MXPA06006506A - Modulation of immunostimulatory properties by small oligonucleotide-based compounds - Google Patents

Abstract

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art 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 appended claims.

Description

MODULATION OF IMMUNOESTIMULATING PROPERTIES BY MEANS OF COMPOUNDS BASED ON SMALL OLIGONUCLEOTIDE RELATED REQUEST This application claims the benefit of the provisional application of E.U. No. 60 / 528,277, filed on December 5, 2003, which is incorporated as a reference in its entirety.

FIELD OF THE INVENTION The invention relates to immunology and immunotherapy applications that employ oligonucleotides as immunostimulatory agents.

BACKGROUND OF THE INVENTION Oligonucleotides have become an indispensable tool of modern molecular biology, being used in a wide variety of techniques ranging from diagnostic diagnostic methods for PCR, to antisense inhibition of gene expression and immunotherapy applications. This widespread use of oligonucleotides has led to an increasing demand for fast, cheap and efficient methods of oligonucleotide synthesis. Currently, the synthesis of oligonucleotides for antisense and diagnostic applications can be performed routinely; see, for example, "Methods in Molecular Biology", Vol. 20: "Protocols for Oligonucleotides and Analogs" p. 165-189 (S. AgrawaI, ed., Humana Press, 1993); "Oligonucleotides and Analogues, A Practical Approach", p. 87-108 (F. Eckstein, ed., 1991); and Uhlmann and Peyman, above; AgrawaI e lyer, Curr. Op. In Biotech. 6:12 (1995); and "Antisense Research and Applications" (Crooke and Lebleu, ed., CRC Press, Boca Raton, 1993). The first synthetic approaches included phosphodiester and phosphotriester chemistry. For example, Khorana et al., J. Molec Biol. 72: 209 (1972) describe phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for the synthesis of oligonucleotides and polynucleotides. These first approaches have given way to more efficient phosphoramidite and H-phosphonate synthesis approaches. For example, Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), describe the use of deoxyribonucleoside phospho-ramites in the synthesis of polynucleotide. AgrawaI and Zamecnik, patent of E.U. No. 5,149,798 (1992), describe the optimized synthesis of oligonucleotides by the H-phosphonate approach. These two modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. AgrawaI and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teach the synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry.

Connolly and others, Biochem. 23: 3443 (1984), describe the synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochem. 27: 7237 (1988), describe the synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. AgrawaI et al., Proc. Nati Acad. Sci. (USA) 85: 7079-7083 (1988), describe the synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry. More recently, several researchers have demonstrated the validity of the use of oligonucleotides as immunostimulatory agents in immunotherapy applications. The observation that phosphodiester and phosphorothioate oligonucleotides can induce immune stimulation has created interest in the development of this side effect as a therapeutic tool. These efforts have focused on phosphorothioate-oligonucleotides that contain the natural CpG dinucleotide. Kuramoto and others, Jpn. J Cancer Res. 83: 1128-1131 (1992) teach that phosphodiester oligonucleotides containing a palindrome including a CpG dinucleotide can induce the synthesis of interferon alpha and gamma and increase the natural destructive activity. Krieg et al., Nature 371: 546-549 (1995) disclose that phosphorothioate oligonucleotides containing CpG are immunostimulatory. Liang et al., J. Clin. Invest. 98: 1119-1129 (1996) describe that such oligonucleotides activate human B cells. Moldoveanu and others, Vaccine 16: 1216-124 (1998) teach that phosphorothioate oligonucleotides containing CpG increase the immune response against the influenza virus. McCluskie and Davis, J. Immunol. 161: 4463-4466 (1998) teach that oligonucleotides containing CpG act as potent adjuvants, increasing the immune response against the surface antigen of hepatitis B. Other modifications of phosphorothioate oligonucleotides can also affect their ability to act as response modulators. immune. See, for example, 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 and others, Bioorg. Med Chem. Lett. (1999) 9: 3453-3458; Zhao and others, Bioorg. Med Chem. Lett. (2000) 10: 1051-1054; Yu and others, Bioorg. Med. Chem. Lett. (2000) 10: 2585-2588; Yu and others, Bioorg. Med. Chem. Lett. (2001) 11: 2263-2267; and Kandimalla and others, Bioorg. Med. Chem. (2001) 9: 807-813. One response that can modulate the oligonucleotides that contain CpG, is asthma. An allergic asthma response is characterized by the activation of helper T lymphocytes type 2 (Th2). The responses induced by Th2 lymphocytes have a major role in the pathogenesis and spread of allergic inflammation in asthma. The cytokine of Th2 IL-5 increases the generation and survival of eosinophils, increasing the eosinophilia of the respiratory tract. Other Th2 cytokines (IL-4, IL-9 and IL-13) also have critical functions in allergic inflammation, inducing the production of allergen-specific IgE, mast cell proliferation, expression of endothelial cell adhesion molecules, and the hypersensitivity of the respiratory tract. Currently corticosteroids are the only widely used treatment for allergic asthma. However, treatment with steroids is only effective to minimize the manifestations of inflammation and does not cure the disease. Continuous therapy is required to prevent the advance of allergic asthma. These reports make it evident that the need to increase and modify the immune response caused by immunostimulatory oligonucleotides persists.

BRIEF DESCRIPTION OF THE INVENTION The invention provides methods for increasing and modifying the immune response caused by oligonucleotide compounds. The methods according to the invention make it possible to increase the immunostimulatory effect of immunostimulatory oligonucleotides for immunotherapy applications. The present inventors have surprisingly discovered that modifying an immunostimulatory oligonucleotide to optimally present its 5 'end significantly increases its immunostimulatory capacity. Such an oligonucleotide is referred to herein as a "immunomer." Therefore, in a first aspect, the invention provides immunomers comprising at least two oligonucleotides linked at their 3 'ends, an internucleotide linkage, or a nucleobase or sugar functionalized by means of a non-nucleotide linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having a 5 'end accessible. In one embodiment, the immunomestimulatory oligonucleotide immunomer comprises the sequence of SEQ ID NO 170. In a second aspect, the invention provides a immunomodulatory composition, comprising the immunomodulatory immunomodulatory oligonucleotide comprising the sequence of SEQ ID NO 170; and further comprising a costimulatory molecule selected from the group consisting of cytokines, chemokines, protein ligands, transactivation factors, peptides, and peptides comprising a modified amino acid. In this aspect of the invention, optionally the costimulatory molecule is conjugated to the immunomodulatory oligonucleotide, and the immunomodulatory composition also optionally comprises a pharmaceutically acceptable adjuvant or vehicle. In a third aspect, the invention provides a immunomodulatory composition, comprising the immunomodulatory immunomodulatory oligonucleotide comprising the sequence of SEQ ID NO 170; and further comprising an antigen, wherein the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins, polysaccharides, and lipids, or wherein the antigen is an allergen. In this aspect of the invention, optionally the immunomodulatory composition also comprises a pharmaceutically acceptable adjuvant or vehicle.

In another embodiment, the immunomestimulatory oligonucleotide immunomer comprises the sequence of SEQ ID NO 171. In a fourth aspect, the invention provides an immunomodulatory composition, comprising the immunomodulatory immunomodulatory oligonucleotide comprising the sequence of SEQ ID NO 171; and further comprising a costimulatory molecule selected from the group consisting of cytokines, chemokines, protein ligands, transactivation factors, peptides, and peptides comprising a modified amino acid. In this aspect of the invention, optionally the costimulatory molecule is conjugated to the immunomodulatory immunomodulatory oligonucleotide, and optionally the immunomodulatory composition also comprises a pharmaceutically acceptable adjuvant or vehicle. In a fifth aspect, the invention provides an immunomodulatory composition, comprising the immunomodulatory immunomodulatory oligonucleotide comprising the sequence of SEQ ID NO 171; and further comprising an antigen, wherein the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins, polysaccharides and lipids, or wherein the antigen is an allergen. In this aspect of the invention, optionally the immunomodulatory composition also comprises a pharmaceutically acceptable adjuvant or vehicle. In another embodiment, the invention provides a method of therapeutically treating a patient having inflammation of the respiratory tract, inflammatory disorders, allergy, or asthma, said method comprising administering to the patient an immunomer. In a sixth aspect, the invention provides a method for treating a patient therapeutically, wherein the immunomer comprises at least two oligonucleotides linked by means of a non-nucleotide linker and has more than one 5 'end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5 'end and comprising an immunostimulatory dinucleotide. The immunostimulatory dinucleotide is selected from the group consisting of CpG, C * pG, CpG *, and C * pG *, where C is cytidine or 2'-deoxycytidine; C * is 2'-deoxythymidine, arabinocytidine, 1- (2'-deoxy-β-D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine, 2'-deoxy-2'-substituted arabinocytidine, 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N-4-alky1-cytidine, 2'-deoxy-4-thiouridine or another pyrimidine nucleoside unnatural; G is guanosine or 2'-deoxyguanosine; G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxyinosine, 2'-deoxy-2'-substituted arabinoguanosine, 2'-0-substituted arabinoguanosine. In a seventh aspect, the invention provides a method for treating a patient therapeutically, wherein the immunomer comprises the sequence of SEQ ID NO 170, or the sequence of SEQ ID NO 171, or the sequence of SEQ ID NO 172, or the sequence of SEQ ID NO 173. In an eighth aspect, the invention provides a method for treating a patient therapeutically, which comprises administering an antigen associated with said disease or disorder, wherein the immunomer or the antigen or both are linked to an immunogenic protein or a non-immunogenic protein, and which further comprises administering an adjuvant. In another embodiment, the invention provides a method for modulating an immune response in a patient having airway inflammation, inflammatory disorders, allergy, or asthma, which comprises administering to the patient an immunomer, wherein the immune response is a response immune of Th1 or Th2. In a ninth aspect, the invention provides a method for modulating an immune response in a patient, wherein the immunomer comprises at least two oligonucleotides linked by means of a non-nucleotide linker and having more than one 5 'end, in wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5 'terminus and comprising an immunostimulatory dinucleotide. The immunostimulatory dinucleotide is selected from the group consisting of CpG, C * pG, CpG *, and C * pG *, wherein C is cytidine or 2'-deoxycytidine; C * is 2'-deoxythymidine, arabinocytidine, 1- (2'-deoxy-β-D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine, 2'-deoxy-2'-substituted arabinocytidine , 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N-4-alkyl-cytidine, 2'-deoxy-4-thiouridine or another unnatural pyrimidine nucleoside; G is guanosine or 2'-deoxyguanosine; G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxyinosine, 2'-deoxy-2'-substituted arabinoguanosine, 2'-O-substituted arabinoguanosine.

