig. 6A, 6B, 6C, 6D, 6E and 6F demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides alter their TLR 9-mediated B cell proliferation activity, and that this activation profile can be dose-dependent depending on the chemical modification. More generally, these data demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides can be used to modulate B cell proliferation.

The TLR9 agonists exemplified in table 1 were tested for in vivo immunostimulatory activity in C57Bl/6 and BALB/C mice as described in example 5. The results shown in figures 7A, 7B, 7D, 7E and 8F demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides alter their TLR 9-mediated immune activation profile in vivo in a mouse model. More generally, these data demonstrate that specific chemical modifications to 3 '-3' linked oligonucleotides can alter cytokine and/or chemokine concentrations in vivo, which may find application in a number of diseases.

As described above, in a first aspect, the invention provides oligonucleotide-based synthetic agonists of TLR 9. Based on certain chemical modifications to bases, sugars, linkages, or linkers, agonists of TLR9 may possess increased stability when combined and/or duplexed (duplex) with other TLR9 agonist molecules, while retaining an accessible 5' -terminus.

In some embodiments, the non-nucleotide linker may include, but is not limited to, those listed in table 2.

Table 2: representative non-nucleotide linkers

Table 2: continuously for

Table 2: continuously for

Table 2: continuously for

Table 2: continuously for

Table 2: continuously for

In a second aspect, the present invention provides a pharmaceutical formulation comprising an oligonucleotide-based TLR9 agonist ("compound") according to the invention and a pharmaceutically acceptable carrier.

The amount of active compound included in the pharmaceutically acceptable carrier or diluent is sufficient to deliver a pharmaceutically effective amount to the patient without causing serious toxic effects in the treated patient. The effective dosage range of a pharmaceutically acceptable derivative can be calculated based on the weight of the parent compound to be delivered or by other means known to those skilled in the art. If the derivative itself exhibits activity, the effective dose can be estimated as described above using the weight of the derivative or by other means known to those skilled in the art.

In a third aspect, the invention provides a vaccine. The vaccine according to this aspect comprises a pharmaceutical formulation according to the invention and further comprises an antigen. Antigens direct molecules that elicit specific immune responses. Such antigens include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and any complexes or combinations thereof. Any such antigen may optionally be linked to an immunogenic protein or peptide, such as Keyhole Limpet Hemocyanin (KLH), cholera toxin B subunit, or any other immunogenic carrier protein.

Vaccines according to the present invention may further include any of a number of known adjuvants, including but not limited to Freund's complete adjuvant, Keyhole Limpet Hemocyanin (KLH), monophosphoryl lipid A (MPL), alum, and saponins, including QS-21, imiquimod, R848, TLR agonists, or combinations thereof.

In a fourth aspect, the invention provides a method for generating a TLR 9-mediated immune response in an individual, such method comprising administering to the individual a compound, pharmaceutical formulation or vaccine according to the invention. In some embodiments, the subject is a mammal. In a preferred embodiment, the compound, pharmaceutical formulation or vaccine is administered to an individual in need of immune stimulation.

In the methods according to this aspect of the invention, administration of the compound, pharmaceutical formulation composition or vaccine according to the invention may be by any suitable route, including but not limited to parenteral, oral, intratumoral, sublingual, transdermal, topical (topical), intranasal, aerosol, intraocular, intratracheal, intrarectal, mucosal, vaginal, by gene gun, dermal patch or in the form of eye drops or mouthwash. Administration of the compound, pharmaceutical formulation or vaccine can be carried out using known procedures at dosages and for periods of time effective to alleviate symptoms of the disease or to replace markers. In systemic administration, the compound, pharmaceutical formulation or vaccine is preferably administered in a sufficient dose to achieve blood levels of the compound according to the invention of from about 0.0001 micromolar to about 10 micromolar. For topical application, concentrations much lower than this can be effective, and much higher concentrations can be tolerated without toxic effects. Preferably, the total dose of the compounds according to the invention ranges from about 0.001mg per patient per day to about 200mg per kg body weight per day. It may be desirable to administer to an individual, either simultaneously or sequentially, a therapeutically effective amount of one or more of the therapeutic compositions of the present invention as a single treatment event.

