HK1128239A - Novel synthetic agonists of toll-like receptors containing cg dinucleotide modifications - Google Patents

Description

Novel synthetic antagonists of Toll-like receptors containing CG dinucleotide modifications

RELATED APPLICATIONS

This application claims benefit of U.S. provisional application serial No. 60/752,335 filed on 20/12/2005 and U.S. provisional application serial No. 60/821,458 filed on 4/8/2006. The teachings of the above application are incorporated herein in their entirety by reference.

Background

Technical Field

The present invention relates generally to the field of immunization and immunotherapy applications using oligonucleotides as immunomodulators. More particularly, the present invention relates to novel chemical compositions and methods of using them. These compounds can efficiently generate unique cytokine/chemokine profiles (cytokines/chemokines) through TLR 9-mediated immune responses.

Brief description of the related Art

Immune responses include both innate and adaptive responses, depending on the subpopulation of cells involved in the response. For example, T helper (Th) cells involved in classical cell-mediated functions such as delayed-type hypersensitivity and Cytotoxic T Lymphocyte (CTL) activation are Th1 cells; while the Th cells that act as B cell activation helper cells are Th2 cells. The type of immune response is influenced by cytokines produced in response to antigen exposure. The difference in cytokines secreted by Th1 and Th2 cells may be due to the different biological functions of these two subsets.

Th1 cells are involved in the body's innate response to antigens (e.g., viral infections, intracellular pathogens, and tumor cells). The result is secretion of IL-2 and IFN-gamma and concomitant activation of CTL. Th2 cells are known to be activated in response to bacteria and parasites and may mediate adaptive immune responses in the body (e.g. IgE production and eosinophil activation) by secreting IL-4 and IL-5.

For example, in mammals, a Th1 immune response may be induced by the introduction of bacterial or synthetic DNA containing unmethylated CpG dinucleotides as a result of specific oligonucleotide sequences (e.g., unmethylated CpG) being presented to receptors on specific immune cells, referred to as Pattern Recognition Receptors (PRRs). Some of these PRRs are Toll-like receptors (TLRs).

Toll-like receptors (TLRs) are closely associated with innate immune responses. In vertebrates, a family known as Toll-like receptors (TLRs) comprising ten proteins is known to recognize pathogen-associated molecular patterns. Among these ten proteins, TLRs 3, 7, 8, and 9 are known to localize in endosomes within cells, recognizing nucleic acids (DNA and RNA) and small molecules such as nucleosides and nucleic acid metabolites. TLR3 and TLR9 are known to recognize nucleic acids such as dsRNA and unmethylated CpG dinucleotides present in viral and bacterial DNA and synthetic DNA, respectively. Bacterial DNA has been shown to activate the immune system and anti-tumor activity (Tokunaga T et al, J.Natl.cancer Inst. (1984)72: 955-. Other studies using antisense oligonucleotides containing CpG dinucleotides have shown to stimulate immune responses (Zhao Q, et al, biochem. pharmacol.1996, 26, 173-82). Subsequent studies have shown that TLR9 recognizes unmethylated CpG motifs present in bacterial and synthetic DNA (Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S.A Toll-like receiver recogninizationbacerial DNA. Nature. (2000); 408: 740-5). Other modifications to CpG-containing phosphorothioate oligonucleotides may also affect their ability to function as modulators of immune response via TLR9 (see, e.g., Zhao et al, Biochem.Pharmacol. (1996)51: 173. 182; Zhao et al, Biochem Pharmacol. (1996)52: 1537. 1544; Zhao et al, Antisense Nucleic acid drug Dev. (1997)7: 495. 502; Zhao et al, bioorg.Med.Chem.Lett. (1999)9: 3453. 3458; Zhao et. Bioorg.Med.Lett. (1999), bioorg.Med.Chem.Lett. (2000)10: 1051. 1054; Yu et al, bioorg.Chem.Lett. (2000)10: 2585. med.chem.10. Lett. (2000; Yo.2585. chem.2001. 9: 11. chem. 9. chem.chem.10: 11. chem., Bioorg.9. chem. 9. 11. chet. 9. 11. chem. 9). In addition, a variety of synthetic motifs and novel DNA-based structures have been identified through structural activity relationship studies that induce a distinct immune response profile (profile) distinct from non-methylated CpG dinucleotide production [ kandimilla ER, Bhagat L, Li Y, Yu D, Wang D, Cong YP, Song SS, Tang JX, Sullivan T, Agrawal s.proc Natl Acad Sci U S s.2005; 6925-30.Kandimalla ER, Bhagat L, Zhu FG, Yu D, Cong YP, Wang D, Tang JX, Tang JY, Knetter CF, Lien E, Agrawal S.Proc Natl Acad Sci U S.2003; 14303-8.Cong YP, Song SS, Bhagat L, Pandey RK, Yu D, KandimallaraER, Agrawal S.Biochem Biophys Res Commun.2003; 1133-9.Kandimalla ER, Bhagat L, Cong YP, Pandey RK, Yu D, ZHao Q, Agrawal S.Biochem Biophys ResCommun.2003; 948-53.Kandimalla ER, Bhagat L, Wang D, Yu D, Zhu FG, Tang J, Wang H, Huang P, Zhang R, Agrawal S.nucleic Acids Res.2003; 2393-400 Yu D, Kandimilla ER, ZHao Q, Bhagat L, Cong Y, Agrawal S.Bioorg Med chem.2003; 459-64.Bhagat L, Zhu FG, Yu D, Tang J, Wang H, Kandimalla ER, Zhang R, Agrawal S.Biochem Biophys Res Commun.2003; 853-61.Yu D, Kandimalla ER, Bhagat L, Tang JY, Cong Y, Tang J, Agrawal S.nucleic Acids Res.2002; 4460-9.Yu D, Kandimalla ER, Cong Y, Tang J, Tang JY, Zhao Q, Agrawal S.J Med chem.2002; 45:4540-8.Yu D, Zhu FG, Bhagat L, Wang H, Kandimalla ER, Zhang R, Agrawal S.Biochem Biophys ResCommun.2002; 297:83-90.Kandimalla ER, Bhagat L, Yu D, Cong Y, Tang J, Agrawal S.Bioconjug Chem.2002; yu D, Kandimalla ER, ZHao Q, Cong Y, Agrawal S.nucleic Acids Res.2002; 1613-9.Yu D, Kandimalla ER, ZHao Q, Cong Y, Agrawal S.Bioorg Med chem.2001; yu D, Kandimallara ER, Zhao Q, Cong Y, Agrawal S.Bioorg Med Chem Lett.2001; 2263-7. Kandimilla ER, Yu D, ZHao Q, Agrawal S.Bioorg Med chem.2001; 807-13.YuD, Zhao Q, Kandimalla ER, Agrawal S.Bioorg Med Chem Lett.2000; 2585-8, Putta MR, Zhu F, Li Y, Bhagat L, Cong Y, Kandimalla ER, Agrawal S.Nucleicacids Res.2006, 34: 3231-8. In addition, other modifications to CpG-containing phosphorothioate oligonucleotides may also affect their ability to function as modulators of immune response. See, e.g., Zhaoet et al, biochem. Pharmacol (1996)51: 173-; zhao et al, Biochem Pharmacol (1996)52: 1537-; zhao et al, Antisense Nucleic Acid Drug Dev (1997)7: 495-502; zhao et al, bioorg.med.chem.lett. (1999)9: 3453-; zhao et al, bioorg.med.chem.lett. (2000)10: 1051-; yu et al, bioorg.med.chem.lett. (2000)10: 2585-; yu et al, bioorg.Med.chem.Lett. (2001)11: 2263-; and Kandimalla et al, bioorg.Med.chem. (2001)9: 807-813.

Oligonucleotides and oligodeoxynucleotides have been widely used in a variety of fields, including but not limited to diagnostic probing, PCR priming, antisense inhibition of gene expression, siRNA, aptamers, ribozymes, and Toll-like receptor (TLR's) based immunotherapeutic agents. Recently, a number of publications have demonstrated the use of oligodeoxynucleotides as immunomodulators, and their use alone or as adjuvants in immunotherapeutic applications for a variety of diseases such as allergy, asthma, autoimmunity, cancer and infectious disease.

These reports indicate that there is still a need to create new chemical entities capable of generating unique immune responses. However, creating new chemical entities that can generate unique cytokine/chemokine-mediated immune responses and that are also recognized as ligands of TLR9 remains a challenge. Ideally, this challenge can be addressed by introducing unique chemical groups into new chemical entities, thereby generating new immunotherapeutic agents, and by generating unique cytokine/chemokine profiles following administration.

Brief description of the invention

The present invention provides novel chemical entities and their use for generating unique cytokine/chemokine-mediated immune responses. The novel chemical entities are useful for modulating the immune response elicited by oligonucleotide compounds. The method according to the invention enables the modification of the cytokine/chemokine profile produced by immunomodulatory oligonucleotides for immunotherapy applications. The present inventors have surprisingly found that modifications to immunomodulatory dinucleotides allow flexibility in the spectrum of immune responses generated.

In a first aspect, the present invention provides an immunomodulatory oligonucleotide comprising an immunostimulatory dinucleotide of the general formula CG, wherein C is cytosine, 2' -deoxycytosine, N³-methyl-dC, dF or Ψ -iso-dC, G is guanosine, 2' -deoxyguanosine or N¹-methyl-dG; with the proviso that when C is cytosine or 2' -deoxycytosine, G is N¹-methyl-dG; with the further proviso that when G is guanosine or 2' -deoxyguanosine, C is N³-methyl-dC, dF or Ψ -iso-dC.

In a second aspect, the present invention provides a pharmaceutical composition. These compositions comprise the components disclosed in the first aspect of the invention and a pharmaceutically acceptable carrier.

In a third aspect, the invention provides a method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate an immunomodulatory oligonucleotide according to the first or second aspect of the invention.

In a fourth aspect, the invention provides a method for treating a vertebrate suffering from cancer, an autoimmune disorder, airway inflammation, an inflammatory disorder, a skin disorder, allergy, asthma or a disease caused by a pathogen, the method comprising administering to the patient an immunomodulatory oligonucleotide according to the first or second aspect of the invention.

In a fifth aspect, the invention provides a method of preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, the method comprising administering to the vertebrate an immunomodulatory oligonucleotide according to the first or second aspect of the invention.

Brief Description of Drawings

FIG. 1 depicts a typical set of small molecule linkers suitable for linear synthesis of immunomodulatory oligonucleotides of the invention.

FIG. 2 depicts a typical set of small molecule linkers suitable for parallel synthesis of the immunomodulatory oligonucleotides of the invention.

FIG. 3 is a synthesis scheme for linear synthesis of immunomodulatory oligonucleotides of the invention. DMTr ═ 4, 4' -dimethoxytrityl; CE ═ cyanoethyl.

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

FIGS. 5A-5D show IL-12 and IL-6 levels in C57BL/6 mouse splenocyte culture following administration of an immunomodulatory oligonucleotide of the invention. More generally, FIGS. 5A-5D show that administration of immunomodulatory oligonucleotides containing new bases can produce unique profiles of IL-12 and IL-6.

FIGS. 6A and 6B show IL-6 and IL-10 levels in human PBMC cultures following administration of immunomodulatory oligonucleotides of the invention. More generally, FIGS. 6A-6B show that administration of immunomodulatory oligonucleotides containing new bases can produce unique IL-6 and IL-10 profiles.

Figure 7 shows TLR9 activation in HEK293 cells following administration of an immunomodulatory oligonucleotide of the invention, activation being measured by NF-kB activity of these cells. More generally, figure 7 shows that administration of immune modulatory oligonucleotides containing novel bases can generate a unique profile of TLR9 activation.

FIG. 8 IL-12 levels in C57BL/6 mice following subcutaneous (s.c.) administration of an immunomodulatory oligonucleotide of the invention. More generally, FIG. 8 shows that in vivo administration of an immunomodulatory oligonucleotide containing a new base can produce a unique profile of IL-12.