In a tenth aspect, the invention provides a method for modulating an immune response in a patient, wherein the immunomer comprises the sequence of SEQ ID NO 170, or the sequence of SEQ ID NO 171, or the sequence of SEQ ID NO 172, or the sequence of SEQ ID NO 173. In an eleventh aspect, the invention provides a method for treating a patient therapeutically, which comprises administering an antigen associated with said disease or disorder, wherein the immunomer or the antigen or both are linked to an immunogenic protein or a non-immunogenic protein, and further comprising administering an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of representative immunomers of the invention. Figure 2 represents several representative immunomers of the invention. Figure 3 represents a group of representative small molecule linkers, suitable for the linear synthesis of the invention immunomers. Figure 4 represents a group of representative small molecule linkers, suitable for the parallel synthesis of the immunomers of the invention. Figure 5 is a synthetic scheme of the linear synthesis of the immunomers of the invention; DMTr = 4,4'-dimethoxytrityl; CE = cyanoethyl. Figure 6 is a synthetic scheme of the parallel synthesis of the immunomers of the invention; DMTr = 4,4'-dirnetoxitrityl; CE = cyanoethyl. Figure 7A is a graphical representation of the induction of IL-12 with immunomers 1-3 in cultures of BALB / c mouse spleen cells. These data suggest that immunomer 2, which has accessible 5 'ends, is a stronger IL-12 inducer than monomeric oligo 1, and that immunomer 3, which has no accessible 5' ends, has an equal or greater capacity weak to produce an immune stimulation compared to oligo 1. Figure 7B is a graphic representation of the induction of IL-6 with immunomers 1-3 (top to bottom, respectively) in cultures of BALB / spleen cells c. These data suggest that immunomer 2, which has accessible 5 'ends, is a stronger IL-6 inducer than monomeric oligo 1, and that immunomer 3, which has no accessible 5' ends, has an equal or greater capacity weak to produce an immune stimulation compared to oligo 1. Figure 7C is a graphic representation of the induction of IL-10 with immunomers 1-3 (top to bottom, respectively) in cultures of BALB / spleen cells c. Figure 8A is a graphic representation of the proliferation induction of BALB / c mouse spleen cells in cell cultures with different concentrations of immunomers 5 and 6, which have inaccessible and accessible 5 'ends, respectively. Figure 8B is a graphical representation of BALB / c mouse spleen enlargement with immunomers 4-6, which have an immunogenic chemical modification in the 5 'flanking sequence of the CpG motif. Again, the immunomer having accessible 5 'ends (6), has a higher spleen enlargement capacity compared to immunomer 5, which has no accessible 5' end and monomeric oligo 4. Figure 9A is a graphic representation of the induction of IL-12 with different concentrations of oligo 4 and immunomers 7 and 8 in cultures of BALB / c mouse spleen cells. Figure 9B is a graphical representation of the induction of IL-6 with different concentrations of oligo 4 and immunomers 7 and 8 in cultures of BALB / c mouse spleen cells. Figure 9C is a graphic representation of the induction of IL-10 with different concentrations of oligo 4 and immunomers 7 and 8 in cultures of BALB / c mouse spleen cells. Figure 10A is a graphical representation of the induction of cell proliferation with immunomers 14, 15 and 16 in cultures of BALB / c mouse spleen cells. Figure 10B is a graphical representation of the proliferation induction of cells by IL-12 with different concentrations of immunomers 14 and 16 in cultures of BALB / c mouse spleen cells. Figure 10C is a graphical representation of the induction of cell proliferation by IL-6 with different concentrations of immunomers 14 and 16 in cultures of BALB / c mouse spleen cells. Figure 11A is a graphical representation of the induction of proliferation of cells with oligo 4 and 17 and immunomers 19 and 20 in cultures of BALB / c mouse spleen cells. Figure 11 B is a graphic representation of the induction of cell proliferation by IL-12, with different concentrations of oligo 4 and 17 and immunomers 19 and 20 in cultures of BALB / c mouse spleen cells. Figure 11C is a graphic representation of the induction of cell proliferation by IL-6, with different concentrations of oligo 4 and 17 and immunomers 19 and 20 in cultures of BALB / c mouse spleen cells. Figure 12 is a graphical representation of BALB / c mouse spleen enlargement using oligonucleotide 4 and immunomers 14, 23 and 24. Figure 13 is a schematic representation of the 3 'terminal nucleoside of an oligonucleotide, showing that can add a non-nucleotide link in the nucleoside nucleobase, in the 3 'position or in the 2' position. Figure 14 shows the chemical substitutions used in example 13. Figures 15A-15B show the cytokine profiles obtained using the modified oligonucleotides of example 13. Figures 16A-16B show the relative cytokine induction of the glycerol linkers in comparison with the amino linkers. Figures 17A-17B show the relative induction of cytokine of various linkers and linker combinations. Figures 18A-E show the relative nuclease resistance of various PS and PO immunomers and oligonucleotides. Figures 19A-19B show the relative induction of cytokine of PO immunomers compared to PSnmunomers in cultures of BALB / c mouse spleen cells. Figures 20A-20B show the relative induction of cytokine of PO immunomers as compared to PS immunomers in cultures of mouse spleen cells C3H / Hej. Figures 21A-21B show the relative induction of cytokine of PO immunomers compared to PSnmunomers in cultures of C3H / Hej mouse spleen cells at high concentrations of immunomers. Figure 22 shows the sequences and chemical modifications of the immunomers (IMO's). Figures 23A1-23B5 show the prevention of the Th2-induced immune response by OVA with IMO in mice, demonstrated by the cytokine response in spleen cell cultures. Figures 24A1-24B5 show the prevention of the Th2-induced immune response by OVA with IMO in mice, demonstrated by the serum antibody response. Figures 25A-25C show the dose-dependent effects of IMO's 1 and 2 on established allergic asthma, induced by OVA in mice. Cytokine secretion is in the cultures of spleen cells in response to OVA-memory. The IMO's 1 and 2 significantly inhibited the secretion of IL-5 in a dose-dependent manner. IL-13 was significantly inhibited by the two IMO compounds. The two IMO compounds induced dose-dependent IFN-g secretion. Figures 26A-26D show total serum antibody and antigen specific. The IMO's 1 and 2 produced a dose-dependent reduction of OVA-specific IgE and an increase in OVA-specific IgG2a. Figures 27A-27F show the effect of a single high dose compared to lower multiple doses of IMO compounds on the local and systemic Th1 cytokine concentration in unaffected mice. A single dose of 100 mg induced a higher degree of systemic cytokine response. In contrast, three smaller doses (3 x 33 mg) induced higher local induced responses (BALF) of cytokine. Figures 28A-28D show the dose-dependent effects of low multiple administrations of IMO's on the local and systemic concentration of cytokine in unaffected mice. IMO 1 increased the local concentration (BALF) of cytokine but not the systemic concentration of cytokine in mice, when administered multiple times in small doses. This effect is dose dependent. Figures 29A-29D compare the effects of IMO and corticosteroid in vitro. Both the IMO 1 and the budesonide suppressed the secretion of Th2 cytokine induced by OVA. Nevertheless, only IMO 1 showed strong induction of Th1 cytokine.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents for immunotherapy applications. The patents issued, 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 case of inconsistencies between any teaching of any cited reference and the present specification, the latter shall prevail for the purposes of the invention. The invention provides methods for increasing and modifying the immune response caused by immunostimulatory compounds used for immunotherapy applications, such as, for example, without limitation, the treatment of cancer, autoimmune diseases, asthma, respiratory allergies, food allergies, bacterial infections, parasitic and viral, in human adult and pediatric and veterinary applications. Allergic asthma is a particularly preferred condition for treatment by means of the methods and compounds herein. In this way, the invention also provides compounds having optimal levels of immunostimulatory effect for immunotherapy, and methods for preparing and using said compounds. In addition, the immunomers of the invention are useful as adjuvants in combination with DNA vaccines, antibodies, allergens, chemotherapeutic agents, and antisense oligonucleotides. The present inventors have surprisingly discovered that the modification of an immunostimulatory oligonucleotide to optimally present its 5 'ends, greatly affects its immunostimulatory capacity. Such an oligonucleotide is referred to herein as an "immunomer." In a first aspect, the invention provides immunomers comprising at least two oligonucleotides linked at their 3 'ends, or an internucleoside linkage or a nucleobase or sugar functionalized to a non-nucleotide linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having a 5 'end accessible. As used herein, the term "accessible 5 'terminus" means that the 5' end of the oligonucleotide is sufficiently available, such that factors that recognize and bind immunomers and stimulate the immune system have access thereto. In oligonucleotides having an accessible 5 'end, the 5'-OH position of the terminal sugar is not covalently linked to more than two nucleoside residues. Optionally, 5'-OH may be linked to a portion of phosphate, phosphorothioate or phosphorodithioate, an aromatic or aliphatic linker, cholesterol, or other entity that does not affect accessibility. For the purposes of the invention, the term "immunomer" refers to any compound comprising at least two oligonucleotides linked at their 3 'ends or intemucleoside linkages, or nucleobase or functionalized sugar, either directly or via a non-nucleotide linker, at least one of the oligonucleotides (in the context of the immunomer) being an immunostimulatory oligonucleotide and having an accessible 5 'end, wherein the compound induces an immune response when administered to a vertebrate. In some modalities, the vertebrate is a mammal, including a human being. In some embodiments, the immunomer comprises two or more immunostimulatory oligonucleotides (in the context of the immunomer) which may be the same or different. Preferably, each immunostimulatory oligonucleotide has at least one accessible 5 'end. In some embodiments, in addition to the immunostimulatory oligonucleotide (s), the immunomer also comprises at least one oligonucleotide that is complementary to a gene. As used herein, the term "complementary to" means that the oligonucleotide hybridizes under physiological conditions to a region of the gene. In some embodiments, the oligonucleotide negatively regulates the expression of a gene. Said negative-regulated oligonucleotides are preferably selected from the group consisting of antisense oligonucleotides, ribozyme oligonucleotides, small inhibitory RNAs and decoy oligonucleotides. As used herein, the term "negatively regulates a gene" means that it inhibits the transcription of a gene or the translation of a product of the gene. In this manner, the immunomers according to these embodiments of the invention can be used to direct them to one or more specific disease targets, also by stimulating the immune system. In some embodiments, the immunomer includes a ribozyme or a decoy oligonucleotide. As used herein, the term "ribozyme" refers to an oligonucleotide that possesses catalytic activity. Preferably, the ribozyme binds to a specific target nucleic acid and cuts it. As used herein, the term "decoy oligonucleotide" refers to an oligonucleotide that binds to a transcription factor in a sequence-specific manner and stops transcription activity. Preferably, the ribozyme or decoy oligonucleotide exhibits a secondary structure including, without limitation, stem and coil or hairpin structures. In some embodiments, at least one oligonucleotide comprises poly (l) -poly (dC). In some embodiments, at least one group of Nn includes a stretch of 3 to 10 dGs or Gs, or 2'-substituted ribo- or arabino-Gs. For the purposes of the invention, the term "oligonucleotide" refers to a polynucleoside formed from a plurality of linked nucleoside units. Such oligonucleotides can be obtained from existing sources of nucleic acid, which include genomic DNA or cDNA, but are preferably produced by synthetic methods. In preferred embodiments, each nucleoside unit includes a heterocyclic base and a pentofuranosyl, trehalose, arabinose, 2-deoxy-2'-substituted arabinose, 2'-0-substituted arabinose or hexose sugar group. The nucleoside residues can be coupled together by any of the many known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borane, thioether, bridge phosphoramidate, bridge methylene phosphonate, phosphorothioate bridge and internucleoside sulfone bonds. The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkages (e.g., (Rp) - or (Sp) -phosphorothioate, alkylphosphonate or phosphotriester linkages). As used herein, the terms "oligonucleotide" and "dinucleotide" expressly include polynucleosides and dinucleosides having any such internucleoside linkages, whether the linkage comprises a phosphate group or not. In some preferred embodiments, these internucleoside linkages may be phosphodiester, phosphorothioate, or phosphorodithioate linkages, or combinations thereof. In some embodiments, each oligonucleotide has from about 3 to about 35 nucleoside residues, preferably from about 4 to about 30 nucleoside residues, most preferably from about 4 to about 20 nucleoside residues. In some embodiments, the oligonucleotides have from about 5 to about 18, or from about 5 to about 14, nucleoside residues. As used herein, the term "approximately" implies that the exact number is not critical. In this way, the number of nucleoside residues in the oligonucleotides is not critical, and oligonucleotides having one or two less nucleoside residues, or one to several additional nucleoside residues, are contemplated as equivalents of each, one of the modalities previously described. In some embodiments, one or more of the oligonucleotides have 11 nucleotides. The term "oligonucleotide" also encompasses polynucleosides having additional substituents that include, without limitation, protein groups, lipophilic groups, intercalation agents, diamines, folic acid, cholesterol and adamantane. The term "oligonucleotide also encompasses any other polymer containing nucleobase, including without limitation peptidonucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), closed nucleic acids (LNA), oligonucleotides with morpholino backbone, and oligonucleotides having main chain sections with alkyl linkers or amino linkers As used herein, the term "secondary structure" refers to intramolecular and intermolecular hydrogen bonds The intramolecular hydrogen bond results in the formation of a stem and loop structure. The intermolecular hydrogen bonding results in the formation of a double helical nucleic acid molecule.

The oligonucleotides of the invention can include natural nucleosides, modified nucleosides, or mixtures thereof. As used herein, the term "modified nucleoside" is a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or a combination thereof. In some embodiments, the modified nucleoside is an unnatural 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'-fluoroarabinoside. For purposes of the invention, the term "2'-substituted ribonucleoside" includes ribonucleosides wherein the hydroxyl group at the 2 'position of the pentose portion is substituted to produce a 2'-0-substituted ribonucleoside. Preferably, said substitution is with a saturated or unsaturated lower alkyl group containing 1-6 carbon atoms, or with an aryl group having 6-10 carbon atoms, wherein said alkyl or aryl group may be unsubstituted or may be substituted. to be substituted, for example, with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy or amino groups. Examples of these 2'-0-substituted ribonucleosides include, without limitation, 2'-0-methylribonucleosides and 2'-0-methoxyethylribonucleosides. The term "2'-substituted ribonucleoside" also includes ribonucleosides wherein the 2'-hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halogen group. Examples of these 2'-substituted ribonucleosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl and 2'-propargyl. The term "oligonucleotide" includes hybrid and chimeric oligonucleotides. A "chimeric oligonucleotide" is an oligonucleotide that has more than one type of internucleoside linkage. A preferred example of said chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region, and nonionic bonds such as alkylphosphonate or alkylphosphonothioate linkages (see, for example, Pederson et al., U.S. Patent Nos. 5,635,377 and 5,366,878). A "hybrid oligonucleotide" is an oligonucleotide that has more than one type of nucleoside. A preferred example of said hybrid oligonucleotide comprises a 2'-substituted ribonucleotide or ribonucleotide region, and a deoxyribonucleotide region (see, for example, Metelev and AgrawaI, U.S. Patent Nos. 5,652,355, 6,683,167, 6,346,614 and 6,143,881). For the purposes of the invention, the term "immunostimulatory oligonucleotide" refers to an oligonucleotide, as described above, that induces an immune response when administered to a vertebrate such as a fish, domestic bird or mammal. As used herein, the term "mammal" includes without limitation rats, mice, cats, dogs, horses, cattle, cows, pigs, rabbits, non-human and human primates. Useful immunostimulatory oligonucleotides can be found in AgrawaI et al., WO 98/49288, published November 5, 1998; WO 01/12804, published February 22, 2001; WO 01/55370, published on August 2, 2001; PCT / US01 / 13682, filed on April 30, 2001; and PCT / US01 / 30137, filed September 26, 2001. Preferably, the immunostimulatory oligonucleotide comprises at least one intemucleoside phosphodiester, phosphorothioate or phosphorodithioate linkage. In some embodiments, the immunostimulatory oligonucleotide comprises an immunostimulatory dinucleotide of the formula 5'-Pyr-Pur-3 ', wherein Pyr is a natural or synthetic pyrimidine nucleoside and Pur is a natural or synthetic purine nucleoside. As used herein, the term "pyrimidine nucleoside" refers to a nucleoside wherein the base component of the nucleoside is a pyrimidine base. Similarly, the term "purine nucleoside" refers to a nucleoside wherein the base component of the nucleoside is a purine base. For purposes of the invention, a "synthetic" pyrimidine or purine nucleoside includes an unnatural pyrimidine or purine base, an unnatural sugar portion, or a combination thereof. The preferred pyrimidine nucleosides according to the invention have structure (I): (i) where: D is a hydrogen bond donor; D 'is selected from the group consisting of hydrogen, a hydrogen bond donor, a hydrogen bond receptor, a hydrophilic group, a hydrophobic group, an electron-withdrawing group and an electron-donor group; A is a hydrogen bond receptor or a hydrophilic group; A 'is selected from the group consisting of a hydrogen bonding receptor, a hydrophilic group, a hydophobic group, an electron attracting group and an electron donor group; X is carbon or nitrogen; and S 'is a ring of pentose sugar or hexose, or a non-natural sugar. Preferably, the sugar ring is modified with a phosphate moiety, a modified phosphate moiety, or other suitable linker portion for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog. Preferred hydrogen bond donors include, without limitation, -NH-, -NH2-, -SH and -OH. Preferred hydrogen bonding receptors include, without limitation, C = 0, C = S, and the ring nitrogen atoms of an aromatic heterocycle, eg, cytosine N3. In some embodiments, the base portion in (I) is an unnatural pyrimidine base. Examples of preferred non-natural pyrimidine bases include, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, and 4-thiouracil. However, in some embodiments, 5-bromocytosine is specifically excluded. In some embodiments, the sugar portion S 'in (I) is a portion of unnatural sugar. For the purposes of the present invention, a "natural sugar portion" is a portion of sugar that occurs naturally as part of a nucleic acid, for example ribose and 2'-deoxyribose, and a "non-natural sugar portion" is any sugar that does not occur naturally as part of a nucleic acid, but that can be used in the backbone of an oligonucleotide, for example hexose. Arabinose and arabinose derivatives are examples of preferred sugar portions. The preferred purine nucleoside analogs according to the invention have structure (II): (¡I) where: D is a hydrogen bond donor; D 'is selected from the group consisting of hydrogen, a hydrogen bond donor, and a hydrophilic group; A is a hydrogen bond receptor or a hydrophilic group; X is carbon or nitrogen; each L is independently selected from the group consisting of C, O, N and S; and S 'is a ring of pentose sugar or hexose, or a non-natural sugar. Preferably, the sugar ring is modified with a phosphate moiety, a modified phosphate moiety, or other suitable linker portion for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog. Preferred hydrogen bond donors include, without limitation, -NH-, -NH2, -SH and -OH. Preferred hydrogen bonding receptors include, without limitation, C = 0, C = S, -N02, and the ring nitrogen atoms of an aromatic heterocycle, eg, guanine N1. In some embodiments, the base portion in (II) is a non-natural purine base. Examples of preferred non-natural purine bases They include, without limitation, 6-thioguanine and 7-deazaguanine. In some embodiments, the sugar portion S 'in (II) is a portion of natural sugar, as described above for structure (I). In preferred embodiments, the immunostimulatory dinucleotide is selected from the group consisting of CpG, C * pG, CpG * and C * pG *, wherein C is cytidine or 2'-deoxycytidine; C * is 2'-deoxythymidine, 1- (2'-deoxy-β-D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine, arabinocytidine, 2'-deoxythymidine, arabinocytidine 2'-deoxy- 2'-substituted, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other non-natural pyrimidine nucleoside or nucleoside of pyrimidine that occurs rarely; G is guanosine or 2'-deoxyguanosine; G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, 2'-0-substituted arabinoguanosine, 2'-deoxyinosine, or another nucleoside of unnatural purine or purine nucleoside which occurs rarely, and p is an intemucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate and phosphorodithioate. In some preferred embodiments, the immunostimulatory dinucleotide is not CpG. The immunostimulatory oligonucleotides can include immunostimulatory portions on one or both sides of the immunostimulatory dinucleotide. Thus, in some embodiments, the immunostimulatory oligonucleotide comprises in the immunostimulatory domain the structure (III): 5'-Nn-N1-YZ-N1-Nn-3 '(III) wherein: Y is cytidine, 2' -deoxythymidine, 2'-deoxycytidine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-deoxythymidine, 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy- N4-alkylcytidine, 2'-deoxy-4-thiouridine or another non-natural pyrimidine nucleoside; Z is guanosine or 2'-deoxyguanosine, G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, arabinoguanosine 2'-O- substituted, 2'-deoxyinosine, or other non-natural purine nucleoside. N1, in each occurrence, is preferably a natural or synthetic nucleoside or an immunostimulatory portion selected from the group consisting of abbasic nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and nucleosides linked by a phosphodiester linkage or a modified internucleoside linkage with the adjacent nucleoside on the 3 'side, the modified internucleotide linkage being selected, without limitation, from a linker having a length from about 2 angstroms to about 200 angstroms, an alkyl linker of C2 -C18, a poly (ethylene glycol) linker, a 2-aminobutyl-1,3-propanediol linker, a glyceryl linker, a 2'-5 'internucleoside linkage, and an internucleoside linkage of phosphorothioate, phosphorodithioate or methylphosphonate; Nn, in each occurrence, is preferably a natural nucleoside or an immunostimulatory portion selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, 2'-0-substituted ribonucleosides, and nucleosides linked by a internucleoside linkage modified with the adjacent nucleoside on the 3 'side, the modified internucleotide linkage preferably selected from the group consisting of an amino linker, a 2'-5' internucleoside linkage, and an internucleoside methyl phosphonate linkage; with the proviso that at least one of N1 or Nn is an immunostimulatory portion; where n is a number from 0 to 30; and wherein the 3 'end, an internucleoside linker, or a nucleobase or modified sugar, is linked directly or via a non-nucleotide linker with another oligonucleotide, which may or may not be immunostimulatory. In some preferred embodiments, YZ is arabinocytidine or 2'-deoxy-2'-substituted arabinocytidine and arabinoguanosine or 2'-deoxy-2'-substituted arabinoguanosine. Preferred immunostimulatory portions include modifications to the phosphate backbones, including without limitation, methylphosphonates, methylphosphonothioates, phosphotriesters, phosphotriesters, phosphorothioates, phosphorodithioates, prodrugs of triester, sulfones, sulfonamides, sulfamates, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidates, especially primary amino phosphoramidates, N3-phosphoramidates and N5-phosphoramidates, and stereospecific linkages (for example, (Rp) - or (Sp) -phosphorothioate, alkylphosphonate or phosphotriester linkages). Preferred immunostimulatory portions according to the invention also include nucleosides having sugar modifications, including, without limitation, 2'-substituted pentose sugars including, without limitation, 2'-0-methylribose; 2'-0-methoxyethylribose, 2'-0-propargylribose and 2'-deoxy-2'-fluororribose; 3'-substituted pentose sugars, which include, without limitation, 3'-0-methylribose; 1 ', 2'-dideoxyribose; arabinose; substituted arabinose sugars, including without limitation, 1'-methylorabinose, 3'-hydroxymethylarabinose, 4'-hydroxymethylarabinose and 2'-substituted arabinose sugars; hexose sugars including, without limitation, 1,5-anhydrohexitol; and alpha anomers. In embodiments wherein the modified sugar is a 3'-deoxyribonucleoside or a 3'-0-substituted ribonucleoside, the immunostimulatory portion is linked to the adjacent nucleoside via a 2'-5 'intemucleoside linkage. Preferred immunostimulatory portions according to the invention also include oligonucleotides having other modifications and replacements in the carbohydrate backbone, including peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), closed nucleic acids (LNA), oligonucleotides with morpholino backbone, and oligonucleotides having backbone linker sections having a length of about 2 angstroms to about 200 angstroms, including without limitation, alkyl linkers or amino linkers. The alkyl linker may be branched or unbranched, substituted or unsubstituted, and may be chirally pure or a racemic mixture. Most preferably, said alkyl linkers have from about 2 to about 18 carbon atoms. In some preferred embodiments, said alkyl linkers have from about 3 to about 9 carbon atoms. Some alkyl linkers include one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thioether. Some of these functionalized alkyl linkers are poly (ethylene glycol) linkers of the formula -0- (CH2-CH2-0-) n (A? = 1-9). Some other functionalized alkyl linkers are peptides or amino acids. Preferred immunostimulatory portions according to the invention also include DNA isoforms, which include without limitation β-L-deoxyribonucleosides and α-deoxyribonucleosides. Preferred immunostimulatory portions according to the invention incorporate 3 'modifications and further include nucleosides having non-natural internucleoside linking positions, including without limitation, 2'-5', 2'-2 ', 3'-3' and 5'-5 '. Preferred immunostimulatory portions according to the invention also include nucleosides having heterocyclic modified bases including, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitropyrrole, C5-propynylpyrimidine and diaminopurines, including, without limitation, 2,6-diaminopurine. By way of specific illustration and not limitation, for example, in the immunostimulatory domain of structure (III), an internucleoside linkage of methylphosphonate at position N1 or Nn is an immunostimulatory portion, a linker having a length of about 2 angstroms at about 200 angstroms, a C2-C18 alkyl linker at position X1 is an immunostimulatory portion, and a β-L-deoxyribonucleoside at position X1 is an immunostimulatory portion. See table 1 below to see the positions and structures representative of the immunostimulatory portions. It is understood that reference to a linker as an immunostimulatory portion at a specific position means that the nucleoside residue at that position is substituted at its 3'-hydroxyl with the indicated linker, thereby creating an internucle bond between that nucleoside residue. and the adjacent nucleoside on the 3 'side. Similarly, the reference to a modified internucleoside linkage as the immunostimulatory portion at a specific position, means that the nucleoside residue at that position is linked to the adjacent nucleoside on the 3 'side by means of the aforementioned linkage.

