Title of Invention

A PHARMACEUTICAL COMPOSITION COMPRISING A THERAPEUTICALLY EFFECTIVE SYNERGISTIC AMOUNT OF AT LEAST ONE IMMUNOMER COMPOUND

Abstract

The invention provides optimized meincds and compositions for enhancing the immunc responce caosed by immunostimulaiory compoands used for the acarment of cisiase such as, but not limited to, treatment of cancer, auloimmune disordes, asthma, respiratory allergies, food allergies and infections diseases in a patienL The optimized methods according to the invention provide synergy berween the therapentic effects of immtmostimulatory oligonucleotides and immunomer compounds in accordancc with the invention, and the therapeutic effect of cytokire immunothcrapy and/or chemothcrapeulic agents and/or radiation.

Full Text

Related Applications This application claims the benefit of U.S. Provisional Application No. 60/487,529, filed July 15, 2003, and U.S. Provisional Application No. 60/503,242, September 15, 2003, which are incorporated by reference in their entirety. 10 BACKGROUND OF THE INVENTION. Field of the Invention The invention relates to the use of immunomer compounds and immunostimulatory oligonucleotides as therapeutic agents. Summary of the Related Ar: 15 Recently, several researcher s 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 developing these compounds as a therapeutic tool. These efforts haw focused on phosphorothioate oligonucleotides 20containing the natural dinucleotide CpG. Kuramoto et al.,Jpn. J. Cancer Res. 83:1128-1131 (1992) teaches that p hosphodiester oligonucleotides containing a palindrome that includes a CpG dir ucleotide can induce interferon-alpha and gamma synthesis and enhance natural killer activity. Krieg et al., Nature 371:546-549 (1995) discloses that phosphorothioate CpG-containing oligonucleotides are 25immunostimulatory. Liang et al.,J Clin. Invest. 98:1119-1129 (1996) discloses that such oligonucleotides activate human B cells. Moldoveanu et al., Vaccine 16:1216- 124 (1998) teaches that CpG-containing phosphorothioate oligonucleotides enhance immune response against influenza virus. McCluskie and Davis, J. Immunol. 161:4463-4466 (1998) teaches that CpG-containing oligonucleotides act as potent adjuvants, enhancing immune response against hepatitis B surface antigen. Other modifications of CpG-containing phosphorothioate oligonucleotides can also affect their ability to act as modulators of immune response. See, e.g., Zhao et 5al., Biochem. Pharmacol. (1996) 51:173-182; Zhao et al., Biochem Pharmacol. (1996) 52:1537-1544; Zhao et al., Antisense Nucleic Acid Drug Dev. (1997) 7:495- 502; Zhao et al., Bioorg. Med. Chem. Lett. (1999) 9:3453-3458; Zhao et al., Bioorg. Med. Chem. Lett. (2000) 10:1051-1054; Yu et al., Bioorg. Med. Chem. Lett. (2000) 10:2585-2588; Yu et al., Bioorg. Med. Chem. Lett. (2001) 11:2263-2267; and lOKandimalla et al., Bioorg. Med. Chem. (2001) 9:807-813. US Patent No. 6,426,334 shows the promise of these compounds as anti-cancer agents. Another means by which at immune response may be modulated is through the therapeutic use of cytokines. Cytokines are soluble molecules that cells of the immune system produce to control reactions between other cells. Thus, cytokines are 15regulators of humoral and cellular mmunity. An understanding of how T cells undiate the immune response is cr tical in order to modulate the response. CD4+ T helper (Th) cells differentiate along; either the Thl or Th2 pathway. The Thl pathway is important for the generation of cell-mediated immunity and is characterized by the production of, for example, • inter eron and interleukin-2 (IL-2). The Th2 response is 20important for the generation of humoral immunity and is characterized by the production of, for example, IL-4 ar d IL-5. The Thl response is known to be critical for immune system defense againsi infections, e.g., viral infections, and immune system surveillance of the body for the removal of neoplastic cells. Krieg, A., M. et al. (U.S. Patent No. 6,429,199) and Krieg, A., M. et al. (U.S. 25Patent No. 6,218,371) purport to teach the co-administration of immunostimulatory CpG oligonucleotides and cytokines, particularly GM-CSF. Decker et al. {Experimental Hematology 28:558 565 (2000)), demonstrate that the co-adminstration of IL-2 with CpG oligonucleotides increases TNF- and IL-6 production in B-chronic lymphocytic (B-CLL) cells but not in normal B-cells. These reports make clear that there remains a need to be able to further optimize the therapeutic effectiveness of immunostimulatory oligonucleotides for the treatment of disease and enhance the anticancer activity of immunostimulatory oligonucleotides. BRIEF SI MMARY OF THE INVENTION The invention provides opt mized methods, compositions and treatment regimens for enhancing the immune response caused by immunostimulatory compounds used for the treatment of disease such as, but not limited to, treatment of 5cancer, autoimmune disorders, asthma, respiratory allergies, food allergies and infectious diseases in a patient. The optimized methods according to the invention provide synergy between the therapeutic effects of immunostimulatory oligonucleotides in accordance with the invention, and the therapeutic effect of cytokine immunotherapy and/or chemotherapeutic agents. Modification of an 10immunostimulatory oligonucleotide to optimally present 5' ends dramatically enhances its anti-cancer activity. S uch an oligonucleotide is referred to herein as an "immunomer", which may contain one or more immunostimulatory oligonucleotide. In a first aspect, therefore, the invention provides methods for treating cancer in a cancer patient comprising adn inistering to the patient an immunostimulatory 15oligonucleotide and/or immunomer compound in combination with a chemotherapeutic agent, wherein the immunostimulatory oligonucleotide and/or immunomer compound and the chemotherapeutic agent create a synergistic therapeutic effect. In a further aspect, the invention provides a method for synergistically 20stimulating an immune response it; a patient. The method comprises administering to a patient a combination of a therapeutically effective synergistic amount of at least one immunomer compound or immunostimulatory oligonucleotide in accordance with the invention and a therapeutically effective synergistic amount of IL-2 (and/or an agent that induces IL-2 production in situ, such as a DNA vaccine or expression 25vector expressing IL-2), wherein administration of said combination synergistically stimulates the production of cytok nes in a patient. Preferred cytokines that are synergistically stimulated in accor lance with the invention are selected from the group consisting of, IL-12 and inttiferon- (IFN- ),IFN-β ,IFN-β or combinations thereof. In accordance with the inv ention, an "immunomer" refers to any compound comprising at least two oligonucleotides linked directly at their 3' ends, or directly via internucleoside linkages, or directly at a functionalized nucleobase or sugar, or that are indirectly linked together via a noa-nucleotidic linker, wherein at least one of the 5oligonucleotides, in the context 0f the immunomer compound, is an immunostimulatory oligonucleoti ie having an accessible 5' end. In the context of the invention, an immunostimulatory oligonucleotide is an oligonucleotide that comprises at least one of an immunostimulaiory CpG dinucleotide, an immunostimulatory domain, or other immunostimulat Dry moiety. As used herein, the term "accessible 5' l0end" means that the 5' end of the oligonucleotide is sufficiently available such that the factors that recognize and bind to immunomer compounds or immunostimulatory oligonucleotides and stimulate the immune system have access to the 5' end. Such immunostimulatory oligonucleoti ies may include secondary structures, provided that the 5' end remains accessible. 15 In some embodiments, the immunostimulatory oligonucleotide and/or immunomer compound used in the method according to the invention comprises an immunostimulatory dinucleotide 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, 2'-deoxy-2'-substituted arabinocytidine, 2'-O- 20substitutedarabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other non -natural pyrimidine nucleosides, or l-(2'-deoxy-# - D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; G is guanosine or 2'-deoxyguanosine, G* is 2' deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted-arabinoguanosine, 2'-O-substituted- 25arabinoguanosine, or other non-natural punne nucleoside, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate. In certain preferred embodiments, the immunostimulatory dinucleotide is not CpG. In some embodiments, the immunostimulatory oligonucleotide and/or immunomer compound used in the method according to the invention comprises an immunostimulatory domain of fo mula (III): 5'-Nn-Nl-Y-Z-Nl-Nn-3' (III) 5 wherein: Y is cytidine, 2'-deoxythymidine, 2'-deoxycytidine, arabinocytidine, 2'-deoxy-2'-substitutedarabinocytidine, 2'-O-substitutedarabinocytidine, 2'-deoxy-5- hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other non- natural pyrimidine nucleosides, or l-(2'-deoxy-*-D-ribofuranosyl)-2-oxo-7-deaza-8- 10methyl-purine; Z is guanosine or 2'-deoxyguanosine, is 2' deoxy-7-deazaguanosine, 2'-deoxy- 6-thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted-arabinoguanosine, 2'-O- substituted-arabinoguanosine, 2'- deoxyinosine, or other non-natural purine nucleoside 15 N1, at each occurrence, is preferably a naturally occurring or a synthetic nucleoside or an immunostimulalory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, -deoxyribonucleosides, -L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified internucleoside linkage to the adjacent nucleoside on the 3' side, the modified 20internucleotide linkage being selected from, without limitation, a linker having a length of from about 2 angstrom; to about 200 angstroms, C2-C18 alkyl linker, poly (ethylene glycol) linker, 2-amino3utyl-l,3-propanediol linker, glyceryl linker, 2'-5' internucleoside linkage, and phosphorothioate, phosphorodithioate, or methylphosphonate internucleoside linkage; 25 Nn, at each occurrence, is independently a naturally occurring nucleoside or an immunostimulatory moiety, preferably selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, -deoxyribonucleosides, 2'-O- substituted ribonucleosides, and nucleosides linked by a modified internucleoside linkage to the adjacent nucleosicU on the 3' side, the modified internucleotide linkage being selected from the group consisting of amino linker, C2-C18 alkyl linker, poly (ethylene glycol) linker, 2-aminobutyl-l,3-propanediol linker, glyceryl linker, 2'-5' internucleoside linkage, and methylphosphonate internucleoside linkage; 5 provided that at least one Nl or Nn is an immunostimulatory moiety; wherein n is a number from 0-30; wherein the 3'nucleoside is optionally linked directly or via a non-nucleotidic linker to another oligonucleotide, which may or may not be immunostimulatory. In a second aspect, the invention provides a method for treating cancer in a l0cancer patient comprising admit istering an immunostimulatory oligonucleotide and/or immunomer conjugate, which comprises an immunostimulatory oligonucleotide and/or immunomer compound, as described above, and a cancer antigen conjugated to the immu lostimulalory oligonucleotide and/or immunomer compound at a position other than the accessible 5' end, in combination with a 15chemotherapeutic agent. In a third aspect, the invention provides pharmaceutical formulations comprising an immunostimulatory oligonucleotide or immunostimulatory oligonucleotide conjugate and/or an immunomer compound or immunomer conjugate according to the invention, a chemotherapeutic agent and a physiologically acceptable 20carrier. In a fourth aspect, the invention provides a method for sensitizing cancer cells to ionizing radiation. The method according to this aspect of the invention comprises administering to a mammal an immunostimulatory oligonucleotide or an immunomer compound according to the indention and treating the animal with ionizing radiation. 