USOO7427405B2

(12) United States Patent (10) Patent No.: US 7.427.405 B2 Agrawal et al. (45) Date of Patent: Sep. 23, 2008

(54) IMMUNOSTIMULATORY Tokunaga et al., “Antitumor Activity of Deoxyribonucleic Acid Frac OLGONUCLEOTDE MULTIMERS tion from Mycobaterium bovis BCG. I. Isolation. Physicochemical Characterization, and Antitumor Activity”. J. Natl. Cancer Inst. 72: (75) Inventors: Sudhir Agrawal, Shrewsbury, MA (US); 955-962(1984). Pisetsky et al., “Stimulation of in vitro proliferation of murine lym Ekambar Kandimala, Southboro, MA phocytes by synthetic oligodeoxynucleotides'. Molecular Biology (US); Dong Yu, Westboro, MA (US) Reports 18:217-221 (1993). Krieg et al., “CpG motifs in bacterial DNA trigger direct B-cell (73) Assignee: Idera Pharmaceuticals, Inc., activation”, Nature 374: 546-549 (1995). Cambridge, MA (US) Sato et al., “Immunostimulatory DNA Sequence Necessary for Effective Intradermal Gene Immunization', Science 273: 352-354 (*) Notice: Subject to any disclaimer, the term of this (1996). patent is extended or adjusted under 35 Krieg et al., “CpG Motifs in Bacterial DNA and their Immune U.S.C. 154(b) by 209 days. Effects”, Annu. Rev. Immunol. 20: 709-760 (2002). Dalpke et al., “Immunopharmacology of CpG DNA'. Biol. Chem. (21) Appl. No.: 11/174,282 383: 1491-1500 (2002). Kandimala et al., “Towards Optimal Design of Second-Generation (22) Filed: Jul. 1, 2005 Immunomodulatory Oligonucleotides'. Curr. Opin. Mol. Ther. 4(2): 122-129 (2002). (65) Prior Publication Data Kandimala et al., “Immunomers-novel 3'-3'-Linked CpG Oligodeoxyribonucleotides as Potent Immunomodulatory Agents'. US 2006/OO19919 A1 Jan. 26, 2006 Nucleic Acids Res. 30: 4460-4469 (2002). Kandimala et al. “Secondary Structures in CpG Oligonucleotides Related U.S. Application Data affect Immunostimulatory Activity”. Biochem. Biophys. Res. Com mun. 306:948-953 (2003). (63) Continuation of application No. 10/892,550, filed on Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Jul. 15, 2004, and a continuation of application No. Mack Publishing Co., Easton, PA, 1990, ISBN: 0-912734-04-3. 11/153,054, filed on Jun. 15, 2005. Burgstaller, P. et al., "Aptamers and AptaZymes: Accelerating Small (60) Provisional application No. 60/579,985, filed on Jun. Molecule Drug Discovery'. Curr Opin Drug Discov Devel. 5(5): 15, 2004, provisional application No. 60/599,362, 690-700 (2002). Iyer R.P. et al., “3H-1,2-Benzodithiole-3-one 1,1-Dioxide as an filed on Aug. 6, 2004. Improved Sulfurizing Reagent in the Solid-Phase Synthesis of Oligodeoxyribonucleoside Phosphorothioates'. J. Am. Chem. Soc. (51) Int. Cl. 112: 1253-1254 (1990). A6 IK 39/00 (2006.01) Zhao, Q., et al., “Effect of Different Chemically Modified C7H 2L/02 (2006.01) Oligodeoxynucleotides on Immune Stimulation'. Biochem (52) U.S. Cl...... 424/277.1; 424/277.1536/23.1 Pharmacol. 51: 173-182 (1996). (58) Field of Classification Search ...... 424/277.1; Branda, R.F., et al., “Immune Stimulation by an antisense oligomer 536/23.1 complementary to the rev Gene of HIV-1'. Biochem. Pharmacol. See application file for complete search history. 45(10): 2037-2043 (1993). Bhagat L., et al., "CpG penta- and Hexadeoxyribonucleotides as (56) References Cited potent immunomodulatory agents'. Biochem. BiophyS. Res. Com mun. 300: 853-861 (2003). U.S. PATENT DOCUMENTS Hemmi H., et al., “A Toll-Like Receptor Recognizes Bacterial DNA'. 5,366,878 A 11/1994 Pederson et al. Nature 408: 740–745 (2000). 5,635,377 A 6, 1997 Pederson et al. Yi A.K., et al., “Rapid Induction of Mitogen-Activated Protein 5,652,355 A 7, 1997 Metelev et al. Kinases by Immune Stimulatory CpG DNA'. J. Immunol. 161:4493 5,912,332 A 6/1999 Agrawal et al. 4497 (1998). 6,143,881 A 11/2000 Metelev et al. Stacey, K.J., et al., “Macrophages Ingest and Are Activated by Bac 6,346,614 B1 2/2002 Metelev et al. terial DNA”, J. Immunol. 157: 21 16-2122 (1996). Yu D., et al., “Potent CpG Oligonucleotides Containing OTHER PUBLICATIONS Phosphodiester Linkages: in vitro and in vivo immunostimulatory properties”. Biochem. Biophys. Res. Commun. 297: 83-90 (2002). Moldoveanu et al (Vaccine 16:1216-1224, 1998).* Kuramoto etal (Jpn. J. Cancer Res. 83:1128-1131, 1992).* * cited by examiner Krieg etal (Nature 371:546-549, 1995).* Liang etal (J. Clin. Invest. 98: 1119-1129, 1996).* Primary Examiner Brandon J Fetterolf McCluskie and Davis (J. Immunol. 161:4463-4466, 1998).* (74) Attorney, Agent, or Firm—Keown & Zucchero, LLP: Baral et al (Cancer Immunol. Immunother. 52(3):317-327, 2003).* Wayne A. Keown; Joseph C. Zucchero Kandimala et al (Bioconjugate Chem. 13, 966-974, 2002; Nucleic Acid Research vol. 31, No. 9, 2003).* (57) ABSTRACT Kandimala et al (PNAS 100(24): 14303-14308, 2003).* Donnelly et al (Nature Medicine, 9(11): 1354-1356, 2003).