TABLE 1 Position TYPICAL IMMUNOSTIMULATING PORTIONS N1 Natural nucleosides, nucleic acid, arabinonucleoside, 2'-deoxyuridine, β-L-deoxyribonucleoside-C2-C18 alkyl linker, poly (ethylene glycol) linker, 2-aminobutyl-l, 3-propanediol linker ( amino linker), internucleoside link 2'-5 ', internucleoside linkage of methylphosphonate Nn Natural nucleosides, abasic nucleoside, arabinonucleosides, 2'-deoxyuridine, 2'-O-substituted ribonucleoside, 2'-5' internucleoside link, methylphosphonate intemucleoside linkage , with the proviso that N1 and N2 can not be both abasic links Table 2 shows representative positions and structures of immunostimulatory portions within an immunostimulatory oligonucleotide having a 5 'enhancement domain. As used herein, the term "spacer 9" refers to a poly (ethylene glycol) linker of formula -0- (CH2CH2-0) p-, where n is 3. The term "spacer 18" refers to a poly (ethylene glycol) linker of formula -O- (CH2CH2-0) n-, wherein n is 6. As used herein, the term "C2-C18 alkyl linker" refers to a linker of formula - 0- (CH2) q-0-, where q is an integer from 2 to 18. Accordingly, the terms "C3 linker" and "C3 alkyl linker" refer to a linker of formula -O- (CH2) ) 3-0-. For each of the spacer 9, spacer 18, and C2-C18 alkyl linker, the linker is attached to the adjacent nucleosides via phosphodiester, phosphorothioate, or phosphorodithioate linkages.

TABLE 2 Table 3 shows representative positions and structures of immunostimulatory portions within an immunostimulatory oligonucleotide having a 3 'enhancement domain.

TABLE 3 The immunomers according to the invention comprise at least two oligonucleotides linked at their 3 'ends, or an internucleoside linkage or a nucleobase or sugar functionalized by means of a non-nucleotide linker. For purposes of the invention, a "non-nucleotide linker" is any portion that can be linked to the oligonucleotides by means of covalent or non-covalent linkages. Preferably, said linker is from about 2 angstroms to about 200 angstroms in length. Several examples of preferred linkers are indicated below. Non-covalent bonds include, without limitation, electrostatic interaction, electrophobic interactions, p-stacking interactions, and hydrogen bonds. The term "non-nucleotide linker" does not refer to an internucleoside linkage as described above, for example a phosphodiester, phosphorothioate, or phosphorodithioate functional group, which directly binds the 3'-hydroxyl groups of two nucleosides. For the purposes of this invention, said 3'-3 'direct link is considered a "nucleotide link". In some embodiments, the non-nucleotide linker is a metal, including, without limitation, gold particles. In some other embodiments, the non-nucleotide linker is a soluble or insoluble biodegradable polymer globule. In other embodiments, the non-nucleotide linker is an organic moiety that has functional groups that allow binding to the oligonucleotide. Said binding is preferably by means of any stable covalent bond. As a non-limiting example, the linker can be attached at any suitable position in the nucleoside, as illustrated in Figure 13. In some preferred embodiments, the linker is attached to the 3'-hydroxyl. In such embodiments, the linker preferably comprises a hydroxyl functional group, which is preferably linked to the 3'-hydroxyl by means of a phosphodiester, phosphorothioate, phosphorodithioate, or non-phosphate-based linkage. In some embodiments, the non-nucleotide linker is a biomolecule, including, without limitation, polypeptides, antibodies, lipids, antigens, allergens and oligosaccharides. In some other embodiments, the non-nucleotide linker is a small molecule. For purposes of the invention, a small molecule is an organic portion 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, any of which may optionally include, in the linear chain joining the oligonucleotides or annexes thereto, one or more functional groups selected from the group consisting of hydroxy, amino , thioether, ether, amide, thioamide, ester, urea and thiourea. The small molecule can be cyclic or acyclic. Examples of small molecule linkers include, without limitation, amino acids, carbohydrates, cyclodextrins, adamantane, cholesterol, haptens and antibiotics. However, for the purposes of the description of the non-nucleotide linker, the term "small molecule" does not include a nucleoside. In some embodiments, the small molecule linker is glycerol or a glycerol homolog of the formula HO- (CH2) 0-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- (CH2) mC (0) NH-CH2-CH (OH) -CH2-NHC (0) - (CH2) m-OH, where m is an integer from 0 to about 10 , from 0 to about 6, from 2 to about 6, or from 2 to about 4. Some non-nucleotide linkers according to the invention allow the joining of two or more oligonucleotides, as schematically depicted in Figure 1. For example, the small molecule linker glycerol has three hydroxyl groups to which the oligonucleotides can be covalently linked. Some immunomers according to the invention, therefore, comprise more than two oligonucleotides linked at their 3 'ends with a non-nucleotide linker. Some such immunomers comprise at least two immunostimulatory oligonucleotides, each having an accessible 5 'end. The immunomers of the invention can be conveniently synthesized using an automatic synthesizer and the phosphoramidite approach as schematically depicted in Figures 5 and 6, and further described in the examples. In some embodiments, the immunomers are synthesized by a linear synthesis approach (see Figure 5). As used herein, the term "linear synthesis" refers to a synthesis that begins at one end of the immunomer and proceeds linearly to the other end. The linear synthesis allows the incorporation of identical or non-identical monomeric units (in terms of length, base composition or incorporated chemical modifications) into the immunomers. An alternative mode of synthesis is the "parallel synthesis", in which the synthesis proceeds externally from a central linker portion (see Figure 6). A linker attached to a solid support can be used for parallel synthesis, as described in the US patent. No. 5,912,332. Alternatively a universal solid support (such as a controlled pore glass support linked to phosphate) can be used. The parallel synthesis of immunomers has several advantages over linear synthesis: (1) parallel synthesis allows the incorporation of identical monomer units; (2) unlike linear synthesis, both (or all) monomer units are synthesized at the same time, so that the number of synthetic steps and the time required for the synthesis are the same as for a monomer unit; and (3) the reduction in the synthetic steps improves the purity and yield of the final immunomer product. At the end of the synthesis, either linear synthesis or parallel synthesis, conveniently the immunomers can be deprotected with concentrated ammonia solution or as recommended by the phosphoramidite supplier, if a modified nucleoside is incorporated. The product immunomer is preferably purified by means of reverse phase HPLC, the triphenyl is removed, desalted and dialyzed. Tables 4A and 4B show representative immunomers according to the invention. Additional immunomers are described in the examples.

TABLE 4A Examples of immunomer sequences r-NHCOC4H6- = Larger symmetric branches; = Symmetric glycerol branches (short) L-NHCOC.Hß- L = C3 alkyl linker; X = 1 ', 2'-d-deoxyriboside; Y = 50HdC; R = 7-desaza-dG TABLE 4B SEQ ID Sequences and Modification (5"-3 ') NO. Izs 5'-CTGTC TTCTC-X-CirTTRCTGTC-5s 171 S'-TCRTCRlTG Í-GTTRCTRCT-S'. 172 S ^ TC? GTCRTTCT-X-TCTTRCTGTCTÓ1 173 5 , -tcrrGTR, GTTCT-x-trttrt "R 'TGTCT-S-174 S ^ TCRTCRTI? -X-GITRCTRCT-S' 175 5 '-YYCTGACGTTCTCTGT-X-TÜTCTCTTGC AGTCYY-5: X = glycerol linker; R = arabinoguanosine; R = 2'-deoxy-7'-deazaguanosine; R '= 1 - (2'-deoxy-b-D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine; Y = linker of C3. In another aspect, the invention provides an immunostimulatory nucleic acid comprising at least two oligonucleotides, wherein the immunostimulatory nucleic acid has a secondary structure.