25 In a fifth aspect, the invention provides a method for synergistically stimulating an immune response in a patient comprising administering to a patient a therapeutically effective synergistic amount of at least one immunomer compound or immunostimulatory oligonucleotide in combination with a therapeutically effective synergistic amount of IL-2, (aad optionally an antigen), wherein administration of said combination synergistically stimulates the production cytokines in a patient. Preferred cytokines that are synergistically stimulated in accordance with the invention are selected from the group consisting of IL-12 and interferon-* ,IFN-» ,IFN-β or combinations thereof. In certain smbodiments of this second aspect of the invention, 5the antigen is operationally associated with the immunomer compound at a position other than the accessible 5' end. In a sixth aspect of the invention, at least one immunostimulatory oligonucleotide that is not an immunomer compound is used in combination with a therapeutically effective amount of IL-2 to selectively and synergistically stimulate the l0production cytokines in a patient. Preferred cytokines that are synergistically stimulated in accordance with the invention are selected from the group consisting of IL-12 and IFN-* ,IFN- ,IFN-β of combinations thereof. In accordance with the present invention, preferred imminostimulatory oligonucleotides that are not immunomer compounds include those containing at least one immunostimulatory 15CpG dinucleotide wherein C is nc t cytosine or deoxycytosine and/or G is not guanosine or 2-deoxyguanosine. other preferred immunostimulatory oligonucleotides the invention that are not immunomer compounds are those that include alternative immunostimulatory moieties that are not CpG. Examples of such alternative immunostimulatory moieties inch de but are not limited to nucleosides comprising 20non-naturally occurring bases and or sugar and secondary structures of the oligonucleotide itself such as hairpin structures that stabilize the oligonucleotide. In a seventh aspect, the in\ ention provides therapeutic compositions comprising a therapeutically effec ive synergistic amount of at least one immunomer compound, or immmunostimulatory oligonucleotide, a therapeutically effective 25synergistic amount of IL-2 (and/or an agent that induces IL-2 production in situ, such as a DNA vaccine or expression vector expressing IL-2) and optionally an antigen wherein administration of said combination synergistically stimulates the production of cytokines in a patient. Preferred cytokines that are synergistically stimulated in accordance with the invention are selected from the group consisting of IL-12 and 30IFN-* ,IFN- JFN-β or combinations thereof. The methods and compositions according to all aspects of the invention are useful in therapeutic approaches to human or veterinary diseases involving immune system modulation and immune-based therapies. Particularly preferred disease targets include cancer, infectious diseases, asthma and allergies. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of representative immunomer compounds of the invention. Figure 2 depicts several representative immunomer compounds of the 5 invention. Figure 3 depicts a group of lepresentative small molecule linkers suitable for linear synthesis of immumomers of the invention. Figure 4 depicts a group of lepresentative small molecule linkers suitable for parallel synthesis of immunomer compounds of the invention. 10 Figure 5 is a synthetic scheme for the linear synthesis of immunomer compounds of the invention. DMT1 := 4,4'-diraethoxytrityl; CE = cyanoethyl. Figure 6 is a synthetic scheme for the parallel synthesis of immunomer compounds of the invention. DMTi =4,4'-dimethoxytrityl; CE = cyanoethyl. Figure 7A is a graphic representation of the induction of IL-12 by l5Oligonucleotide (Oligo) 1 and Immunomers 2-3 in BALB/c mouse spleen cell cultures. These data suggest that Itrmunomer 2, which has accessible 5'-ends, is a stronger inducer of IL-12 than moncmeric Oligo 1, and that Immunomer 3, which does not have accessible 5'-ends, has equal or weaker ability to produce immune stimulation compared with Oligo 1. 20 Figure 7B is a graphic repret entation of the induction of IL-6 (top to bottom, respectively) by Oligo 1 and Immunomers 2-3 in BALB/c mouse spleen cells cultures. These data suggest that Immunomet 2, which has accessible 5'-ends, is a stronger inducer of IL-6 than monomeric Oligo 1, and that Immunomer 3, which does not have accessible 5'-ends, has equal or weaker ability to induce immune stimulation 25compared with Oligo I. Figure 7C is a graphic representation of the induction of IL-10 by Oligo 1 and Immunomers 2-3 (top to bottom, respectively) in BALB/c mouse spleen cell cultures. Figure 8 A is a graphic rearesentation of the induction of BALB/c mouse spleen cell proliferation in cell cultures by different concentrations of Immunomers 5 and 6, which have inaccessible and accessible 5'-ends, respectively. Figure 8B is a graphic representation of BALB/c mouse spleen enlargement by 5Oligo 4 and Immuaomevs 5-6, which have an immunogenic chemical modification in the 5'-flanking sequence of the CpG motif. Again, the immunomer compound, which has accessible 5'-ends (6), has a greater ability to increase spleen enlargement compared with Immunomer 5, which does not have accessible 5'-end and with monomeric Oligo 4. 10 Figure 9A is a graphic representation of induction of IL-12 by different concentrations of Oligo 4 and Imnrunomers '7 and 8 in BALB/c mouse spleen cell cultures. Figure 9B is a graphic repre sentation of induction of IL-6 by different concentrations of Oligo 4 and Imminomers 7 and 8 in BALB/c mouse spleen cell I5cultures. Figure 9C is a graphic representation of induction of IL-10 by different concentrations of Oligo 4 and Immunomers 7 and 8 in BALB/c mouse spleen cell cultures. Figure 10A is a graphic repr mentation of the induction of cell proliferation by 20lmmunomers 14, 15, and 16 in BALB/c mouse spleen cell cultures. Figure 10B is a graphic representation of the induction of cell proliferation by IL-12 by different concentrations of Immunomers 14 and 16 in BALB/c mouse spleen cell cultures. Figure 10C is a graphic representation of the induction of cell proliferation by 25IL-6 by different concentrations of Immunomers 14 and 16 in BALB/c mouse spleen cell cultures. Figure 11A is a graphic representation of the induction of cell proliferation by Oligo 4 and 17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures. Figure 11B is a graphic representation of the induction of cell proliferation IL- 12 by different concentrations of Oligo 4 and 17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures. Figure 11C is a graphic resresentation of the induction of cell proliferation IL- 56 by different concentrations of Otigo 4 and 17 and Immunomers 19 and 20 in BALB/c mouse spleen cell cultures, Figure 12 is a graphic representation of BALB/c mouse spleen enlargement using Oligo 4 and Immunomers 14, 23, and 24. Figure 13 shows the effect of a method according to the invention on rumor l0growth in a nude mouse model for prostate cancer. Figure 14 shows the effect of a method according to the invention on body weight of the mice used in the stud /. Figure 15A is a graphic representation demonstrating the synergistic effect on 1L-12 production after BALB/c spleaocytes were treated with Oligo 1 and IL-2. 15 Figure 15B is a graphic reprsentation demonstrating the synergistic effect on IL-12 production after BALB/c spleenocytes were treated with Oligo 2 and IL-2. Figure 15C is a graphic representation demonstrating the synergistic effect on IL-12 production after BALB/c spleenocytes were treated with Oligo 3 and IL-2. Figure 15D is a graphic representation demonstrating the synergistic effect on 20IL-12 production after BALB/c spleonocytes were treated with Oligo 4 and IL-2. Figure 16A is a graphic representation demonstrating the effect on IL-6 production after BALB/c spleenocytes were treated with Oligo 1 and IL-2. Figure 16B is a graphic representation demonstrating the effect on IL-6 production after BALB/c spleenocytes were treated with Oligo 2 and IL-2. 25 Figure 16C is a graphic representation demonstrating the effect on IL-6 production after BALB/c spleenocytes were treated with Oligo 3 and IL-2. Figure 16D is a graphic representation demonstrating the effect on IL-6 production after BALB/c spleenoeytes were treated with Oligo 4 and IL-2. Figure 17 is a graphic representation demonstrating the synergistic effect on IL-12 production after BALB/c spleenocytes were treated with Oligo 5 and IL-2. 5 Figure 18A is a graphic representation demonstrating the effect on IFN-* production after BALB/c spleenocytes were treated with Oligo 1 and IL-2. Figure 18B is a graphic representation demonstrating the effect on IFN- production after BALB/c spleenocytes were treated with Oligo 2 and IL-2. Figure 18C is a graphic representation demonstrating the effect on IFN-* lOproduction after BALB/c spleenocytes were treated with Oligo 3 and IL-2. Figure 18D is a graphic repiesentation demonstrating the effect on IFN-* production after BALB/c spleenocytes were treated with Oligo 4 and IL-2. Figure 19 is a graphic representation demonstrating the effect on IFN-* production after BALB/c spleenocy es were treated with Oligo 5 and IL-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention relates to optimized methods and compositions for enhancing the immune response caused by immunostimulatory compounds used in immune- based therapies. The optimized methods according to the invention result in synergy 5between the therapeutic effect of immunostimulatory compounds such as immunostimulatory oligonucleotides and immunomer compounds and the therapeutic effect of cytokine immunotherapy and/or chemotherapeutic agents. The issued patents, patent applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and l0individually indicated to be incorporated by reference. In the event of inconsistencies between any teaching of any reference cited herein and the present specification, the latter shall prevail for purposes of the invention. The invention provides methods for enhancing the anti-cancer effect caused by immunostimulatory compounds used for immunotherapy applications for the I5treatment of cancer. In the method; according to the invention, immunostimulatory oligonucleotides and/or immunomer compounds provide a synergistic therapeutic effect when use in combination with chemotherapeutic agents. This result is surprising in view of the fact that ir imunostimulatory oligonucleotides and immunomer compounds cause cell division of immune system cells, whereas 20chemotherapeutic agents normally kill actively dividing cells. In a first aspect, the inventic n provides a method for treating cancer in a cancer patient comprising administering, it. combination with chemotherapeutic agents, immunostimulatory oligonucleotides and/or immunomer compounds, the latter comprising at least two oligonucleo ides linked together, such that the immunomer 25compound has more than one accessible 5' end, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide. As used herein, the term "accessible 5' end" means that the 5 end of the oligonucleotide is sufficiently available such that the factors that recognize and bind to immunomer compounds and stimulate the immune system have acess to it. Optionally, the 5' OH can be linked to 30a phosphate, phosphorothioate, or p1 osphorodithioate moiety, an aromatic or aliphatic linker, cholesterol, or another emity which does not interfere with accessibility. Immunostimulatory oligonucleot des and irnmunomer compounds induce an immune response when administered to a vertebrate. When used in combination with chemotherapeutic agents, a synergistie therapeutic effect is obtained. 5 Preferred chemotherapeutic agents used in the method according to the invention include, without limitation Gemcitabine, methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorub cin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS l0famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, C1-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/M troxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682,9-AC, AG334), AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, 15PD183805, DX8951f, Lemonal DP 2202, FK317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Pladitaxel, Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Dox fluridine, Cyclopax/oral paelitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/ 20RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/7"6C85/5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, DoxiVliposomal doxorubicin, Caely dliposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt ZD1839, LU 79553/Bis-Naphtalimide, LU 25103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4S09/Dexifosamide, ifes/Mesnex/lfosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisolatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel, prodrug of guarine arabinoside, Taxane Analog, nitrosoureas, 30alkylating agents such as melphelan and cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carbophtin, Chlosrombucil, Cytarabine HC1, Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor Sanalogue), Lomustine (CCNU), ]V echlorethamine HCI (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Stieprozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Intsrleukin 2, Mitoguazone (methyl-GAG; methyl lOglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyi-CCNU), Teniposide (VM-26) and Vindesine sulfate. In the methods according tc this aspect of the invention, administration of immunostimulatory oligonucleotidos and/or itnmunomer compounds can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, I5topical, intranasal, aerosol, intraoctlar, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or topical cream or in eye drop or mouthwash form. Administration of the therapeutic compositions of imnunostimulatory oligonucleotides and/or immvmomer compounds can be canied out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease. 20When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of immunostimulatory oligonucleotide and/or immunomer compound from about 0.0001 micromolar to about 10 micromolar. For localized administration, much tower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a 25total dosage of immunostimulatory cligonucleotide and/or immunomer compound ranges from about 0.0001 mg per patient per day to about 200 mg per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode. For 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 immunostimulatory oligonucleotide and/or immunomer compound and/or the chemotherapeutic agent in any order, including simultaneous 5administration, as well as temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of the immunostimulatory oligonucleotkle and/or immunomer compound, and/or independently the chemotherapeutic agent. The administration of the immunostimulatory oligonucleotide