In certain preferred embodiments, a compound, pharmaceutical formulation or vaccine according to the present invention is administered or co-administered in combination with other agents including, but not limited to, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, siRNA, aptamers, ribozymes, targeted therapies, kinase inhibitors, peptides, proteins, gene therapy vectors, DNA vaccines, and/or adjuvants (to enhance the specificity or strength of the immune response).

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

In a fifth aspect, the invention provides a method of therapeutically treating a patient having a disease or disorder, such method comprising administering to the patient a compound, pharmaceutical formulation or vaccine according to the invention. In various embodiments, the disease or disorder to be treated is cancer, an autoimmune disorder, an infectious disease, airway inflammation, an inflammatory disorder, allergy, asthma, or a disease caused by a pathogen or allergen. Pathogens include, for example, bacteria, parasites, fungi, viruses, viroids, and prions. Administration is carried out as described in relation to the fourth aspect of the invention.

In a sixth aspect, the present invention provides a method for preventing a disease or disorder, such method comprising administering to a patient a compound, pharmaceutical formulation or vaccine according to the present invention. In various embodiments, the disease or disorder to be prevented is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, allergy, asthma, or a disease caused by a pathogen. Pathogens include, but are not limited to, bacteria, parasites, fungi, viruses, viroids, and prions. Administration is carried out as described in relation to the fourth aspect of the invention.

In any of the methods according to the present invention, a compound, pharmaceutical formulation or vaccine according to the present invention may be administered or co-administered in combination with any other agent useful for preventing or treating a disease or condition and which does not abrogate the immunostimulatory effect of a compound, pharmaceutical formulation or vaccine according to the present invention. In any of the methods according to the invention, agents useful for preventing or treating a disease or condition include, but are not limited to, vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, kinase inhibitors, peptides, proteins, gene therapy vectors, DNA vaccines and/or adjuvants (to enhance the specificity or strength of the immune response), or co-stimulatory molecules such as cytokines, chemokines, protein ligands, transactivators, peptides and peptides comprising modified amino acids. For example, in the prevention and/or treatment of cancer, it is contemplated that a compound, pharmaceutical formulation or vaccine according to the present invention may be administered or co-administered in combination with a chemotherapeutic compound or monoclonal antibody.