FIG. 9 shows the weight of the spleen of C57BL/6 mice after administration of an immunomodulatory oligonucleotide of the invention. More generally, FIG. 9 shows that in vivo administration of an immunomodulatory oligonucleotide containing a new base results in a unique immune response profile.

FIGS. 10A-10D show IL-5, IL-12, IL-13, and IFN- γ levels in OVA-sensitized mouse splenocytes following administration of an immunomodulatory oligonucleotide of the invention. More generally, FIGS. 10A-10D show that administration of an immunomodulatory oligonucleotide containing a new base produces a unique cytokine/chemokine profile even in the presence of an immune system activator (e.g., ovalbumin), which varies depending on the amount and base composition (base composition) of the oligonucleotide administered.

FIG. 11 shows the activation of HEK293 cells expressing mouse TLR9 using immunomodulatory oligonucleotides and a control compound at a concentration of 10. mu.g/ml. Figure 11 shows in more general terms that administration of an immunomodulatory oligonucleotide containing a nucleobase can result in a unique profile of TLR9 activation.

FIGS. 12A-12B show the induction of cytokine secretion by immunomodulatory oligonucleotides of the invention in C57BL/6 mouse splenocyte culture. FIGS. 12A-12B show more generally that administration of an immunomodulatory oligonucleotide containing a novel base can produce unique IL-6 and IL-12 profiles that vary depending on the amount and base composition of the oligonucleotide administered.

FIGS. 13A and 13B show splenomegaly in animals 72 hours after receiving subcutaneously administered immunomodulatory oligonucleotide, control compound or PBS (FIG. 13A), and IL-12 secretion induced by immunomodulatory oligonucleotide after subcutaneous administration (FIG. 13B). More generally, FIGS. 13A and 13B show that in vivo administration of immunomodulatory oligonucleotides containing new bases can generate unique immune response profiles.

FIG. 14 shows the human B cell proliferation induced by immunomodulatory oligonucleotides. More generally, FIG. 14 shows that administration of an immunomodulatory oligonucleotide containing a new base can produce a unique cell proliferation profile that varies depending on the amount and base composition of the oligonucleotide administered.

Detailed description of various embodiments

The present invention relates to the therapeutic use of oligonucleotides as immunomodulators for immunotherapeutic applications. The entire contents of the issued patents, patent applications, and references cited herein are incorporated by reference as if they were specifically and individually indicated to be incorporated by reference. In the event that any of the teachings of any of the documents cited herein are inconsistent with this specification, the specification should preferably be taken for the purposes of the present invention.

The present invention provides methods of enhancing the immune response elicited by immunostimulatory compounds that are useful in immunotherapeutic applications such as, but not limited to, the treatment of cancer, autoimmune disorders, asthma, respiratory allergies, food allergies, and bacterial, parasitic, and viral infections in both adult and juvenile human and veterinary applications. Thus, the invention also provides compounds having optimal levels of immunostimulatory effect on immunotherapy, and methods of making and using these compounds. In addition, the compounds of the present invention may be used as adjuvants in combination with DNA vaccines, antibodies and allergens; and in combination with chemotherapeutic agents and/or antisense oligonucleotides.

In a first aspect, the present invention provides an immunomodulatory oligonucleotide comprising at least one immunomodulatory dinucleotide of formula CG, wherein C is cytosine, 2' -deoxycytosine, N³-methyl-dC, dF or Ψ -iso-dC, G is guanosine, 2 '-deoxyguanosine, 2' -deoxy-7-deazaguanosine, arabinoguanosine (arabinoguanosine) or N¹-methyl-dG; with the proviso that when C is cytosine or 2' -deoxycytosine, G is N¹-methyl-dG; with the further proviso that when G is guanosine or 2' -deoxyguanosine, C is N³-methyl-dC, dF or Ψ -iso-dC.

In one embodiment of this aspect, the invention provides an immunomodulatory oligonucleotide that is separate or that comprises at least two oligonucleotides linked at their 3 'end, or internucleoside linkage, or functionalized nucleobase or sugar to a non-nucleotidic linker, wherein at least one oligonucleotide is an immunomodulatory oligonucleotide and has an accessible 5' end. Oligonucleotides linked to each other by non-nucleotide linkers may have the same nucleotide sequence or may have different nucleotide sequences, provided that at least one of the oligonucleotides contains at least one immunomodulatory dinucleotide of the invention.

The term "accessible 5 'end" as used herein means that the 5' end of the oligonucleotide is sufficiently available that factors recognizing and binding to the oligonucleotide and stimulating the immune system can access it. In oligonucleotides with accessible 5 ' ends, the 5 ' OH position of the terminal sugar is not covalently linked to more than two nucleoside residues or any other moiety that interferes with the interaction with the 5 ' end. The 5' OH may optionally be linked to a phosphate, phosphorothioate, or phosphorodithioate moiety, an aromatic or aliphatic linker, cholesterol, or other entity that does not interfere with accessibility.

For the purposes of the present invention, the term "immunostimulatory oligonucleotide" or "immunomodulatory oligonucleotide" means a compound comprising at least one immunomodulatory dinucleotide, which compound, if not comprising such an immunomodulatory dinucleotide, has no immunomodulatory effect. An "immunomodulatory dinucleotide" is a dinucleotide having the general formula 5 '-CpG-3', wherein "C" is a pyrimidine nucleoside or synthetic derivative thereof naturally occurring in mammals and "G" is a purine nucleoside or synthetic derivative thereof naturally occurring in mammals. The immunomodulatory oligonucleotide of the invention may have one immunomodulatory dinucleotide or several immunomodulatory dinucleotides. For example, each immunomodulatory oligonucleotide can have 2,3, 4, or more immunomodulatory dinucleotides that are the same or can independently be modified as described herein.

The terms "CpG" and "CpG dinucleotide" refer to the dinucleotide 5 '-deoxycytidine-deoxyguanosine-3', where p is an internucleoside linkage, including but not limited to phosphodiester, phosphorothioate and phosphorodithioate linkages.

For the purposes of the present invention, the term "oligonucleotide" refers to a polynucleotide (polynucleotide) formed from a plurality of linked nucleoside units. These oligonucleotides may be obtained from existing nucleic acid sources, including genomic or cDNA sources, but are preferably produced by synthetic methods. In some embodiments, each nucleoside unit comprises a heterocyclic base and a pentofuranosyl (pentofuranosyl), trehalose, arabinose, 2 ' -deoxy-2 ' -substituted arabinose, 2 ' -O-substituted arabinose, or hexose sugar group. The nucleoside residues may be coupled to each other by any of a number of known internucleoside linkages. These internucleoside linkages include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy (carboalkoxy), acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" also includes oligonucleotides having one or more stereospecific internucleoside linkages (e.g., (R)_P) -or (S)_P) Phosphorothioate, alkylphosphate or phosphotriester linkages). The terms "oligonucleotide" and "dinucleotide" as used herein are expressly intended to include polynucleotides and dinucleosides having any such internucleoside linkage, whether or not the linkage comprises a phosphate group. In particular embodiments, these internucleoside linkages can be phosphodiester, phosphorothioate or phosphorodithioate linkages, or combinations thereof.

In some embodiments, each oligonucleotide has from about 3 to about 35 nucleoside residues, or from about 4 to about 30 nucleoside residues, or from about 4 to about 18 nucleoside residues. In some embodiments, the immunomodulatory oligonucleotide comprises an oligonucleotide having from about 1 to about 18, or from about 1 to about 15, or from about 5 to about 14 nucleoside residues. The term "about" as used herein means that the precise number is not critical. Thus, the number of nucleoside residues in the oligonucleotide is not critical, oligonucleotides that are 1 or 2 nucleoside residues less, or oligonucleotides that are 1 to several nucleoside residues more, are considered equivalents of each of the embodiments described above. In some embodiments, one or more of the oligonucleotides have 11 nucleotides or 18 nucleotides. In the context of an immunomodulatory oligonucleotide, particular embodiments have from about 13 to about 35 nucleotides, or from about 13 to about 26 nucleotides, or from about 11 to about 22 nucleotides.

The term "oligonucleotide" also includes polynucleotides having other substituents including, but not limited to, proteinyl, lipophilic, intercalating agents, diamines, folic acid, cholesterol, and adamantane. The term "oligonucleotide" also includes any other nucleobase-containing polymer, including, but not limited to, Peptide Nucleic Acids (PNAs), peptide nucleic acids having phosphate groups (PHONAs), morpholino-backbone oligonucleotides, and oligonucleotides having a backbone portion comprising an alkyl linker or an amino linker.

The oligonucleotides of the invention may include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. The term "modified nucleoside" as used herein refers to nucleosides that include a modified heterocyclic base, a modified sugar moiety, or a combination thereof. In some embodiments, the modified nucleoside is a non-natural pyrimidine or purine nucleoside as described herein. In some embodiments, the modified nucleoside is a 2 ' -substituted ribonucleoside, an arabinonucleoside, or a 2 ' -deoxy-2 ' -substituted-arabinoside.

For the purposes of the present invention, the term "2 ' -substituted ribonucleoside" or "2 ' -substituted arabinoside" 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. These substitutions are with lower alkyl groups containing 1 to 6 saturated or unsaturated carbon atoms, or with aryl groups having 6 to 10 carbon atoms, wherein such alkyl or aryl groups may be unsubstituted or may be substituted, for example with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, alkoxycarbonyl or amino. Examples of 2 '-O-substituted ribonucleosides or 2' -O-substituted arabinosides include, but are not limited to, 2 '-O-methyl ribonucleosides or 2' -O-methyl arabinosides and 2 '-O-methoxyethyl ribonucleosides or 2' -O-methoxyethyl arabinosides.

The term "2 ' -substituted ribonucleoside" or "2 ' -substituted arabinoside" also includes ribonucleosides or arabinosides wherein the 2 ' -hydroxyl group is replaced by a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or an amino or halogen group. Examples of such 2 '-substituted ribonucleosides or 2' -substituted arabinosides include, but are not limited to, 2 '-amino, 2' -fluoro, 2 '-allyl, and 2' -propargyl ribonucleosides or arabinosides.

The term "oligonucleotide" includes hybrid oligonucleotides and chimeric oligonucleotides. "chimeric oligonucleotide" refers to an oligonucleotide having more than one type of internucleoside linkage. An example of such a chimeric oligonucleotide is one comprising a phosphorothioate, phosphodiester, or phosphorodithioate region and a non-ionic linkage, such as an alkyl phosphate or alkyl thiophosphate linkage (see, e.g., Pederson et al, U.S. patent nos. 5,635,377 and 5,366,878).

A "hybridizing oligonucleotide" is an oligonucleotide having more than one type of nucleoside. One example of such a hybridizing oligonucleotide comprises a ribonucleotide or 2' -substituted ribonucleotide region, as well as a deoxyribonucleotide region (see, e.g., Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

For the purposes of the present invention, the term "immunostimulatory oligonucleotide" or "immunomodulatory oligonucleotide" refers to an oligonucleotide of the type described above that modulates (e.g., induces) an immune response when administered to a vertebrate, e.g., fish, poultry, or mammal. The term "mammal" as used herein includes, but is not limited to, rats, mice, cats, dogs, horses, cows (cats), cows (cows), pigs, rabbits, non-human primates, and humans.

For the purposes of the present invention, a "natural" nucleoside refers to a nucleoside comprising one of the five bases (e.g., adenosine, guanosine, thymidine, cytosine, and uridine) commonly found in DNA or RNA, with either deoxyribose or ribose. For purposes of the present invention, a "modified" or "non-natural" nucleoside refers to a nucleoside that includes a modified naturally occurring base and/or a modified naturally occurring sugar moiety. Examples of modified naturally occurring bases include, but are not limited to, those compositions represented by formula I or formula II. For the purposes of the present invention, a "dinucleotide analogue" is an immunostimulatory dinucleotide as described above, wherein the pyrimidine and/or purine nucleosides are non-natural nucleosides. The term "C^*pG "and" CpG^*"refers to immunostimulatory dinucleotide analogs comprising a cytidine analog (non-natural pyrimidine nucleoside) or a guanosine analog (non-natural purine nucleoside), respectively.