* The invention provides an immunostimulatory nucleic acid. DeGruijl et al. Nature Medicine, 51.10: 1124-1125, 1999.* Bitton R.J. (Current Opinion in Molecular Therapeutics 611: 17-26, In certain embodiments according to this aspect of the inven 2004).* tion, the sequence of the immunostimulatory oligonucleotide Weiner (J. Leukoc. Biol. 68: 455-463, 2000).* and/or immunomer is at least partially self-complementary. Ballas et al (J. Immunol. 167: 4878-4886, 2001).* Agrawaletal (Trends in Molecular Medicine 8(3): 114-120, 2002).* 7 Claims, 6 Drawing Sheets U.S. Patent Sep. 23, 2008 Sheet 1 of 6 US 7.427.405 B2 U.S. Patent Sep. 23, 2008 Sheet 2 of 6 US 7.427.405 B2 U.S. Patent US 7.427.405 B2

U.S. Patent US 7.427.405 B2

U.S. Patent Sep. 23, 2008 Sheet 6 of 6 US 7.427.405 B2 US 7,427,405 B2 1. 2 IMMUNOSTIMULATORY ythymidine, 1-(2'-deoxy-f-D-ribofuranosyl)-2-oxo-7-deaza OLGONUCLEOTDE MULTIMERS 8-methyl-purine, 2-dideoxy-5-halocytosine, 2'-dideoxy-5- nitrocytosine, arabinocytidine, 2'-deoxy-2-substituted RELATED APPLICATIONS arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy 5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4- This application is a continuation-in-part of U.S. patent thiouridine, or other pyrimidine nucleoside analogs, G is application Ser. No. 10/892,550, filed Jul. 15, 2004 and a 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, ara continuation of U.S. patent application Ser. No. 1 1/153,054, binoguanosine. 2'-deoxy-2'substituted-arabinoguanosine, filed Jun. 15, 2005, which claims the benefit of U.S. Provi 2'-O-substituted-arabinoguanosine. 2'-deoxyinosine, or other sional Application Ser. No. 60/579,985, filed Jun. 15, 2004, 10 purine nucleoside analogs, and p is an internucleoside linkage and U.S. Provisional Application Ser. No. 60/599,362, filed selected from the group consisting of phosphodiester, phos on Aug. 6, 2004. The entire teachings of the above-referenced phorothioate, and phosphorodithioate. Applications are incorporated herein by reference. In Some embodiments, the immunostimulatory nucleic acid is from about 2 to about 50 nucleotides in length. In BACKGROUND OF THE INVENTION 15 certain embodiments the immunostimulatory nucleic acid is from about 12 to about 26 nucleotides in length. In some 1. Field of the Invention embodiments, the oligonucleotides each have from about 3 to The invention relates to immune stimulation by oligo about 35 nucleoside residues, in further embodiments from nucleotide analogs. about 4 to about 30 nucleoside residues, in even further 2. Summary of the Related Art embodiments from about 4 to about 20 nucleoside residues. Tokunaga et al., J. Natl. Cancer Inst. 72 (1984) 955-96. In some embodiments, the oligonucleotides have from about Pisetsky et al.; Reich et al., Mol. Biol. Rep. 18 (1993) 217 5 to about 18, or from about 5 to about 14, nucleoside resi 221; Krieg et al., Yi et al., Nature 374 (1995) 546-549 and dues. As used herein, the term “about implies that the exact Sato et al., Science 273 (1996) 352-354 teach that bacterial number is not critical. Thus, the number of nucleoside resi DNA, synthetic oligodeoxynucleotides, and DNA vaccines 25 dues in the oligonucleotides is not critical, and oligonucle containing unmethylated CpG-dinucleotides in specific otides having one or two fewer nucleoside residues, or from sequence contexts (CpG DNA) activate the vertebrate one to several additional nucleoside residues are contem immune system. plated as equivalents of each of the embodiments described Krieg et al., Annu. Rev. Immunol. 20 (2002) 709-760; above. In some embodiments, one or more of the oligonucle Dalpke et al., Biol. Chem. 383 (2002) 1491-1500 and Kan 30 otides have 11 nucleotides. dimala et al., Curr. Opin. Mol. Ther. 4 (2002) 122-129 teach In a second aspect the invention provides an immunomer that CpG DNAS induce innate immune cells to produce Th1 comprising at least two oligonucleotides linked by a non cytokines that promote cytotoxic T lymphocyte (CTL) nucleotide linker, wherein the sequences of the immuno responses and production of immunoglobulins by B cells. stimulatory oligonucleotides are at least partially self The immunostimulatory properties of CpG DNAs have 35 complementary. In certain embodiments according to this allowed their use as therapeutic agents for a broad spectrum aspect of the invention at least one of the oligonucleotides of disease indications including cancers, viral and bacterial contains at least one dinucleotide selected from the group infections, inflammatory disorders and as adjuvant in immu consisting of CpG, CpG, CpG* and CpG*, wherein C is notherapy. cytidine or 2'-deoxycytidine, G is guanosine or 2'-deoxygua In addition to chemical modifications, a number of struc 40 nosine, C* is 2'-deoxythymidine, 1-(2'-deoxy-f-D-ribofura tural modifications influenced the activity of CpG DNAs. nosyl)-2-oxo-7-deaza-8-methyl-purine, 2-dideoxy-5-halo Kandimalla et al., Nucleic Acids Res. 30 (2002) 4460-4469 cytosine. 