Preferred chemotherapeutic agents include, but are not limited to, Gemcitabine (Gemcitabine) methotrexate (methotrexate), vincristine (vincristine), doxorubicin (adriamycin), cisplatin (cissplatin), non-sugar-containing chloroethylnitrosoureas (non-sugar-containing chloronitrosoureas), 5-fluorouracil (5-fluorouracil), mitomycin C (mitomycin C), bleomycin (bleomycin), doxorubicin (doxorubicin), dacarbazine (dacarbazine),fragyline, Meglamine GLA, valrubicin, carmustine (carmustine) and polifeprosan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitor (famesyl transferase inhibitor), farnesyltransferase inhibitor (famesyltransferase inhibitor), MMP, MTA/LY231514, LY 264618/Lometenol, Glamolec, CI-994, TNP-470, and methin (Hycamtin)/Topotecan (Topotecan), PKC412, valpristan (Valspadar)/PSC 833, and so on,Mitoxantrone (Mitroxantrone), Metaret/Suramin (Suramin), Batimastat (Batimastat), E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516/Maristat, BB 2516/Maristat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, imatinib mesylate (imatinib mesylate) mesylatePicibanil/OK-432, AD 32/Valrubicin (Valrubicin),Strontium derivatives, Temodal/Temozolomide (Temozolomide), Evacet/Liposomal doxorubicin, Yewtaxan/paclitaxel (Placlixel),Peritaxel (Paclitaxel), Hexoda (Xeload)/Capecitabine (Capecitabine), Fluoroxylon (Furtulon)/deoxyfluorouridine (Doxifluridine), Cyclopaax/oral Paclitaxel, oral Paclitaxel (Taxoid), SPU-077/Cisplatin (Cisplatin), HMR 1275/Flavopiridol, CP-358(774)/EGFR, CP-609(754)/RAS oncogene inhibitor, UFBMS-182751/oral platinum, UFT T^TM(Tegafur/Uracil) (Uracil)),levamisole (Levamisole), Eniluracil (Eniluracil)/776C85/5FU enhancer, Campto/Levamisole (Levamisole),Irinotecan (Irinotecan), Tumodex/Ralitrexed,(iii) Cladribine (Cladripine), Paxex/Paclitaxel (Paclitaxel),Liposomal doxorubicin, Caelyx/Liposomal doxorubicin,(iii) Fludarabine (Fludarabine), Pharmarubicin/Epirubicin (Epirubicin),ZD1839, LU79553/Bis-Naphtalimide, LU 103793/dolastatin (Dolastain), Caetyx/liposomal doxorubicin,/Gemcitabine (Gemcitabine), ZD 0473YM 116, luodineseeds (lodineseeds), CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes @Ifosfamide (Ifosamide),Teniposide (Teniposide),Carboplatin, plantainol/cisplatin, Vepeside/Etoposide (Etoposide), ZD 9331, Carboplatin,Docetaxel (Docetaxel), guanine cytosine prodrug (prodruductof arabinoside), Taxane Analog (Taxane Analog), nitrosourea (nitrosoureas), alkylating agent (alkylating agents), such as melphalan (melphelan) and cyclophosphamide (cyclophosphamide), Aminoglutethimide (Aminoglutethimide), Asparaginase (asparagenase), Busulfan (Busufan), Carboplatin (Carboplatin), chlorambucil (Chlorombustil), Cytarabine hydrochloride (Cytarabine HCl), actinomycin D (Danomycin), Daunorubicin hydrochloride (Daunoluubicin HCl), estramustine sodium phosphate (estramustine hydrochloride), Etoposide (Etoposide 16), guanine cytosine hydrochloride (LHRH), flunomide (D-365), Flutamide (D-o.p), Flutamide (D-o.p), Flutamide (flunomide), flunomide (D), flunomide (D) (flunomide), flunomide (D) (flunomide), flunomide (D) (flunomide), flunomide (D) (flunomide), flunomide (D) (flunomide), flunomide (rone HCl), Octreotide (Octreotide), Plicamycin (Plicamycin), Procarbazine hydrochloride (Procarbazine HCl), chain star (Streptozocin), Tamoxifen citrate (Tamoxifen citrate), Thioguanine (Thioguanine), Thiotepa (Thiotepa), Vinblastine sulfate (Vinblastine sulfate), Amsacrine (Amsacrine) (m-AMSA), Azacitidine (Azacitidine), erythropoietin (ertropoitine), altretamine (Hexamethylmelamine) (HMM), interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-amidinohydrazone; MGBG), Pentostatin (pentastatin) (2 '-deoxysynomycin (2' deoxynomycin)), Semustine (Semustine) (methyl-CCNU), Teniposide (Teniposide) (VM-26), and Vindesine sulfate (Vindesine sulfate). Preferred monoclonal antibodies include, but are not limited to(Glaxo-Welicome)、(IDEC/Genentech/Hoffmanla Roche)、(Wyeth)、(Millennium)、(IDEC andSchering AG)、(Corixa/GSK)、(Imclone/BMS)、(Genentech)(Genentech/Hoffman la Roche)、(OSIPharmaceuticals/Genentech)。

Alternatively, an agent useful for preventing or treating a disease or condition may comprise a DNA vector encoding an antigen or allergen. In these embodiments, the compounds, pharmaceutical formulations or vaccines according to the invention can act variously as adjuvants and/or produce a direct immunomodulatory effect.