In different cases, the dinucleotides are denoted as R' pG, C^*pG or YZ, in these cases, R', C^*Or Y independently represents a synthetic or non-natural pyrimidine, such as, but not limited to N³-methyl-dC, pseudo-iso-deoxycytidine (i.e., Ψ -iso-dC), and deoxyfuranosyl (i.e., dF). In other cases, the dinucleotide is represented as CpR, CpG^*Or YZ, in these cases R, G^*Or Z independently represents a synthetic purine, such as, but not limited to N¹-methyl-dG or 7-deaza-dG. The term "pyrimidine nucleoside" as used herein refers to a nucleoside in which the base component of the nucleoside is a monocyclic nucleobase. Similarly, the term "purine nucleoside" refers to a nucleoside in which the base component of the nucleoside is a bicyclic nucleobase. For purposes of the present invention, "synthetic" pyrimidine or purine nucleosides include non-naturally occurring pyrimidine or purine bases, non-naturally occurring sugar moieties, or combinations thereof.

The pyrimidine nucleosides of the present invention have structure (I):

wherein:

d is a hydrogen bond donor;

d' is selected from hydrogen, hydrogen bond donor, hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;

d and D' may be part of a 5-or 6-membered ring;

a is nitrogen or a heteroatom, substituted or unsubstituted;

a' is selected from hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group;

a' is carbon or nitrogen

X is carbon or nitrogen; and

s' is a pentose or hexose sugar ring, or a non-naturally occurring sugar.

In some embodiments, the sugar ring is derivatized with a phosphate moiety, a modified phosphate moiety, or other linker moiety suitable for linking a pyrimidine nucleoside to another nucleoside or nucleoside analog.

Hydrogen bond donors include, but are not limited to, -NH-, -NH-₂-SH and-OH. Hydrogen bond acceptors include, but are not limited to, C O, C ═ S and the ring nitrogen atom of aromatic heterocycles, e.g., N3 of cytosine.

In some embodiments, the base moiety in (I) is a non-naturally occurring pyrimidine base. Examples of non-naturally occurring pyrimidine bases include, but are not limited to, 5-hydroxycytosine, 5-hydroxymethylcytosine, N³-methyl-dC, pseudo-iso-deoxycytidine (i.e., Ψ -iso-dC); deoxyfuranosyl (i.e., dF), 4-thiouracil, and N4-alkylcytosine, such as N4-ethylcytosine. However, in some embodiments, 5-bromocytosine is specifically excluded.

In some embodiments, the sugar module S' in (I) is a modified naturally occurring sugar module. For purposes of the present invention, a "naturally occurring sugar module" is a sugar module that occurs naturally as part of a nucleic acid, e.g., ribose and 2' -deoxyribose, while a "modified naturally occurring sugar module" refers to any sugar that does not occur naturally as part of a nucleic acid, but can be used in the backbone of an oligonucleotide, e.g., hexose. Arabinose and arabinose derivatives are examples of sugar moieties.

The purine nucleoside analogs of the present invention have structure (II):

wherein:

d is nitrogen or a heteroatom, substituted or unsubstituted;

d' is selected from hydrogen, hydrogen bond donors and hydrophilic groups;

a is a hydrogen bond acceptor or a hydrophilic group;

x is carbon or nitrogen;

each L is an atom independently selected from C, O, N and S; and

s' is a pentose or hexose sugar ring, or a non-naturally occurring sugar.

In some embodiments, the sugar ring is derivatized with a phosphate moiety, a modified phosphate moiety, or other linker moiety suitable for linking a pyrimidine nucleoside to another nucleoside or nucleoside analog.

Hydrogen bond donors include, but are not limited to, -NH-, -NH-₂-SH and-OH. Hydrogen bond acceptors include, but are not limited to, C O, C ═ S, -NO₂And the ring nitrogen atom of aromatic heterocycles, for example, N1 of guanine.

In some embodiments, the base module of (II)Is a non-naturally occurring purine base. Examples of non-naturally occurring purine bases include, but are not limited to, 2-amino-6-thiopurine, 7-deazaguanosine, N¹-methyl-dG and 2-amino-6-oxo-7-deazapurine. In some embodiments, the sugar module S' in (II) is a naturally occurring sugar module or a modified naturally occurring sugar module, as described above for structure (I).

In some embodiments, the immunostimulatory dinucleotide is selected from C^*pG、CpG^*And C^*pG^*Wherein the base of C is cytosine, C^*The base of (A) is thymine, 5-hydroxycytosine, N³-methyl-dC, N4-alkylcytosine, pseudo-iso-deoxycytidine; deoxyfuranosyl, 4-thiouracil or other non-natural pyrimidine, or 2-oxo-7 deaza-8-methylpurine, wherein when the base is 2-oxo-7-deaza-8-methylpurine, it is preferably covalently bound to the 1' -position of the pentose through the 1-position of the base; the base of G is guanosine, G^*The base is 2-amino-6-oxo-7-deazapurine, 2-oxo-7-deaza-8-methylpurine, 6-thioguanine, 7-deazaguanosine, inosine, N¹-methyl-dG, 6-oxopurine or other non-natural purine nucleoside, p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate and phosphorodithioate, with the proviso that at least one C or G is not cytosine or guanosine, respectively.

The immunomodulatory oligonucleotide may include an immunostimulatory moiety on one or both sides of the immunostimulatory dinucleotide. Thus, in some embodiments, the immunostimulatory oligonucleotide comprises an immunostimulatory domain of structure (III):

5’-Nn-N1-Y-Z-N1-Nn-3’(III)

wherein:

the base of Y is cytosine, thymine, 5-hydroxycytosine, N4-alkyl-cytosine, N³-methyl-cytosine, Ψ -iso-dC, dF, 4-thiouracil or other non-natural pyrimidine nucleoside, or 2-oxo-7-deaza-8-methyl-purine, wherein when the base is 2-oxo-7-deaza-8-methyl-purineIt is preferably covalently bound to the 1' position of the pentose through the 1 position of the base;

the base of Z is guanine, 2-amino-6-oxo-7-deazapurine, 2-oxo-7-deaza-8-methylpurine, 2-amino-6-thio-purine, 7-deazaguanosine, N¹-methyl-dG, 6-oxopurine or other non-natural purine nucleoside;

n1 and Nn, independently for each occurrence, are preferably a naturally occurring or non-natural or synthetic nucleoside or immunostimulatory moiety selected from the group consisting of abasic nucleosides, N³-methyl-dC, N¹-methyl-dG, arabino nucleosides, 2 '-deoxyuridine, α -deoxyribonucleosides, β -L-deoxyribonucleosides and nucleosides linked to the 3' side adjacent nucleoside by phosphodiester or modified internucleoside linkages selected from, but not limited to, linkers from about 2 angstroms to about 200 angstroms in length, C2-C18 alkyl linkers, poly (ethylene glycol) linkers, 2-aminobutyl-1, 3-propanediol linkers, glyceryl linkers, 2 '-5' internucleoside linkages, and phosphorothioate, phosphorodithioate, or methylphosphonate internucleoside linkages;

provided that at least one of N1 or Nn is optionally an immunostimulatory moiety;

with the further proviso that at least one Y or Z is not cytosine or guanosine, respectively;

wherein n is a number from 0 to 30; and

wherein the 3' end, internucleoside linker, or derivatized nucleobase or sugar is linked directly or through a non-nucleotidic linker to another oligonucleotide, which may or may not be immunostimulatory.

In some embodiments, YZ is cytosine, Ψ -iso-dC, dF, or N³-methyl-dC and guanosine or N¹-methyl-dG. The immunostimulatory moiety includes a natural phosphodiester backbone and modifications in the phosphate backbone including, but not limited to, methylphosphonates, methylphosphonothioates, phosphotriesters, phosphothiotriesters (phosphothiotriesters)ters), phosphorothioates, phosphorodithioates, triester prodrugs, sulfones, sulfonamides, sulfamates, formals (formacetals), N-methylhydroxylamines, carbonates, carbamates, morpholinos, boranophosphates, phosphoramidates, particularly primary amino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, and stereospecific linkages (e.g., (R)_P) -or (S)_P) Phosphorothioate, alkylphosphonate or phosphotriester linkages).

In some embodiments, the immunostimulatory oligonucleotides of the invention also include nucleosides with sugar modifications, including, but not limited to, 2 '-substituted pentoses, including, but not limited to, 2' -O-methyl ribose, 2 '-O-methoxyethyl ribose, 2' -O-propargyl ribose, and 2 '-deoxy-2' -fluoro ribose; 3 '-substituted pentoses, including, but not limited to, 3' -O-methyl ribose; 1 ', 2' -dideoxyribose; arabinose; substituted arabinits including, but not limited to, 1 ' -methyl arabinose, 3 ' -hydroxymethyl arabinose, 4 ' -hydroxymethyl arabinose, 3 ' -hydroxy arabinose, and 2 ' -substituted arabinose; hexoses, including, but not limited to, 1, 5-anhydrohexitols; and alpha anomers (anomers). In embodiments where the modified sugar is a 3 '-deoxyribonucleoside or a 3' -O-substituted ribonucleoside, the immunostimulatory module is linked to the adjacent nucleoside by a 2 '-5' internucleoside linkage.

In some embodiments, immunostimulatory oligonucleotides of the invention also include oligonucleotides with other carbohydrate backbone modifications and substitutions, including Peptide Nucleic Acids (PNAs), morpholino backbone oligonucleotides, and oligonucleotides with backbone linker moieties ranging from about 2 angstroms to about 200 angstroms in length, including, but not limited to, alkyl linkers or amino linkers. The alkyl linker may be branched or unbranched, substituted or unsubstituted, and chirally pure or racemic mixtures. In some embodiments, such alkyl linkers have from about 2 to about 18 carbon atoms. In some embodiments such alkyl linkers have from about 3 to 9 carbon atoms. Some alkyl linkers include those selected from the group consisting of hydroxy, amino, thiol, thioether, etherAmide, thioamide, ester, urea and thioether. Some such functionalized alkyl linkers are of the formula-O- (CH)₂-CH₂-O-)_nA poly (ethylene glycol) linker represented by (n-1-9) or glycerol. Some other functionalized alkyl linkers are peptides or amino acids.

In some embodiments, the immunostimulatory oligonucleotides of the invention also include DNA subtypes, including, but not limited to, β -L-deoxyribonucleosides and α -deoxyribonucleosides. In some embodiments, the immunostimulatory oligonucleotides of the invention include 3 ' modifications, and also include nucleosides having non-natural internucleoside linkage positions (including, but not limited to, 2 ' -5 ', 2 ' -2 ', 3 ' -3 ', and 5 ' -5 ' linkages).

In some embodiments, immunostimulatory oligonucleotides of the invention also include nucleosides with modified heterocyclic bases, including, but not limited to, 5-hydroxycytosine, 5-hydroxymethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitropyrrole, C5-propynyl pyrimidine, N4-alkylcytosine such as N4-ethylcytosine, and diaminopurine, including, but not limited to, 2, 6-diaminopurine.

By way of specific illustration, and not limitation, in an immunostimulatory domain having structure (III), for example, the linkage between methylphosphonate nucleosides at positions N1 or Nn is an immunostimulatory moiety, a linker having a length of about 2 angstroms to about 200 angstroms-the C2-C18 alkyl linker at position X1 is an immunostimulatory moiety, and the β -L-deoxyribonucleoside at position X1 is an immunostimulatory moiety. Typical positions and structures of the immunostimulatory module are given in table 1 below. It is understood that reference to a linker as an immunostimulatory moiety at a particular position means that the nucleoside residue at that position is substituted at its 3 '-hydroxyl group with the referenced linker, thereby creating a modified internucleoside linkage between the nucleoside residue and the adjacent nucleoside on its 3' side. Similarly, reference to a modified internucleoside linkage as an immunostimulatory module at a particular position means that the nucleoside residue at that position is linked to the 3' adjacent nucleoside by the linkage.