In this aspect, the immunostimulatory nucleic acid comprises a structure as detailed in formula (I). Domain A-Domain B-Domain C (I) Domains can be from about 2 to about 12 nucleotides in length. Domain A can be DNA, RNA, RNA-DNA, DNA-RNA 5'-3 '. or 3'-5 ', or 2'-5', which has or does not have a palindromic or self-complementary domain, which contains or does not contain at least one dinucleotide selected from the group consisting of CpG, YpG, YpR, CpR, R * pG and R * pR, wherein C is cytidine or 2'-deoxycytidine, G is guanosine or 2'-deoxyguanosine, Y is cytidine, 2'-deoxythymidine, 2'-deoxycytidine, 2'-dideoxy-5-halocytosine, 2 '. -dideoxy-5-nitrocytosine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N-4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other unnatural pyrimidine nucieosides, R * is 1- (2'-deoxy-β-D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine; R is guanosine or 2'-deoxyguanosine, 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, 2'-0-substituted arabinoguanosine, 2 ' -deoxyinosine, or other unnatural purine nucleoside, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate and phosphorodithioate. In some preferred embodiments, the immunostimulatory dinucleotide is not CpG. In some embodiments, the A domain will have more than one dinucleotide selected from the group consisting of CpG, YpG, YpR, CpR, R * pG and R * pR, located at the 5 'end of the domain A oligonucleotide. The B domain is a linker linking domains A and C, which may be a 3'-5 'link, a 2'-5' link, a 3'-3 'link, a phosphate group, a nucleoside, or a non-nucleoside linker that it may be aliphatic, aromatic, aryl, cyclic, chiral, achiral, a peptide, a carbohydrate, a lipid, a fatty acid, mono-, tri-, or hexapolyethylene glycol, or a heterocyclic moiety. Domain C can be DNA, RNA, RNA-DNA, DNA-RNA, Poly 1-Poly C 5'-3 'or 3'-5', 2'-5 ', having or not a palindromic or self-complementary sequence, which may or may not have a dinucleotide selected from the group consisting of CpG, YpG, YpR , CpR, R * pG and R * pR, where C is cytidine or 2'-deoxycytidine, G is guanosine or 2'-deoxyguanosine, Y is cytidine, 2'-deoxythymidine, 2'-deoxycytidine, 2'- dideoxy-5-halocytosine, 2'-dideoxy-5-halocytosine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy N-4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, R * is 1- (2'-deoxy-β-D-ribofuranosyl) -2-oxo-7-deaza- 8-methyl-purine; R is guanosine or 2'-deoxyguanosine, 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted arabinoguanosine, 2'-0-substituted arabinoguanosine, 2 '-deoxyinosine, or another non-natural purine nucleoside, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate and phosphorodithioate. In some preferred modalities, the immunostimulatory dinucleotide is not CpG. In some embodiments, domain B is preferably a non-nucloetidic linker that binds the oligonucleotides of domain A and domain C that are referred to as "immunomers." In some preferred embodiments, the C domain does not have the CpG dinucleotide, YpG, YpR, CpR, R * pG or R * pR. In some embodiments, the oligonucleotides contained in formula (I) are from about 12 to about 50 nucleotides in length. In some embodiments, the oligonucleotides contained in formula (I) are from about 12 to about 26 nucleotides in length. By way of non-limiting example, in some embodiments of this aspect the immunostimulatory nucleic acid will have a structure as indicated by formula (II).

As the person skilled in the art will recognize, there is an element of secondary structure at the 3 'end of the molecule in the form of an intramolecular stem-coil. By way of non-limiting example, in some embodiments of this aspect the immunostimulatory nucleic acid will have a structure as indicated by formula (III).

The structure represented in formula (III) is referred to herein as a "terminal dimer", since the 3 'ends of the two molecules are blocked because the sequences of the two 3' ends are complementary, allowing the binding of intermolecular hydrogen. In addition, domains A and A 'may or may not be identical, domains B and B' may or may not be identical, and domains C and C may or may not be identical. By way of non-limiting example, in some embodiments of this aspect the immunostimulatory nucleic acid will have a structure as indicated by formula (IV).

As the person skilled in the art will recognize, the 3 'end of the molecule represented has a secondary structure because the complementary sequence of its 3' end is linked by hydrogen to this region. In some embodiments a molecule may be attached to the 3 'end, such as a ligand, to facilitate cellular incorporation or improve the stability of the molecule. Tables 24B-C and 25B-C present non-limiting examples of some nucleic acid molecules of the invention (see below). In a second aspect, the invention provides immunomer conjugates comprising an immunomer as described above, and an antigen conjugated to the immunomer at a position different from the accessible 5 'end. In some embodiments, the non-nucleotide linker comprises an antigen that is conjugated to the oligonucleotide. In some other embodiments, the antigen is conjugated to the oligonucleotide at a position different from its 3 'end. In some embodiments, the antigen produces a vaccine effect. The antigen is preferably selected from the group consisting of antigens associated with a pathogen, antigens associated with a cancer, antigens associated with an autoimmune disorder and antigens associated with other diseases, such as, for example, without limitation, veterinary or pediatric diseases, or in where the antigen is an allergen. For purposes of the invention, the term "associated with" means that the antigen is present when the pathogen, cancer, autoimmune disorder, food allergy, skin allergy, respiratory allergy, asthma, or other disease is present; but when the pathogen, cancer, autoimmune disorder, food allergy, skin allergy, respiratory allergy or disease is absent, the antigen is not present or is present in small amounts. The immunomer is covalently linked to the antigen, or is otherwise associated with the antigen. As used herein, the term "operatively associated with" refers to any association that maintains the activity of both the immunomer and the antigen. Non-limiting examples of such operational associations include those that are part of the same liposome or other such delivery vehicles or reagents. In embodiments wherein the immunomer is covalently bound to the antigen, preferably such a covalent bond is in a position on the different immunomer of an accessible 5 'end of an immunostimulatory oligonucleotide. For example, the antigen may be linked to an internucleoside linkage or may be linked to the non-nucleotide linker. Alternatively, the antigen alone can be the non-nucleotide linker. In a third aspect, the invention provides pharmaceutical formulations comprising an immunomer or an immunomer conjugate according to the invention and a physiologically acceptable carrier. As used herein, the term "physiologically acceptable" refers to a material that does not affect the effectiveness of the immunomer 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. As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the vehicle, excipient or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described, for example, in "Remington's Pharmaceutical Sciences", 18th edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pennsylvania, 1990. In a fourth aspect, the invention provides methods for generating an immune response in a vertebrate, such methods comprising administering to the vertebrate an immunomer or immunomer conjugate according to the invention . In some modalities, the vertebrate is a mammal. For the purposes of this invention, the term "mammal" expressly includes human beings. In preferred embodiments, the immunomer or conjugate immunomer is administered to a vertebrate in need of immunostimulation. In the methods according to this aspect of the invention, the administration of the immunomers can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intramuscular, intraperitoneal, subcutaneous, intradermal , aerosol, intraocular, intrathecal, intrarectal, vaginal, by means of a gene gun, dermal patch or in the form of ophthalmic drops or mouth rinses. Administration of the therapeutic compositions of the immunomers can be done using known methods and doses, and for effective periods to reduce the symptoms or subrogate markers of the disease. When administered systemically, the therapeutic composition is preferably administered at a dose sufficient to achieve an immunomer concentration in the blood of about 0.0001 micromolar to about 10 micromolar. For localized administration, much lower concentrations may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of immunomer ranges from about 0.001 mg per patient per day, to about 200 mg per kg of body weight per day. It may be desirable to administer to a subject simultaneously or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention as a single episode of treatment. In some preferred embodiments, the immunomers according to the invention are administered in combination with vaccines, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, peptides, proteins, gene therapy vectors, DNA vaccines, or adjuvants, to increase the specificity or magnitude of the immune response. In these embodiments, the immunomers of the invention may act variously as adjuvants or produce direct immunostimulatory effects. The immunomer or the vaccine, or both, may optionally be linked to an immunogenic protein, such as limpet hemocyanin (KLH), subunit B of cholera toxin, or any other immunogenic carrier protein or non-immunogenic carrier protein. Any of the adjuncts of a plethora can be used including, without limitation, Freund's complete adjuvant, incomplete Freund's adjuvant, KLH, monophosphoryl lipid A (MPL), alum and saponins, including QS-21, imiquimod, R848, or combinations thereof. Toll-like receptors (TLRs) function as sensors of infection and induce the activation of innate and adaptive immune responses. TLRs recognize a wide variety of ligands called molecular patterns associated with pathogens (PAMPs). After recognizing conserved molecular products associated with pathogens, TLRs activate host defense responses through their intracellular signaling domain, the toll receptor domain -interleucin 1 (TIR), and the MyD88 cascade adapter protein. Dendritic cells and macrophages normally respond to toll-like receptor (TLR) ligands and cytokines (eg, interleukin 1β); IL-6 and tumor necrosis factor, TNF), which also produce; Natural killer cells (NK) and T cells are also involved in the proinflammatory circuit. After the stimulation of the TLR produced by bacterial compounds, the innate immune cells release a range of cytokines. Some examples of TLR ligands include, without limitation, lipoproteins; peptidoglycan, zymosan (TLR2), double-stranded RNA, poly I: poly C (TLR3), lipopolysaccharide, heat shock proteins, taxol (TLR4), flagellin (TLR5) and imidazoquinolines-R848, resiquimod, imiquimod; ARNcs (TLR7 / 8). For the purposes of this aspect of the invention, the term "in combination with" means in the course of the treatment of the same disease in the same patient, and includes administering the immunomer or the vaccine or the adjuvant in any order, including simultaneous administration. , as well as also spaced temporarily in the order of up to several days of separation. Said combination treatment may also include more than a single administration of the immunomer, or independently of the vaccine, or independently of the adjuvant. The administration of the immunomer or the vaccine or the adjuvant can be by the same route or by different routes. The methods according to this aspect of the invention are useful for model studies of the immune system. The methods are also useful for the prophylactic or therapeutic treatment of human or animal disease. For example, the methods are useful for applications of pediatric and veterinary vaccines. In a fifth aspect, the invention provides methods for therapeutically treating a patient having a disease or disorder, such methods comprising administering to the patient an immunomer or immunomer conjugate according to 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 effected as described in the fourth aspect of the invention. For the purposes of the invention, the term "allergy" includes, without limitation, food allergies, atopic dermatitis, allergic rhinitis (also known as hay fever), allergic conjunctivitis, urticaria, respiratory allergies and allergic reactions to other substances such as latex, medications and insect bites, or problems that commonly originate from allergic rhinitis, sinusitis and otitis media. The term "inflammation of the respiratory tract" includes, without limitation, asthma. Specific examples of asthma include, without limitation, allergic asthma, non-allergic asthma, exercise-induced asthma, occupational asthma, and nocturnal asthma. Allergic asthma is characterized by a blockage of the airways associated with allergies and caused by substances called allergens. Allergic asthma triggers include, without limitation, airborne pollen, molds, animal dander, household dust mites, and cockroach droppings. Non-allergic asthma is caused by viral infections and some medications or irritants found in the air, which irritate the nose and respiratory tract. Non-allergic asthma triggers include, without limitation, airborne particles (eg, hard coal, chalk dust), air pollutants (eg, tobacco smoke, wood smoke), strong odors or aerosols (eg perfumes, household cleaners, kitchen smoke, paints or varnishes), viral infections (eg colds, viral pneumonia, sinusitis, nasal polyps), sensitivity to aspirin and gastroesophageal reflux disease (GERD). Exercise-induced asthma (EIA) is triggered by vigorous physical activity. The symptoms of EIA occur to varying degrees in most people with asthma, and are probably caused as a result of breathing dry, cold air during exercise. The provocateurs of the EIA include, without limitation, breathe pollen in the air during exercise, breathe air pollutants during exercise, exercise with viral infections of the respiratory tract, and exercise in dry, cold air. Occupational asthma is directly related to the inhalation of irritants and other potentially hazardous substances found in the workplace. The triggers of occupational asthma include, without limitation, vapors, chemical agents, gases, resins, metals, powders and insecticides. As used herein, the term "autoimmune disorder" refers to disorders in which "own proteins" suffer an attack by the immune system. This term includes autoimmune asthma. Without wishing to be limited by any particular theory, a decrease in exposure to bacteria may be partially responsible for a higher incidence of severity and mortality of allergic diseases such as asthma, atopic dermatitis and rhinitis in developing countries. This hypothesis is supported by evidence that infections or bacterial products can inhibit the development of allergic disorders in experimental animal models and clinical studies. Bacterial DNA or synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides, in some sequence contexts (CpG DNA) potently stimulate innate immune responses and thus acquired immunity. The immune response to CpG DNA includes activation of innate immune cells, proliferation of B cells, induction of Th1 cytokine secretion and production of immunoglobulins (Ig). Activation of immune cells with CpG DNA occurs by means of the toll-like receptor 9 (TLR9), a molecular pattern recognition receptor. The CpG DNAs induce strong Th1-dominant immune responses characterized by secretion of IL-12 and IFN-α. Immunomeres (IMO's), alone or as allergen conjugates, decrease the production of IL-4, IL-5 and IgE, and reduce eosinophilia in mouse models of allergic asthma. The IMO compounds also effectively reverse the established atopic eosinophilic disease of the airways, converting a Th2 response into a Th1 response.

OVA is commonly used with alum to establish a Th2-dominant immune response in several mouse and rat models. The Th2 immune response includes increased production of IL-4, IL-5 and IL-13, high serum concentration of total and antigen-specific IgE, IgG1, and lower IgG2a concentration. The IMO compounds prevent and reverse the Th2-dominant immune responses established in mice. Coadministration of IMO compounds with OVA / alum to mice reduces the production of IL-4, IL-5 and IL-13, and induces the production of IFN-α. in cultures of spleen cells subjected to restimulation with antigen. In addition, the IMO compounds inhibit total and antigen-specific IgE and increase the production of IgG2a in these mice. The injection of OVA / alum and IMO compounds induces a lymphocyte antigen (Th1 type) memory response in mice, characterized by low concentrations of cytokines associated with Th2, IgE and IgG1, and high concentrations of cytokines associated with Th1 and IgG2a . The co-administration of IMO compounds with other types of antigens, such as S. masoni egg lysozyme and chicken egg, also causes reversion of the Th2 response to a Th1-dominant response in in vitro and in vivo studies. As described herein, IMO compounds effectively prevent the development of a Th2 immune response and allow a strong Th1 response. Although Th2 cytokines cause a change of Ig isotype towards the production of IgE and IgG1, the cytokine of Th1 IFN-? induces the production of IgG2a by B lymphocytes. Mice that received injections of OVA alum and IMO compounds produced lower concentrations of IL-4, IL-5 and IL-13 and higher concentrations of IFN-α, accompanied by lower concentrations of IgE and IgG1 and higher concentrations of IgG2a, than mice that received only OVA / alum injection. This suggests the existence of a close link between the Th1 cytokine induction and the immunoglobulin isotype change in the mice that received antigen and IMO compounds. Serum concentrations of antigen-specific and total IgE are significantly lower in the mice that received OVA / alum and compounds I O, than in the mice that received only OVA / alum. In contrast, the OVA-specific IgG1 concentrations changed significantly, and the total IgG1 concentrations barely decreased slightly compared to the mice that received OVA / alum injection alone (data not shown). The different response may originate from the different mechanisms involved in the control of the class change of IgE and IgG1, although both isotypes are affected by IL-4 and IL-13. For example, IL-6 promotes B lymphocytes synthesizing IgG1 in the presence of IL-4. In any of the methods according to the invention, the immunomer or conjugate immunomer can be administered in combination with any other agent useful in the treatment of the disease or condition, which does not diminish the immunostimulatory effect of the immunomer. For the purposes of this aspect of the invention, the term "in combination with" means in the course of treating the same disease in the same patient, and includes administering the immunomer and an agent, in any order that includes simultaneous administration and also any temporarily spaced order; for example, sequentially with one immediately after the other, up to several days apart. Such a combination treatment may also include more than a single administration of the immunomer, and independently the agent. The administration of the immunomer and the agent can be by the same route or by different routes. In any of the methods according to the invention, the agent useful for the treatment of the disease or condition includes, without limitation, an antigen, an allergen, or costimulatory molecules such as cytokines., chemokines, protein ligands, transactivation factors, peptides and peptides comprising modified amino acids. Additionally, the agent can include DNA vectors that encode an antigen or allergen. The invention provides a kit comprising immunostimulatory oligonucleotides or immunomers, the latter comprising at least two oligonucleotides linked together, such that the immunomer has more than one accessible 5 'end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide. In another aspect, the kit comprises an immunostimulatory oligonucleotide, or an immunostimulatory oligonucleotide conjugate, or an immunomer, or an immunomer conjugate according to the invention, and a physiologically acceptable carrier. Generally the team will also include a series of instructions for use. The following examples further illustrate some preferred embodiments of the invention and are not considered to limit the scope of the invention.