and/or immunomer compound and/or l0chemotherapeutic agent may be by the same or different routes. In some embodiments, the immunomer compound used in the method according to the invention comprises two or more immunostimulatory oligonucleotides, (in the context of the immunomer) which may be the same or different. Preferably, each such imiounostimulatory otigonucleotide has at least one 15accessible5'end. In certain embodiments of the method according to the invention, in addition to the immunostimulatory oligonuc)eotide(s), the immunomer compound also comprises at least one oiigonucleoti ie that is complementary to a gene. As used herein, the term "complementary to' means that the oligunucleotide hybridizes under 20physiological conditions to a region of the gene, in some embodiments, the oligonucleotide downregulates expression of a gene. Such downregulatory otigomicleotides preferably are selected from the group consisting of antisense oligonucleotides, ribozyme oligonuc teotides, small inhibitory RNAs and decoy oligonucleotides. As used herein, the term "downregulate a gene" means to inhibit the 25transcription of a gene or translation of a gene product. Thus, the immunomer compounds used in the method according to the invention can be used to target one or more specific disease targets, while also stimulating the immune system. in certain embodiments, the mmunostimulatory oligonucleotide and/or immunomer compound used in the rnethod according to the invention includes a 30ribozyme 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 nucleic acid target and cleaves the target. As used herein, the term "decoy oligonucleotide" refers to an oligonucleotide that binds to a transcription factor in a sequence-specific manner and arrests transcription activity. Preferably, the ribozyme 5or decoy oligonucleotide exhibits secondary structure, including, without limitation, stem-loop or hairpin structures. In certain embodiments, at least one oligonucleotide comprises poly(I)-poly(dC). In certain embodiments, at least one set of Nn includes a string of 3 to 10 dGs and/or Gs or 2'-substituted ribo or arabino Gs. For purposes of the invention, the term "oligonucleotide" refers to a 1 0polynucleoside formed from a plurality of linked nucleoside units. Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods. In preferred embodiments each nucleoside unit includes a heterocyclic base and a pentofuranosyl, trehalose, arabinose, 2'-deoxy-2' substituted arabinose, 2'-O-substituted arabinose or 15hexose sugar group. The nucleoside residues can be coupled to each other by any of the numerous known internucleouide linkages. Such internucleoside linkages include, without limitation, phosphodiestt r. phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy. acetamidate, carbamate, morpholino, borano, 20thioether, bridged phosphoramidtte, bridged methylene phosphonate, bridged phosphorothioate, and sulfone intsmucleoside linkages. The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, the ter us "oligonucleotide" and "dinucleotide" are 25expressly intended to include pohnucleosides and dinucleosides having any such internucleoside linkage, whether or not the linkage comprises a phosphate group. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate linkages, or combinations thereof. In some embodiments, the immunomer compound comprises oligonucleotides each having from about 3 to about 35 nucleoside residues, preferably from about 4 to about 30 nucleoside residues, more preferably from about 4 to about 20 nucleoside residues. In some embodiments, the oligcnucleotides have from about 5 or 6 to about 518, or from about 5 or 6 to about 14, nucleoside residues. As used herein, the term "about" implies that the exact number is not critical. Thus, the number of nucleoside residues in the oligonucleotides is not critical, and oligonucleotides having one or two fewer nucleoside residues, or from one to several additional nucleoside residues are contemplated as equivalents of each of the embodiments described above, for l0purposes of this invention. In some embodiments, one or more of the oligonucleotides have 11 nucleotides. The term "oligonucleotide" also encompasses polynucleosides having additional substituents including, without limitation, protein groups, lipophilic groups, intercalating agents, diamines, folic acid, cholesterol and adamantane. The term 15"oligonucleotide" also encompasses any other nucleobase containing polymer, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino-backbone oligonucleotides , and oligonuc eotides having backbone sections with alkyl linkers or amino linkers. 20 The immunostimulatory oligonucleotides and/or immunomer compounds used in the method according to the invention can include naturally occurring nucleosides, modified nucleosides, or mixtuies 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 25nucleoside is a non-natural pyri nidine or purine nucleoside, as herein described. In some embodiments, the modified nucleoside is a 2'-substituted ribonucleoside an arabinonucleoside or a 2'-deoxy -2'-fiuoroarabinoside. For purposes of the invention, the term "2'-substituted ribonucleoside" includes ribonucleosides in which the hydroxyl group at the 2' position of the pentose 30moiety is substituted to produce a 2'-O-substituted ribonucleoside. Preferably, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group ha /ing 6-10 carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino 5groups. Examples of such 2'-0-substituted ribonucleosides include, without limitation 2'-O-methylribonucle osides and 2'-O-methoxyethylribonucleosides. The term "2'-substituted ribonucleoside" also includes ribonucleosides in which the 2'-hydroxyl group is "eplaced with a lower alkyl group containing 1 -6 saturated or unsaturated carbon atoms, or with an amino or halo group. Examples of lOsuch 2'-substituted ribonucleosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonacleosides. The term "oligonucleotide" includes hybrid and chimeric oligonucleotides. A "chimeric oligonucleotide" is an oligonucleotide having more than one type of internucleoside linkage. One preferred example of such a chimeric oligonucleotide is 15a chimeric oligonucleotide con prising a phosphorothioate, phosphodiester or phosphorodithioate region and non-ionic linkages such as alkylphosphonate or alkylphosphonothioate linkage:, (see e.g., Pederson et al. U.S. Patent Nos. 5,635,377 and 5,366,878). A "hybrid oligonucleotide" is an oligonucleotide having more than one type of 20nucleoside. One preferred example of such a hybrid oligonucleotide comprises a ribonucleotide or 2'-substituted ribonucleotide region, and a deoxyribonucleotide region (see, e.g., Metelev and Agrawal, U.S. Patent No. 5,652,355, 6,346,614 and 6,143,881). For purposes of the invention, the term "immunostimulatory oligonucleotide" 25refers to an oligonucleotide as described above that induces an immune response when administered to a vertebrate, such as a fish, bird, or mammal. As used herein, the term "mammal" includes, without limitation rats, mice, cats, dogs, horses, cattle, cows, pigs, rabbits, non-humar primates, and humans. Useful immunostimulatory oligonucleotides can be found described in Agrawal et al, WO 98/49288, published November 5, 1998; WO 01/12804, published February 22, 2001; WO 01/55370, published August 2, 2001; PCI/US01/13682, filed April 30, 2001; and PCT/US01/30137, filed September 26, 2001. Preferably, the immunostimulatory oligonucleotide comprises at least one phosphodiester, phosphorothioate, 5methylphosphonate, orphosphordithioate internucleoside linkage. In a further aspect, the i nvention provides a method for synergistically stimulating an immune respond e in a patient. The method comprises administering to a patient, a combination of a therapeutically effective synergistic amount of at least one immunomer compound or immunostimulatory oligonucleotide in accordance with l0the invention and a therapeutically effective synergistic amount of IL-2 (and/or an agent that induces IL-2 produc ion in situ, such as a DNA vaccine or expression vector expressing IL-2), where in administration of said combination synergistically stimulates the production of cytokines in a patient. Preferably, the cytokines that are synergistically stimulated in accordance with the invention are selected from the 15group consisting of, IL-12 and interferon-* (IFN-* ),IFN-» ,IFN-B or combinations thereof. The term "effective synergistic amount" is used herein to denote known concentrations of immunomer compound or immunostimulatory oligonucleotide and of IL-2 administered for an eflective period of time such that the combined 20stimulatory effect of the immi nomer compound or immunostimulatory oligonucleotide and IL-2 are more than additive, i.e. the combined stimulatory effect is greater than the expected to;al stimulatory effect calculated on the basis of the sum of the individual stimulatory t fleets. As used herein, the term "cytokine" refers to any of many soluble molecules 25that cells of the immune system produce to control reactions between other cells. The term "cytokine" includes, for example, interleukins (e.g., IL-1, IL-2, IL-3, IL-6, IL-10, IL12, etc.), interferons (e.g., IFN-.alpha., IFN-.beta., IFN-.gamma.), chemokines, hematopoietic growth factors (e.g. erythropoietin), tumor necrosis factors, colony stimulating factors (e.g., G-C SF, M-CSF, GM-CSF) and transforming growth factors 30(TGF-alpha). In accordance with the i nvention, an "immunomer" refers to any compound comprising at least two oligontcleotides linked directly at their 3' ends, or directly via internucleoside linkages, or directly at a fiinctionalized nucleobase or sugar, or that are indirectly linked together via a non-nucleotidic linker, wherein at least one of the 5oligonucleotides, in the contex of the immunomer compound, is an immunostimulatory oligonucleotide having an accessible 5' end. In the context of the invention, an immunostimulatory oligonucleotide is an oligonucleotide that comprises at least one of an immunostimulatory "CpG" dinucleotide, an immunostimulatory domain, or other immunostimulatory moiety. As used herein, the term "accessible 5' l0end" means that the 5' end of the oligonucleotide is sufficiently available such that the factors that recognize and bind to immunomer compounds and immunostimulatory oligonucleotides and stimulate the immune system have access to the 5' end. in some embodiments, at least one immunostimulatory oligonucleotide of the immunomer compound comprises an immunostimulatory dinucleotide of formula 155'-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 20invention, a "synthetic" pyrimidine or purine nucleoside includes a non-naturally occurring pyrimidine or purine base, a non-naturally occurring sugar moiety, or a combination thereof. Preferred pyrimidine r ucleosides in the immunostimulatory oligonucleotides and/or immunomer compounds used in the method according to the invention have 2 5 the structure (1): wherein: D is a hydrogen bond c onor; D is selected from the group consisting of hydrogen, hydrogen bond donor, 5hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron donating group; A is a hydrogen bond acceptor or a hydrophilic group; A' is selected from the group consisting of hydrogen bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing group and electron lOdonating group; X is carbon or nitroge r. and S' is a pentose or hexese sugar ring, or a non-naturally occurring sugar. Preferably, the sugar J ing is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine 15nucleoside to another nucleoside or nucleoside analog. Preferred hydrogen bond donors include, without limitation, -NH-, -NH2, -SH and -OH. Preferred hydroge 1 bond acceptors include, without limitation, C=O, C=S, and the ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of cytosine. In some embodiments, the base moiety in (1) is a non-naturally occurring 20pyrimidine base. Examples of preferred non-naturally occurring pyrimidine bases include, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, and 4-thiouracil. In some embodiments, the sugar moiety S1 in (1) is a non-naturally occurring sugar moiety. For purposes of the present invntion, a "naturally occurring sugar moiety" is a sugar moiety that occurs naturally as part of nucleic acid, e.g., ribose and 2'-deoxyribose, and a "non-naturally occurring sugar moiety" is any sugar that does not occur naturally as part of a nucleic acid, but w rich can be used in the backbone for an 5oligonucleotide, e.g, hexose. Axabinose and arabinose derivatives are examples of preferred sugar moieties. Preferred purine nucleoside analogs in immunostimulatory oligonucleotides and/or immunomer compounds used in the method according to the invention have the structure (II): wherein: D is a hydrogen bond donor; D' is selected from the group consisting of hydrogen, hydrogen bond donor, 15and hydrophilic group; A is a hydrogen bone acceptor or a hydrophilic group; X is carbon or nitrogen; each L is independer tly selected from the group consisting of C, O, N and S; and 20 S' is a pentose or hexose sugar ring, or a non-naturally occurring sugar. Preferably, the sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other inker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog. Preferred hydrogen bor d donors include, without limitation, -NH-, -NH2, -SH and -OH. Preferred hydrogen pond acceptors include, without limitation, C=O, C=S, -NO2 and the ring nitrogen atons of an aromatic heterocycle, e.g., Nl of guanine. In some embodiments, the