2'-dideoxy-5-nitrocytosine, arabinocytidine, teaches that CpG DNAs that contained two freely accessible 2'-deoxy-2-substituted arabinocytidine, 2'-O-substitutedara 5'-ends through a 3'-3'-linkage had greater activity than did binocytidine. 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4 conventional CpG DNAs containing multiple copies of CpG 45 alkyl-cytidine, 2'-deoxy-4-thiouridine, or other pyrimidine motifs and a single 5'-end. nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine, Kandimalla et al. Biochem. Biophys. Res. Commun. 306 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy (2003) 948-953 teaches that the presence of a secondary 2'substituted-arabinoguanosine, 2'-O-substituted-ara structure in CpG DNAs significantly affected their activity binoguanosine. 2'-deoxyinosine, or other purine nucleoside depending on the position and nature of the secondary struc 50 analogs, and p is an internucleoside linkage selected from the ture, that the presence of a hairpin structure at the 5'-end group consisting of phosphodiester, phosphorothioate, and abrogated Stimulatory activity, and that the same structure at phosphorodithioate. the 3'-end had an insignificant effect on stimulatory activity In Some embodiments, the immunostimulatory nucleic but caused lower IL-6 secretion and contributed to higher acid is from about 2 to about 50 nucleotides in length. In stability against nucleases. 55 certain embodiments the immunostimulatory nucleic acid is There remains a need to “customize' the immune response from about 12 to about 26 nucleotides in length. In some through modification of oligonucleotide analogs. embodiments, the oligonucleotides each have from about 3 to about 35 nucleoside residues, or from about 4 to about 30 BRIEF SUMMARY OF THE INVENTION nucleoside residues, or from about 4 to about 20 nucleoside 60 residues. In some embodiments, the oligonucleotides have In a first aspect the invention provides an immunostimula from about 5 to about 18, or from about 5 to about 14, tory oligonucleotide the sequence of which is at least partially nucleoside residues. As used herein, the term “about implies self-complementary. The immunostimulatory nucleic acid that the exact number is not critical. Thus, the number of comprises an oligonucleotide sequence containing at least nucleoside residues in the oligonucleotides is not critical, and one dinucleotide selected from the group consisting of CpG. 65 oligonucleotides having one or two fewer nucleoside resi CpG, CpG* and CpG*, wherein C is cytidine or 2'-deoxy dues, or from one to several additional nucleoside residues are cytidine, G is guanosine or 2'-deoxyguanosine, C is 2'-deox contemplated as equivalents of each of the embodiments US 7,427,405 B2 3 4 described above. In some embodiments, one or more of the sistencies between any teaching of any reference cited herein oligonucleotides have 11 nucleotides. and the present specification, the latter shall prevail for pur In a third aspect the invention provides pharmaceutical poses of the invention. compositions. These compositions comprise any one of the The invention relates to the therapeutic use of oligonucle compositions disclosed in the first and second aspects of the otides as immunostimulatory agents for immunotherapy invention either alone or in combination and a pharmaceuti applications. The invention also provides methods for gener cally acceptable carrier. ating, enhancing and modifying the immune response caused In a fourth aspect the invention provides a method for by immunostimulatory compounds used for immunotherapy generating an immune response in a vertebrate. This method applications such as, but not limited to, treatment and/or comprises administering to the vertebrate any one of the 10 prevention of cancer, autoimmune disorders, asthma, respi compositions, alone or in combination, disclosed in the first, ratory allergies, food allergies, and bacteria, parasitic, and second and third aspects of the invention. The compositions viral infections in adult and pediatric human and Veterinary disclosed herein can be administered through any Suitable applications. Allergic asthma is a certain embodied condition route of administration including, but not limited to, for treatment by the present methods and compounds. Thus, parenteral, oral, Sublingual, transdermal, topical, intranasal, 15 the invention further provides compounds having optimal aerosol, intraocular, intratracheal, intrarectal, vaginal, gene