EXAMPLES EXAMPLE 1 Synthesis of oligonucleotides containing immunomodulatory portions Oligonucleotides were synthesized at a scale of 1 μmol using an automatic DNA synthesizer (Expedite 8909; PerSeptive Biosystems, Framingham, Massachusetts), following the procedures of linear synthesis or parallel synthesis described in figures 5 and 6. Deoxyribonucleoside was obtained. phosphoramidites from Applied Biosystems (Foster City, California). 1 ', 2'-dideoxyribose-phosphoramidite, propyl-1-phosphoramidite, 2-deoxyuridine-phosphoramidite, 1,3-bis- [5- (4,4'-dimethoxytrityl) pentylamidyl] -2-propanol- phosphoramidite and methyl-phosphoramidite from Glen Research (Sterling, Virginia). ß-L-2'-deoxyribonucleoside-phosphoramidite, a-2'-deoxyribonucleoside-phosphoramidite, mono-DMT-glycerol-phosphoramidite and di-DMT-glycerol-phosphoramidite were obtained from ChemGenes (Ashland, Massachusetts). (4-aminobutyl) -1,3-propanediol-phosphoramidite was obtained from Clontech (Palo Alto, California). Arabinocytidine phosphoramidite, arabinoguanosine, arabinothymidine and arabinouridine were obtained from Reliable Pharmaceutical (St. Louis, Missouri). Arabinoguanosine-phosphoramidite, arabinothymidine-phosphoramidite and arabinouridine-phosphoramidite were synthesized in Hybridon, Inc. (Cambridge, Mass.) (Noronha et al. (2000) Biochem., 39: 7050-7062). All nucleoside phosphoramidites were characterized by means of 31 P and 1 H NMR spectra. The modified nucleosides were incorporated at specific sites using normal coupling cycles. After the synthesis, the oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reverse phase HPLC, followed by dialysis. Purified oligonucleotides such as the sodium salt were lyophilized before use. The purity was tested by CGE and MALDI-TOF MS.

EXAMPLE 2 Analysis of Spleen Cell Proliferation The proliferation of splenocytes was analyzed in vitro using standard procedures as previously described (see, for example, Zhao et al., Biochem Pharma 51: 173-182 (1996)). The results are shown in Figure 8A. These results demonstrate that at the highest concentrations, immunomer 6, which has two accessible 5 'ends, produces more proliferation of splenocytes than immunomer 5, which has no accessible 5' end, or oligonucleotide 4, with only one terminus. 'accessible. Immunomer 6 also causes more proliferation of splenocytes than the positive control of LPS.

EXAMPLE 3 Tests of splenomegaly in vivo To test the applicability of in vitro results in an in vivo model, selected oligonucleotides were administered to mice and the degree of splenomegaly was measured as an indicator of immunostimulatory activity. BALB / c mice (females 4-6 weeks of age, Harlan Sprague Dawley Inc, Baltic, Connecticut) were intraperitoneally administered a single dose of 5 mg / kg. The mice were sacrificed 72 hours after the administration of the oligonucleotide and the spleens were harvested and weighed. The results are shown in Figure 8B. These results demonstrate that immunomer 6, which has two accessible 5 'ends, has a much greater immunostimulatory effect than oligonucleotide 4 or immunomer 5.

EXAMPLE 4 Analysis of cytokines Secretion of IL-12 and IL-6 in vertebrate cells, preferably BALB / c mouse spleen cells or human PBMCs, was measured by means of sandwich ELISA. The required reagents, including cytokine antibodies and cytokine standards, were purchased from PharMingen, San Diego, California. ELISA plates (Costar) were incubated with appropriate antibodies at 5 μg / mL in PBSN buffer (PBS / 0.05% sodium azide, pH 9.6) overnight at 4 ° C, and then blocked with PBS / BSA at 1 ° C. % at 37 ° C for 30 minutes. Cell culture supernatants and cytokine standards were appropriately diluted with 10% PBS / FBS, added to the plates in triplicate and incubated 2 hours at 25 ° C. Plates were covered with 1 μg / ml of the appropriate biotinylated antibody and incubated 1.5 hours at 25 ° C. After, the plates were thoroughly washed with PBS-T buffer (PBS / 0.05% Tween 20) and incubated 1.5 hours more at 25 ° C, after adding peroxidase conjugated with streptavidin (Sigma, St. Louis Missouri). The plates were developed with Sure Blue ™ chromogenic reagent (Kirkegaard and Perry) and the reaction was terminated by adding Stop Solution (Kirkegaard and Perry). The color change was measured in a Ceres 900 HDI spectrophotometer (Bio-Tek Instruments). The results are shown in Table 5A below. Human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers by means of Ficoll-Paque density gradient centrifugation (Histopaque-1077, Sigma, St. Louis, Missouri). Briefly, heparinized blood was stratified on Histopaque-1077 (equal volume) in a conical centrifuge and centrifuged at 400 x g for 30 minutes at room temperature. The buffy coat, which contains the mononuclear cells, was carefully removed and washed twice with phosphate buffered saline (PBS) by centrifugation at 250 x g for 10 minutes. The resulting cell pellet was then resuspended in RPMI 1640 medium containing L-glutamine (MediaTech, Inc., Herndon, Virginia) and supplemented with 10% heat-inactivated FCS and penicillin-streptomycin (100 U / ml). Cells were grown in 24-well plates for different periods at 1 X 106 cells / ml / well in the presence or absence of oligonucleotides. At the end of the incubation period, supernatants were harvested and stored frozen at -70 ° C until analyzed by sandwich ELISA to determine several cytokines including IL-6 (BD Pharmingen, San Diego, California), IL-10 (BD Pharmingen), IL-12 (BioSource International, Camarillo, California), IFN-a (BioSource International) and -? (BD Pharmingen) and TNF-a (BD Pharmingen). The results are shown in tables 5 and 5A below. In all cases the concentrations of IL-12 and IL-6 were calculated in the cell culture supernatants of the standard curve constructed under the same experimental conditions for IL-12 and IL-6, respectively.

The concentrations of IL-10, IFN-gamma and TNF-a in the cell culture supernatants were calculated from the standard curve constructed under the same experimental conditions for IL-10, IFN-gamma and TNF-a, respectively.

TABLE 5 Structure of immunomer and immunostimulatory activity in human PBMC cultures D1 and D2 are donors 1 and 2.

TABLE 5A Structure of immunomer and immunostimulatory activity in cultures of BALB / c mouse spleen cells Normal letters represent a phosphorothioate link; the italicized letters represent a phosphodiester bond.

In addition, the results shown in Figures 7A-C demonstrate that oligonucleotide 2, with two accessible 5 'ends, elevates IL-12 and IL-6 but not IL-10 at lower concentrations than oligonucleotides 1 or 3, with one and zero accessible 5 'ends, respectively.

EXAMPLE 5 Effect of chain length on immunostimulatory activity of immunomers To study the effect of the chain length of the oligonucleotides, immunomers containing 18, 14, 11 and 8 nucleotides were synthesized in each chain, and their immunostimulatory activity, measured by their ability to induce the secretion of the IL-cytokines, was tested. 12 and IL-6 in cultures of BALB / c mouse spleen cells (Tables 6-8). In this and all subsequent examples, cytokine tests were performed on cultures of BALB / c mouse spleen cells as described in example 4.

TABLE 6 Structure of immunomer and immunostimulatory activity TABLE 7 Structure of immunomer and immunostimulatory activity TABLE 8 Immunomer structure and immunostimulatory activity The results suggest that the immunostimulatory activity of the immunomers increases as the length of the oligonucleotide chains decreases from 18 nucleotides to 7 nucleotides. Immunomers having short oligonucleotide chain lengths of 6 nucleotides or 5 nucleotides, show immunostimulatory activity comparable to that of the oligonucleotide of 18 nucleotides with a single 5 'end. However, immunomers having short oligonucleotide chain lengths of 6 nucleotides or 5 nucleotides, have a greater immunostimulatory activity when the linker is in the length of about 2 angstroms to about 200 angstroms.

EXAMPLE 6 Immunostimulatory activity of immunomers containing an unnatural pyrimidine or purine nucleoside As shown in Tables 9-11, the immunostimulatory activity was maintained with immunomers of various lengths, which have an unnatural pyrimidine nucleoside or an unnatural purine nucleoside in the immunostimulatory dinucleotide motif.

TABLE 9 Structure of immunomer and immunostimulatory activity TABLE 10 Structure of immunomer and immunostimulatory activity TABLE 11 Structure of immunomer and immunostimulatory activity EXAMPLE 7 Effect of the linker on immunostimulatory activity To examine the effect of the length of the linker linking the two oligonucleotides, immunomers containing the same oligonucleotides but different linkers were synthesized, and their immunostimulatory activity was tested. The results shown in table 12 suggest that the length of the linker has a role in the immunostimulatory activity of the immunomers. The best immunostimulatory effect was achieved with C6 alkyl linkers or abasic linkers having interspersed phosphate charges.

TABLE 12 Immunomer structure and immunostimulatory activity EXAMPLE 8 Effect of the oligonucleotide backbone on immunostimulatory activity In general, immunostimulatory oligonucleotides containing natural phosphodiester backbones are less immunostimulatory than oligonucleotides of the same length with phosphorothioate backbones. This lower degree of immunostimulatory activity could be due in part to the rapid degradation of the phosphodiester oligonucleotides under the experimental conditions. The degradation of the oligonucleotides is mainly the result of 3'-exonucleases, which digest the oligonucleotides from the 3 'end. The immunomers of this example do not contain a free 3 'end. In this way, the immunomers with phosphodiester backbones would have a longer half life under the experimental conditions than the corresponding monomeric oligonucleotides, and would therefore exhibit improved immunostimulatory activity. The results presented in Table 13 demonstrate this effect, with the immunomers 84 and 85 exhibiting immunostimulatory activity, determined by induction of cytokine in cultures of BALB / c mouse spleen cells.

TABLE 13 Structure of immunomer and immunostimulatory activity L = C3 Linker EXAMPLE 9 Synthesis of immunomers 73-92 Oligonucleotides were synthesized at a scale of 1 μmol using an automated DNA synthesizer (Expedite 8909; PerSeptive Biosystems). Deoxynucleoside phosphoramidites were obtained from Applied Biosystems (Foster City, California). 7-Deaza-2'-deoxyguanosine phosphoramidite was obtained from Glen Research (Sterling, Virginia). 1,3-bis-DMT-glycerol-CPG was obtained from ChemGenes (Ashland, Massachusetts). The modified nucleosides were incorporated into the oligonucleotides at the specific site using normal coupling cycles. After the synthesis, the oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reverse phase HPLC, followed by dialysis. Purified oligonucleotides such as the sodium salt were lyophilized before use. The purity of the oligonucleotides was verified by CGE and MALDI-TOF MS (Bruker Proflex III mass spectrometer MALDI-TOF).

EXAMPLE 10 Stability of the immunomer The oligonucleotides were incubated at 37 ° C for 24 or 48 hours in PBS containing 10% bovine serum. The intact oligonucleotide was determined by capillary gel electrophoresis. The results are shown in table 14.

TABLE 14 Digestion of oligonucleotides in PBS solution with 10% bovine serum X = Linker of C3, U, C = 2'-0-Me-ribonucleoside EXAMPLE 11 Effect of accessible 5 'ends on immunostimulatory activity BALB / c mouse spleen cells (4-8 weeks) were cultured in complete RPMI medium. Murine macrophage-like cells, J774 (American Type Culture Collection, Rockville, Maryland) were cultured in Dulbecco's modified Eagle medium, supplemented with 10% (v / v) FCS and antibiotics (100 IU / mL penicillin G / streptomycin). ). All other culture reagents were purchased from Mediatech (Gaithersburg, Maryland). ELISA's for IL-12 and IL-6. BALB / c or J774 mouse spleen cells were cultured in 24-well plates at a density of 5x106 or 1x106 cells / mL, respectively. The IMO compounds dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) were added at a final concentration of 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 μg / mL to the cell cultures of mouse spleen, and 1.0, 3.0, or 10.0 μg / mL to J774 cell cultures. The cells were then incubated 24 h at 37 ° C and the supernatants were collected for the ELISA tests. The experiments were done two or three times for each IMO compound in triplicate for each concentration. The secretion of IL-12 and IL-6 was measured by sandwich ELISA. The required reagents, including cytokine antibodies and standards, were purchased from PharMingen. ELISA plates (Costar) were incubated overnight at 4 ° C with the appropriate antibodies at 5 μg / mL in PBSN buffer (PBS / 0.05% sodium azide, pH 9.6), and then blocked with PBS / 1% of BSA at 37 ° C for 30 min. Cell culture supernatants and cytokine standards were appropriately diluted with PBS / 1% BSA, added to the plates in triplicate, and incubated at 25 ° C for 2 h. Plates were washed and incubated with 1 μg / mL of appropriate biotinylated antibody and incubated at 25 ° C for 1.5 h. The plates were washed extensively with PBS / 0.05% Tween 20, and incubated at 25 ° C for 1.5 h after the addition of streptavidin-conjugated peroxidase (Sigma). The plates were developed with the Sure Blue ™ chromogenic reagent (Kirkegaard and Perry), and the reaction was terminated by adding Stop Solution (Kirkegaard and Perry). The color change was measured on a Ceres 900 HDI spectrophotometer (Bio-Tek Instruments) at 450 nm. The concentration of IL-12 and IL-6 in cell culture supernatants was calculated from the standard curve constructed under the same experimental conditions for IL-12 and IL-6, respectively. The results are shown in table 15.