base moiety in (II) is a non-naturally occurring 5purine base. Examples of pre erred non-naturally occurring purine bases include, without limitation, 6-thioguanine and 7-deazaguanine. In some embodiments, the sugar moiety S1 in (II) is a naiurally occurring sugar moiety, as described above for structure (I). In preferred embodiments, the immunostimulatory dinucleotide in the l0immunostimulatory oligonuc eotides arid/or immunomer compound used in the method according to the invention 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, 2'-deoxythymidine, 2'-deoxy- 2'-substitutedarabinocytidim, 2'-O-substitutedarabinocytidine, 2'-deoxy-5- 15hydroxycytidine, 2-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other non- natural pyrimidine nucleosides, or l-(2'-deoxy- -D-ribofuranosyl)-2-oxo-7-deaza-8- methyl-purine; G is guanosine or 2'-deoxyguanosine, G* is 2' deoxy-7- deazaguanosine, 2'-deoxy-6 thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted- arabinoguanosine, 2'-O-sub:.tituted-anibinoguanosine, 2'-deoxyinosine, or other non- 20natural purine nucleoside, and p is an internucleoside linkage selected from the group consisting of phosphodieste •, phosphorothioate, and phosphorodithioate. In certain preferred embodiments, the immunostimulatory dinucleotide is not CpG. The immunostimula tory oligonucleotides may include immunostimulatory moieties on one or both sidus of the immunostimulatory dinucleotide. Thus, in some 25embodiments, the immunostimulatory oligonucleotide comprises an immunostimulatory domair of structure (III): 5'-Nn-Nl-Y-Z-Nl-Nn-3' (III) wherein: Y is cytidine, 2'deoxythymidine, 2' deoxycytidine arabinocytidine, 2'-deoxy- 2'-substitutedarabinocytidine,2'-deoxythymidine, 2'-O-substitutedarabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides, orl-(2'-deoxy-* -D-ribofuranosyl)-2-oxo-7-deaza- 5 8-methyl-purine; Z is guanosine or 2'-deoxyguanosine, 2' deoxy-7-deazaguanosine, 2'-deoxy-6- thioguanosine, arabinoguanosme, 2'-deoxy-2'substituted-arabinoguanosine, 2'-O- substituted-arabinoguanosine. 2'deoxyinosine, or other non-natural purine nucleoside; 10 N1, at each occurrence, is preferably a naturally occurring or a synthetic nucleoside or an immunostirr ulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, -deoxyribonucleosides, -L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or modified internucleoside linkage to the adjacent nucleoside on the 3' side, the modified 15internucleotide linkage being; selected from, without limitation, a linker having a length of from about 2 angsfoms to about 200 angstroms, C2-C18 alkyl linker, poly (ethylene glycol) linker, 2-ammobutyl-l,3-propanediol linker, glyceryl linker, 2'-5' internucleoside linkage, and phosphorothioate, phosphorodithioate, or methylphosphonate internuc feoside linkage; 20 Nn, at each occurrence, is preferably a naturally occurring nucleoside or an immunostimulatory moiety selected from the group consisting of abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, -deoxyribonucleosides, 2'-O-substituted ribonucleosides, and nuclee sides linked by a modified internucleoside linkage to the adjacent nucleoside on the 3' side, the modified internucleoside linkage preferably 25being selected from the group consisting of amino linker, C2-C18 alkyl linker, poly (ethylene glycol) linker, 2-aminobutyl-l,3-propanediol linker, glyceryl linker, 2'-5' internucleoside linkage, an i methylphosphonate internucleoside linkage; provided that at least one Nl or Nn is an immunostimulatory moiety; wherein each n is independently a number from 0 to 30; and wherein, in the case of an immunomer compound, the 3'end is linked directly or via a non-nucleotidic linker tc another oligonucleotide, which may or may not be immunostimulatory. In some preferred embodiments, YZ is arabinocytidine or 2'-deoxy- 52'-substituted arabinocytidine and arabinoguanosine or 2'deoxy-2'-substituted arabinoguanosine. Preferred immunostimulatory moieties include modifications in the phosphate backbones, includ ng, without limitation, methylphosphonates, methylphosphonothioates, phosphotriesters,, phosphothiotriesters, phosphorothioates, phosphorodithioates, triester prodrugs, sulfones, sulfonamides, sulfamates, lOformacetal, N-methylhydroxylarr ine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidates, especially primary amino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, and stereospecific linkages (e.g., (Rp)- or (Sp)-phosphorothioate, akylphosphonate, or phosphotriester linkages). Preferred immunostimula ory moieties according to the invention further 15include nucleosides having sugar modifications, including, without limitation, 2'-substituted pentose sugars including, without limitation, 2'-O-methylribose, 2'-O-methoxyethylribose, 2'-O-p opargylribose, and 2'-deoxy-2'-fluororibose; 3'-substituted pentose sugars, including, without limitation, 3'-O-methylribose; r,2'-dideoxyribose; arabinose; substituted arabinosc sugars, including, without 201imitation, l'-methylarabinose, 3'hydroxymethylarabinose, 4'-hydroxymethyl- arabinose, and 2'-substituted arab nose sugars; hexose sugars, including, without limitation, 1,5-anhydrohexitol; and alpha-anomers. In embodiments in which the modified sugar is a 3'-deoxyribonucleoside or a 3'-O-substituted ribonucleoside, the immunostimulatory moiety is attached to the adjacent nucleoside by way of a 2'-5' 25internucleoside linkage. Preferred immunostimulatory moieties in immunostimulatory oligonucleotides and/or immunomer compounds used in the method according to the invention further include oligonucleotides having other carbohydrate backbone modifications and replacements, including peptide ni cleic acids (PNA), peptide nucleic acids with 30phosphate groups (PHONA), lockt d nucleic acids (LNA), morpholino backbone oligonucleotides, and oligonucleotides having backbone linker sections having a length of from 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 chirally pure or a racemic mixture. 5Most preferably, such alkyl linkos have from about 2 to about 18 carbon atoms. In some preferred embodiments such alkyl linkers have from about 3 to about 9 carbon atoms. Some alkyl linkers includ; one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thioether. Some such fi nctionalized alkyl linkers are poly(ethylene glycol) lOlinkers of formula -O-(CH2-CH2-O-)n (n = 1 -9). Some other functionalized alkyl linkers are peptides or amino acids. Preferred immunostimulatory moieties in immunostimulatory oligonucleotides and/or immunomer compounds used in the method according to the invention further include DNA isoforms, including, without limitation, -L-deoxyribonucleosides and 15 -deoxyribonucleosides. Preferred immunostimulatory moieties incorporate 3' 'notifications, and further include nucleosides having unnatural internucleoside mkage positions, including, withe ut limitation, 2'-5\ 2'-2\ 3'-3' and 5'-5' linkages. Preferred immunostimulatory moieties in immunostimulatory oligonucleotides and/or immunomer compounds used in the method according to the invention further 20include nucleosides having modified heterocyclic bases, including, without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil, 6-tliioguanine, 7-deazaguanine, inosine, nitropyrrole, C5-propynylpyrimidine, and diaminopurines, including, without limitation, 2,6-diaminopurine. 25 By way of specific illustrat on and not by way of limitation, for example, in the immunostimulatory domain of structure (III), a methylphosphonate internucleoside linkage at position Nl or Nn is an immunostimulatory moiety, a linker having a length of from about 2 an gstroms to about 200 angstroms, C2-C18 alkyl linker at position XI is an immunostimulatory moiety, and a -L-deoxyribonucleoside at position XI is an immunostimulatory moiety. See Table 1 below for representative positions and structures of immunostimulatory moieties. It is to be understood that reference to a linker as the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is substituted at its 3'-hydroxyl with the 5 indicated linker, thereby creating a modified internucleoside linkage between that nucleoside residue and the adjacen nucleoside on the 3' side. Similarly, reference to a modified internucleoside linkage a;; the immunostimulatory moiety at a specified position means that the nucleoside residue at that position is linked to the adjacent nucleoside on the 3' side by way of the recited linkage. 10 Table 1 Position TYPICAL IMMUNOSTIMULATORY MOIETIES ' Nl Naturally-occurring nucleosides, abasic nucleoside, arabinonucleoside, ■ 2'-deoxyuridine, -L-deoxyribonucleoside C2-C18 alkyl linker, poly (ethylene glycol) linkage, 2-aminobutyl-l,3-propanediol linker (amino linker), 2'-5' internucleoside linkage, methylphosphonate internucleoside linkage Nn ; Naturally-occurring nucleosides, abasic nucleoside, arabinonucleosides, 2'-deoxyuridine, 2'-O-: ubstituted ribonucleoside, 2'-5' internucleoside linkage, methylphosphonate intemucleoside linkage, provided that Nl and N2 cannot both be abasic linkages Table 2 shows representative positions and structures of immunostimulatory moieties within an immunostimulacory oligonucleotide having an upstream potentiation domain. As used herein, the term "Spacer 9" refers to a poly(ethylene glycol) linker of formula -O(CH2CH2-0),r, wherein n is 3. The term "Spacer 18" 15refers to a poly(ethylene glycol) linker of formula -O-(CH2CH2-O),,-, wherein n is 6. As used herein, the term "C2-C18 ilkyl linker refers to a linker of formula -O-(CH2) ,rO-, where q is an integer from 2 to 18. Accordingly, the terms "C3-linker" and "C3- alkyl linker" refer to a linker of for mila -O-(CH2)3-O-. For each of Spacer 9, Spacer 18, and C2-C18 alkyl linker, the linker is connected to the adjacent nucleosides by 20way of phosphodiester, phosphoroihioate, phosphorodithioate or methylphosphonate linkages. Table 2 Positiqn TYPICAL IMMUNOSTIMULATORY MOIETY 5'_N2 ! Naturally-occurring nucleosides,L2-aminobutyl-1,3-propanediol linker 5'N1 Naturally-occurrinlg nucleosides -L-deoxyribonucleoside, C2-C18 alkyl linker, poly(ethylt;ne glycol), abasic linker, 2-aminobutyl-l,3-propanediol ! linker 3'N1 Naturally-occurring nucleosides, 1,2'-dideoxyribose, 2'-6-methyl- 1 ribonucleoside, C2-C18 alky linker, Spacer 9,Spacer 18 3' N2 Naturally-occurriiig nucleosides, 1 ',2'-dideoxyribose, 3' -deoxyribonuch oside, -L-deoxyribonucleoside, 2' -O-propargyl- | ribonucleoside, C 2-C 18 alkyl linker, Spacer 9, Spacer 18, methylphosphona. e internucleoside linkage ! 3'N3 Naturally-occurring nucleosides, 1 ',2 '-dideoxyribose, C2-C18 alkyl ' linker, Spacer 9, Spacer 18, methylphosphonate internucleoside linkage, f 2'-5Mntemucleosidelinkage,d(G)n,polyI-polydC 3'N2+3'N3 1,2'-dideoxyribose, -L-deoxyribonucleoside, C2-C18 alkyl linker, d(G) | n, polyl-polydC 3'N3+3'N4 : 2'-O-methoxyethyl -ribonucleoside, methylphosphonate internucleoside linkage, d(G)n, polyl-polydC 3'N5+ 3' N 6 1 ',2'-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyl-polydC :5'N1+3'N3 1,2'-dideoxyribose, d(G)n, polyl-polydC Table 3 shows representatire positions and structures of immunostimulatory moieties within an immunostimulatory oligonucleotide having a downstream potentiation domain. 5 Table 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5'N2 methylphosphonate into nucleoside linkge 5'N1 methylphosphonate into nucleoside linkge 3N1 The immunomer compoum Is used in the method according to the invention comprise at least two oligonucleotides linked directly or via a non-nucleotidic linker. For purposes of the invention, a "n )n-nucleoi:idic linker" is any moiety that can be linked to the oligonucleotides by w ay of covalent or non-covalent linkages. lOPreferably such linker is from aboi t 2 angstroms to about 200 angstroms in length. Several examples of preferred linkers are set forth below. Non-covalent linkages include, but are not limited to, electrostatic interaction, hydrophobic interactions, -stacking interactions, and hydrogen bonding. The term "non-nucleotidic linker" is not meant to refer to an internuclcoside linkage, as described above, e.g., a 5phosphodiester, phosphorothioatc, or phosphorodithioate functional group, that directly connects the 3'-hydroxyl groups of IAVO nucleosides. For purposes of this invention, such a direct 3'-3' linkage is considered to be a "nucleotidic linkage." In some embodiments, the non-nucleotidic linker is a metal, including, without limitation, gold particles. In somt other embodiments, the non-nucleotidic linker is a l0soluble or insoluble biodegradable polymer bead. In yet other embodiments, the non-nucleotidic linker is an organic moiety having functional groups that permit attachment to the oligonucleotide. Such attachment preferably is by any stable covalent linkage. In some embodiments, the non-nucleotidic linker is a biomolecule, including, 15without limitation, polypeptides, antibodies, lipids, antigens, allergens, and oligosaccharides. In some other embodiments, the non-nucleotidic linker is a small molecule. For purposes of the invention, a small molecule is an organic moiety having a molecular weight of less han 1,000 Da. In some embodiments, the small molecule has a molecular weight of less than 750 Da. 20 In some embodiments, the small molecule is an aliphatic or aromatic hydrocarbon, either of which optionally can include, either in the linear chain connecting the oligonucleotides or appended to it, one or more functional groups selected from the group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thiourea. The small molecule can be cyclic or acyclic. 