TABLE 1

Position of	Exemplary immunostimulatory Module
		N1	Naturally occurring nucleosides, abasic nucleosides, N3-methyl-dC, N1-methyl-dG, arabino nucleosides, 2 ' -deoxyuridine, β -L-deoxyribonucleosides, C2-C18 alkyl linker, poly (ethylene glycol) linkage, 2-aminobutyl-1, 3-propanediol linker (amino linker), 2 ' -5 ' internucleoside linkage, methylphosphonate internucleoside linkage
Nn	Naturally occurring nucleosides, abasic nucleosides, N3-methyl-dC, N1-methyl-dG, arabino-nucleosides, 2 '-deoxyuridine, 2' -O-substituted ribonucleosides, 2 '-5' internucleoside linkages, methylphosphonic acid

Ester internucleoside linkages provided that neither N1 nor N2 can be a base-free linkage

Table 2 shows typical positions and structures of immunostimulatory modules within immunomodulatory oligonucleotides with upstream enhancing domains. The term "spacer group 9" as used herein refers to a compound of formula-O- (CH)₂CH₂-O)_n-a poly (ethylene glycol) linker of formula (i) wherein n is 3. The term "spacer group 18" refers to a compound of formula-O- (CH)₂CH₂-O)_n-a poly (ethylene glycol) linker of formula (i) wherein n is 6. The term "C2-C18 alkyl linker" as used herein refers to a linker of formula-O- (CH)₂)_q-O-wherein q is an integer from 2 to 18. Thus, the terms "C3-linker" and "C3-alkyl linker" refer to the chemical formula-O- (CH)₂)₃-a linker of O-, which may be substituted or unsubstituted, branched or unbranched (e.g. 1, 2,3, glycerol). For each of spacer 9, spacer 18, and C2-C18 alkyl linker, the linker is connected to the adjacent nucleoside by a phosphodiester, phosphorothioate, or phosphorodithioate linkage.

TABLE 2

Position of	Exemplary immunostimulatory Module
		5’N2	Naturally occurring nucleoside, 2-aminobutyl-1, 3-propanediol linker
5’N1	Naturally occurring nucleosides, beta-L-deoxyribonucleosides, C2-C18 alkyl linkers, poly (ethylene glycol), abasic linkers, 2-aminobutyl-1, 3-propanediol linkers
		3’N1	Naturally occurring nucleosides, 1 ', 2 ' -dideoxyribose, 2 ' -O-methyl-ribonucleosides, C2-C18 alkyl linker, spacer 9, spacer 18
3’N2	Naturally occurring nucleosides, 1 ', 2' -dideoxyribose, 3 '-deoxyribonucleoside, beta-L-deoxyribonucleoside, 2' -O-propargyl-ribonucleoside, C2-C18 alkyl linker, spacer 9, spacer 18, methylphosphonate internucleoside linkage
		3’N3	Naturally occurring nucleosides, 1 ', 2' -dideoxyribose, C2-C18 alkyl linker, spacer 9, spacer 18, methylphosphonate internucleoside linkages, 2 '-5' internucleoside linkages, d (G) n, poly I-poly C
3’N2+3’N3	1 ', 2' -dideoxyribose, beta-L-deoxyriboside, C2-C18 alkyl linker, d (G) n, poly I-poly C
		3’N3+3’N4	2' -O-methoxyethyl-ribonucleosides,Methylphosphonate internucleoside linkages, d (G) n, poly I-poly C
3’N5+3’N6	1 ', 2' -dideoxyribose, C2-C18 alkyl linker, d (G) n, poly I-poly C
		5’N1+3’N3	1 ', 2' -dideoxyribose, d (G) n, poly I-poly C

Table 3 shows typical positions and structures of immunostimulatory modules within immunomodulatory oligonucleotides with downstream enhancing domains.

TABLE 3

Position of	Exemplary immunostimulatory Module
		5’N2	Methylphosphonate internucleoside linkages
5’N1	Methylphosphonate internucleoside linkages
		3’N1	1 ', 2 ' -dideoxyribose, methylphosphonate internucleoside linkages, 2 ' -O-methyl
3’N2	1 ', 2 ' -dideoxyribose, beta-L-deoxyribonucleoside, C2-C18 alkyl linker, spacer 9, spacer 18, 2-aminobutyl-1, 3-propanediol linker, methylphosphonate internucleoside linkage, 2 ' -O-methyl
		3’N3	3 ' -deoxyribonucleosides, 3 ' -O-substituted ribonucleosides, 2 ' -O-propargyl ribonucleosides
3’N2+3’N3	1 ', 2' -dideoxyribose, beta-L-deoxyriboside

The immunomodulatory oligonucleotides of the invention comprise at least two oligonucleotides linked at their 3' ends or internucleoside linkages, or at functionalized nucleobases, or at sugars by a non-nucleotidic linker. For the purposes of the present invention, a "non-nucleotidic linker" refers to any moiety capable of linking to an oligonucleotide by covalent or non-covalent bonds. Such linkers range in length from about 2 angstroms to about 200 angstroms. Examples of several linkers are listed below. Non-covalent associations include, but are not limited to, electrostatic interactions, hydrophobic interactions, pi stacking interactions, and hydrogen bonding. The term "non-nucleotidic linker" is not intended to refer to an internucleoside linkage as described above directly linking the 3' hydroxyls of two nucleosides, e.g., a phosphodiester, phosphorothioate or phosphorodithioate functionality. For the purposes of the present invention, such a direct 3 '-3' linkage (without the participation of a linker) is considered to be a "nucleotide linkage".

In some embodiments, the non-nucleotide linker is a metal, including but not limited to a gold particle. In some other embodiments, the non-nucleotide linker is a soluble or insoluble biodegradable polymer bead.

In other embodiments, the non-nucleotide linker is an organic moiety with a functional group that allows attachment to the oligonucleotide. The attachment is by any stable covalent bond. As a non-limiting example, a linker may be attached at any suitable position on the nucleoside. In some embodiments, the linker is attached to the 3' -hydroxyl. In such embodiments, the linker comprises a hydroxyl functional group attached to the 3' -hydroxyl group by a phosphodiester, phosphorothioate, phosphorodithioate, or non-phosphate based linkage (non-phosphate-basedlinkage).

In some embodiments, the non-nucleotide linker is a biomolecule, including, but not limited to, polypeptides, antibodies, lipids, antigens, allergens, and oligosaccharides. In some other embodiments, the non-nucleotide linker is a small molecule. For the purposes of the present invention, small molecules are organic moieties 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.

In some embodiments, the small molecule is an aliphatic or aromatic hydrocarbon that may optionally include one or more functional groups selected from the group consisting of hydroxyl, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thiourea, either in a linear chain with the oligonucleotide attached thereto. The small molecule may be cyclic or acyclic. Examples of small molecule linkers include, but are not limited to, amino acids, sugars, cyclodextrins, adamantane, cholesterol, haptens, and antibiotics. However, for the purpose of describing non-nucleotide linkers, the term "small molecule" is intended to exclude nucleosides.

In some embodiments, the small molecule linker is of formula HO- (CH)₂)_o-CH(OH)-(CH₂)_p-OH, wherein o and p are independently 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 of these derivatives have the formula HO- (CH)₂)_m-C(O)NH-CH₂-CH(OH)-CH₂-NHC(O)-(CH₂)_m-OH, wherein m is an integer between 0 to about 10, 0 to about 6, 2 to about 6 or 2 to about 4.

Some non-nucleotide linkers of the invention allow for the attachment of more than two oligonucleotides. For example, the small molecule linker glycerol has 3 hydroxyl groups for covalent attachment of oligonucleotides. Thus, some immunomodulatory oligonucleotides of the invention comprise more than two oligonucleotides linked at their 3' ends to a non-nucleotide linker.

The immunomodulatory oligonucleotides of the invention can be conveniently synthesized using an automated synthesizer and phosphoramidite methodology (as shown in the schematic diagrams of FIGS. 3 and 4, further described in the examples). In some embodiments, the immunomodulatory oligonucleotide is synthesized by linear synthesis methods (see fig. 3). The term "linear synthesis" as used herein refers to synthesis starting from one end of an immunomodulatory oligonucleotide and progressing 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 in the immunomodulatory oligonucleotide.

An alternative synthesis is "parallel synthesis", in which synthesis proceeds outward from the central junction module (see fig. 4). Linkers attached to solid supports can be used for parallel synthesis as described in U.S. Pat. No. 5,912,332. In addition, a general purpose solid support (e.g., phosphate-attached controlled pore glass) can be used.

Parallel synthesis of immunomodulatory oligonucleotides has several advantages over linear synthesis (1) parallel synthesis allows incorporation of identical monomer units; (2) unlike linear synthesis, all monomer units are synthesized simultaneously, so the number of synthesis steps and the time required for synthesis are the same as for one monomer unit; and (3) the reduction of synthetic steps results in increased purity and yield of the final immunomodulatory oligonucleotide product.

At the end of the synthesis according to linear synthesis or parallel synthesis protocols, if modified nucleosides are incorporated, the immunomodulatory oligonucleotide can be deprotected conveniently using concentrated ammonia solution or according to the recommendations of the phosphoramidite supplier. The product immunomodulatory oligonucleotides can be purified by reverse phase HPLC, detritylated, desalted and dialyzed.

Table 4 shows typical immunomodulatory oligonucleotides of the invention.

TABLE 4A. examples of immunomodulatory oligonucleotide sequences

SEQ ID NO.	Sequences and modifications
		1	5’-CTATCTGAC1GTTCTCTGT-3’
2	5’-CTATCTGACG1TTCTCTGT-3’
		3	5’-CTATCTGTC1GTTCTCTGT-3’
4	5’-CTATCTGTCG1TTCTCTGT-3’
		5	5’-CTATCTGAGC1TTCTCTGT-3’
6	5’-CTATCTGAG1CTTCTCTGT-3’
		7	5’-TCTGAC1GTTCT-X-TCTTGC1AGTCT-5’
8	5’-TCTGACG1TTCT-X-TCTTG1CAGTCT-5’
		9	5’-TCTGTC1GTTCT-X-TCTTGC1TGTCT-5’
10	5’-TCTGTCG1TTCT-X-TCTTG1CTGTCT-5’
		11	5’-TCTGAGC1TTCT-X-TCTTC1GAGTCT-5’
12	5’-TCTGAG1CTTCT-X-TCTTCG1AGTCT-5’
		13	5’-CTATCTGACGTTCTCTGT-3’
14	5’-CTATCTGTCGTTCTCTGT-3’
		15	5'-CTATCTCACCTTCTCTG-3' (control)
16	5’-TCTGACGTTCT-X-TCTTGCAGTCT-5’
		17	5’-TCTGACG2TTCT-X-TCTTG2CAGTCT-5’
18	5 '-TCTCACCTTCT-X-TCTTCCACTCT-5' (control)
		19	5 '-ACACACCAACT-X-TCAACCACACA-5' (control)
20	5’-TCTGTCG2TTCT-X-TCTTG2CTGTCT-5’