levels of immunostimulatory effect for immunotherapy and gun, dermal patch, eye drop and mouthwash. methods for making and using such compounds. In addition, In a fifth aspect the invention provides a method for thera immunostimulatory oligonucleotides/immunomers of the peutically treating a vertebrate having cancer, an autoimmune invention are useful as adjuvants in combination with DNA disorder, airway inflammation, inflammatory disorders, skin vaccines, antibodies, allergens, chemotherapeutic agents, and disorders, allergy, asthma or a disease caused by a pathogen. antisense oligonucleotides. This method comprises administering to the vertebrate any In a first aspect the invention provides an immunostimula one of the compositions, alone or in combination, disclosed in tory oligonucleotide the sequence of which is at least partially the first, second and third aspects of the invention. The com self-complementary. The immunostimulatory nucleic acid positions disclosed herein can be administered through any 25 comprises an nucleic acid sequence containing at least one Suitable route of administration including, but not limited to, dinucleotide selected from the group consisting of CpG. parenteral, oral, Sublingual, transdermal, topical, intranasal, CpG, CpG* and CpG*, wherein C is cytidine or 2'-deoxy aerosol, intraocular, intratracheal, intrarectal, vaginal, gene cytidine, G is guanosine or 2'-deoxyguanosine, C is 2'-deox gun, dermal patch, eye drop, ear drop and mouthwash. ythymidine, 1-(2'-deoxy-f-D-ribofuranosyl)-2-oxo-7-deaza In a sixth aspect the invention provides a method for pre 30 8-methyl-purine, 2-dideoxy-5-halocytosine, 2'-dideoxy-5- venting cancer, an autoimmune disorder, airway inflamma nitrocytosine, arabinocytidine, 2'-deoxy-2-substituted tion, inflammatory disorders, skin disorders, allergy, asthma arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy ora disease caused by a pathogen in a vertebrate. This method 5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4- comprises administering to the vertebrate any one of the thiouridine, or other pyrimidine nucleoside analogs, G is compositions, alone or in combination, disclosed in the first, 35 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, ara second and third aspects of the invention. The compositions binoguanosine. 2'-deoxy-2'substituted-arabinoguanosine, disclosed herein can be administered through any Suitable 2'-O-substituted-arabinoguanosine. 2'-deoxyinosine, or other route of administration including, but not limited to, purine nucleoside analogs, and p is an internucleoside linkage parenteral, oral, Sublingual, transdermal, topical, intranasal, selected from the group consisting of phosphodiester, phos aerosol, intraocular, intratracheal, intrarectal, vaginal, gene 40 phorothioate, and phosphorodithioate. gun, dermal patch, eye drop, ear drop and mouthwash. In some embodiments, the immunostimulatory oligonucle otide is from about 2 to about 50 nucleotides in length. In BRIEF DESCRIPTION OF THE DRAWINGS certain embodiments the immunostimulatory oligonucle otide is from about 12 to about 26 nucleotides in length. In FIGS. 1A and 1B are representations of various embodi 45 Some embodiments, the oligonucleotides are from about 3 to ments of the invention (SEQID NO: 41). In FIG. 1B, mand about 35 nucleoside residues, or from about 4 to about 30 in are independently 0-1000. nucleoside residues, or from about 4 to about 20 nucleoside FIG. 2 is a synthetic scheme for the parallel synthesis of residues. In some embodiments, the oligonucleotides have immunomers of the invention. DMTr 4,4'-dimethoxytrityl: from about 5 to about 18, or from about 5 to about 14, CE-cyanoethyl. 50 nucleoside residues. As used herein, the term “about implies FIG. 3 depicts a group of representative small molecule that the exact number is not critical. Thus, the number of linkers suitable for linear synthesis of immumomers of the nucleoside residues in the oligonucleotides is not critical, and invention. oligonucleotides having one or two fewer nucleoside resi FIG. 4 is a synthetic scheme for the linear synthesis of dues, or from one to several additional nucleoside residues are immunomers of the invention. DMTr 4,4'-dimethoxytrityl: 55 contemplated as equivalents of each of the embodiments CE-cyanoethyl. described above. In some embodiments, one or more of the FIG. 5 depicts a group of representative small molecule oligonucleotides have 11 nucleotides. linkers suitable for parallel synthesis of immunomers of the As would be recognized by one skilled in the art, the invention. complementary sequence of the oligonucleotides allows for 60 intermolecular hydrogen bonding thereby giving