25Examples of small molecule linkers include, but are not limited to, amino acids, carbohydrates, cyclodextrins, adamantane, cholesterol, haptens and antibiotics. However, for purposes of describir g the non-nucleotidic linker, the term "small molecule" is not intended to include a nucleoside. In some embodiments, the small molecule linker is glycerol or a glycerol homolog of the formula HO-(CH))-CH(OH)-(CH2)P-OH, wherein o andp independently are integers from 1 to about 6, from 1 to about 4, or from 1 to about 3. In some other embodiments, the small molecule linker is a derivative of 1,3-diamino- 52-hydroxypropane. Some such derivatives have the formula HO-(CH2)m-C(O) NH-CH2-CH(OH)-CH2-NHC(O)- (CH2)m-OH, wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 to about 6, or from 2 to about 4. Some non-nucleotidic linkers in immunomer compounds used in the method according to the invention permit attachment of more than two oligonucleotides, as l0schematically depicted in Figure 1 For example, the small molecule linker glycerol has three hydroxyl groups to which oligonucleotides may be covalently attached. Some immunomer compounds according to the invention, therefore, comprise more than two oligonucleotides linked at their 3' ends to a non-nucleotidic linker. Some such immunomer compounds comprise at least two immunostimulatory 15oligonucleotides, each having an accessible 5' end. The immunostimulatory ol gonucleotides and/or immunomer compounds used m the method according to the invention may conveniently be synthesized using an automated synthesizer and phosphoramidite approach as schematically depicted in Figures 5 and 6, and further described in the Examples. In some embodiments, the 20immunostimulatory oligonucleotidss and/or immunomer compounds are synthesized by a linear synthesis approach (see Figure 5). As used herein, the term "linear synthesis" refers to a synthesis that starts at one end of the immunomer compound and progresses linearly to the other end. Linear synthesis permits incorporation of either identical or un-identical (in terms of length, base composition and/or chemical 25modifications incorporated) monomeric units into the immunostimulatory oligonucleotides and/or immunomcr compounds. An alternative mode of syn hesis for immunomer compounds is "parallel synthesis", in which synthesis proceeds outward from a central linker moiety (see Figure 6). A solid support attachec. linker can be used for parallel synthesis, as is described in U.S. Patent No. 5,912 332. Alternatively, a universal solid support, such as phosphate attached to controlled pore glass support, can be used. Parallel synthesis of immunomer compounds has several advantages over linear synthesis: (1) parallel synthesis permits the incorporation of identical 5monomeric units; (2) unlike in liner synthesis, both (or all) the monomeric units are synthesized at the same time, thereoy the number of synthetic steps and the time required for the synthesis is the same as that of a monomeric unit; and (3) the reduction in synthetic steps improves purity and yield of the final immunomer product. 10 At the end of the synthesis by either linear synthesis or parallel synthesis protocols, the immunostimulatory oligonucleotides or immunomer compounds used in the method according to the invent on may conveniently be deprotected with concentrated ammonia solution or is recommended by the phosphoramidite supplier, if a modified nucleoside is incorpo "ated. The product immunostimulatory 15oligonucleotides and/or immunomor compound is preferably purified by reversed dose HPLC, detritylated, desalted and dialyzed. Immunostimulatory oligonucleotides suitable for use as a component of an immunomer compound, or in acco dance with the fourth aspect of the invention , are described in the following U.S. patents and pending U.S. patent applications and are 20incorporated herein by reference: J.S. Patent Numbers 6,426,334 and 6,476,000; and U.S. Patent Application Numbers 09/770,602, 09/845,623, 09/965,116, 60/440,587, 10/361,111, 60/471,247, 60/477. Preferred immunostimulatory oligonucleotides and immunomer compounds of the invention are described in pending U.S. Patent Application Number 10/279,684. Table 4 shows representative immunomer 25compounds used in the method according to the invention. Additional immunomer compounds are found described in the Examples and in U.S. Patent Application No. 10/279,684. L = C3-alkyl linker; X = 1',2-dide oxyribosido; Y = 50H dC; R = 7-deaza-dG R = arabinoguanosine; X1 = glycerol linker; \ ^.o 5 A further aspect of the invention provides an immunostimulatory nucleic acid comprising at least two oligonuc leotides, wherein the immunostimulatory nucleic acid has a secondary structure. In certain embodiments, the immunostimulatory nucleic acid has a 3'-end stem loop secoidary structure by way of hydrogen bonding with a complementary sequence. In certain embodiments the nucleic acid that has reduced 1 Oimmunostimulatory activity forms a 5'-end stem loop secondary structure by way of hydrogen bonding with a complementary sequence. In this aspect, immunostimulatory nucleic acid comprises a structure as detailed in formula (I). Domain A-Domain B-Domain C (I) Domains may be from about 2 to about 12 nucleotides in length. Domain A 15may be 5'-3' or 3'-5' or 2'-5' DNA, RNA, RNA-DNA, DNA-RNA having or not having a palindromic or self-complementary domain containing or not containing at least one dinucleotide selected from the group consisting of CpG, C*pG, C*pG* and CpG*, wherein C is cytidine or 1'• deoxycytidine, G is guanosine or 2'-deoxyguanosine, C* is 2'-deoxythymidine, l-(2'-deoxy-β-D-ribofuranosyl)-2-oxo- 207-deaza-8-methyl-purine, 2'-didt oxy-5-halocytosine, 2'-deoxy-5-nitrocytosine, arabinocytidine, 2'-deoxy-2'-subs titutedarabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydr sxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4- thiouridine, or other pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted-arabinoguanosine, 252'-O-substituted-arabinoguanosir e, 2'- deoxyinosine, or other purine nucleoside analogs, and p is an internucleoside linkage: selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate. In certain preferred embodiments, the immunostimilatory dinucleotide is not CpG. In certain embodiments, Domain A will have more than one dinucleotide selected from the group consisting of CpG, C*pG, C*pG* and CpG*. 5 Domain B, as depicted by an "X" below, is a linker joining Domains A and C that may be a 3'-'5' linkage, a 2 '-5' linkage, a 3'-3' linkage, a phosphate group, a nucleoside, or a non-nucleoside linker that may be aliphatic, aromatic, aryl, cyclic, chiral, achiral, a peptide, a carbohydrate, a lipid, a fatty acid, mono- tri- or hexapolyethylene glycol, or a hoterocyclic moiety. 10 Domain C maybe 5'-3' or 3'-5', 2'-5' DNA, RNA, RNA-DNA, DNA-RNA Poly I-Poly C having or not ha\ ing a palindromic or self-complementary sequence, which can or cannot have a din lcleotide selected from the group consisting of CpG, C*pG, C*pG*, CpG*, wherein C is cytidme or 2'-deoxycytidine, G is guanosine or 2'-deoxyguanosine, C* is 2'-deoxythymiciine, l-(2'-deoxy-B-D-ribofuranosyl)-2-oxo- 157-deaza-8-methyl-purine, 2' dideoxy-5-halocytosine, 2'-deoxy-5-halocytosine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4- thiouridine, other pyrimidine n icleoside analogs, G* is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted-arabinoguanosine, 202'-O-substituted-arabinoguano: ine, 2'-deoxyinosine, or other purine nucleoside analogs, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate. In certain preferred embodiments, the immunostin ulatory dinucleotide is not CpG. In some embodiments, Domain B is pnferably a non-nucloetidic linker connecting 25oligonucleotides of Domain A and Domain C, which are referred to as "immunomers." In certain preferred embodiments, Domain C does not have the dinucleotide CpG, C*pG, C*pG* or CpG*. By way of non-limiting example, in certain embodiments of this aspect the immunostimulatory nucleic acid will have a structure as detailed in formula (II). The structure depicted in formula (III) is referred to herein as a "terminal dimmer," l0since the ends of the two molecules are blocked because the sequences of the two ends are complementary allowing for intermolecular hydrogen bonding. 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 e cample, in certain embodiments of this aspect the 15immunostimulatory nucleic acid will have a structure as detailed in formula (IV). As would be recognized by one skilled in the art, the terminal end of the depicted molecule has a secondary structure because the complementary sequence of its end is hydrogen bonded to this region. In certain embodiments, a molecule such as 5a ligand may be attached to the terminal end in order to facilitate cellular uptake or improve stability of the moleculr. Non-limiting examples of some nucleic acid molecules of the invention are presented in Table 5. 35Italic phase represents a phosphodi ester linkage, other linkages are phosphorothioate unless otherwise indicated Underline = 2'-OMe-nucleoside; X = C3 linker R= 2'-deoxy-7-deazaguanosine Gr = 2'-deoxy-7-deazaguanoise 40 Another aspect of the invention provides an immunostimulatory nucleic acid wherein the sequence of the immutiostimulatory oligonucleotide and/or immunomer is at least partially self-complementaiy. A self-complementary sequence as used herein refers to a base sequence which, upon suitable alignment, may form intramolecular or, more typically, intermolecular basepairing between G-C, A-T, A-U and/or G-U 45wobble pairs. In one embodiment he extent of self-complementarity is at least 50 percent. For example an 8-mer that is at least 50 percent self-complementary may have a sequence capable of forming 4, 5, 6, 7, or 8 G-C, A-T, A-U and/or G-U wobble basepairs. Such basepairs may but need not necessarily involve bases located at either end of the self-complementary immunostimulatory oligonucleotide and/or 50immunomer. Where nucleic acid stabilization may be important to the immunostimulatory oligonucleotids and/or immunomer, it may be advantageous to "clamp" together one or both ends of a double-stranded nucleic acid, either by basepairing or by any other suitabl•; means. The degree of self-complementarity may depend on the alignment between immunostimulatory oligonucleotide and/or 55 immunomer, and such alignment may or may not include single- or multiple- nucleoside overhangs. In other embodiments, the degree of self-complementarity is at least 60 percent, at least 70 percent, at least 30 percent, at least 90 percent, or even 100 percent. By way of non-limiting example, in certain embodiments of this aspect the irnmunostimulatory nucleic acid will have a structure as detailed in formula (V) 5' —X ' X C IIIMIIIIIIIIIIIIIIIIIIIIIIIIIII v IIIIIIIIIMIIIIIIIIIIIIIIMIIIM HIIIIIIIIIIIIIIIIIIIIIIIIIIIIII y (V) As would be recognized by one sk lled in the art, the depicted immunomer compounds have secondary structure because the sequences of the domains are complementary allowing for intermolecular hydrogen bonding. Domains A and A' l0may or may not be identical, domains A and C may or may not be identical, domains A and C may or may not be identical, domains A and C may or may not be identical, domains A and C may or may noi be identical, domains B and B' may or may not be identical and domains C and C may or may not be identical. Moreover, additional immunomers can bind through intrmolecular hydrogen bonding thereby creating a 15chain, or multimers, of immunomers according to the invention, n can be any number of continuous self complementary immunomer compounds. As used herein, the term "complementary" means having the ability to hybridize to a nucleic acid. Such hybridization is ordinarily the result of hydrogen bonding between complementary strands, preferably to form Watson-Crick or 20Hoogsteen base pairs, although other modes of hydrogen bonding, as well as base stacking can also lead to hybridiza ion. As used herein, the term "secondary structure" refers to intermolecular hydrogen bonding. Intermolecular hydrogen bonding results in the formation of a duplexed nucleic acid molecule. 25 Non-limiting representative nucleic acid molecules are presented in Table 8. Table 8 173 B'-TCGiAACGiTTCGrX-GiCTTG^AAGiCT-S' 174 : S'-TCGiAACCTTCG-X-GCTTGiCAAG^T-S' 175 ; S'-TCTCACCTTCT^-TCTTCCACTCT-S1 I , „ „.. . . .1 . - . . . . . . . - . . _ The methods and composi ions according to all aspects of the invention are useful in therapeutic approaches to treating diseases wherein the treatment involves immune system modulation and ir lmune-based therapies. Particularly preferred 5disease targets include cancer, infectious diseases and allergies. In certain embodiments, th 5 therapeutic method is for the treatment of cancer. Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; cho "iocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lOlung cancer (e.g. small cell and no 1-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas. In some embodiments, the herapeutic method is for the treatment of an 15infection. By way of non-limiting example, viruses that have been found to infect humans include but are not limited to: Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III), and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. corona viruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, -abies viruses); Coronaviridae (e.g. corona viruses); 5Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses lOand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (paoilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); anc Iridoviridae (e.g. African swine fever virus); and 15unclassified viruses (e.g. the etiokgical agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the its of non-A, non-B hepatitis (class l=internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses). In certain embodiments, therapeutic methods of the invention are directed to 20the treatment of an allergy. An "allergen" refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is enormous and can include pollens., insect venoms, animal dander dust, fungal spores and drugs (e.g. penicillin). Examples of natural, animal and plant allergens include but are not limited to proteins specific to the following genuses: Canine 25(Canis familiaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felis domesticus)', Ambrosia (Ambrosia arterniisfolia); Lolium (e.g. Loliwn perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosd)\ Quercus (Quercus alba); Olea (Olea europc); Artemisia (Artemisia vulgaris); Plantago (e.g. 30Plantago lanceolata); Parietaria (e g. Parietaria offtcinalis or Parietaria judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus sabino'des, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis 5obtusa); Periplaneta (e.g. Periphneta americana); Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avena saliva); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis l0(e.g. Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g. Parpalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g. Bromus inennis). Specific allergens may be purchased commercially (e.g., INDOOR Bictechnologies Inc., Charlottesville, VA 22903). 15 In a second aspect, the inv ention provides a method for treating cancer in a cancer patient comprising administering to the patient a chemotherapeutic agent in combination with an immunostimulatory oligonucleotide and/or immunomer conjugate, which comprises an immunostimulatory oligonucleotide and/or immunomer compound, as described above, and an antigen conjugated to the 20immunostimulatory oligonucleotide and/or immunomer compound at a position other than the accessible 5' end. In some embodiments, the non-nucleotidic linker comprises an antigen associated with cancer, which is conjugated to the oligonucleotide. In some other embodiments, the antigen is conjugated to the oligonucleotide at a position other than its 3' end. In some embodiments, the antigen 25produces a vaccine effect. For put poses of the invention, the term "associated with" means that the antigen is present vhen the cancer, is present, but either is not present, or is present in reduced amounts, when the cancer is absent. The immunostimulatory ohgonucleotides and/or immunomer compound is covalently linked to the antigen, OR it is otherwise operatively associated with the 30antigen. As used herein, the term 'operatively associated with" refers to any association that maintains the activity of both immunostimulatory oligonucleotide and/or immunomer compound and antigen. Nonlimiting examples of such operative associations include being part of the same liposome or other such delivery vehicle or reagent. Additionally, a nucleic acid molecule encoding the antigen can be cloned 5into an expression vector and administered in combination with the immunostimulatory oligonucleotide and/or immunomer compound. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Preferred vectors are those capable of autonomous replication and expression of nucleic acids to which they are linked (e.g., l0an episome). Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors." In general, expression vectors of utility in recoombinant DNA techniques are often in the form of "plasmids" which refer generally :o circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. In the present specification, 15"plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto. In embodiments wherein the immunostimulatory oligonucleotide and/or 20immunomer compound is covalently linked to the antigen, such covalent linkage preferably is at any position on the immunostimulatory oligonucleotide and/or immunomer compound other than an accessible 5' end of an immunostimulatory oligonucleotide. For example, the antigen may be attached at an internucleoside linkage or may be attached to the non-nucleotidic linker. Alternatively, the antigen 25may itself be the non-nucleotidic 1 nker. In a third aspect, the invem ion provides pharmaceutical formulations comprising an immunostimulatory oligonucleotide and/or immunostimulatory oligonucleotide conjugate and/or inmunomer compound or immunomer conjugate according to the invention, a chemotherapeutic agent and a physiologically acceptable 30carrier. As used herein, the term " ohysiologically acceptable" refers to a material that does not interfere with the effec iveness of the immunomer compound and is compatible with a biological system such as a cell, cell culture, tissue, or organism. Preferably, the biological systerr is a living organism, such as a vertebrate. Preferred chemotherapeutic agents include, without limitation Gemcitabine methotrexate, 5vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5- fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitcr, famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Loinetexol, Glamolec, CI-994, TNP-470, lOHycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7 )70, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101. 1SI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D:!163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Vilrubicin, Metastron/strontium derivative, 15Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitibine, Furuilon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077'Cisplatm HMR 1275/Flavopiridol, CP-358 (774)/ EGFR, CP-609 (754)/RAS oncog^ne inhibitor, BMS-182751/oral platinum, UFT (Tegafur/Uracil), Ergamisol/Leva insole, Eniluracil/776C85/5FU enhancer, 20Campto/Levamisole, Camptosar/lrinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Pacli axel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fl ldara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal doxorubicin, Gt mzar/Gemcitabine, ZD 0473/Anormed, YM 116, 251odine seeds, CDJC4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/T ;niposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Anal Dg, nitrosoureas, alkylating agents such as melphelan and cyclophosphamide Aminoglutethimide, Asparaginase, Busulfan, 30Carboplatin, Chlorombucil, Cytanbine HC1, Dactinomycin, Daunorubicin HC1, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5- FU), Flutamide, Hydroxyurea (h/droxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHllH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HC1 (nitrogen imstard), Mercaptopurine, Mesna, Mitotane 5(o.p'-DDD), Mitoxantrone HC1, Octreotide. Plicamycin, Procarbazine HC1, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (metbyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl-CCNU), Teniposide 10(VM-26) and Vindesine sulfate. In yet another embodimen:, the formulations include a cancer vaccine selected from the group consisting of EFG Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/new, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic 15vaccine, Melacine, peptide antigen vaccines, toxin/antigen vaccines, MVA-vased vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus and ImmunCyst/TheraCys. In a further aspect, the invention provides a method for treating cancer in a cancer patient comprising adminis ering to the patient a monoclonal antibody ia 20combination with an immunostimi latory oligonucleotide and/or immunomer compound, as described herein. Passive immunotherapy in the form of antibodies, and particularly monoclonal antibodies, has been the subject of considerable research and development as anti-cancer agents. The term "monoclonal antibody" as used herein refers to an antibody molecile of single molecular composition. A monoclonal 25antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline imm moglobulin sequences. Examples of anti-cancer agents include, but are not limited to, Panorex (Glaxo-Welcome), Rituxan 30(IDEC/Genentech/Hoffman la Rod e), Mylotarg (Wyeth), Campath (Millennium), Zevalin (IDEC and Schering AG;, Bexxar (Corixa/GSK), Erbitux (Imclone/BMS), Avastin (Genentech) and Herceplin (Genentech/Hoffman la Roche). Antibodies may also be employed in active immunotherapy utilising anti-idiotype antibodies which appear to mimic (in an immunological sense) cancer antigens. Monoclonal antibodies 5can be generated by methods known to those skilled in the art of recombinant DNA technology. As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for use in pharmaceutical formulations. I: will be understood that the characteristics of the lOcarrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, e.g , Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990. Toll-like receptors (TLRs) function as sensors of infection and induce the 15activation of innate and adaptive immune responses. TLRs recognize a wide variety of iligands, called pathogen-associa ted molecular patterns (PAMPs). Upon recognizing conserved pathogen-associated molecular products, TLRs activate host defense responses through their intracellular signalling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88. Dendritic cells 20and macrophages normally respond to Toll-like receptor (TLR) ligands and cytokines (for example, interleukin-1 β; IL-6 and tumour necrosis factor, TNF), which they also produce; natural killer (NK) cells and T cells are also involved. After TLR stimulation by bacterial compound;, innate immune cells release a range of cytokines. Some examples of TLR ligands include, but are not limited to, lipoproteins; 25peptidoglycan, zymosan (TLR2), double-stranded RNA, polyI:polyC (TLR3), lipopolysaccharide, heat shock proteins, taxo) (TLR4), flagellin (TLR5), and imidazoquinolines- R848, resiquitnod, uniquimod; ssRNA (TLR7/8). In a fourth aspect, the inver tion provides a method for sensitizing cancer cells to ionizing radiation. The method according to this aspect of the invention comprises 30administering to a mammal an immunostimulatory oligonucleotide or an immunomer compound according to the invention and treating the animal with ionizing radiation. In certain preferred embodiments, •-Irradiation is administered at 1.56 Gy/min. In certain preferred embodiments, adiation therapy is administered from about 0.1 to about 10.0 Gy, preferably from about 0.25 to about 8.0 Gy, more preferably from 5about 0.5 to about 5.0 Gy, or as 3.0 Gy of radiation either twice for one week, four times for one week, or three time s on Days 2, 4, and 9. In certain embodiments pre- treatment with an immunostimuktory oligonucleotide or an immunomer compound is from about 2 to about 6 h prior to • -irradiation. In a fifth aspect, the invention provides a method for synergistically l0stimuIating an immune response in a patient comprising administering to a patient a therapeutically effective synergist c amount of an immunomer compound in combination with a therapeutically effective synergistic amount of IL-2, and an antigen, wherein administration o "said combination synergistically stimulates the production of cytokines in a patient Preferred cytokines stimulated in accordance 15with the invention include but are not limited to one or more of, IL-12, interferon-* , IFN-* and IFN-β. In certain embodiments, the method is for the treatment of cancer and the antigen is one specific to or associated with a cancer. In some embodiments, the method is for the treatment of an i itection and the antigen is an antigen associated 20with the infection. In certain embodiments, the method is for the treatment of an allergy and the antigen is associated with the allergy. As used herein, the term "associated with" means that the antigen is present when the cancer, allergen or infectious disease is present, but e ther is not present, or is present in reduced amounts, when the cancer, allergen or infectious disease is absent. 