21	5’-TCTGACGTTCT-X-TCTTGCAGTCT-5’
		22	5’-TCTGAC2GTTCT-X-TCTTGC2AGTCT-5’
23	5’-TCTGAC3GTTCT-X-TCTTGC3AGTCT-5’
		24	5’-TCTGAGC2TTCT-X-TCTTC2GAGTCT-5' (control)
25	5’-TCTGAGC3TTCT-X-TCTTC3GAGTCT-5' (control)
		26	5’-TCTGTCGTTCT-X-TCTTGCTGTCT-5’
27	5’-TCTGTC3GTTCT-X-TCTTGC3TGTCT-5’
		28	5’-TCTGTC2GTTCT-X-TCTTGC2TGTCT-5’
29	5 '-ACACACCAACT-X-TCAACCACACA-5' (control)
		30	5’-TC3G2AAC3G3TTC3G3-X-G2C3TTG3C3AAG2C3T-5’
31	5’-TC4G2AAC4G3TTC4G2-X-G2C4TTG3C4AAG2C4T-5’
		32	5’-TC3G2AAC3G2TTCG2-Y-TCTTG3C3TGTCT-5’
33	5’-TC4G2AAC4G2TTC4G2-Y-TCTTG3C4TGTCT-5’

C₁＝N³-methyl-dC; c₂＝dF；C₃Ψ -iso-dC; c₄1- (2' -deoxy- β -D-ribofuranosyl) -2-oxo-7-deaza-8-methylpurine; g₁＝N¹-methyl-dG; g₂7-deaza-dG; g₃Guanosine arabinoside; x ═ glycerol linker; y ═ C3 linker

Particular embodiments of this aspect of the invention provide immunomodulatory oligonucleotide conjugates comprising an immunostimulatory oligonucleotide as described above and a compound coupled to the immunostimulatory oligonucleotide at a position other than the accessible 5' end. In some embodiments, the compound is coupled to a non-nucleotide linker. In some other embodiments, the compound is coupled to the oligonucleotide at a position other than its 5' end. Suitable compounds capable of being coupled to the immunomodulatory oligonucleotides of the invention include, but are not limited to, cholesterol, polyethylene glycols of varying lengths, peptides, antibodies, proteins, vaccines, lipids, antigens, and any immunostimulatory small molecule, such as, but not limited to, imiquimod, R848, loxoribine, isatoribine, and chemotherapeutic agents.

Antigens include, but are not limited to, antigens associated with pathogens, antigens associated with cancer, antigens associated with autoimmune disorders, and antigens associated with other diseases, such as, but not limited to, veterinary or pediatric diseases. In some embodiments, the antigen produces a vaccine effect. The term "associated with" means, for purposes of the present invention, that an antigen is present when a pathogen, cancer, autoimmune disorder, food allergy, respiratory allergy, asthma or other disease is present, but is absent or reduced when the pathogen, cancer, autoimmune disorder, food allergy, respiratory allergy or disease is absent.

The immunostimulatory oligonucleotide is covalently linked to the antigen, or it is otherwise operably associated with the antigen. The term "operably associated with" as used herein refers to any association that preserves the activity of both the immunostimulatory oligonucleotide and the antigen (association). Non-limiting examples of such "operable binding" include moieties that constitute the same liposome or other such delivery vehicle or agent (the following part of the same liposome or other such delivery vehicle or agent). In embodiments where the immunostimulatory oligonucleotide is covalently linked to the antigen, such covalent linkage is preferably located anywhere on the immunostimulatory oligonucleotide except at the accessible 5' end of the immunostimulatory oligonucleotide. For example, the antigen may be attached to an internucleoside linkage or may be linked to a non-nucleotidic linker. Furthermore, the antigen itself may be a non-nucleotide linker.

In a second aspect, the invention provides a pharmaceutical formulation comprising an immunomodulatory oligonucleotide or an immunomodulatory oligonucleotide conjugate of the invention and a physiologically acceptable carrier. The term "physiologically acceptable" as used herein means that the material does not interfere with the effectiveness of the immunomodulatory oligonucleotide 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 "carrier" as used herein includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid or other material known in the art for use in pharmaceutical formulations. It will be appreciated that the nature of the carrier, excipient or diluent will depend on the route of administration for the particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described, for example, in Remington's pharmaceutical Sciences, 18th Edition, ed.a. gennaro, Mack Publishing co., Easton, PA, 1990.

In a third aspect, the invention provides a method of generating an immune response in a vertebrate, such method comprising administering to the vertebrate an immunomodulatory oligonucleotide or an immunomodulatory oligonucleotide conjugate of the invention. In some embodiments, the vertebrate is a mammal. For the purposes of the present invention, the term "mammal" is expressly intended to include humans. In certain embodiments, the immunomodulatory oligonucleotide or immunomodulatory oligonucleotide conjugate is administered to a vertebrate in need of immune stimulation.

In the methods of this aspect of the invention, the administration of the immunomodulatory oligonucleotide or immunomodulatory oligonucleotide conjugate can be by any suitable route, including but not limited to parenteral, oral, sublingual, transdermal, topical, mucosal, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch, or in the form of eye drops or mouthwash. Administration of the immunomodulatory oligonucleotide therapeutic composition can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms of the disease or surrogate markers. When administered systemically, it is preferred to administer a sufficient dose of the therapeutic composition such that blood levels of the immunomodulatory oligonucleotide reach from about 0.0001 micromolar to about 10 micromolar. For topical application, concentrations much lower than this may also be effective, while much higher concentrations may also be tolerable. Preferably, the total dose of the immunomodulatory oligonucleotide ranges from about 0.001mg per patient per day to about 200mg per kilogram of body weight per day. It may be desirable to administer to an individual, simultaneously or sequentially, a therapeutically effective amount of one or more of the therapeutic compositions of the present invention as a single treatment session.

In particular embodiments, the immunomodulatory oligonucleotide or immunomodulatory oligonucleotide conjugate of the invention is administered in combination with a vaccine, antibody, cytotoxic agent, allergen, antibiotic, antisense oligonucleotide, peptide, protein, gene therapy vector, DNA vaccine and/or adjuvant to enhance the specificity or magnitude of the immune response. In these embodiments, the immunomodulatory oligonucleotides of the invention may act in different ways as adjuvants and/or produce a direct immunostimulatory effect.

The immunomodulatory oligonucleotide or immunomodulatory oligonucleotide conjugate and/or vaccine may optionally be linked to an immunogenic protein, such as Keyhole Limpet Hemocyanin (KLH), cholera toxin B subunit, or any other immunogenic carrier protein. Any of a variety of adjuvants may be used, including, but not limited to, freund's complete adjuvant, KLH, monophosphoryl lipid a (mpl), alum, and saponins, including QS-21, imiquimod, R848, or combinations thereof.

For the purposes of the present invention, the term "in combination with" means in the course of treating the same disease in the same patient, including the administration of the immunomodulatory oligonucleotides and/or vaccines and/or adjuvants in any order, including simultaneously, and in temporally separated orders (up to several days apart). Such combination therapy may also include the administration of an immunomodulatory oligonucleotide, and/or the independent administration of a vaccine, and/or the independent administration of an adjuvant more than once. The immunomodulatory oligonucleotide and/or the vaccine and/or the adjuvant may be administered by the same or different routes.

The methods of this aspect of the invention are useful for model studies of the immune system. The method can also be used for preventing or treating human or animal diseases. For example, the method may be used for pediatric and veterinary vaccine applications.

In a fourth aspect, the invention provides a method of therapeutically treating a patient having a disease or disorder, the method comprising administering to the patient an immunomodulatory oligonucleotide or an immunomodulatory oligonucleotide conjugate of the invention. In various embodiments, the disease or disorder to be treated is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, allergy, asthma, or a disease caused by a pathogen. Pathogens include bacteria, parasites, fungi, viruses, viroids, and prions. Administration is carried out as described for the third aspect of the invention.

For the purposes of the present invention, the term "allergy" includes, but is not limited to, food allergy (food allergy) and respiratory allergy. The term "airway inflammation" includes, but is not limited to, asthma. The term "autoimmune disorder" as used herein refers to a disorder in which the "self" protein is subject to attack by the immune system. The term includes autoimmune asthma.

In a fifth aspect, the invention provides methods for preventing a disease or disorder, the methods comprising administering to a patient an immunomodulatory oligonucleotide or an immunomodulatory oligonucleotide conjugate of the invention. In various embodiments, the disease or disorder to be prevented is cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, allergy, asthma, or a disease caused by a pathogen. Pathogens include bacteria, parasites, fungi, viruses, viroids, and prions. Administration is carried out as described for the third aspect of the invention.

In any of the methods of this aspect of the invention, the immunomodulatory oligonucleotide or immunomodulatory oligonucleotide conjugate may be administered in combination with any other agent useful for treating a disease or disorder without reducing the immunostimulatory effect of the immunomodulatory oligonucleotide. In any of the methods of the invention, agents useful for treating a disease or disorder include, but are not limited to, vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, peptides, proteins, gene therapy vectors, DNA vaccines and/or adjuvants that enhance the specificity or size 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 treatment of cancer, it is contemplated that an immunomodulatory oligonucleotide or an immunomodulatory oligonucleotide conjugate may be administered in combination with a chemotherapeutic compound or monoclonal antibody. Alternatively, the agent may comprise a DNA vector encoding an antigen or allergen. In these embodiments, the immunomodulatory oligonucleotides of the invention may act in different ways as adjuvants and/or produce a direct immunomodulatory effect.