the oligo DETAILED DESCRIPTION OF THE PREFERRED nucleotides secondary structure. Additional oligonucleotides EMBODIMENTS can bind together thereby creating a chain, or multimers, of oligonucleotides according to the invention. The issued patents, patent applications, and references that In a second aspect the invention provides an immunomer are cited herein are hereby incorporated by reference to the 65 comprising at least two oligonucleotides linked by a non same extent as if each was specifically and individually indi nucleotide linker, wherein the sequences of the immuno cated to be incorporated by reference. In the event of incon stimulatory oligonucleotides are at least partially self US 7,427,405 B2 5 6 complementary. In certain embodiments according to this purine nucleoside analogs, and p is an internucleoside linkage aspect of the invention at least one of the oligonucleotides selected from the group consisting of phosphodiester, phos contains at least one dinucleotide selected from the group phorothioate, and phosphorodithioate. In certain embodi consisting of CpG, CpG, CpG* and CpG*, wherein C is ments, the immunostimulatory dinucleotide is not CpG. In cytidine or 2'-deoxycytidine, G is guanosine or 2'-deoxygua Some embodiments, Domain B is preferably a non-nucleo nosine, C* is 2'-deoxythymidine, 1-(2'-deoxy-f-D-ribofura tidic linker connecting oligonucleotides of Domain A and nosyl)-2-oxo-7-deaza-8-methyl-purine, 2-dideoxy-5-halo Domain C, which are referred to as “immunomers.” In certain cytosine. 2'-dideoxy-5-nitrocytosine, arabinocytidine, embodiments, Domain C does not have the dinucleotide CpG. 2'-deoxy-2-substituted arabinocytidine, 2'-O-substitutedara CpG, CpG* or CpG*. binocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4 10 In some embodiments, the oligonucleotides contained in alkyl-cytidine, 2'-deoxy-4-thiouridine, or other pyrimidine formula (I) are from about 2 to about 50 nucleotides in length. nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine, In certain embodiments the oligonucleotides contained in 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy formula (I) are from about 12 to about 26 nucleotides in 2'substituted-arabinoguanosine, 2'-O-substituted-ara length. In some embodiments, the oligonucleotides each have binoguanosine. 2'-deoxyinosine, or other purine nucleoside 15 from about 3 to about 35 nucleoside residues, preferably from analogs, and p is an internucleoside linkage selected from the about 4 to about 30 nucleoside residues, more preferably from group consisting of phosphodiester, phosphorothioate, and about 4 to about 20 nucleoside residues. In some embodi phosphorodithioate. ments, the oligonucleotides have from about 5 to about 18, or In this aspect, immunostimulatory nucleic acid comprises from about 5 to about 14, nucleoside residues. As used herein, a structure as detailed in formula (I). the term “about implies that the exact number is not critical. Thus, the number of nucleoside residues in the oligonucle Domain A-Domain B-Domain C (1) otides is not critical, and oligonucleotides having one or two Domains may be from about 2 to about 12 nucleotides in fewer nucleoside residues, or from one to several additional length. Domain A may be 5'-3' or 3'-5" or 2'-5' DNA, RNA, nucleoside residues are contemplated as equivalents of each RNA-DNA, DNA-RNA having a palindromic or self 25 of the embodiments described above. In some embodiments, complementary domain containing or not containing at least one or more of the oligonucleotides have 11 nucleotides. one dinucleotide selected from the group consisting of CpG. A self-complementary sequence as used herein refers to a CpG, CpG* and CpG*, wherein C is cytidine or 2'-deoxy base sequence which, upon Suitable alignment, may form cytidine, G is guanosine or 2'-deoxyguanosine, C is 2'-deox intramolecular or, more typically, intermolecular basepairing ythymidine, 1-(2'-deoxy-f-D-ribofuranosyl)-2-oxo-7-deaza 30 between G-C, A-T, A-U and/or G-U wobble pairs. In one 8-methyl-purine, 2'-deoxy-5-halocytosine, 2'-deoxy-5- embodiment the extent of self-complementarity is at least 50 nitrocytosine, arabinocytidine, 2'-deoxy-2'-substituted percent. For example an 8-mer that is at least 50 percent arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy self-complementary may have a sequence capable of forming 5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4- 4, 5, 6, 7, or 8 G-C, A-T, A-U and/or G-U wobble basepairs. thiouridine, or other pyrimidine nucleoside analogs, G is 35 Such basepairs may but need not necessarily involve bases 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, ara located at either end of the self-complementary immuno binoguanosine. 