The following examples are intended to further illustrate certain preferred embodiments of the invention and are not intended to limit the scope of the invention in any way.

Example 1: synthesis of oligonucleotide-based compounds containing immunostimulatory moieties

Chemical entities according to the invention were synthesized on a 1. mu. mol to 0.1mM scale using an automated DNA synthesizer (Oligopilot II, AKTA, (Amersham) and/or Expedite 8909(Applied biosystems)) following the linear synthesis or parallel synthesis protocol outlined in FIGS. 1 and 2.

5 '-DMT dA, dG, dC and T iminophosphate were purchased from Prologo (Boulder, CO), 5' -DMT 7-deaza-dG and araG iminophosphate from Chemsenes (Wilmington, MA). DiDMT-glycerol linker solid support was obtained from Chemsenes.1- (2 '-deoxy- β -D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purinylimide (amidite) from Glen Research (Sterling, VA), 2' -O-methylribonucleoside imide from Promega (Obispo, CA). all compounds according to the invention were modified with a phosphorothioate backbone.

By passing³¹P and¹h NMR spectra were used to characterize all nucleoside phosphoramido esters. Modified nucleosides were incorporated at specific sites using the normal coupling cycle recommended by the supplier. After synthesis, the compound was deprotected using concentrated ammonium hydroxide and detritylated by reverse phase HPLC (detrityla)Station), followed by dialysis. The purified compound in the form of the sodium salt was lyophilized for use. Purity was tested by CGE and MALDI-TOF MS. The LAL test determined endotoxin levels below 1.0 EU/mg.

Example 2: cell culture conditions and reagents

HEK293 cells or HEK293XL cells (Invivogen, San Diego, Calif.) expressing mouse TLR9 in 5% CO₂Cultures were grown in 48-well plates in an incubator, 250 μ l DMEM per well and supplemented with 10% heat-inactivated FBS. At 80% confluence, cultures were transiently transfected with 400ng/ml SEAP (secreted human embryo alkaline phosphatase) reporter plasmid (pNifty2-Seap) (Invivogen) in the presence of 4. mu.l/ml Lipofectamine (Invitrogen, Carlsbad, Calif.) in the medium. Plasmid DNA and Lipofectamine were separately diluted in serum-free medium and incubated for 5 minutes at room temperature. After incubation, the diluted DNA and Lipofectamine were mixed and the mixture was incubated at room temperature for 20 minutes. 25 u l containing 100ng plasmid DNA and 1 u l Lipofectamine DNA/Lipofectamine mixture aliquots were added to each well of the cell culture plate, and the culture was continued for 4 hours.

Cytokine induction by compounds exemplified in table 1 in HEK293 cells expressing mouse TLR9

After transfection, the medium was replaced with fresh medium, the compounds exemplified in table 1 were added to the culture at a concentration of 0, 0.1, 0.3, 1.0, 3.0, or 10.0 μ g/ml, and the culture was continued for 18 hours. At the end of compound treatment, NF-. kappa.B levels were determined using the SEAP (secreted human embryo alkaline phosphatase) assay according to the manufacturer's protocol (Invivogen). Briefly, 30. mu.l of culture supernatant from each treatment was removed and incubated with a p-nitrophenyl phosphate substrate and the resulting yellow color was measured at 405nm with a plate reader (Putta MR et al, Nucleic Acids Res.,2006,34: 3231-8).

Example 3: induction of cytokines in human PBMC, pDC, and mouse splenocytes with compounds exemplified in Table 1

Isolation of human PBMC

Peripheral Blood Mononuclear Cells (PBMC) were isolated from freshly collected healthy volunteer blood by Ficoll density gradient centrifugation (Histopaque-1077, Sigma) (CBR Laboratories, Boston, Mass.).