Chemotherapeutic agents useful in the methods of the invention include, but are not limited to, Gemcitabine (Gemcitabine), methotrexate, vincristine, doxorubicin, cisplatin, non-saccharide-containing chloroethyl nitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin (doxorubicin), dacarbazine (dacarbazine), paclitaxel, fradazine (fragyline), meglumine GLA (Meglamine GLA), valrubicin (valrubicin), carmustine (carmustine) and polifeprosan (polifeprosan), BAY12-9566, RAS farnesyl transferase inhibitors, MMP, MTA/231514, LY 264618/lometrexol (Lometexol), glatiramer (Glamolec), CI-994, TNP-470, and tomotecan/potriosol (hymexane)/xanthaxanthrin (Timentin), Varacanthine (Timentin)/valtrolene (sodium), valtrolene (sodium citrate)/gent (valtrolene), valtrolene (valrubine)/gent (TM)/gent), and valtrexone (TM)/gent (TM)/Na) (Metatrolene (MMI)/MMI) inhibitors), and (MMI) (Metataraxanthrin)/TM)/Na (TM)/Na) (TM)/Na-3, and D, wherein, Batimastat (Batimastat), E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516/mamistat (Maristat), BB 2516/mamitat (Maristat), CDP 845, D2163, PD183805, DX8951f, Lactobacillus formulation DP2202(Lemonal DP 2202), FK317, Streptomycete formulation (Picibanil)/OK-432, AD 32/valrubicin, strontium chloride (Metastron)/strontium derivative, Tatarian (modal)/Temozolomide (Temozolomide), Adriamycin liposome (Evacet)/Doxorubicin, Youwultan/taxol (Paclitaxel)/Paclitaxel, Paclitaxel/Paciferol (Paciferol), Paclitaxel/Pacific acid (Pacific), oral administration (Paclitaxel)/doxepirubine (Paclitaxel), oral administration (Paclitaxel)/doxepidine (Paclitaxel)/Paclitaxel), oral administration (Paclitaxel)/doxepidine (Pacifloxine), oral administration (Paclitaxel)/doxepidine), and oral administration (Paclitaxel)/doxepidine (oral administration (Paclitaxel)/oral administration (Paclitaxel) Oral taxane (Oral Taxoid), SPU-077/cisplatin, HMR 1275/fusidic (Flavopiridol), CP-358(774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/Oral platinum, UFT (Tegafur/uracil), Levamisole (Ergamisol)/Levamisole (Levamisole), Eniluracil (Eniluracil)/776C85/5FU enhancer, Irinotecan (Campto)/Levamisole, Irinotecan hydrochloride and sorbitol injection (Camptosar)/Irinotecan (Irinotecan), Dosimox/Ralitrexed (Ralitrexed), rilipine (Leustridine)/Cladribine (dribine), Paxex/taxol, Doxil/Doxix/Doelyix/Doelyx/Doxix, Fludara (Fludara)/Fludarabine (Fludarabine), Epirubicin (pharmorubicin)/Epirubicin (Epirubicin), liposomal cytarabine injection (DepoCyt), ZD1839, LU 79553/Bis-naphthalimide (Bis-naphalimide), LU 103793/dolastatin (Dolastain), caltix (Caetyx)/doxorubicin liposome, Gemcitabine hydrochloride/difluorine (Gemzar)/Gemcitabine (Gemcitabine), 0473/Anormed, YM 116, etodolac inoculation (lodine seeds), CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/dexfosasamide, Ifes/sodium acetate injection (Mesnex)/ifosfamide (ixasamide), wilford (vum)/niposide (Teniposide), cisplatin (paclitaxel)/paclitaxel (Docetaxel), Etoposide (ZD 1), Etoposide (ZD)/paclitaxel), Docetaxel (paclitaxel)/paclitaxel (Docetaxel (paclitaxel), Etoposide (fluvastatin (taetoposide)/Docetaxel (933), Etoposide (e), paclitaxel (paclitaxel)/Docetaxel (paclitaxel)/cisplatin), paclitaxel (paclitaxel)/Docetaxel (Docetaxel), Docetaxel (paclitaxel)/Docetaxel (Docetaxel), Docetaxel (Docetaxel), Docetaxel (Docetaxel), guanine arabinoside prodrugs, taxane analogs, nitrosoureas, alkylating agents such as melphelan and cyclophosphamide, Aminoglutethimide (Aminoglutethimide), asparaginase, Busulfan, carboplatin, chlorambucil (Chlorambucil), Cytarabine hydrochloride (Cytarabine HCl), Dactinomycin (Dactinomycin), Daunorubicin hydrochloride (Daunorubicin HCl), estramustine phosphate (Estrastine phosphate sodium), etoposide (VP16-213), Floxuridine (Floxuridine), fluorouracil (5-FU), Flutamide (Flutamide), Hydroxyurea (Hydroxyurea) (Hydroxyurea (Hydroxycarbamide)), Ifosfamide (Isfamide), interferon alpha-2 a, alpha-2 b, Leuprolide acetate (Leuconolide RH), cysteine hydrochloride (Louvolfactor), cysteine hydrochloride (cysteine ethyl acetate), cysteine hydrochloride (cysteine hydrochloride), cysteine acetate (cysteine acetate), cysteine acetate (L-D (L-D), cysteine (L-D, L-D (L-D, L-D, D-D (L-D, D-D, mitotane (Mitotane) (ortho-para-trichomonas (o.p '-DDD)), Mitoxantrone hydrochloride (Mitoxantrone HCl), Octreotide (Octreotide), Plicamycin (Plicamycin), Procarbazine hydrochloride (Procarbazine HCl), Streptozocin (Streptozocin), tamoxifen citrate (tamoxifen), thioguanine, Thiotepa (Thiotepa), Vinblastine (Vinblastine sulfate), Amsacrine (Amsacrine) (amicrine (m-AMSA)), Azacitidine (Azacitidine), erythropoietin (Erthropoetin), Hexamethylmelamine (Hexamethymelamine) (HMM), interleukin 2, Mitoguazone (Mitoguazone) (methyl-GAG; methyl-hydrazone (methyl-guanidium BG), vinpocetine-2 (methyl-doxycycline sulfate)), vincamine (2' -methyl-doxycycline) (MGN (2)), vincamine (2 '-doxycycline sulfate (MCN-2 (methyl-GAG (methyl-doxycycline), Vindesine (2' -methyl-doxycycline) (MGN (2 (methyl-doxycycline), Vinblastine (2, vinpocetine-2, and (N-2-doxycycline) (methyl-2, and (N-2, wherein, Tyrosine kinase inhibitors, such as EGFR and VEGF inhibitors, including, but not limited to, Lapatinib (Lapatinib) (EGFR and ErbB-2(Her2/neu) dual tyrosine kinase inhibitor (GSK)), Gefitinib (Gefitinib) (ZD1839/Iressa (AstraZeneca)), Erlotinib (Erlotinib) (Tarceva) -EGFR/HER1 inhibitor (genech)), Thalidomide (Thalidomide) -antiangiogenic drugs), Imatinib (Imatinib) (Glivec) and Vatalanib (Vatalanib) (VEGFR tyrosine kinase inhibitors), Sorafenib (Sorafenib) (Raf kinase inhibitor (Bayer)), VX-680(Aurora kinase inhibitors), sotentan (Sutent) (receptor tyrosine kinase (RTKs) inhibitors (izer)), Bortezomib (Bortezomib) inhibitors (Bortezomib), interferon alpha (Teolog A) inhibitors (Teolog A), interferon (Teolog A) inhibitors (Teolog A), rosmarin (Roferon a)).

Passive immunotherapy of antibodies, particularly in the form of monoclonal antibodies, as anti-cancer agents is the subject of a great deal of research and development. The term "monoclonal antibody" as used herein refers to an antibody molecule of single molecular composition. Monoclonal antibody compositions exhibit unique binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Examples of anti-cancer agents include, but are not limited to, monoclonal antibody 17-1A (Panorex) (Glaxo-welome), B cell monoclonal antibody (Rituxan) (IDEC/Genentech/Hoffman laRoche), Mylotarg (Wyeth), Campath (Campath) (Millennium), Jewel (Zevalin) (IDEC and Schering AG), Bexxar (Bexxar) (Corixa/GSK), Erbitux (Erbitux) (Imclone/BMS), Avastin (Avastin) (Genentech), Herceptin (Herceptin) (Genentech/Hoffman Roche), Cetuximab (Cetuximab) (clone), and Panitumumab (Imgenix/Amgen). Antibodies can also be used in active immunotherapy using anti-idiotype antibodies, apparently mimicking (immunologically) cancer antigens. Monoclonal antibodies can be prepared by methods known to those skilled in the art of recombinant DNA technology.

The following examples are intended to further illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention.

Examples

Example 1 Synthesis of an oligonucleotide containing an immunostimulatory Module

Oligonucleotides were synthesized on a scale of 1. mu. mol to 0.1mM using an automated DNA synthesizer (Oligopilot II, AKTA, (Amersham) and/or Expedite8909(Applied biosystems)) following the linear or parallel synthesis procedure outlined in FIGS. 3 and 4.

5' -DMT dA, dG, dC and T phosphoramidite were purchased from Prooligo (Boulder, CO). 5' -DMT 7-deaza-dG and araG phosphoramidites were obtained from Chemmentes (Wilmington, MA). Dmt-glycerol linker solid support was obtained from Chemgenes. 1- (2 '-deoxy- β -D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine phosphoramidite (1- (2' -deoxy- β -D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine amide) was obtained from Glen Research (Sterling, VA), 2 '-O-methylribonucleoside phosphoramidite (2' -O-methylribonucleoside amides) was obtained from Promega (Obispo, CA). All oligonucleotides are phosphorothioate backbone modified.

All nucleoside phosphoramidites are prepared by³¹P and¹and H nuclear magnetic resonance spectrum characterization. Modified nucleosides were incorporated at specific sites using conventional coupling cycles recommended by the supplier. After synthesis, the oligonucleotides were deprotected with concentrated ammonium hydroxide, then purified by reverse phase HPLC, detritylated, followed by dialysis. The purified oligonucleotides were lyophilized as sodium salts prior to use. Purity was checked by CGE and MALDI-TOF MS. Endotoxin levels were below 1.0EU/mg as determined by the LAL assay.

Example 2 mouse spleen cell culture

4-8 week-old C57BL/6 and BALB/C mice were obtained from Taconic Farms, Germannown, NY, and are maintained according to Idera's IACUC-approved animal protocols. All animal studies reported herein were performed following idea's IACUC guidelines and approved operating protocols. Splenocytes from 4-8 week old BALB/C or C57BL/6 mice were prepared and cultured in RPMI complete medium. Mouse splenocytes were treated at 5X10⁶Cells/ml were seeded in 24-well culture dishes. IMOs in TE buffer (10mM Tris-HCl, pH 7.5, 1mM EDTA) were added to the cell culture to a final concentration of 0.03, 0.1, 0.3, 1.0, 3.0 or 10. mu.g/ml. Cells were then incubated at 37 ℃ for 24 hours and supernatants were collected for ELISA analysis.

The supernatant was analyzed for IL-12 and IL-6 levels by sandwich ELISA. The results are shown in FIGS. 5A-5D. Reagents required, including cytokine antibodies and standards, were purchased from BD Pharmingen. The streptavidin-peroxidase and the substrate were from KPL.

Example 3 human PBMC isolation

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

Example 4 cytokine ELISA

Human PBMC were treated with 5X10⁶Cells/ml were seeded in 48-well plates. IMOs in DPBS (pH 7.4; Mediatech) were added to the cell culture to a final concentration of 10. mu.g/ml. Cells were then incubated at 37 ℃ for 24 hours and supernatants were collected for ELISA analysis. Experiments were performed in 3-well replicates. The supernatant was analyzed for IL-6 and IL-10 levels by sandwich ELISA. The results are shown in FIGS. 6A and 6B. Reagents required, including cytokine antibodies and standards, were purchased from PharMingen.

Example 5 HEK293 cell culture:

HEK293/mTLR9 cells (Invivogen, San Diego, Calif.) were cultured in 5% CO₂In 48 well plates in incubators, 250. mu.l DMEM per well, supplemented with 10% heat-inactivatedFBS。

Example 6 reporter Gene transformation

At 80% confluence, cultures were transiently transformed with 400ng/ml of a Seap reporter plasmid (pNifty2-Seap) (San Diego CA) in the presence of 4 μ l/ml Lipofectamine (Lipofectamine) (Invitrogen, CA) in the medium. Plasmid DNA and lipofectin were diluted separately in serum-free medium and incubated for 5 minutes at room temperature. After incubation, the diluted DNA and lipofectin were mixed and the mixture was incubated at room temperature for 20 minutes. Mu.l of DNA/lipofectin mixture containing 100ng plasmid DNA and 1. mu.l lipofectin was added to each well of the cell culture plate and incubation was continued for 4 hours.

Example 7 immunomodulatory oligonucleotide treatment

After transfection, the medium was replaced with fresh medium, and stimulatory oligo (stimulating oligo) immunomodulatory oligonucleotides were individually added to each culture, and the culture was continued for 18 hours.

Example 8 SEAP analysis

At the end of the oligo immunomodulatory oligonucleotide treatment, 30 μ l of culture supernatant from each treatment was taken for SEAP analysis. The analysis was performed according to the manufacturer's protocol (Invivogen). The signal was detected with a microplate reader at 405 nm. The results are shown in figure 7, demonstrating that administration of an immune modulatory oligonucleotide containing a neobase results in a unique profile of TLR9 activation.

Example 9 evaluation of mouse serum cytokine levels

5-6 week old female C57BL/6 mice were obtained from Taconnic Farms, Germanown, NY, and were bred according to Idera Pharmaceutical IACUC approved animal protocols. Mice (n-2-3) were injected subcutaneously with the individual immunomodulatory oligonucleotides at 25 or 100 μ g doses or 1mg/kg (single dose). Serum was collected by retro-orbital bleeding 4 hours after the administration of the immunomodulatory oligonucleotides and analyzed for IL-12 by sandwich ELISA. The results are shown in FIG. 8, which illustrates that in vivo administration of an immunomodulatory oligonucleotide containing a novel base can produce a unique profile of IL-12. All reagents, including cytokine antibodies and standards, were purchased from PharMingen (San Diego, CA).