2'-deoxy-2'substituted-arabinoguanosine, stimulatory oligonucleotide and/or immunomer. Where 2'-O-substituted-arabinoguanosine. 2'-deoxyinosine, or other nucleic acid stabilization may be important to the immuno purine nucleoside analogs, and p is an internucleoside linkage stimulatory oligonucleotide and/or immunomer, it may be selected from the group consisting of phosphodiester, phos 40 advantageous to “clamp' together one or both ends of a phorothioate, and phosphorodithioate. In certain embodi double-stranded nucleic acid, either by basepairing or by any ments, the immunostimulatory dinucleotide is not CpG. other suitable means. The degree of self-complementarity In certain embodiments, Domain A will have more than may depend on the alignment between immunostimulatory one dinucleotide selected from the group consisting of CpG. oligonucleotide and/or immunomer, and Such alignment may CpG, CpG* and CpG*. 45 or may not include single- or multiple-nucleoside overhangs. Domain B, as depicted by an “X” below, is a linker joining In other embodiments, the degree of self-complementarity is Domains A and C that may be a 3’-5’ linkage, a 2'-5' linkage, at least 60 percent, at least 70 percent, at least 80 percent, at a 3'-3' linkage, a phosphate group, a nucleoside, or a non least 90 percent, or even 100 percent. nucleoside linker that may be aliphatic, aromatic, aryl, cyclic, Similar considerations apply to intermolecular basepairing chiral, achiral, a peptide, a carbohydrate, a lipid, a fatty acid, 50 between immunostimulatory oligonucleotides and/or immu mono-tri- or hexapolyethylene glycol, or a heterocyclic moi nomers of different base sequence. Thus, where a plurality of ety. immunostimulatory oligonucleotides and/or immunomers Domain C may be 5'-3' or 3'-5', 2'-5' DNA, RNA, RNA are used together, the plurality of immunostimulatory oligo DNA, DNA-RNA Poly I-Poly C having a palindromic or nucleotides and/or immunomers may, but need not, include self-complementary sequence, containing or not containing a 55 sequences which are at least partially complementary to one dinucleotide selected from the group consisting of CpG. another. In one embodiment the plurality of immunostimula CpG, CpG*, CpG*, wherein C is cytidine or 2'-deoxycy tory oligonucleotides and/or immunomers includes an immu tidine, G is guanosine or 2'-deoxyguanosine, C is 2'-deox nostimulatory oligonucleotide and/or immunomer having a ythymidine, 1-(2'-deoxy-f-D-ribofuranosyl)-2-oxo-7-deaza first sequence and an immunostimulatory oligonucleotide 8-methyl-purine, 2'-deoxy-5-halocytosine. 2'-dideoxy-5- 60 and/or immunomer having a second sequence, wherein the nitrocytosine, arabinocytidine, 2'-deoxy-2-substituted first sequence and the second sequence are at least 50 percent arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy complementary. For example, as between two 8-mers that are 5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4- at least 50 percent complementary, they may form 4, 5, 6, 7, thiouridine, other pyrimidine nucleoside analogs, G* is or 8 G-C, A-T, A-U, and/or G-U wobble basepairs. Such 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, ara 65 basepairs may but need not necessarily involve bases located binoguanosine. 2'-deoxy-2'substituted-arabinoguanosine, at either end of the complementary immunostimulatory oli 2'-O-substituted-arabinoguanosine. 2'-deoxyinosine, or other gonucleotides and/or immunomers. The degree of comple US 7,427,405 B2 7 8 mentarity may depend on the alignment between immuno rothioate, phosphorodithioate, alkylphosphonate, alky stimulatory oligonucleotides and/or immunomers, and Such lphosphonothioate, phosphotriester, phosphoramidate, alignment may or may not include single- or multiple-nucleo siloxane, carbonate, carboalkoxy, acetamidate, carbamate, side overhangs. In other embodiments, the degree of comple morpholino, borano, thioether, bridged phosphoramidate, mentarity is at least 60 percent, at least 70 percent, at least 80 bridged methylene phosphonate, bridged phosphorothioate, percent, at least 90 percent, or even 100 percent. and Sulfone internucleoside linkages. The term "oligonucle By way of non-limiting example, in certain embodiments otide' also encompasses polynucleosides having one or more of this aspect the immunostimulatory nucleic acid will have a stereospecific internucleoside linkage (e.g., (RP)- or (SP)- structure as detailed in formula (II). phosphorothioate, alkylphosphonate, orphosphotriesterlink 10 ages). As used herein, the terms "oligonucleotide' and “dinucleotide' are expressly intended to include polynucleo (II) sides and dinucleosides having any Such internucleoside link 5'-X-5 age, whether or not the linkage comprises a phosphate