Isolation of human pDC

Human plasmacytoid dendritic cells (pdcs) were isolated by positive selection from freshly obtained healthy human volunteer blood PBMCs using the BDCA4 cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions.

Isolation of mouse splenocytes

Mix 5x10⁶Individual cells/ml of human PBMC were dispensed into 48-well plates. Will be 1x10⁶In some experiments IFN- α, IL-6, and/or IL-12 levels were measured by sandwich ELISA the required reagents (including cytokine antibodies and standards) were purchased from PharMingen.

Cytokine Luminex Multiplex

In some experiments, IL-1R α, IL-6, IL-10, IL-12, IFN- α, IFN-. gamma., MIP-1 α, MIP- β, MCP-1, and IL-12p40p70 levels in culture supernatants were measured by the Luminex Multiplex assay, performed on a Luminex 100 instrument using the Biosource human multiple cytokine assay kit, and data were analyzed using StarStation software supplied by applied cytometry Systems (Sacramento, Calif.).

Activation of human immune cells

Human plasmacytoid dendritic cells (pDCs) were isolated from freshly obtained healthy human blood PBMCs and incubated with 50 μ g/ml TLR9 agonistOr control incubated for 24 hours. Cells were stained with fluorescently conjugated antibodies (CD123, CD80, CD86) and data were collected on a FC500MPL cytometer. Analysis of CD123 Using FlowJo software⁺CD80 and CD86 fluorescence intensity averages on cells and are expressed as fold changes relative to PBS control.

Human myeloid dendritic cells (mdcs) were isolated from freshly obtained healthy human blood PBMCs and incubated with 50 μ g/ml TLR9 agonist or control for 24 hours. Cells were stained with fluorescently conjugated antibodies (CD11c, CD80, CD40) and data were collected on a FC500MPL cytometer. Analysis of CD11c Using FlowJo software⁺CD80 and CD40 fluorescence intensity averages on cells and are expressed as fold changes relative to PBS control.

Example 4: human B cell proliferation assay in the presence of compounds exemplified in Table 1

Human B cells were isolated from PBMCs by positive selection using the CD19 cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

The media composition used for this assay was as follows: RPMI 1640 medium, supplemented with 1.5mM glutamine, 1mM sodium pyruvate, 0.1mM non-essential amino acids, 50. mu.M 2-mercaptoethanol, 100IU/ml penicillin-streptomycin mixture and 10% heat-inactivated fetal bovine serum.

A total of 0.5X 10 stimulation was performed in 96-well flat-bottom plates in triplicate with different concentrations of the exemplified compounds of table 1⁶B cells/ml (i.e., 1X 10)⁵200. mu.l/well) for a total of 68 hours. After 68 hours, 20. mu.l of RPMI 1640 medium (serum-free) was used in each well to prepare a suspension of 0.75. mu. Ci [ sic ], [ solution ]³H]Thymidine (1Ci 37 GBq; Perkin Elmer Life Sciences) pulsed cells, harvested 6-8 hours later. Plates were then harvested using a cell harvester and assayed for radioactive incorporation using standard liquid scintillation techniques. In some cases, the corresponding [ 2 ]³H]-T (cpm) is converted to proliferation index and reported as such.

Example 5: in vivo cytokine secretion in mouse model treated with TLR9 agonist compounds

5-6 week old C57BL/6 mouse BALB/C mice were obtained from Taconic Farms (Germantown, NY) and were bred in accordance with the Idera Pharmaceutical IACUC approved animal protocol mice (n ═ 3) were injected subcutaneously (s.c) with 0.25 or 1.0mg/kg (single dose) of each of the ge immunomodulatory compounds of Table 1. serum was collected by post-orbital bleeding after 2 hours of administration of the immunomodulatory compounds and IL-12, IL-10, IL-6, IP-10, KC, MCP1, MIG, MIP-1 α and TNF- α concentrations were determined by sandwich ELISA or Luminex multiplex assay.7A, 7B, 7C, 7D, 7E and 8F demonstrate that administration of the new chemical compositions containing the immunomodulatory compounds in vivo produced unique cytokines and all agents (including cytokines and chemokines) and all chemotactic products (Mineg. Ming) from standard preparations, Ming Co., Mino).