Example 10 mouse splenocyte cultures:

splenocytes from C57BL/6 mice were prepared and cultured in RPMI complete medium consisting of RPMI 1640 containing 10% Fetal Calf Serum (FCS), 100U/ml penicillin, 100. mu.g/ml streptomycin, and 2mM L-glutamine (HyClone, Logan, UT). Mouse splenocytes were treated at 5X10⁶Cells/ml were seeded in 24-well plates. Cell cultures were supplemented with TE buffer [10mM Tris-HCl (pH 7.5) and 1mM EDTA]To a final concentration of 3 or 10. mu.g/ml of the individual immunomodulatory oligonucleotides. The cells were then incubated at 37 ℃ for 24 hours and the supernatant was collected for cytokine analysis by enzyme-linked immunosorbent assays (ELISAs).

IL-12 and IL-6 levels in the supernatant were measured by sandwich ELISA. Reagents required, including cytokine antibodies and standards, were purchased from BD Pharmingen (San Diego, CA). Streptavidin-peroxidase and TMB substrates were from Sigma (St. Louis, MO) and KPL (Gaithersburg, MD), respectively.

Example 11 human B cell proliferation assay:

purification of approximately 1X10 from human PBMCs⁵B cells, stimulated with different concentrations of an immunomodulatory oligonucleotide for 64 hours, then treated with 0.75. mu. Ci of³H]Thymidine pulse stimulation (pulse), harvest after 8 hours. The incorporation is measured by means of a scintillation counter³H]Thymidine, data expressed as readings per minute (c.p.m.).

Example 12 human multiple cytokine ELISA:

human PBMCs were treated at 5x10⁶The concentration of cells/ml was seeded in 96-well plates. The cell culture was supplemented with immunomodulatory oligonucleotides dissolved in phosphate buffered saline to a final concentration of 10 μ g/ml. However, the device is not suitable for use in a kitchenCells were then incubated at 37 ℃ for 24 hours. The supernatants were then analyzed for the listed cytokines using the Luminex-multiplex ELISA system. Human multiplex kits were obtained from invitrogen.

Example 13 mouse splenomegaly test

Female BALB/c mice (4-6 weeks, 19-21gm) were divided into three groups. The immunomodulatory oligonucleotide DNA was dissolved in sterile PBS and administered Subcutaneously (SC) at a dose of 5mg/kg to mice. After 72 hours, the mice were sacrificed and the spleens were harvested and weighed. The results are shown in FIG. 9, which illustrates the unique immune response profile that can be generated by in vivo administration of an immunomodulatory oligonucleotide containing a new base.

Example 14 OVA-sensitized mouse spleen cell culture assay

BALB/c female mice 4-6 weeks old were obtained from Taconic (Germantown, NY). Mice were injected intraperitoneally with 20 μ g of chicken ovalbumin (OVA; Sigma) mixed with 100 μ L ImjectAllum adjuvant (Pierce) in 100 μ L PBS on day 0, day 7 and challenged intranasally with 10 μ g OVA in 40 μ L PBS on day 14 and day 21. Mice were sacrificed 72 hours after the last challenge by inhalation of CO 2.

The spleen was excised and a single cell suspension was prepared as described above. Splenocytes were treated with different concentrations of immunomodulatory oligonucleotides for 2 hours followed by treatment with 100 μ g/mL OVA.

After 72 hours, the supernatant was collected and IL-5, IL-13, IL-12 and IFN-. alpha.levels were determined by ELISA as described above. The results are shown in FIGS. 10A-10D, demonstrating that administration of an immunomodulatory oligonucleotide containing a new base can produce a unique cytokine/chemokine profile even in the presence of an immune system activator (e.g., ovalbumin), which varies depending on the amount and base composition of the oligonucleotide administered.

Example 15 in vivo anti-cancer Activity of immunomodulatory oligonucleotides in combination with chemotherapeutic Agents

Can be stored in 100U/ml penicillin and 100 mu g/ml streptomycinIn the following, PC3 cells were cultured in 90% Ham's, F12K medium containing 10% Fetal Bovine Serum (FBS) to establish a human prostate cancer model (PC 3). Male athymic nude mice (Frederick Cancer Development Center, Frederick, MD) of 4-6 weeks of age can be acclimatized for 6 days prior to the study. Cultured PC3 cells can be harvested from monolayer cultures, washed twice with Ham's, F12K medium (10% FBS), resuspended in Ham's, F12K medium without FBS, Matrigel (Matrigel) basement membrane matrix (Becton Dickinson Labware, Bedford, MA) (5: 1; v/v), injected subcutaneously (5X 10)⁶Cells, total volume 0.2ml) to the inguinal region of each mouse. Routine clinical observations, body weight and tumor growth of the animals can be monitored. Tumor growth can be monitored by measuring two perpendicular diameters of the implant with calipers. Can be represented by the formula 1/2aXb²Tumor mass (weight in grams) was calculated, where 'a' is the long diameter (cm) and 'b' is the short diameter (cm). When the mean tumor size reached-80 mg, animals carrying human cancer xenografts were randomly divided into treatment and control groups (5 animals/group). The control group may receive sterile saline (0.9% NaCl) only. The immunomodulatory oligonucleotides of the invention can be aseptically dissolved in physiological saline and administered by subcutaneous injection at a dose of 0.5 or 1.0 mg/kg/day, three weekly doses. The chemotherapeutic agent may be provided by intraperitoneal injection of 160mg/kg on days 0 and 3.

Example 16 Synthesis of oligonucleotides containing immunomodulatory moieties

Immunomodulatory oligonucleotides containing 2 '-deoxy-pyridine [2, 3-d ] pyrimidine-2, 7(8H) -dione (2' -deoxy-pyrido [2, 3-d ] pyrimidine-2, 7(8H) -dione) (dF) or 2 '-deoxy pseudoisocytidine (2' -deoxy sedoisocytidine) (Ψ -iso-dC) modifications were synthesized on a 2- μmol scale using β -cyanoethyl phosphoramidite chemistry on a PerSeptive Biosystem 8909 expedite DNA synthesizer. Di-DMT protected glyceryl linker attached to CPG solid support was obtained from Chemgenes Corporation (Wilmington, Mass.). dA. The 3' -phosphoramidites for dG, dC and T were obtained from Applied Biosystems, while the dmf-dG phosphoramidites were obtained from Glen Research (Sterling, Va.). Phosphoramidites of dF and Ψ -iso-dC were obtained from Berry & Associates (Dexter, MI). Beaucage reagent was used as an oxidizing agent to obtain phosphorothioate backbone modifications. dF and Ψ -iso-dC phosphoramidite incorporation and deprotection were performed using the synthesis protocol recommended by the supplier. After synthesis, the immunomodulatory oligonucleotides were deprotected, purified by "trityl on" RP-HPLC, detritylated, and dialyzed against washing sterile water (Braun, Irvine, CA) of the quality of the united states pharmacopoeia (united states Pharmacopea). The immunomodulatory oligonucleotides were lyophilized, redissolved in distilled water, and the concentration determined by measuring UV absorbance at 260 nm. The purity of all synthesized compounds was determined by denaturing PAGE and sequence integrity was characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for molecular mass. All immunomodulatory oligonucleotides were synthesized (table 4A) and purified under identical conditions to minimize endotoxin contamination.

Example 17 immune modulatory oligonucleotides containing dF or Ψ -iso-dC in CpG motifs activate TLR9

HEK293 cells expressing mouse TLR9 were activated with immune modulatory oligonucleotides at a concentration of 10. mu.g/ml and a control compound. The ability of immune regulatory oligonucleotides containing dF or Ψ -iso-dC modifications to activate TLR9 was studied in HEK293 cells stably expressing mouse TLR 9. Human Secreted Embryonic Alkaline Phosphatase (SEAP) gene was used as NF-. kappa.B receptor. Results are expressed as fold increase in NF- κ B activation relative to the PBS control (FIG. 11). Immunomodulatory oligonucleotides 27 and 28(SEQ ID NOs 27 and 28) containing dF or Ψ -iso-dC (table 4A) activated TLR9 as shown by the increase in NF- κ B activity. These results indicate that dF or Ψ -iso-dC modifications are tolerated at the C-position and functional, further demonstrating that administration of an immunomodulatory oligonucleotide containing a novel base can generate a unique TLR9 activation profile (fig. 11).

Example 18 Induction of cytokine secretion in mouse splenocyte cultures by immunomodulatory oligonucleotides containing dF or Ψ -iso-dC in CpG motifs

Cytokine secretion was induced by IMO in C57BL/6 mouse splenocyte cultures. C57BL/6 mouse spleen cells were cultured in medium (M) alone or in the presence of different concentrations of immunomodulatory oligonucleotides for 24 hours, and the levels of secreted IL-12 (FIG. 12A) and IL-6 (FIG. 12B) in the culture supernatants were measured by ELISA. Data shown are at immunomodulatory oligonucleotide concentrations of 3 and 10 μ g/ml (FIGS. 12A and 12B). The immunomodulatory oligonucleotides 27(SEQ ID NO27) and 28(SEQ ID NO28) containing dF or Ψ -iso-dC induced secretion of IL-12 and IL-6 in C57BL/6 mouse splenocyte cultures compared to the control immunomodulatory oligonucleotide 29(SEQ ID NO 29) (FIGS. 12A and 12B). These results indicate that the immune cells are tolerant to and active against immune cells containing dF or Ψ -iso-dC modified immunomodulatory oligonucleotides, further indicate that administration of an immunomodulatory oligonucleotide containing a new base can produce a unique profile of IL-6 and IL-12 that varies depending on the amount and base composition of the oligonucleotide administered.

Example 19 immunomodulatory oligonucleotides containing dF or Ψ -iso-dC in CpG motifs induce splenomegaly and cytokines in mice

Splenomegaly in C57BL/6 mice that received a subcutaneously administered dose of 5mg/kg of immunomodulatory oligonucleotide, control compound, or PBS (FIG. 13A). Changes in spleen weight were determined 72 hours after administration of the immunomodulatory oligonucleotide. After subcutaneous administration of a 1mg/kg dose, the immunomodulatory oligonucleotides induced IL-12(13.B) secretion in C57BL/6 mice. Blood was collected 4 hours after the administration of the immunomodulatory oligonucleotide, and the IL-12 level in serum was determined by ELISA. The increase in spleen weight in mice after administration of CpG oligomers is a measure of immunomodulatory activity. Both mice containing dF or Ψ -iso-dC and human specific immunomodulatory oligonucleotides showed an increase in spleen compared to mice receiving control immunomodulatory oligonucleotides 4 or 5 (fig. 13A). Mice that received mouse specific immunomodulatory oligonucleotide 22(SEQ ID NO 22) or 23(SEQ ID NO23) containing dF or Ψ -iso-dC produced a greater increase in spleen weight than mice injected with human specific immunomodulatory oligonucleotide 27(SEQ ID NO27) or 28(SEQ ID NO 28). These results also demonstrate that mice receiving the dF-modified immunomodulatory oligonucleotide 22(SEQ ID NO 22) or 28(SEQ ID NO28), respectively, produced a greater increase in spleen weight compared to mice injected with Ψ -iso-dC-modified immunomodulatory oligonucleotide 23(SEQ ID NO23) or 27(SEQ ID NO 27). Further examination of cytokine induction profiles in vivo 4 hours after administration of the immunomodulatory oligonucleotide revealed that both mouse and human specific immunomodulatory oligonucleotides containing dF or Ψ -iso-dC modifications induced an increase in IL-12 in mice (FIG. 13B). As seen in the splenomegaly test, mouse-specific immunomodulatory oligonucleotide 22(SEQ ID NO 22) induced higher levels of IL-12 than immunomodulatory oligonucleotide 23(SEQ ID NO 23). These results indicate that both modifications (dF or Ψ -iso-dC) are tolerated and both activate TLR9, but that the level of immune response varies and that in vivo administration of an immunomodulatory oligonucleotide containing a new base results in a unique immune response profile.

Example 20 immune modulatory oligonucleotide-induced proliferation of human B cells

Human B cells isolated from PBMCs obtained from healthy human volunteers were stimulated with different concentrations of immunomodulatory oligonucleotides and 3H-thymidine uptake was determined by scintillation counting (fig. 14). FIG. 14 demonstrates that administration of an immunomodulatory oligonucleotide containing a new base produces a unique cell proliferation profile that varies depending on the amount and base composition of the oligonucleotide administered.