TABLE 15 Sequences of CpG DNA phosphorothioate and modifications used in the study! 1 DNA Sequence End Length 5 'End 3' CpG # 89 5'-TCCATGACGTTCCTGATGC-3 '19 nucí. eleven 90 5'-TCCATGACGTTCCTGATGC-3'-b 19 nucí. 1 blocked 91 5'-TCCATGACGTTCCTGATGC-3'-3'-g-5 '20 nucí. 2 blocked 92 5'-TCCATGACGTTCCTGATGC-3'-3'-h-5 '23 nucí. 2 blocked 93 5'-TCCATGACGTTCCTGATGC-3'-3'-i-5 '27 nucí. 2 blocked 94 5'-TCCATGACGTTCCTGATGC-3'-3'-j-5 '38 nucí. 2 blocked 95 b-5'-TCCATGACGTTCCTGATGC-3 '19 nucí. blocked 1 96 3'-c-5'-5'-TCCATGACGTTCCTGATGC-3 '20 nuci. blocked 2 97 3'-d-5'-5'-TCCATGACGTTCCTGATGC-3 '23 nucí. blocked 2 98 3'-e-5'-5'-TCCATGACGTTCCTGATGC-3 '27 nuc !. blocked 2 99 3'-f-5'-5'-TCCATGACGTTCCTGATGC-3 '38 nuc !. blocked 2 100 5'-TCCATGACGTTCCTGATGC-3'-k 19 nucí. 1 blocked 101 l-5'-TCCATGACGTTCCTGATGC-3 * 19 nucí. blocked 1 a: See diagram I for chemical structures b-I; the 5'-CG-3 'dinucleotide is underlined DIAGRAM 1 TABLE 16 Induction of secretion of IL-12 and IL-6 icon conjugates of CpG DNA in cultures of mouse spleen cells BALB / c DNA IL-12 (pg / mL) d: SD IL-6 (pg / mL) ± SD CpG #a 0.1 0.3 1.0 3.0 10.0 0.1 0.3 1.0 μg / mL 3.0 μg / mL 10.0 μg / mL μg / mL μg / mL μg / mL μg / mL μg / mL μg / mL μg / mL 89 991 + 121 1820 + 224 2391 ± 175 3507 ± 127 2615 ± 279 652 ± 48 2858 ± 180 13320 + 960 18625 ± 1504 17229 ± 1750 90 526 ± 32 2100 ± 175 1499 ± 191 3019 ± 35 3489 ± 162 1387 ± 152 1426 ± 124 5420 ± 370 19096 ± 484 19381 ± 2313 91 1030 ± 11 1348 ± 102 2060 ± 130 3330 ± 130 3582 ± 259 923 ± 22 2542 ± 81 9054 ± 120 14114 ± 179 13693 ± 264 92 1119 ± 159 1726 ± 207 2434 ± 100 2966 ± 204 3215 ± 464 870 ± 146 1905 ± 56 7841 ± 350 17146 ± 1246 15713 ± 693 ND 93 1175 ± 68 2246 ± 124 1812 ± 75 2388 + 320 2545 + 202 1152 ± 238 3499 ± 116 7142 ± 467 14064 ± 167 13566 ± 477 94 1087 ± 121 1705 ± 163 1797 ± 141 2522 ± 195 3054 ± 103 1039 + 105 2043 ± 157 4848 ± 288 15527 ± 224 21021 ± 1427 95 1173 ± 107 2170 ± 155 2132 + 58 2812 + 203 3689 ± 94 807 ± 0.5 927 ± 0.5 3344 ± 0.5 10233 ± 0.5 9213 ± 0.5 96 866 ± 51 1564 ± 63 1525 ± 63 2666 ± 97 4030 ± 165 750 ± 63 1643 ± 30 5559 ± 415 11549 ± 251 11060 ± 651 97 227 ± 3 495 ± 96 1007 ± 68 897 ± 15 1355 ± 97 302 ± 18 374 ± 22 1000 ± 68 9106 +271 13077 ± 381 98 139 ± 18 211 + 12 452 ± 22 458 + 29 1178 ± 237 220 ± 23 235 ± 18 383 ± 35 1706 ± 33 11530 ± 254 99 181 ± 85 282 ± 105 846 ± 165 2082 ± 185 3185 ± 63 467 ± 122 437 ± 85 1697 ± 283 9781 + 13 11213 ± 294 Medium 86 + 6 60 ± 12 a: See table 1 for the sequences Taken together, these results suggest that an accessible 5 'end of CpG DNA is required for optimal immunostimulatory activity, and that smaller groups such as a phosphorothioate, a mononucleotide or a dinucleotide, do not effectively block the accessibility of the 5' end of the CpG DNA to receptors or factors involved in the immunostimulatory pathway. However, conjugation of large molecules such as fluorescein or larger molecules at the 5 'end of CpG DNA could abolish immunostimulatory activity. These results have a direct impact on the studies of the immunostimulatory activity of the CpG DNA / vaccine / monoclonal antibody (mAb) antigen conjugates. Conjugation of large molecules such as vaccines or mAbs at the 5 'end of a CpG DNA could result in a suboptimal immunostimulatory activity of the CpG DNA. The conjugation of functional ligands at the 3 'end of the CpG DNA not only contributes to greater nuclease stability, but also to a greater immunostimulatory potency of the CpG DNA in vivo.

EXAMPLE 12 Effect of linkers on cytokine secretion For this study, the oligonucleotides given below were synthesized. Each of these modified oligonucleotides can be incorporated into an immunomer.

TABLE 17 CpG DNA sequences showing the position of the substitution CpG DNA Sequence (5 '- »3') to #? 02 CCTACTAGCGTTCTCATC 103 CCTACTAGC2TTCTCATC 104 CCTACT2GCGTTCTCATC 105 CCTA2TAGCGTTCTCATC 106 CCT22TAGCGTTCTCATC 107 22TACTAGCGTTCTCATC 108 CCTACTAGCGT2CTCATC 109 CCTACTAGCGTTC2CATC 110 CCTACTAGCGTTC22ATC 111 CCT6CTAGCGTTCTCATC 112 CCTACTAGCGTTC6CATC 113 CCT7CTAGCGTTCTCATC 114 CCTACTAGCGTTC7CATC 4 CTATCTGACGTTCTCTGT 115 CTAT1 TGACGTTCTCTGT 116 CTA1 CTGACGTTCTCTGT 117 CTATCTG2CGTTCTCTGT 118 CTATC2GACGTTCTCTGT 119 CTA2CTGACGTTCTCTGT 120 22222TGACGTTCTCTGT 121 2222TGACGTTCTCTGT 122 222TGACGTTCTCTGT 123 22TGACGTTCTCTGT 124 2TGACGTTCTCTGT 125 CTAT3TGACGTTCTCTGT 126 CTA3CTGACGTTCTCTGT 127 CTA33TGACGTTCTCTGT 128 33TGACGTTCTCTGT 129 CTAT4TGACGTTCTCTGT 130 CTA4CTGACGTTCTCTGT 131 CTA44TGACGTTCTCTGT 132 44TGACGTTCTCTGT 133 CTAT5TGACGTTCTCTGT 134 CTA5CTGACGTTCTCTGT 135 CTA55TGACGTTCTCTGT 136 55TGACGTTCTCTGT TABLE 17 (Continued) CpG DNA Sequence (5 '- ^ 3')? A # 37 CTA6CTGACGTTCTCTGT 138 CTATCTGACGTTC6CTGT 139 CTA7CTGACGTTCTCTGT 140 CTATCTGACGTTC7CTGT 141 CTATCTG8CGTTCTCTGT 142 CTATCT8ACGTTCTCTGT 143 CTATC8GACGTTCTCTGT 144 CTAT8TGACGTTCTCTGT 145 CTA8CTGACGTTCTCTGT 146 CTATCTGACG8TCTCTGT 147 CTATCTGACGT8CTCTGT 148 CTATCTGACGTT8TCTGT 149 CTATCTGACGTTC8CTGT 150 CTATCTG9CGTTCTCTGT 151 CTATCT9ACGTTCTCTGT 152 CTA9CTGACGTTCTCTGT 153 CTATCTGACGT9CTCTGT 154 CTATCTGACGTTC9CTGT a: See figure 14 for the chemical structures of substitutions 1-9. All CpG's DNAs are modified in the main chain with phosphorothioate To evaluate the optimal size of the linker to enhance the immunostimulatory activity, the secretion of IL-12 and IL-6 induced by modified CpG DNAs was measured in cultures of BALB / c mouse spleen cells. All CpG DNAs induced concentration-dependent IL-12 and IL-6 secretion. Figures 15A-15B show the data obtained at a concentration of 1 μg / mL of the selected CpG's DNAs, 116, 119, 126, 130 and 134, which had a linker in the fifth nucleotide position in the flanking sequence 5 'for the CpG dinucleotide, compared to the original CpG DNA. The CpG's DNAs, which contain the linkers of C2 (1), C3 (2) and C4 (3), induced a secretion of IL-12 similar to that of the original CpG DNA 4. The CpG DNA containing linkers of C6 and C9 (4 and 5) in the fifth nucleotide position of the CpG dinucleotide in the 5 'flanking sequence. induced a lower degree of IL-12 secretion than the original CpG DNA (Figures 15A-15B), suggesting that substitution with linkers larger than a C4 linker results in the induction of lower concentrations of IL-12. The five CpG's DNAs that had linkers induced two to three times more IL-6 secretion than the original CpG DNA. The presence of a linker in these CpG's DNAs showed a significant effect on the induction of IL-6 compared to CpG's DNAs that did not have a linker. However, the present authors did not observe an effect of the linker on the secretion of IL-6 dependent on length. To examine the effect of CpG DNA containing ethylene glycol linkers on the immunostimulatory activity, the CpG's 137 and 138 DNAs were synthesized, in which a triethylene glycol linker (6) was incorporated in the fifth nucleotide position in the 5 'flanking sequence. , and in the fourth nucleotide position in the 3 'flanking sequence of the CpG dinucleotide, respectively. Similarly, CpG's 139 and 140 DNAs contained a hexaethylene glycol linker (7) in the 5 'or 3' flanking sequence of the CpG dinucleotide, respectively. The four CpG DNAs (137-140) were tested in cultures of BALB / c mouse spleen cells to determine the induction of cytokine (IL-12, IL-6 and IL-10) compared to the original CpG DNA 4. All CpG DNAs induced a concentration-dependent cytokine production on the scale of concentrations tested (0.03-10.0 μg / mL, data not shown). Table 18 shows the concentrations of cytokines induced at a concentration of 0.3 μg / mL of the CpG's DNA 137-140. CpG DNAs 137 and 139, which had an ethylene glycol linker in the 5 'flanking sequence, induced higher secretion of IL-12 (2106 ± 143 and 2066 ± 153 pg / mL) and IL-6 (2362 ± 166 and 2507 ± 66 pg / mL) than the original CpG DNA 4 (Table 18). At the same concentration, 137 and 139 induced a slightly lower IL-10 secretion than the original CpG DNA (Table 18). The CpG 138 DNA, which had a shorter ethylene glycol linker (6) in the 3 'flanking sequence, induced a secretion of IL-12 similar to that of the original CpG DNA, but significantly lower concentrations of IL-6 and IL-10. (table 18). The CpG 140 DNA, which had a larger ethylene glycol linker (7), induced significantly lower concentrations of the three cytokines compared to the original CpG DNA (Table 18). Although the triethylene glycol linker (6) has a chain length similar to the C9 linker (5), the CpG DNA containing the triethylene glycol linker has a better immunostimulatory activity than the CpG DNA containing the C9 linker, determined by induction of cytokine secretion in spleen cell cultures. These results suggest that the lower immunostimulatory activity of CpG DNA containing larger alkyl linkers (4 and 5) may not be related to its greater length, but to its hydrophobic characteristics. This observation induced the present authors to examine the substitution of branched alkyl linkers containing hydrophilic functional groups on the immunostimulatory activity.

TABLE 18 Secretion of Cytokine Induced by CpG's DNA Containing an Ethylene Glycol Linker in Spleen Cell Cultures of BALB / c Mice DNA Cytokine, pg / m CpG # IL-12 IL-6 IL-10 4 1887 ± 233 2130 ± 221 86 ± 14 137 21061143 2362 ± 166 78 ± 21 138 1888 ± 259 1082 ± 25 47 ± 14 139 2066 ± 153 2507 ± 66 73 ± 17 140 1318 ± 162 476 ± 13 25 + 5 Medium 84 ± 13 33 ± 6 2 ± 1 To test the effect on the immunostimulatory activity of CpG DNA containing branched alkyl linkers, two branched alkyl linkers containing a hydroxyl (8) or amino (9) functional group were incorporated into the original CpG DNA 4, and the effects on the immunostimulatory activity of the resulting CpG's DNA (150-154 in table 19). Table 19 shows the data obtained from CpG's DNA 150-154, which contain the amino 9 linker at different nucleotide positions, in cultures of BALB / c mouse spleen cells (proliferation) and in vivo (splenomegaly).

TABLE 19 Proliferation of Spleen Cells Induced by CpG DNA Containing an Aminobutyryl-propanediol Linker in Spleen Cell Cultures of BALB / c Mice, and Splenomegaly in BALB / c Mice The original CpG DNA 4 showed a proliferation index of 3,710.8 at a concentration of 0.1 μg / mL. At the same concentration, the modified CpG's DNAs 151-154, which contain the amino 9 linker at different positions, caused a greater proliferation of spleen cells than the original CpG DNA (Table 19). As observed with other linkers, when the substitution was placed adjacent to the CpG (150) dinucleotide, a lower proliferation index was observed compared to the original CpG DNA (Table 19), confirming more than the placement of an adjacent linker substitution. to the CpG dinucleotide, has a deleterious effect on immunostimulatory activity. In general, replacement of an amino linker with 2'-deoxyribonucleoside in the 5 'flanking sequence (151 and 152) resulted in a larger spleen cell proliferation than that found with the 3' flanking sequence substitution (153 and 154). Similar results were observed in the splenomegaly test (table 19), confirming the results observed in the cultures of spleen cells. The modified CpG DNAs containing a glycerol linker (8) showed immunostimulatory activity similar or slightly greater than that observed with a modified CpG DNA containing an amino linker (9) (data not shown). To compare the immunostimulatory effects of CpG DNAs containing linkers 8 and 9, the present authors selected CpG's 145 and 152 DNAs, which had a substitution in the 5 'flanking sequence, and tested their ability to induce the secretion of IL cytokines. -12 and IL-6 in cultures of BALB / c mouse spleen cells. The two CpG's 145 and 152 DNAs induced concentration-dependent cytokine secretion. Figure 4 shows the concentrations of IL-12 and IL-6 induced by 145 and 152 in cultures of mouse spleen cells at a concentration of 0.3 μg / mL, compared to the original CpG DNA 4. Both CpG DNAs induced concentrations of IL-12 and IL-6 greater than the original CpG DNA 4. A CpG DNA containing a glycerol linker (8) induced concentrations of cytokines (especially IL-12) slightly higher than a CpG DNA containing an amino linker (9) (Figures 16A-16B). These results further confirm that linkers containing hydrophilic groups are more favorable for the immunostimulatory activity of CpG DNA. The present authors examined two different aspects of multiple linker substitutions in CpG DNA. In one group of experiments, the length of the nucleotide sequence was maintained at 13 nucleotides, and one to five substitutions of C3 (2) linker at the 5 'end (120-124) were incorporated. These modified CpG's DNA allowed to study the effect of an increase in the length of the linkers without causing problems of solubility. In the second group of experiments two of the same linker substitutions were incorporated (3, 4 or 5) at adjacent positions in the 5 'flanking sequence of the CpG dinucleotide, to study if there is any additive effect on the immunostimulatory activity. We studied the ability of modified CpG DNAs to induce cytokine production in cultures of BALB / c mouse spleen cells, compared to original CpG DNA 4. All CpG's DNA induced concentration-dependent cytokine production. Table 20 shows the data obtained at a concentration of 1.0 μg / mL of CpG DNA. In this test, the CpG 4 DNA induced the secretion of 967128 pg / ml of IL-12, 1593194 pg / ml of IL-6, and 1416 pg / ml of IL-10, at a concentration of 1 μg / ml. The data presented in Table 20 suggest that as the number of linker substitutions decreases, the induction of IL-12 decreases. However, the induction of a lower secretion of IL-12 by the CpG's 123 and 124 DNA could be the result of the shorter length of the CpG's DNA. The present studies with unmodified CpG DNA of less than 15 nucleotides showed negligible immunostimulatory activity (data not shown). Neither the length nor the number of linker substitutions had a minor effect on the secretion of IL-6. Although the secretion of IL-10 increased with the linker substitutions, the overall secretion of IL-10 by these CpG DNAs was minimal. The ability to induce cytokine secretion in cultures of BALB / c mouse spleen cells, CpG DNAs containing two linker substitutions (linker 3 -127) was tested; linker 4 -131; linker 5 -135) in the fourth and fifth positions in the 5 'flanking sequences of the CpG dinucleotide, and the corresponding 5'-truncated versions 128, 132 and 136, respectively. The amounts of IL-12 and IL-6 secreted at a concentration of 1.0 μg / mL are shown in Figures 17A-17B. The results presented in Figures 17A-17B suggest that the immunostimulatory activity depends on the nature of the incorporated linker. Substitution of the fourth and fifth nucleoside with the C4 3 linker (CpG 127 DNA) had a significant effect on cytokine secretion compared to the original CpG DNA 4, suggesting that the nucleobase and the sugar ring at these positions are not necessary for the recognition of the receiver or their union. The deletion of the nucleotides beyond the substitutions of the linker (CpG 128 DNA) caused a greater secretion of IL-12 and IL-6 than that found with the CpG's DNAs 4 and 127. As expected, the substitution of two linkers of C6 (4) resulted in a lower IL-12 secretion and an IL-6 secretion similar to that induced by the original CpG DNA 4. The 5'-truncated CpG DNA 132 induced greater cytokine secretion than the CpG DNA 131. The CpG's 135 and 136 DNA, which had two C9 (5) linkers, induced negligible cytokine secretion, confirming the results obtained with monosubstituted CpG DNA containing the same linker described above.