25 As used herein, the term "antigen" means a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor. Antigens can include peptides, proteins, glycopioteins, polysaccharides, gangliosides and lipids; portions thereof and combinations thereof. The antigens can be those found in nature or can be synthetic. Haptens are included within the scope of "antigen." A hapten is a 301ow molecular weight compound taat is not immunogenie by itself but is rendered immunogenic when conjugated with an immunogenic molecule containing antigenic determinants. In certain embodiments, antigens useful in methods and compositions of the invention are tumor-associated and/or tumor-specific antigens. Non-limiting 5examples include: Prostate Specific Antigen (PSA) and Prostatic Acid Phosphatase (PAP), which are markers normal y present in the blood in small amounts that can be elevated in the presence of prostate cancer; Cancer Antigen 125 (CA-125), which is at elevated levels in patients with ovarian cancer and is sometimes elevated in the presence of other cancers; CA 15-3 and CA 27-29, which are useful in following the lOcourse of breast cancer and its response to treatment; CA 19-9, which is commonly used as a check for the spread of pancreatic cancer and is also elevated in patients with colorectal, stomach and bile duct cancer; Carcinoembryonic Antigen (CEA), which is normally present in small amounts but can be elevated in the blood of patients with a wide variety of cancers; Alpha-Fetoprotein, which is a marker for 15hepatocellular and germ cell (nonseminoma) carcinoma; and Galactosyl Transferase II, an isozyme of galactosyl trans ft rase, that has been shown to be elevated in a variety of malignancies, predominantly gastrointestinal. As known by one skilled in the art, tumor-associated and tumor-specific antigens are available commercially. Also contemplated by the invention are those antigens that can be made by recombinant 20nucleic acid technologies and/or symthetic antigens, e.g., peptides produced by methods known in the art. In certain embodiments of he fifth aspect of the invention, the invention provides a method for treating canoer in a cancer patient comprising administering to the patient a therapeutically effective synergistic amount of 1L-2 in combination with 25an immunomer conjugate, which comprises an immunomer compound, as described above, and an antigen. In certain embodiments, the antigen is conjugated to the immunomer compound at a position other than the accessible 5' end. In some embodiments, the non-nucleotidic linker of the immunomer compound comprises an antigen associated with cancer. In some embodiments, the antigen is conjugated to 30the immunomer compound at a po iition other than its 5' end. In some embodiments, the antigen produces a vaccine effect. For purposes of the invention, the term "associated with" means that the antigen is present when the cancer is present, but either is not present, or is present n reduced amounts, when the cancer is absent. In some embodiments of the fifth aspect of the invention, the immunomer 5compound is covalently linked to the antigen, or it is otherwise operatively associated with the antigen. As used herein, the term "operatively associated with" refers to any association that maintains the activity of the immunomer compound and antigen. Nonlimiting examples of such operative associations include being part of the same liposome or other such delivery vaiicle or reagent. In embodiments wherein the l0immunomer compound is covalent y linked to the antigen, such covalent linkage preferably is at any position on the immunomer compound other than at an accessible 5' end of the immunomer compound. For example, the antigen may be attached at an internucleoside linkage or may be attached to the non-nucleotidic linker. Alternatively, the antigen may itself be the non-nucleotidic linker. 15 In a sixth aspect of the invention, at least one immunostimulatory oliconucleotide that is not an immunomer compound is used in combination with a therapeutically effective amount of IL-2 to selectively and synergistically stimulate the production of cytokines in a patient Preferred cytokines synergistically stimulated in accordance with the invention are s ilected from the group consisting of, IL-12 and 20IFN-* ,IFN-» ,IFN-β or combinations thereof. In accordance with the present invention, preferred immunostimulatory oligonucleotides that are not immunomer compounds include those containingat least one immunostimulatory CpG dinucleotide wherein C is not cytos ne or deoxycytosine and/or G is not guanosine or 2-deoxyguanosine. Other preferred immunosumulatory oligonucleotides of the 25invention that are not immunomer c ompounds are those that include alternative immunostimulatory moieties that are not CpG. Examples of such alternative immunostimulatory moieties include but are not limited to nucleosides comprising non-naturally occurring bases and/or sugar and secondary structures of the oligonucleotide itself such as hairpi in structures that stabilize the oligonucleotide, as 30described in the following U.S. patents and pending U.S. patent applications and are incorporated herein by reference: U.S. Patent Numbers 6,426,334 and 6,476,000; and U.S. Patent Application Numbers 09/770,602,09/845,623,09/965,116,60/440,587, 10/361,111,60/471,247,60/477,6)8. In certain embodiments of the invention, each of the immunomer compound or 5immunostimulatory oligonucleotice and IL-2 is admixed with a pharmaceutically acceptable carrier prior to administration to the patient. In certain embodiments, the immunomer compound or immune stimulatory oligonucleotide are mixed together with a pharmaceutically acceptable carrier prior to administration, or combined as part of a pharmaceutical composition as, described in the fourth aspect of the invention. As lOused herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing I5these materials is described in, e.g.. Remington: The Science and Practice of Pharmacy, 20* Edition, ed. A. L. Gennaro, Lippincott Williams & Wilkins Publishing Co., Philadelphia, PA, 19106 (ISBN: 0683306472). In a seventh aspect, the invention provides therapeutic compositions comprising a pharmaceutically acce ptable carrier, a therapeutically effective 20synergistic amount of an immunomsr compound or immunostimulotory oligonucleotide, a therapeutically effective synergistic amount of IL-2 and optionally, an antigen, wherein administration of said therapeutic composition synergistically stimulates the production of cytokines in a patient. Preferred cytokines that are synergistically stimulated in accord mce with the invention are selected from the 25group consisting of IL-12 and interieron-* ,IFN-» ,IFN-β or combinations thereof. AH aspects of the invention are useful in the treatment of disease, and are particularly useful in immune-based therapies for treating cancer, infectious diseases and allergies. As used herein the tern "treating" or "treatment" of disease includes: prevention of disease; dimunition o • eradication of signs or symptoms of disease after 30onset; and prevention of relapse of disease. In the methods according 10 the invention, administration of an immunomer compound or immmumostimulatcry oligonucleotide in combination with IL-2 can be by any suitable route including, w thout limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, 5by gene gun, dermal patch or in eye drop or mouthwash form. Administration of immunomer compounds, immunostimulatory oligonucleotides, IL-2 or therapeutic compositions thereof can be carried out using known procedures using therapeutically effective synergistic amounts and for periods of time effective to treat disease. The term "in combination with" means in the course of treating the same lOdisease in the same patient, and includes administering the immunomer compound and /or immunostimulatory oligonucleotide and/or IL-2 in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of the immunomer compound and /or immunostimulatory 15oligonucleotide, and/or IL-2, independently. The administration of the immunomer compound and IL-2 may be by the same or different routes. One of skill in the art will aopreciate that such synergistic effect of either the immunomer compound or immunostimulator/ oligonucleotide, IL-2 or both may vary considerably depending on the tissie, organ, the particular disease or the patient to be 20treated in accordance with the invention. Furthermore, one of skill in the art will appreciate that the therapeutically effective synergistic amount of either the immunomer compound or immunostimulatory oligonucleotide or IL-2 may be lowered or increased by fine tuning and altering the amount of the other component. When administered systemioally, the immunomer compound is preferably 25administered at a sufficient dosage :o attain a blood level of immunomer compound from about 0.0001 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of immunostimulatory oligonucleotide and/or immunomer compound ranges from about 0.0001 mg per 30patient per day to about 200 mg per kg body weight per day. It may be desirable to administer simultaneously, or seq .lentially, a therapeutically effective synergistic amount of each of the immunomer compound or IL-2 to an individual as a single treatment episode. Preferably, IL- 2 is administered in an amount of about 750 to about 75,000 units. 5 The invention provides a kit comprising a cytokine and.or chemotherapeutic agent, and immunostimulatory olgonucleotides and/or immunomer compounds, the latter comprising at least two oligonucleotides linked together, such that the immunomer compound has more than one accessible 5' end, wherein at least one of l0the oligonucleotides is an immunostimulatory oligonucleotide. In another aspect, the kit comprises an immunostimulato y oligonucleotide and/or immunostimulatory oligonucleotide conjugate and/or inmunomer compound or immunomer conjugate according to the invention, a cytokine and/or chemotherapeutic agent and a physiologically acceptable carrier. The kit will generally also include a set of 15instructions for use. The examples below are intended to farther illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention, EXAMPLES Example 1: Synthesis of Oligcnucleotides Containing Immunomodulatory 5 Moieties Oligonucleotides were syn :hesized on a 1 μmol scale using an automated DNA synthesizer (Expedite 8909; PerSeptive Biosystems, Framingham, MA), following the linear synthesis or parallel synthesis procedures outlined in Figures 5 and 6. 10 Deoxyribonucleoside phosphoramidites were obtained from Applied Biosystems (Foster City, CA). 1 ;2:'-dideoxyribose phosphoramidite, propyl-1- phosphoramidite, 2-deoxyuridine phosphoramidite, l,3-bis-[5-(4,4'-dimethoxytrityl) pentylamidyl]-2-propanol phospho -amidite and methyl phosponamidite were obtained from Glen Research (Sterling, VA). -L-2'-deoxyribonucleoside phosphoramidite, - 152'-deoxyribonucleoside phosphoramidite, mono-DMT-glycerol phosphoramidite and di-DMT-glycerol phosphoramidite were obtained from ChemGenes (Ashland, MA). (4-Aminobutyl)-l,3-propanediol p\ osphoramidite was obtained from Clontech (Palo Alto, CA). Arabinocytidine phosphoramidite, arabinoguanosine, arabinothymidine and arabinouridine were obtained from Reliable Pharmaceutical (St. Louis, MO). 20Arabinoguanosine phosphoramiditt, arabinothymidine phosphoramidite and arabinouridine phosphoramidite were synthesized at Hybridon, Inc. (Cambridge, MA) (Noronha et al. (2000) Biochem., 39: 7050-7062). All nucleoside phosphoramidites were characterized by 31P and !H NMR spectra. Modified nucleosides were incorporated at specific sites using normal 25coupling cycles. After synthesis, oligonucleotides were deprotected using concentrated ammonium hydroxide and purified by reverse phase HPLC, followed by dialysis. Purified oligonucleotides is sodium salt form were lyophilized prior to use. Purity was tested by CGE and MAI DI-TOF MS. Example 2: Analysis of Spleen Cell Proliferation In vitro analysis of splenoeyte proliferation was carried out using standard procedures as described previousl / (see, e.g., Zhao et al., Biochem Pharma 51 .173- 182 (1996)). The results are shown in Figure 8A. These results demonstrate that at 5the higher concentrations, Immunomer 6, having two accessible 5' ends results in greater splenocyte proliferation than does Immunomer 5, having no accessible 5' end or Oligonucleotide 4, with a single accessible 5' end. Immunomer 6 also causes greater splenocyte proliferation then the LPS positive control. Example 3: In vivo Splenomegaly Assays 10 To test the applicability of the in vitro results to an in vivo model, selected oligonucleotides were administered to mice and the degree of splenomegaly was measured as an indicator of the level of immunostimulatory activity. A single dose of 5 mg/kg was administered to BALB/c mice (female, 4-6 weeks old, Harlan Sprague Dawley Inc, Baltic, CT) intraperito leally. The mice were sacrificed 72 hours after 15oligonucleotide administration, and spleens were harvested and weighed. The results are shown in Figure 8B. These results demonstrate that Immunomer 6, having two accessible 5' ends, has a far greater immunostimulatory effect than do Oligonucleotide 4 or Immunomer 5. Example 4: Cytokine Analysis 20 The secretion of IL-12 and 1L-6 in vertebrate cells, preferably BALB/c mouse spleen cells or human PBMC, was measured by sandwich ELISA. The required reagents including cytokine antibodies and cytokine standards were purchased form PharMingen, San Diego, CA. ELISA plates (Costar) were incubated with appropriate antibodies at 5 g/mL in PBSN buffer (PBS/0.05% sodium azide, pH 9.6) overnight 25at 4°C and then blocked with PBS/1% BSA at 37 °C for 30 minutes. Cell culture supernatants and cytokine standards were appropriately diluted with PBS/10% FBS, added to the plates in triplicate, and incubated at 25 °C for 2 hours. Plates were overlaid with 1 g/mL appropriate biotinylated antibody and incubated at 25 °C for 1.5 hours. The plates were then washed extensively with PBS-T Buffer (PBS/0.05% Tween 20) and further incubated at 25 °C for 1.5 hours after adding streptavidin conjugated peroxidase (Sigma, St. Louis, MO). The plates were developed with Sure Blue™ (Kirkegaard and Perry) chromogenic reagent and the reaction was terminated by adding Stop Solution (Kirkega* rd and Perry). The color change was measured on a SCeres 900 HDI Spectrophotometei (Bio-Tek Instruments). The results are shown in Table 5 A below. Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers by Ficoll-Paque density gradient centrifugation (Histopaque-1077, Sigma, St. Louis, MO). Briefly, heparinized blood was layered lOonto the Histopaque-1077 (equal volume) in a conical centrifuge and centrifuged at 400 x g for 30 minutes at room temperature. The buffy coat, containing the mononuclear cells, was removed carefully and washed twice with isotonic phosphate buffered saline (PBS) by centrifugation at 250 x g for 10 minutes. The resulting cell pellet was then resuspended in RPM1 1640 medium containing L-glutamine 15(MediaTech, Inc., Herndon, VA) ar d supplemented with 10% heat inactivated FCS and penicillin-streptomycin (100U/nl). Cells were cultured in 24 well plates for different time periods at 1 X 106 eells/ml/well in the presence or absence of oligonucleotides. At the end of the mcubation period, supernatants were harvested and stored frozen at -70 • Cuntil assayed for various cytokines including IL-6 (BD 20Pharmingen, San Diego, CA), IL-K (BD Pharmingen), 1L-12 (BioSource International, Camarillo, CA), IFN- (BioSource International) and - (BD Pharmingen) and TNF- (BD Phamingen) by sandwich ELISA. The results are shown in Tables 9 and 9A below. In all instances, the levels ol IL-12 and IL-6 in the cell culture supematants 25were calculated from the standard cirve constructed under the same experimental conditions for IL-12 and IL-6, respectively. The levels of IL-10, IFN-gamma and TNF-* in the cell culture supernatar is were calculated from the standard curve constructed under the same experimental conditions for IL-10, IFN-gamma and TNF- • Respectively. Example 5: Immunostimulatory Activity of Immunomer Compounds Containing A Non-Natural Pyrimidine or Non-Natural Purine Nucleoside As shown in Tables 10-12 immunostimulatory activity was maintained for immunomer compounds of various lengths having a non-natural pyrimidine 5nucleoside or non-natural purine nucleoside in the immunostimulatory dinucleotide motif. Example 6: Effect of the Linker on Immunostimulatory Activity In order to examine the effect of the length of the linker connecting the two 5oligonucleotides, immunomer compaunds that contained the same oligonucleotides, but different linkers were synthesized and tested for immunostimulatory activity. The results shown in Table 13 suggest that: linker length plays a role in the immunostimulatory activity of immunomer compounds. The best immunostimulatory effect was achieved with C3- to C6-ilkyl linkers or abasic linkers having interspersed Is sphate charges. oligonucleotides with a phosphorc thioate backbones. This lower degree of immunostimulatory activity could be due in part to the rapid degradation of phosphodiester oligonucleotides under experimental conditions. Degradation of oligonucleotides is primarily the result of 3'-exonucleases, which digest the 5oligonucleotides from the 3' end. The immunomer compounds of this example do not contain a free 3' end. Thus, immunomer compounds with phosphodiester backbones should have a longer half life under experimental conditions than the corresponding monomeric oligonucleotides, and thould therefore exhibit improved immunostimulatory activity. The resuits presented in Table 14 demonstrate this l0effect, with Immunomers 84 and 85 exhibiting immunostimulatory activity as determined by cytokine induction in BALB/c mouse spleen cell cultures. Table 14 Immunomer Structure and Immunostimulatory Activity ! No. ! Sequences and Modification (5'-3') ' Oligo Length/ IL-12 (pg/mL) : IL-6 (pg/mL) | ■ or Each Chain 0.3 g/mL 1 g/mL 4 5M3TATCTGACGTTCTCTGT-3' 18mer 225 i 1462 I i i i ■ ' ■ 84 ° 5i-CTGACGTfCTCTGT-31-i 4 14mer t 1551 • 159 h-3'-T-5'(PO) S'-CTGACGTTCTCTGT-a'-J | ; 85 i S'-LLCfGACGTTCTCTGT-S'-i 14mer 466 467 ; 5'-LLCTGACGTTCTCTGT-3'-l L = C3-Linker 15Example 8: In vivo anti-cancer activity of immunomer compounds in combination with chemotherapentic agents PC3 cells were cultured in )()% Ham s, F12K Medium with 10% Fetal Bovine Serum (FBS), in presence of 100 I /ml Penicillin and 100 μg/ml Streptomycin to establish the Human Prostate cancer model (PC3). Male athymic nude mice, 4-6 20weeks old (Frederick Cancer Research and Development Center, Frederick, MD), were accommodated for 6 days for environmental adjustment prior to the study. Cultured PC3 cells were harvested from the monolayer cultures, washed twice with Ham's, F12K Medium (10% FBS) resuspended in FBS-free Ham's, F12K Medium: Matrigel basement membrane matrix (Becton Dickinson Labware, Bedford, MA) 25(5:1; V/V), and injected subcutaneously (5 X 106 cells, total volume 0.2 ml) into the left inguinal area of each of the miee. The animals were monitored by general clinical observation, body weight, and turror growth. Tumor growth was monitored by the measurement, with calipers, of two perpendicular diameters of the implant. Tumor mass (weight in grams) was calculated by the formula, l/2a X b2, where 'a' is the long 5diameter (cm) and 'b' is the short diameter (cm). When the mean tumor sizes reached ~80mg, the animals bearing human cancer xenografts were randomly divided into the treatment and control groups (5 an mals/group). The control group received sterile physiological saline (0.9% NaCl) c nly. Immunomers 26 or 194, aseptically dissolved in physiological saline, was administered by subcutaneously injection at dose of 0.5 or 101.0 mg/kg/day, 3 doses/week. Gemcitabine HC1 (Eli Lilly and Company, Indianapolis, IN) was given twice by intraperitoneal injection at 160 mg/kg on Day 0 and 3. The detailed treatment schedule is shown as follows. Gl: Saline G2: Gemcitabint (160 mg/kg/day, IP, Day 0 and 3) 15 G3: 26 (1.0 mg/l g/day, SC, 3 doses /week, for 6 weeks) G4: 26 (0.5 mg/lg/day, SC, 3 doses /week, for 6 weeks) G5: 194 (1.0 mg, kg/day, SC, 3 doses /week, for 6 weeks) G6: 194 (0.5 mg, kg/day, SC, 3 doses /week, for 6 weeks) G7: 26 (0.5 mg/l g/day, SC, 3 doses /week, for 6 weeks) + 20 Gemcitabine (160 mg/kg/day, Day 0 and 3) G8: 194 (0.5 mg kg/day, SC, 3 doses /week, for 6 weeks) + Gemcitabint (160 mg/kg/day, Day 0 and 3) The tumor measurements ifter various treatments are presented in Table 15 and Figure 13. The tumor growth in all Immunomer 26 and 194 treated animals was 25remarkably inhibited compared with saline control (p dose-response relationship in these treatment groups (Fig. 13). There was no significant difference between 26 and 194 (Table 15). The body weight measurer lents after treatments at various times are presented in Table 16 and Figure 14. There was no significant difference in body weight gains among 26 or 194 alone compared .with controls. Gemcitabine treated animals had body weight loss in the first week and recovered in a week afterwards. Combination 5with 26 or 194 did not change the side effect profiles of Gemcitabine. No other clinical abnormality or death was observed in all the groups. In summary, 26 and 194significantly inhibited tumor growth in nude mice bearing human prostate cancer PC3 xenografts with no significant side effects. When 26 or 194 was given in combination with Gemcitabine, each compound significantly 15increased the therapeutic effect of Gemcitabine without changes in side effect profiles, hi addition, there was a tendency in dose dependent response of 26 or 194 treatment. Example 9 In vivo anti-cancer activity of immunomer compounds in combination with chemotherapeutic agents The experiment of Example 8 was repeated using taxotere instead of Gemcitabine. Taxotere was administered on days 0 and 7. 165 was administered 5 days per week. 26 and 194 were administered on days 0, 2, 4, 7, 9 and 11. The results are shown in Table 17 below. These results clearly demonstrate synergy between the 5immunomer compounds and taxotere. Example 10 Administration of Immunostimulatory Oligonucleotides and IL-2 Splenocytes were isolated rom BALB/c mice as described above and were plated in 24-well dishes at a density of 5x10(' cells/mL. CpG oligonucleotides were dissolved in TE buffer (10 mM Tns-HCI, pH 7.5, 1 mM EDTA) was added to a final 5concentration of 0.03,0.1,0.3,1.0 3.0, or 10.0 μg/mL to mouse spleen cell cultures. In order to study the role of IL-2 in CpG oligonucleotide-induced time-dependent cytokine secretion, recombinant human IL-2 (Sigma) was added at a concentration of 10 U/ml at the start of the experiment. The cells were then incubated at 37. Cfor 4, 8, 24 and 48 h in the presence of test oligonucleotides and the supernatants were lOcollected for ELISA assays. Untre ited cells (only IL-2 addition) were taken as controls. The secretion of mouse IL-12, IL-6 and IFN-* was measured by sandwich ELISA. The required regents, including cytokine antibodies and standards were purchases from PharMingen. ELIS A plates (Costar) were incubated with appropriate IScapture antibodies in PBSN (PBS/O.05% sodium azide, pH 9,6) buffer overnight at 4- Cand then blocked with PBS/10/. BSA at 37. Cfor 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* Cfor 2 h. Plates were washed and incubated with the appropriate biotiaylated antibody and incubated at 25. Cfor 1.5 20h. The plates were washed extensi rely with PBS/0.05% Tween 20 and then further incubated at 25. Cfor 1.5 h. after addition of streptavidine-conjugated peroxidase (Sigma). Plates were developed w th Sure Blue™ (Kirkegaard and Perry) chromogenic reagent and the reaction was terminated by adding Stop Solution (Kirkegaard and Perry). The color change was measured on a Ceres 900 HDI 25Spectrophotometer (Bio-Tek Instruments) at 450nm. The levels of IL-12, IL6 and IFN-* in the cell culture supernatants were calculated from the standard curve constructed under the same experimental conditions for IL-12, IL-6 and IFN- • respectively. The oligonucleotides used n this study are presented in Table 18. 30 Table 18 The results are shown in Figs 15-19 Not shown is an assay indicating that the use of SEQ ID NOs 86-90 alone stimulate IFN-* production only negligibly. The results 35demonstrate synergy between SEQ ID NOs 86-90 and IL-2 in generating secretion of IL-6, IL-12andIFN-. . WE CLATM; 1. A pharmaceutical composition comprising at least one immunomer compound wherein the immunomer compound comprises two oligonucleotides linked together at their 3' ends, an imernucleotide linkage, or a functionalized nucleobase or sugar by a non- mcleotidic linker, wherein at least one of the oligonucleotides is an immunostimulatory oligonucleotide having an accessible 5' end and comprising an mrnuntostimulatory dinucleotide selected from the group consisting of CpG, C*pG, CpG*, and C*p*G. and a therapeutically effective synergistic amount of 1L-2, wherein said composition is administrate for stimulating the production of one or more cytokines selected from the group consisting of IL-12, IFN- y, IFN-α, and IFN-β or combinations thereof. 2. A pharmaceutical composition as claimed in claim 1, wherein C is cytidine or 2'-deoxycytidine, C* is 2'-deo:cythymidine, arabinocytidine, 2'-deoxy- 2'-substitutedarabinocytidine, 2'-O-substitutedarabinocytidine, 2'-deoxy-5- hydroxycytidine, 2'-deoxy-N4-alkyl-cyticline, 2'-deoxy-4-thiouridine or other non- natural pyrimidine nucleoside, G is guanosine or 2'-deoxyguanosine, G* is 2'-deoxy-7-deazaguanosine, 2 -deoxy-6-thioguanosine, arabinoguanosine, 2' -deoxy-2' substituted-arabinc guanosine, 2' -O-substituted-arabinoguanosine, or other non-natural purine nucle( iside, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate. 3. A pharmaceutical composition as claimed in claim 1 or 2 for treating cancer in a mammal who is treated with ionizing radiation. 4. A pharmaceutical composition as claimed in claim 3 for treating cancer in a mammal, wherein the mamml is treated with y-irradiation at 1.56 Gy/min. 5. A pharmaceutical composition as claimed in claim 3 for treating cancer in a mammal, wherein the mamma 1 is treated with 3 Gy of radiation either twice for, one week, four times for one week, or three times on Days 2, 4, and 9. 6. A pharmaceutical compositior as claimed in claim 3 for treating cancer in a mammal, wherein the mammal is pre-treated with an immunomer compound from 2 to 6 h prior to y-irradiation. 7. A pharmaceutical compositior as claimed in claim 1 or 2 for stimulating an immune response in a patient. 8. A pharmaceutical compositior as claimed in claim 1, wherein the immunomer compound has the following s ructure: 9. A pharmaceutical composition as claimed in claim 1 or 2, optionally containing an antigen. 10. A pharmaceutical compositic n as claimed in claim 9, wherein the antigen is an antigen associated with cancer, infectious disease or allergy. 11. A pharmaceutical composition as claimed in claim 1 or 2, for treating an allergy in a patient. 12. A pharmaceutical composition as claimed in claim 11, optionally containing an antigen associated with said i llergy. 13. A pharmaceutical composition as claimed in claim 1 or 2, for treating an infectious disease in a patient 14. A pharmaceutical composition as claimed in claim 13, optionally containing an antigen associated with said infectious disease. The invention provides optimized meincds and compositions for enhancing the immunc responce caosed by im- munostimulaiory compoands used for the acarment of cisiase such as, but not limited to, treatment of cancer, auloimmune disordes, asthma, respiratory allergies, food allergies and infections diseases in a patienL The optimized methods according to the invention provide synergy berween the therapentic effects of immtmostimulatory oligonucleotides and immunomer compounds in accordancc with the invention, and the therapeutic effect of cytokire immunothcrapy and/or chemothcrapeulic agents and/or radiation.

Documents:

306-KOLNP-2006-(09-04-2012)-CORRESPONDENCE.pdf

306-KOLNP-2006-(09-04-2012)-FORM-27.pdf

306-KOLNP-2006-FORM-27.pdf

306-kolnp-2006-granted-abstract.pdf

306-kolnp-2006-granted-assignment.pdf

306-KOLNP-2006-GRANTED-CLAIMS.pdf

306-kolnp-2006-granted-correspondence.pdf

306-kolnp-2006-granted-description (complete).pdf

306-kolnp-2006-granted-examination report.pdf

306-kolnp-2006-granted-form 1.pdf

306-kolnp-2006-granted-form 18.pdf

306-kolnp-2006-granted-form 3.pdf

306-kolnp-2006-granted-form 5.pdf

306-kolnp-2006-granted-gpa.pdf

306-kolnp-2006-granted-reply to examination report.pdf

306-kolnp-2006-granted-specification.pdf