Example 21 cytokine/chemokine Induction of immunomodulatory oligonucleotides

The induction of IL-2R, IL-6, IL-8, TNF- α, MIP-1 α, MIP- β and MCP-1 by immunomodulatory oligonucleotide 26(SEQ ID NO 26), 27(SEQ ID NO27), 28(SEQ ID NO28) or control immunomodulatory oligonucleotide 29(SEQ ID NO 29) was assayed in human PBMC cell cultures (Table 5).

TABLE 5

SEQ IDNO.	IL-2R(pg/ml)	IL-6(pg/ml)	TNF-a(pg/ml)	MIP-1a(pg/ml)	MIP-b(pg/ml)	MCP-1(pg/ml)	IL-8(pg/ml)
								Culture medium	96.05	13.66	14.48	34.18	191.42	18.51	125.28
26	178.21	523.42	165.00	115.41	1339.33	2036.87	1632.89
								27	173.69	461.86	114.53	115.01	1225.94	406.18	3324.42
28	197.71	403.29	119.42	108.31	1121.74	443.07	3547.89
								29	96.62	97.01	61.27	67.62	525.34	68.67	2769.96

Example 22 immunomodulatory oligonucleotides containing dF or Ψ -iso-dC in CpG motifs activate human PBMC and B cells

Immunomodulatory oligonucleotides containing dF or Ψ -iso-dC were further tested for their ability to activate human PBMCs and induce cytokine production. In these experiments, immunomodulatory oligonucleotides 27(SEQ ID NO27) and 28(SEQ ID NO28) containing human specific motifs were used (fig. 4A). Both immunomodulatory oligonucleotides 27(SEQ ID NO27) and 28(SEQ ID NO28) induced IL-2R, IL-6, IL-8, TNF- α, MIP-1 α, MIP- β and MCP-1 (Table 5) compared to control 29(SEQ ID NO 29), indicating that both modifications are tolerated and both activate human TLR 9. Both immunomodulatory oligonucleotides 27(SEQ ID NO27) and 28(SEQ ID NO28) induced dose-dependent B cell proliferation compared to control immunomodulatory oligonucleotide 29(SEQ ID NO 29) (fig. 14).

Equivalents of

Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of 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 (47)

1. An immunomodulatory oligonucleotide comprising at least one immunomodulatory dinucleotide of formula CG wherein C is cytosine, 2' -deoxycytosine, N³-methyl-dC, dF or Ψ -iso-dC, and G is guanosine, 2 '-deoxyguanosine, 2' -deoxy-7-deazaguanosine, arabinoguanosine or N¹-methyl-dG;

with the proviso that when C is cytosine or 2' -deoxycytosine, G is N¹-methyl-dG;

with the further proviso that when G is guanosine or 2' -deoxyguanosine, C is N³-methyl radical-dC, dF or Ψ -iso-dC.

2. An immunomodulatory oligonucleotide according to claim 1, having the structure set forth below:

5’-CTATCTGAC₁GTTCTCTGT-3’，

5’-CTATCTGACG₁TTCTCTGT-3’，

5’-CTATCTGTC₁GTTCTCTGT-3’，

5’-CTATCTGTCG₁TTCTCTGT-3’，

5’-TCTGAC₁GTTCT-X-TCTTGC₁AGTCT-5’，

5’-TCTGACG₁TTCT-X-TCTTG₁CAGTCT-5’，

5’-TCTGTC₁GTTCT-X-TCTTGC₁TGTCT-5’，

5’-TCTGTCG₁TTCT-X-TCTTG₁CTGTCT-5’；

5’-TCTGAC₂GTTCT-X-TCTTGC₂AGTCT-5’，

5’-TCTGAC₃GTTCT-X-TCTTGC₃AGTCT-5’，

5’-TCTGTC₃GTTCT-X-TCTTGC₃TGTCT-5’，

5’-TC₃G₂AAC₃G₃TTC₃G₃-X-G₂C₃TTG₃C₃AAG₂C₃t-5' or

5’-TCTGTC₂GTTCT-X-TCTTGC₂TGTCT-5’；

Wherein C is₁＝N³-methyl-dC; c₂＝dF；C₃psi-iso-dC, G₁＝N¹-methyl-dG; and X ═ glycerol linker.

3. A pharmaceutical formulation comprising the oligonucleotide of claim 1 and a physiologically acceptable carrier.

4. A method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate the immunomodulatory oligonucleotide of claim 1.

5. The method according to claim 4, wherein the route of administration is selected from the group consisting of parenteral, oral, sublingual, transdermal, topical, mucosal, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.

6. The method according to claim 4, wherein the immunomodulatory oligonucleotide is selected from the group consisting of:

5’-CTATCTGAC₁GTTCTCTGT-3’，

5’-CTATCTGACG₁TTCTCTGT-3’，

5’-CTATCTGTC₁GTTCTCTGT-3’，

5’-CTATCTGTCG₁TTCTCTGT-3’，

5’-TCTGAC₁GTTCT-X-TCTTGC₁AGTCT-5’，

5’-TCTGACG₁TTCT-X-TCTTG₁CAGTCT-5’，

5’-TCTGTC₁GTTCT-X-TCTTGC₁TGTCT-5’，

5’-TCTGTCG₁TTCT-X-TCTTG₁CTGTCT-5’；

5’-TCTGAC₂GTTCT-X-TCTTGC₂AGTCT-5’，

5’-TCTGAC₃GTTCT-X-TCTTGC₃AGTCT-5’，

5’-TCTGTC₃GTTCT-X-TCTTGC₃TGTCT-5’，

5’-TC₃G₂AAC₃G₃TTC₃G₃-X-G₂C₃TTG₃C₃AAG₂C₃t-5' or

5’-TCTGTC₂GTTCT-X-TCTTGC₂TGTCT-5’；

Wherein C is₁＝N³-methyl-dC; c₂＝dF；C₃psi-iso-dC, G₁＝N¹-methyl-dG; and X ═ glycerol linker.

7. A method for treating a vertebrate suffering from cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen, the method comprising administering to the patient an immunostimulatory oligonucleotide according to claim 1.

8. The method according to claim 7, wherein the route of administration is selected from the group consisting of parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.

9. The method according to claim 7, wherein the immunomodulatory oligonucleotide is selected from the group consisting of:

5’-CTATCTGAC₁GTTCTCTGT-3’，

5’-CTATCTGACG₁TTCTCTGT-3’，

5’-CTATCTGTC₁GTTCTCTGT-3’，

5’-CTATCTGTCG₁TTCTCTGT-3’，

5’-TCTGAC₁GTTCT-X-TCTTGC₁AGTCT-5’，

5’-TCTGACG₁TTCT-X-TCTTG₁CAGTCT-5’，

5’-TCTGTC₁GTTCT-X-TCTTGC₁TGTCT-5’，

5’-TCTGTCG₁TTCT-X-TCTTG₁CTGTCT-5’；

5’-TCTGAC₂GTTCT-X-TCTTGC₂AGTCT-5’，

5’-TCTGAC₃GTTCT-X-TCTTGC₃AGTCT-5’，

5’-TCTGTC₃GTTCT-X-TCTTGC₃TGTCT-5’，

5’-TC₃G₂AAC₃G₃TTC₃G₃-X-G₂C₃TTG₃C₃AAG₂C₃t-5' or

5’-TCTGTC₂GTTCT-X-TCTTGC₂TGTCT-5’；

Wherein C is₁＝N³-methyl-dC; c₂＝dF；C₃psi-iso-dC, G₁＝N¹-methyl-dG; and X ═ glycerol linker.

10. A method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide according to claim 1.

11. The method according to claim 10, wherein the route of administration is selected from the group consisting of parenteral, oral, sublingual, transdermal, topical, mucosal, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.

12. The method according to claim 10, wherein the immunomodulatory oligonucleotide is selected from the group consisting of:

5’-CTATCTGAC₁GTTCTCTGT-3’，

5’-CTATCTGACG₁TTCTCTGT-3’，

5’-CTATCTGTC₁GTTCTCTGT-3’，

5’-CTATCTGTCG₁TTCTCTGT-3’，

5’-TCTGAC₁GTTCT-X-TCTTGC₁AGTCT-5’，

5’-TCTGACG₁TTCT-X-TCTTG₁CAGTCT-5’，

5’-TCTGTC₁GTTCT-X-TCTTGC₁TGTCT-5’，

5’-TCTGTCG₁TTCT-X-TCTTG₁CTGTCT-5’；

5’-TCTGAC₂GTTCT-X-TCTTGC₂AGTCT-5’，

5’-TCTGAC₃GTTCT-X-TCTTGC₃AGTCT-5’，

5’-TCTGTC₃GTTCT-X-TCTTGC₃TGTCT-5’，

5’-TC₃G₂AAC₃G₃TTC₃G₃-X-G₂C₃TTG₃C₃AAG₂C₃t-5' or

5’-TCTGTC₂GTTCT-X-TCTTGC₂TGTCT-5’；

Wherein C is₁＝N³-methyl-dC; c₂＝dF；C₃psi-iso-dC, G₁＝N¹-methyl-dG; and X ═ glycerol linker.

13. The oligonucleotide of claim 1, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent, or adjuvant.

14. The pharmaceutical composition according to claim 3, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

15. The method of claim 4, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

16. The method of claim 7, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

17. The method of claim 10, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

18. An immunomodulatory oligonucleotide compound comprising an immunostimulatory dinucleotide of the general formula 5 '-pyrimidine-purine-3', wherein the pyrimidine is N³-methyl-dC, the purine being natural or modifiedA purine nucleoside.

19. An immunomodulatory oligonucleotide compound comprising an immunostimulatory dinucleotide of the general formula 5 '-pyrimidine-purine-3', wherein the pyrimidine is a natural or modified pyrimidine nucleoside and the purine is N¹-methyl-dG.

20. A pharmaceutical formulation comprising an oligonucleotide according to claim 18 and a physiologically acceptable carrier.

21. A pharmaceutical formulation comprising an oligonucleotide according to claim 19 and a physiologically acceptable carrier.

22. A method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide according to claim 18.

23. A method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide according to claim 19.

24. A method for treating a vertebrate suffering from cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen, the method comprising administering to the patient an immunostimulatory oligonucleotide according to claim 18.

25. A method for treating a vertebrate suffering from cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen, the method comprising administering to the patient an immunostimulatory oligonucleotide according to claim 19.

26. A method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide according to claim 18.

27. A method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide according to claim 19.

28. The oligonucleotide of claim 18, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

29. The pharmaceutical composition according to claim 20, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

30. The method of claim 22, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

31. The method of claim 24, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

32. The method of claim 26, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

33. The oligonucleotide of claim 19, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

34. The pharmaceutical composition according to claim 21, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

35. The method of claim 23, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

36. The method of claim 25, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

37. The method of claim 27, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

38. An immunostimulatory oligonucleotide compound comprising an immunostimulatory dinucleotide of the general formula 5 '-pyrimidine-purine-3', wherein the pyrimidine is N³-methyl-dC, purine is N¹-methyl-dG.

39. A pharmaceutical formulation comprising an oligonucleotide according to claim 38 and a physiologically acceptable carrier.

40. A method for generating an immune response in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide according to claim 38.

41. A method for treating a vertebrate suffering from cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen, the method comprising administering to the patient an immunostimulatory oligonucleotide according to claim 38.

42. A method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, the method comprising administering to the vertebrate an immunostimulatory oligonucleotide according to claim 38.

43. The oligonucleotide of claim 38, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

44. The pharmaceutical composition according to claim 39, further comprising an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

45. The method of claim 40, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

46. The method of claim 41, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.

47. The method of claim 42, further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.