EXAMPLE 13 Effect of phosphodiester bonds on the induction of cytokine To test the effect of phosphodiester linkages on the induction of cytokine induced by immunomer, the following molecules were synthesized.

TABLE 20 PO immunomer sequences and analytical data a The arrows indicate the 5'-3 'directionality of the CpG dinucleotide in each DNA molecule, and the structures of X and Y are shown in boxes. b PS and PO represent main chains of phosphorothioate and phosphodiester, respectively. c Determined by MALDI-TOF mass spectrometry.

The PS-DNA CpG 4 (Table 20) was found to induce an immune response in the mice (data not shown), CpG 155 PO-DNA serving as control. PO-immunomers 156 and 157 contain two identical truncated copies of the DNA Original CpG 155 linked through its 3 'ends by means of a glyceryl linker, X (Table 20). Although 156 and 157 contain the same 14-base oligonucleotide segments, the 5 'ends of 157 were modified by adding two C3, Y linkers (Table 20). All oligonucleotides, 4, 155-157, contain a hexameric motif 'GACGTT which is known to activate the mouse immune system. The stability of the PO-immunomers against the nucleases was determined by incubating the CpG's 155-157 DNA at 37 ° C for 4 h, 24 h and 48 h in a cell culture medium containing 10% bovine fetal serum (FBS). (not inactivated with heat). The intact CpG DNA remaining in the reaction mixtures was then determined by means of CGE. Figures 18A-D show the nuclease digestion profiles of CpG's 4, 155-157 DNAs incubated in 10% FBS for 24 h. The amount of full-length CpG DNA remaining at each time point is shown in Figure 18E. As expected, the original CpG DNA 4 is the most resistant to serum nucleases. About 55% of oligonucleotide 4 of 18 nucleotides remained undegraded after 48 h of incubation. In contrast, only about 5% of the full-length PO-immunomer 155 remained after 4 h under the same experimental conditions, confirming that DNA containing phosphodiester bonds undergoes rapid degradation. As expected, the two PO-immunomers 156 and 157, were more resistant to serum nucleases than 155. After 4 h, about 62% and 73% of 156 and 157, respectively, were intact, compared to 5. % of 155 (figure 18E). Even after 48 h, approximately 23% and 37% of 156 and 157, respectively, remained undegrade. As these studies show that bound 3'-3 'PO-immunomers are more stable against serum nucleases, they also indicate that chemical modifications at the 5' end may further increase the stability against nuclease. The immunostimulatory activity of CpG DNAs was studied in spleen cell cultures of BALB / c and C3H / HeJ mice by measuring the secreted amounts of IL-12 and IL-6. All CpG DNAs induced a concentration-dependent cytokine secretion in cultures of BALB / c mouse spleen cells (Figures 19A-19B). At 3 μg / mL, the PS-DNA CpG 4 induced 26561256 and 1223411180 pg / mL of IL-12 and IL-6, respectively. The original CpG PO-DNA 155 did not raise the cytokine concentration above the basal level, except at a concentration of 10 μg / mL. This observation is consistent with the results of the nuclease stability test. In contrast, PO-immunomers 156 and 157 induced secretion of both IL-12 and I L-6 in cultures of BALB / c mouse spleen cells. The results presented in Figures 19A-19B show a clear distinction in the cytokine induction profiles of the PS- and PO-DNA CpG's. PO-immunomers 156 and 157 induced higher amounts of IL-12 than PS-DNA CpG 4 in cultures of BALB / c mouse spleen cells (Figure 19A). In contrast, at concentrations of up to 3 μg / mL, they produced negligible amounts of IL-6 (Figure 19B). Even at the highest concentration (10 μg / mL), the PO-immunomer 156 induced significantly less IL-6 than the PS-DNA CpG 4. The presence of C3 linkers at the 5 'end of the PO-immunomer 157 gave as resulted in a slightly higher secretion of IL-6 compared to 156.

However, importantly, the amounts of IL-6 produced by the PO-immunomer 157 are much smaller than those induced by the PS-DNA CpG 4. The insert of Figure 19A shows the ratio of IL-12 to secreted IL-6. at a concentration of 3 μg / mL. In addition to increasing the secretion of IL-12, PO-immunomers 156 and 157 induced higher amounts of IFN-α. than PS-DNA CpG 4 in cultures of BALB / c mouse spleen cells (data not shown). The different cytokine profiles induced by PO- and PS-DNA CpGs in cultures of BALB / c mouse spleen cells, prompted the present authors to study the pattern of cytokine induction of CpG DNA in cultures of mouse spleen cells C3H / HeJ (a race sensitive to lower LPS). The three CpG DNAs tested in this test induced concentration-dependent cytokine secretion (Figure 20A and 20B). Since the CpG 155 PO-DNA could not induce cytokine secretion in cultures of BALB / c mouse spleen cells, it was not further tested in cultures of C3H / HeJ spleen cells. The two PO-immunomers, 156 and 157, induced a higher IL-12 production than the PS-DNA CpG 4 (FIG. 20A). However, at concentrations up to 3 μg / mL, none induced the production of IL-6. At the highest tested concentration (10 μg / mL), both induced significantly less IL-6 than the PS-DNA CpG 4 (Figure 20B). The ratio of IL-12 to secreted IL-6 is calculated to distinguish the cytokine secretion profiles of PS- and PO-DNA CpG's (Figure 20A, insert). In addition, the results of the C3H / HeJ spleen cell culture suggest that responses with CpG DNA are not due to LPS contamination. It has been shown that CpG's PS-DNAs induce potent antitumor activity in vivo. Since the CpG's PO-DNA exhibited greater stability against nuclease and induced higher degree of secretion of IL-12 and IFN-α? in in vitro tests, the present authors were interested in seeing if these desirable properties of the PO-immunomers improve the antitumor activity in vivo. Administered PO-immunomer 157 subcutaneously at a dose of 0.5 mg / kg every third day to hairless mice carrying MCF-7 breast cancer cell tumor xenografts, expressing wild type p53, or prostate cancer cells DU-145 expressing mutated p53. PO-immunomer 157 gave 57% inhibition of growth of MCF-7 tumors on day 15, compared to saline control (FIG. 21A). It also produced 52% inhibition of growth of DU-145 tumors on day 34 (Figure 21 B). These antitumor studies suggest that the PO-immunomers of the proposed design exhibit potent antitumor activity in vivo.

EXAMPLE 14 Short Immunomers To test the effect of short immunomers on the induction of cytokine, the immunomers given below were used. These results show that immunomers as short as 4 nucleotides per segment are effective to induce cytokine production.

TABLE 21 Immunomer structure and immunostimulatory activity in cultures of BALB / c mouse spleen cells The normal letters represent a phosphorothioate link.

EXAMPLE 15 Isolation of human B cells and plasmacytoid dendritic cells (pDCs) PBMCs were isolated from freshly harvested blood from healthy volunteers (CBR Laboratories, Boston, Massachusetts) by the Ficoll density gradient centrifugation method (Histopaque-1077, Sigma), and B cells were isolated from PBMCs by positive selection using the kit. of isolation of CD19 cells (Miltenyi Biotec), according to the manufacturer's instructions.

EXAMPLE 16 B Cell Proliferation Test A total of 1 x 105 B cells / 200 μl was stimulated with concentrations of 0.3, 1.0, 3.0 or 10.0 μg / mL of CpG's DNA for 64 h, then boosted with 0.75 μCi of [3 H] -thymidine and harvested 8 h after. The incorporation of radioactivity was measured using a scintillation counter. Table 23 shows an SDI average of B cell proliferation for 6 CpG DNAs, at a final concentration of 10.0 μg / mL.

TABLE 22 Immunomer structure and immunostimulatory activity in the human B cell proliferation test (72 h) Normal letters represent a phosphorothioate bond; the underlined letters represent a 2'-0-Me ribonucleotide; the italicized letters represent a phosphodiester bond.

EXAMPLE 17 Cultures of human pDC and IFN-a ELISA PDC's were isolated from human PBMC's using a BDCA-4 cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. The pDC was cultured in 96 well plates using 1 x 106 cells / mL, 200 μUcavity). CpG DNAs were added at a final concentration of 0.3, 1.0, 3.0 or 10.0 μg / mL to cell cultures and incubated 24 h at 37 ° C. Then, the supernatants were harvested and tested for IFN-a using the ELISA kit of human IFN-a (PBL). Tables 23A-23C show an SD i average of IFN-a for 6 CpG DNAs at a concentration of 1.0 and 10.0 μg / mL.

TABLE 23A Immunomer structure and immunostimulatory activity in a human dendritic cell test (72 h) TABLE 23B Immunomer structure and immunostimulatory activity in a TABLE 23C Immunomer structure and immunostimulatory activity in a human dendritic cell test (24 h) EXAMPLE 18 Mononuclear cells were isolated from human peripheral blood (PBMCs) from the peripheral blood of healthy volunteers, and were prepared as set forth above in Example 4. Tables 24A-24C show an average ± SD of IL-6, IL-12 and IL-? for 5 IMO compounds at concentrations of 10.0 μg / mL.

TABLE 24A Immunomer structure and immunostimulatory activity in a human PBMC test (72 h) TABLE 24B Immunomer structure and immunostimulatory activity in a human PBMC test (24 h) TABLE 24C Immunomer structure and immunostimulatory activity in a human PBMC test (24 h) Only for the purposes of Tables 22, 23A-23C and 24A-24C: Normal letters represent a phosphorothioate link; the underlined letters represent a 2'-0-Me-ribonucleotide; the italicized letters represent a phosphodiester bond, and R, R ^ X and X are as defined below: EXAMPLE 19 Studies in spleen cells BALB / c mice 5-6 weeks old were obtained from Taconic Farms (Germantown, New York) and maintained on an OVA-free diet. Groups of five mice were used in the immunization study. Compounds I O (Figure 22) were synthesized, purified and analyzed as described above. Each mouse was sensitized by subcutaneous (sc) injection of 10 μg of chicken ovalbumin (OVA, Grade V, Sigma, St. Louis, Missouri), in 100 μl of PBS mixed with an equal volume of alum solution (Imject-Alum , Pierce, Rockford, Illinois), on days 0 and 14, and were stimulated (in) with 20 μg of OVA in 40 μl of PBS on days 28, 29 and 30 (figure 2). The IMO's 1 and 2 (30 or 60 μg) dissolved in 200 μl of PBS were injected s.c. to mice on days 33, 37, 40 and 43. Blood samples were taken from the mice under anesthesia 2 h after the first injection of an IMO compound on day 33 by retro-orbital puncture, and the serum was collected for the tests of cytokine Each mouse was stimulated i.n. with 10 μg of OVA in 40 μl of PBS on day 44. The mice were bled and their lungs and spleens were removed 24 h after the last OVA stimulation. Single-spleen cell suspensions were prepared in cold RPMI 1640 medium (Sigma), and concentrated for each experimental group at 5 x 106 cells / ml in RPMI 1640 medium containing 10% FCS (HyClone, Logan, Utah), 100 μg / ml penicillin and 100 U / ml streptomycin (HyClone). Spleen cells (0.2 ml / well) were incubated at 37 ° C in a 5% C02 atmosphere in 96-well flat bottom culture dishes (Costar, Cambridge, Massachusetts), in the presence of 100 μg / ml of OVA After 72 h of incubation, culture supernatants were harvested for cytokine tests. The concentrations of IL-5, IL-10, IL-12 and IFN-Y were determined by means of an enzyme-linked immunosorbent assay (ELISA), with mouse antibodies from BD Biosciences (San Diego, California). The concentrations of IL-6 and IL-13 were determined with ELISA kits for DuoSet mouse (R & amp; amp;; D System, Minneapolis, Minnesota), following the manufacturer's instructions. The total and specific IgA and IgG2a concentrations of OVA were evaluated in the mouse serum by ELISA. For the detection of OVA-specific Ig, 96-well ELISA plates (Immulon 2, Dynatech, Chantilly, Virginia) were coated with 10 μg / ml OVA in PBS, pH 9.6. For the detection of total Ig, the plates were coated with 1 μg / ml of mouse anti-IgE (clone R35-72) and 1 μg / ml of mouse anti-IgG2a (clone R11-89). After incubating overnight at 4 ° C, the plates were blocked with 1% BSA / PBS, pH 7.4, at room temperature for 1 h. Serial dilutions of serum were added to the plates (1: 10 for IgE, 1: 100 for IgG2a), and incubated at room temperature for 2 h. The plates were washed and added 100 μl / well of biotinylated mouse anti-IgE (clone R35-116) at 0.25 μg / ml, or IgG2a (clone R19-15) at 0.25 μg / ml, and incubated room temperature for 2 h. All antibodies were obtained from BD Biosciences. The plates were washed and 100 μl / streptavidin-peroxidase cavity (Sigma) 0.25 μg / ml was added to incubate 1 h at room temperature. The tests were run in 100 μl / cavity of TMB substrate solution (KPL, Gaithersburg, Maryland), followed by 100 μl / cavity of Stop Solution (KPL). A50 (equal to OD450?) Was measured using a microplate reader (Bio-Tek Instruments, Inc. Winooski, Vermont), and the data was analyzed with KC Junior software (High Point, North Carolina) (see figure 24).

EXAMPLE 20 Histology of the lung Lungs removed on day 45 were fixed in neutral formalin and sent to the Mass Histology Service (Warwick, Rhode Island) for processing and staining with hematoxyphyllin and eosin (HE), and periodic acid-Schiff (PAS). The sections of lung tissue were observed under an optical microscope and photographed with a digital camera. A statistical analysis was made using an analysis of variance (ANOVA). The groups immunized with OVA and treated with IMO were compared using Student's t-test. The results were expressed as the mean ± SEM. All comparisons were two-tailed and statistical significance of * p < 0.05.

EXAMPLE 21 Study in prevention model The ability of IMO compounds to prevent allergic inflammation induced by OVA was examined. Each mouse was sensitized by subcutaneous (sc) injection of 20 μg of chicken ovalbumin (OVA, Grade V, Sigma, St. Louis Missouri) in 100 μl of PBS mixed with an equal volume of alum solution (Imject-Alum, Pierce, Rockford, Illinois), on days 0, 7 and 14. The group of unaffected mice received only one injection of alum. The IMO compounds (10 μg) dissolved in the OVA / alum mixture were administered to the mice on days 0, 7 and 14. Fourteen days after the last immunization, the mice anesthetized with isoflurane (Abbott Laboratories, North Chicago, Illinois), they were bled by retro-orbital puncture and then sacrificed to remove the spleens.