Equivalent scheme

Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art upon reading this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and the appended claims.

Claims (15)

1. An immunomodulatory compound selected from the group consisting of:

5’-TCG₁AACG₁TTCG₁-Y₃-TGTTG₁CTGTCTTG₁CT-5'；

5’-TCG₁AACG₁TTCoG-Z-GoCTTG₁CAAG₁CT-5’；

5’-TCG₁AACG₁TTCG₁-Y-TCTTG₁CTGTCTTG₁CT-5’；

5’-TCG₁AACG₁TTCG₁-Y-TCTTG₁CTGUCT-5’；

5’-TCG₁AACG₁ToTCoG-m-GoCToTG₁CAAG₁CT-5’；

5’-TCG₁AACG₁TTCoG-Y₂-GACTTG₂CTGAC-5'；

wherein G is₁7-deaza-dG; g₂＝AraG；G/C/U2' -O-methyl ribonucleotide; o ═ phosphodiester linkage; an m ═ cis, trans-1, 3, 5-cyclohexanetriol linker; y ═ 1, 3-propanediol linker; y is₂A 1, 4-butanediol linker; y is₃A 1, 5-pentanediol linker; and Z is a 1,3, 5-pentanetriol linker.

2. A composition comprising an immunomodulatory compound according to claim 1 and a physiologically acceptable carrier.

3. Use of an immunomodulatory compound of claim 1 for the preparation of a composition for generating an immune response in an individual comprising administering to the individual a pharmaceutically effective amount of a compound according to claim 1.

4. Use of an immunomodulatory compound of claim 1 in the manufacture of a medicament for the therapeutic treatment of an individual suffering from a disease or disorder that benefits from a TLR 9-mediated immune stimulation response, the method comprising administering to the individual a pharmaceutically effective amount of a compound according to claim 1.

5. Use according to claim 4, wherein the disease or condition is an inflammatory condition or a disease caused by a pathogen or allergen.

6. Use according to claim 4, wherein the disease or disorder is cancer, an autoimmune disorder, airway inflammation, infectious disease, a skin disorder, allergy or asthma.

7. The use according to claim 4, further comprising administering one or more chemotherapeutic compounds, targeted therapeutics, vaccines, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, kinase inhibitors, peptides, proteins, adjuvants, co-stimulatory molecules, or combinations thereof.

8. The use according to claim 4, further comprising administering one or more antigens.

9. Use according to claim 7, wherein the protein is an antibody and the vaccine is a DNA vaccine.

10. Use of an immunomodulatory compound of claim 1 in the manufacture of a medicament for the prophylactic treatment of an individual suffering from a disease or disorder that benefits from a TLR 9-mediated immune stimulation response, the method comprising administering to the individual a pharmaceutically effective amount of a compound according to claim 1.

11. The use according to claim 10, wherein the disease or condition is an inflammatory condition or a disease caused by a pathogen or allergen in an individual.

12. The use according to claim 10, wherein the disease or disorder is cancer, an autoimmune disorder, airway inflammation, infectious disease, a skin disorder, allergy or asthma in an individual.

13. The use according to claim 10, further comprising administering one or more chemotherapeutic compounds, targeted therapeutics, vaccines, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, TLR agonists, kinase inhibitors, peptides, proteins, adjuvants, co-stimulatory molecules, or combinations thereof.

14. The use according to claim 10, further comprising administering one or more antigens.

15. Use according to claim 13, wherein the protein is an antibody and the vaccine is a DNA vaccine.