group. X -X- In certain embodiments, these internucleoside linkages may 15 be phosphodiester, phosphorothioate, or phosphorodithioate As would be recognized by one skilled in the art, the depicted linkages, or combinations thereof. immunostimulatory nucleic acid/immunomer compounds The term "oligonucleotide' also encompasses polynucleo have secondary structure because the sequences of the sides having additional Substituents including, without limi domains are complementary allowing for intermolecular tation, protein groups, lipophilic groups, intercalating agents, hydrogen bonding. Domains A and A may or may not be diamines, folic acid, cholesterol and adamantane. The term identical, domains A and C may or may not be identical, "oligonucleotide also encompasses any other nucleobase domains A and C may or may not be identical, domains A containing polymer, including, without limitation, peptide and C may or may not be identical, domains A and C may or nucleic acids (PNA), peptide nucleic acids with phosphate may not be identical, domains B and B' may or may not be groups (PHONA), locked nucleic acids (LNA), morpholino identical and domains C and C may or may not be identical. 25 backbone oligonucleotides, and oligonucleotides having Moreover, as shown in FIG. 1, additional immunomers can backbone sections with alkyl linkers or amino linkers. bind through intermolecular hydrogen bonding thereby cre The oligonucleotides of the invention can include naturally ating a chain, or multimers, of immunomers according to the occurring nucleosides, modified nucleosides, or mixtures invention. n can be any number of continuous, self comple thereof. As used herein, the term “modified nucleoside' is a 30 nucleoside that includes a modified heterocyclic base, a mentary immunomer compounds. modified Sugar moiety, or a combination thereof. In some As used herein, the term “complementary means having embodiments, the modified nucleoside is a non-natural pyri the ability to hybridize to a nucleic acid. Such hybridization is midine or purine nucleoside, as herein described. In some ordinarily the result of hydrogen bonding between comple embodiments, the modified nucleoside is a 2-substituted mentary strands, preferably to form Watson-Crick or Hoogs 35 ribonucleoside an arabinonucleoside or a 2'-deoxy-2-substi teen base pairs, although other modes of hydrogen bonding, tuted-arabinoside. as well as base stacking can also lead to hybridization. For purposes of the invention, the term “2'-substituted ribo As used herein, the term “secondary structure” refers to nucleoside' or "2"-substituted arabinoside' includes ribo intermolecular hydrogen bonding. Intermolecular hydrogen nucleosides or arabinonucleosides in which the hydroxyl bonding results in the formation of a duplexed nucleic acid 40 group at the 2' position of the pentose moiety is substituted to molecule. produce a 2-substituted or 2'-O-substituted ribonucleoside. "Palindromic sequence' shall mean an inverted repeat (i.e., In certain embodiments, such substitution is with a lower a sequence such as ABCDEED'C'B'A' in which A and A, B alkyl group containing 1-6 saturated or unsaturated carbon and B', etc., are bases capable of forming the usual Watson atoms, or with an aryl group having 6-10 carbon atoms, Crick base pairs. In vivo. Such sequences may form double 45 wherein Such alkyl, or aryl group may be unsubstituted or stranded structures. In one embodiment the CpG nucleic acid may be substituted, e.g., with halo, hydroxy, trifluoromethyl, contains a palindromic sequence. A palindromic sequence cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or used in this context refers to a palindrome in which the CpG amino groups. Examples of 2'-O-substituted ribonucleosides is part of the palindrome. In some embodiments the CpG is or 2'-O-substituted-arabinosides include, without limitation the center of the palindrome. In another embodiment the CpG 50 2'-O-methylribonucleosides or 2'-O-methylarabinosides and nucleic acid is free of a palindrome. An immunostimulatory 2'-O-methoxyethoxyribonucleosides or 2'-O-methoxy nucleic acid that is free of a palindrome is one in which the ethoxyarabinosides. CpG dinucleotide is not part of a palindrome. Such an oligo The term '2'-substituted ribonucleoside' or "2"-substituted nucleotide may include a palindrome in which the CpG is not arabinoside' also includes ribonucleosides or arabinonucleo the center of the palindrome. 55 sides in which the 2'-hydroxyl group is replaced with a lower For purposes of the invention, the term "oligonucleotide' alkyl group containing 1-6 saturated or unsaturated carbon refers to a polynucleoside formed from a plurality of linked atoms, or with an amino or halo group. Examples of Such nucleoside units. Such oligonucleotides can be obtained from 2'-substituted ribonucleosides or 2-substituted arabinosides existing nucleic acid sources, including genomic or cDNA, include, without limitation, 2-amino. 