EXAMPLE 22 Effect of the IMO compounds on the inhibition of the Th2 cytokines IL-4, IL-5, IL-12 and IL-13, in an immune response of antigen-specific recall To determine the ability of the IMO compounds to alter the Th2-dominant immune response in mice injected with OVA / alum, IMO compounds together with OVA / alum were injected on days 0 and 14. Spleen cells were isolated from the mice on the day 28 and were incubated with OVA for 72 h (recall of antigen). Spleen cells from mice injected with OVA / alum alone produced larger amounts of Th2-associated cytokines, such as IL-4 (~ 2-fold), IL-5 (130-fold) and IL-13 (28-fold), which the unaffected mice (see Figures 23A1-23B5). Spleen cells from mice that received CpG DNA or IMO compounds and OVA secreted significantly less of these cytokines, particularly IL-5 and IL-13; IL-5 decreased from 20% to 97% and IL-13 decreased from 60% to 95%. These results suggest that the IMO compounds have an inhibitory effect on the secretion of cytokine associated with Th2.

EXAMPLE 23 Effect of IMO compounds on the production of IFN-v in the immune response of antigen-specific recall In the same antigen recall experiments, the spleen cells of the mice injected with OVA / alum and IMO compounds, produced significantly greater amounts of the Th1 IFN-α cytokine. cells from mice not affected or injected with OVA / alum (see Figures 23A1-23B5). Spleen cells from mice injected with OVA / alum and conventional CpG 1 DNA produced amounts of IFN-α. comparable with those produced by unaffected spleen cells. These results demonstrate that the treatment of mice injected with OVA / alum with the IMO compounds can change the Th2-dominant immune response to a predominantly Th1 type, as reflected by the cytokines produced by spleen cells.

EXAMPLE 24 Effect of IMO compounds on total and specific OVA IgE production To determine the effects of the IMO compounds on the production of IgE, serum concentrations of total IgE and specific OVA were examined, 14 days after the last OVA / alum injection.

Sensitization with OVA / alum resulted in a higher production of OVA-specific IgE; OVA-specific IgE concentrations were significantly lower in the mice receiving the IMO compounds together with OVA / alum (compared to those found in the unaffected mice, see Figures 24A1-24B5). Total serum IgE concentrations were also low in mice injected with OVA / alum and the IMO compounds.

EXAMPLE 25 Effect of the IMO compounds on the production of total and specific IgA lgG1 and IgG2a To determine the effect of the IMO compounds on the production of IgG1 and IgG2a, serum concentrations of IgG1 and IgG2a were examined in total and OVA specific. Animals that received OVA / alum injections produced high concentrations of OVA-specific IgG1 and negligible concentrations of OVA-specific IgG2a (see Figures 24A1-24B5). The injection of IMO compounds to the mice had no effect or reduced the IgG-specific IgG1 concentrations compared to the concentrations found in mice injected with OVA / alum. The concentrations of OVA-specific IgG2a antibody in the sera of mice receiving IMO compounds were significantly higher than in those receiving only OVA / alum. These concentrations of IgG1 and IgG2a result in higher IgA-specific IgG2a / IgG1 ratios in mice injected with OVA / alum plus IMO compounds, than in mice that received only OVA / alum. Similar results were found in the production of total serum Ig. Serum from mice injected with OVA / alum and IMO compounds had lower total lgG1 concentrations and higher total lgG2a concentrations than mice that received only OVA / alum.

EXAMPLE 26 Study in therapeutic model The therapeutic potential of IMO'S 1 and 2 was tested in a mouse asthma model. Mice were sensitized and stimulated with OVA and treated with IMO's 1 and 2 as described in the protocol above. To determine the effects of treatment with IMO's 1 and 2 on the local immune response, the histology of the lungs of unaffected mice and mice sensitized and stimulated with OVA / alum, with or without treatment with IMO's 1 and 2, was examined. 48 h after the last injection of the IMO's 1 and 2. The mice sensitized and stimulated with OVA that were not treated with the IMO's 1 and 2, showed a severe infiltration of inflammatory cells and epithelial hyperplasia of the respiratory tract, in comparison with the unaffected mice (data not shown). In contrast, mice treated with IMO's 1 and 2 had less infiltration of inflammatory cells and less hyperplasia of the airways than untreated mice (data not shown). These results demonstrate the ability of IMO's 1 and 2 to revert OVA-induced lung inflammation in mice sensitized and stimulated with OVA.

EXAMPLE 27 Effect of treatment with IMO's 1 and 2 on Th2 and Th1 cytokines in the immune response of antigen-specific recall To determine the ability of IMO's 1 and 2 to reverse the Th2-dominant immune responses in mice sensitized and stimulated with OVA, the present authors isolated spleen cells from the mice on day 45 and incubated them with OVA for 72 h. After restimulation of the spleen cells with OVA, marked differences were observed in the production of Th2 cytokines (IL-5 and IL-13) between the treatment groups. Spleen cells from mice injected with OVA alone produced high concentrations of Th2-associated cytokines (Figures 25A-25C). Mice treated with IMO's 1 and 2 produced significantly less O2-induced Th2 cytokines (Figures 25A-25C). In the antigen recall experiments, only low concentrations of IL-12 and IFN-α were induced. in cultures of spleen cells of unaffected mice and of mice sensitized and stimulated with OVA / alum. Spleen cells from mice treated with IMO's 1 and 2 after stimulation with OVA, produced significantly higher concentrations of IFN-α. (Figure 25A-25C).

EXAMPLE 28 Effect of the treatment with the IMO's 1 and 2 on the production of total serum IgE and specific OVA in mice sensitized and stimulated with OVA To determine the effects of IMO's 1 and 2 on IgE production, the present authors examined serum concentrations of total and specific OVA IgE. Mice that were sensitized and stimulated with OVA but were not treated with IMO's 1 and 2, had high concentrations of OVA-specific IgE in the serum (Figures 26A-26D), while mice treated with IMO's 1 and 2 had significantly lower concentrations of OVA-specific IgE. Serum total IgE levels were also low in the mice that received treatment with IMO's 1 and 2.

EXAMPLE 29 Effect of the treatment with the IMO's 1 and 2 on the production of total and specific IgG2a of OVA in mice sensitized and stimulated with OVA / alum To determine the effect of the treatment with the IO's 1 and 2 on the production of IgG2a, the present authors examined the serum concentrations of total and specific IgA2 IgG2a. Without treatment with the IMO's 1 and 2, the mice sensitized and stimulated with OVA had negligible concentrations of OVA-specific IgG2a (Figures 26A-26D). The mice sensitized and stimulated with OVA and treated with the IMO's 1 and 2 had an increase in the concentrations of IgA-specific IgG2a antibody (FIGS. 26A-26D). Similar results were found for the production of total Ig (Figures 26A-26D): the mice sensitized and stimulated with OVA and treated with the IMO's 1 and 2, had higher total IgG2a concentrations than the mice that were not treated with the IMO's 1 and 2.

EXAMPLE 30 Effect of a single high dose against lower multiple doses of IMO compounds on local and systemic concentrations of Th1 cytokine in unaffected mice To determine the effects of the dose on the treatment with IMO's, mice were treated intranasally with 33 μg of IMO 1 or 3, on days 1, 2 and 3, or treated with a single intranasal administration of 100 μg of IMO 1 or 3 The mice were bled and their lungs were removed 5 hours after the treatment. A single dose of 100 μg induced higher systemic cytokine responses; however, the three smaller doses (3 x 33 μg) induced larger local cytokine responses (BALF) (Figures 27A-27F). To determine the dose-dependent effects of low multiple administrations of the IMO compounds on local and systemic cytokine concentrations in unaffected mice, mice were treated intranasally with 2.5 μg, 10.0 μg or 40.0 μg of IMO 1 on days 1, 2 and 3. The mice were bled and their lungs were removed 5 hours after the last treatment. The IMO compounds increased local cytokine concentrations (BALF) but not the systemic cytokine concentrations in the mice when small doses were administered multiple times (Figures 28A-28D). This effect was dose dependent.

EXAMPLE 31 Comparison of the effects of IMO compounds and a corticosteroid in vitro Mice were sensitized by intraperitoneal (ip) injection of 10 mg of OVA pm on days 0 and 14, and were stimulated intranasally with 10 μg of OVA in 40 μl of PBS on day 28. Spleen cells were harvested on day 30 and they were incubated for 72 hours with 100 μg / ml of OVA with or without 1 μg / ml to 10 μg / ml of IMO compounds or budesonide. Both IMO 1 and budesonide suppressed the secretion of Th2 cytokine induced by OVA (IL-5, ILB) (Figures 29A-29D). However, only IMO 1 showed strong induction of Th1 cytokine (IL-12, FN-8). Although the invention has been described in some detail for purposes of clarity and understanding, the person skilled in the art will appreciate, from a reading of this description, that various changes can be made in form and detail without departing from the true scope of the invention and the appended claims.

Claims (42)

NOVELTY OF THE INVENTION CLAIMS

1. An immunostimulatory oligonucleotide immunomer comprising the sequence of SEQ ID NO: 170.

2. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunogen of claim 1, and further comprising a costimulatory molecule selected from the group consisting of cytokines, chemokines, ligands. of protein, transactivation factors, peptides, and peptides comprising a modified amino acid.

3. The immunomodulatory composition according to claim 2, further characterized in that the costimulatory molecule is conjugated to the immunomodulatory oligonucleotide.

4. The immunomodulatory composition according to claim 2, further characterized in that it additionally comprises an adjuvant.

5. The immunomodulatory composition according to claim 2, further characterized in that it additionally comprises a pharmaceutically acceptable carrier.

6. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer of claim 1, and further comprising an antigen.

7. The immunomodulatory composition according to claim 6, further characterized in that the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins, polysaccharides and lipids.

8. The immunomodulatory composition according to claim 6, further characterized in that the antigen is an allergen.

9. The immunomodulatory composition according to claim 6, further characterized in that it additionally comprises an adjuvant.

10. The immunomodulatory composition according to claim 6, further characterized in that it additionally comprises a pharmaceutically acceptable carrier.

11. An immunostimulatory oligonucleotide immunomer comprising the sequence of SEQ ID NO: 171.

12. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomodulator of claim 11, and further comprising a costimulatory molecule selected from the group consisting of cytokines, chemokines, protein ligands, transactivation factors, peptides, and peptides comprising a modified amino acid.

13. The immunomodulatory composition according to claim 12, further characterized in that the costimulatory molecule is conjugated to the immunomodulatory oligonucleotide immunomer.

14. The immunomodulatory composition according to claim 12, further characterized in that it additionally comprises an adjuvant.

15. The immunomodulatory composition according to claim 12, further characterized in that it additionally comprises a pharmaceutically acceptable carrier.

16. An immunomodulatory composition comprising the immunomodulatory oligonucleotide immunomer of claim 11, and further comprising an antigen.

17. The immunomodulatory composition according to claim 16, further characterized in that the antigen is selected from the group consisting of peptides, glycoproteins, lipoproteins, polysaccharides and lipids.

18. The immunomodulatory composition according to claim 16, further characterized in that the antigen is an allergen.

19. The immunomodulatory composition according to claim 16, further characterized in that it additionally comprises an adjuvant.

20. The immunomodulatory composition according to claim 16, further characterized in that it additionally comprises a pharmaceutically acceptable carrier.

21. The use of an immunomer for the preparation of a medicament to treat a patient who has inflammation of the respiratory tract, inflammatory disorders, allergy or asthma.

22. The use as claimed in claim 21, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5 'end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5 'end and comprising an immunostimulatory dinucleotide.

23. The use as claimed in claim 21, wherein the immunostimulatory dinucleotide is selected from the group consisting of CpG, C * pG, CpG *, and C * pG *, wherein C is cytidine or 2 ' -deoxycytidine; C * is 2'-deoxythymidine, arabinocytidine, 1- (2'-deoxy-β-D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine, 2'-deoxy-2'-substituted arabinocytidine, 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycinthine, 2'-deoxy-N-4-alkyl-cytidine, 2'-deoxy-4-thiouridine or another non-natural pyrimidine nucleoside; G is guanosine or 2'-deoxyguanosine; G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxyinosine, 2'-deoxy-2'-substituted arabinoguanosine, 2'-0-substituted arabunoguanosine, or other nucleoside of non-natural purine.

24. The use as claimed in claim 21, wherein the immunomer comprises the sequence of SEQ ID NO: 170.

The use as claimed in claim 21, wherein the immunomer comprises the sequence of SEQ ID NO: 171.

26. - The use as claimed in claim 21, wherein the immunomer comprises the sequence of SEQ ID NO: 172.

27.- The use as claimed in claim 21, wherein the immunomer comprises the sequence of SEQ ID NO: 173.

28.- The use as claimed in claim 21, wherein said method also comprises administering an antigen associated with said disease or disorder.

29. The use as claimed in claim 21, wherein the immunomer or the antigen, or both, are linked to an immunogenic protein or a non-immunogenic protein.

30. The use as claimed in claim 21, wherein the medicament additionally comprises an adjuvant.

31.- The use of an immunomer to prepare a medicament for modulating an immune response in a patient who has inflammation of the respiratory tract, inflammatory disorders, allergy or asthma.

32. The use as claimed in claim 31, wherein the immune response is an immune response of Th1.

33. The use as claimed in claim 31, wherein the immune response is an immune response of Th2.

34. The use as claimed in claim 31, wherein the immunomer comprises at least two oligonucleotides linked by a non-nucleotide linker and having more than one 5 'end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5 'end and comprising an immunostimulatory dinucleotide.

35. The use as claimed in claim 31, wherein the immunostimulatory dinucleotide is selected from the group consisting of CpG, C * pG, CpG *, and C * pG *, where C is cytidine or 2 ' -deoxycytidine; C * is 2'-deoxythymidine, arabinocytidine, 1- (2'-deoxy-β-D-ribofuranosyl) -2-oxo-7-deaza-8-methyl-purine, 2'-deoxy-2'-substituted arabinocytidine, 2'-0-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N-4-alkyl-cytidine, 2'-deoxy-4-thiouridine or another unnatural pyrimidine nucleoside; G is guanosine or 2'-deoxyguanosine; G * is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxyinosine, 2'-deoxy-2'-substituted arabinoguanosine, 2'-0-substituted arabinoguanosine.

36. The use as claimed in claim 31, wherein the immunomer comprises the sequence of SEQ ID NO: 170.

37. The use as claimed in claim 31, wherein the immunomer comprises the sequence of SEQ ID NO: 171.

38.- The use as claimed in claim 31, wherein the immunomer comprises the sequence of SEQ ID NO: 172.

39.- The use as claimed in the claim 31, wherein the immunomer comprises the sequence of SEQ ID NO: 173.

40. The use as claimed in claim 31, wherein the medicament further comprises an antigen associated with said disease or disorder.

41. - The use as claimed in claim 31, wherein the immunomer or the antigen, or both, are linked to an immunogenic protein or a non-immunogenic protein.

42. The use as claimed in claim 31, wherein the medicament additionally comprises an adjuvant.