2'-fluoro. 2'-allyl, and but are preferably produced by synthetic methods. In some 60 2'-propargyl ribonucleosides or arabinosides. embodiments each nucleoside unit includes a heterocyclic The term "oligonucleotide' includes hybrid and chimeric base and a pentofuranosyl, 2'-deoxypentfuranosyl, trehalose, oligonucleotides. A "chimeric oligonucleotide' is an oligo arabinose. 2'-deoxy-2-substituted arabinose. 2'-O-substi nucleotide having more than one type of internucleoside link tuted arabinose or hexose Sugar group. The nucleoside resi age. One non-limiting example of Such a chimeric oligo dues can be coupled to each other by any of the numerous 65 nucleotide is a chimeric oligonucleotide comprising a known internucleoside linkages. Such internucleoside link phosphorothioate, phosphodiester or phosphorodithioate ages include, without limitation, phosphodiester, phospho region and non-ionic linkages such as alkylphosphonate or US 7,427,405 B2 10 alkylphosphonothioate linkages (see e.g., Pederson etal. U.S. N4-alkylcytosine, or N4-ethylcytosine, and 4-thiouracil. In Pat. Nos. 5,635,377 and 5,366,878). Some embodiments, the Sugar moiety S' in (III) is a non A “hybrid oligonucleotide' is an oligonucleotide having naturally occurring Sugar moiety. For purposes of the present more than one type of nucleoside. One non-limiting example invention, a “naturally occurring Sugar moiety' is a Sugar of Such a hybrid oligonucleotide comprises a ribonucleotide moiety that occurs naturally as part of nucleic acid, e.g., or 2 substituted ribonucleotide region, and a deoxyribonucle ribose and 2'-deoxyribose and a “non-naturally occurring otide region (see, e.g., Metelev and Agrawal, U.S. Pat. No. Sugar moiety' is any Sugar that does not occur naturally as 5,652,355, 6,346,614 and 6,143,881). part of a nucleic acid, but which can be used in the backbone Alternatively, the nucleic acid molecule of the invention for an oligonucleotide, e.g., hexose. Arabinose and arabinose can be two immunomers linked by way of a non-nucleotidic 10 derivatives are non-limiting examples of Sugar moieties. linker. In some embodiments purine nucleoside analogs in immu In certain embodiments of the invention, at least one immu nostimulatory oligonucleotides and/or immunomers used in nostimulatory oligonucleotide of the invention comprises an the method according to the invention have the structure (IV): immunostimulatory dinucleotide of the formula 5'-Pyr-Pur 3', wherein Pyr is a natural pyrimidine nucleoside or analog 15 thereof and Pur is a natural purine nucleoside or analog (IV) thereof. As used herein, the term "pyrimidine nucleoside' A refers to a nucleoside wherein the base component of the L nucleoside is a pyrimidine base. Similarly, the term “purine D nucleoside' refers to a nucleoside wherein the base compo nent of the nucleoside is a purine base. For purposes of the Kll2 invention, a “synthetic' pyrimidine or purine nucleoside i N D includes a non-naturally occurring pyrimidine or purine base, S. a non-naturally occurring Sugar moiety, or a combination thereof. 25 wherein: In certain embodiments pyrimidine nucleosides in the D is a hydrogen bond donor, immunostimulatory oligonucleotides and/or immunomers D' is selected from the group consisting of hydrogen, used in the method according to the invention have the struc hydrogen bond donor, and hydrophilic group; ture (III): A is a hydrogen bond acceptor or a hydrophilic group; 30 X is carbon or nitrogen; (III) each L is independently selected from the group consisting of C, O, N and S; and S' is a pentose or hexose Sugar ring, or a non-naturally 35 occurring Sugar. In certain embodiments, the Sugar ring is derivatized with a phosphate moiety, modified phosphate moiety, or other linker moiety suitable for linking the pyrimidine nucleoside to another nucleoside or nucleoside analog. 40 In certain embodiments hydrogen bond donors include, without limitation, NH , NH, -SH and —OH. Pre wherein: ferred hydrogen bond acceptors include, without limitation, D is a hydrogen bond donor; C=O, C=S. —NO and the ring nitrogen atoms of an aro D' is selected from the group consisting of hydrogen, matic heterocycle, e.g., N1 of guanine. hydrogen bond donor, hydrogen bond acceptor, hydrophilic 45 In some embodiments, the base moiety in (IV) is a non group, hydrophobic group, electron withdrawing group and naturally occurring purine base. Examples of preferred non electron donating group; naturally occurring purine bases include, without limitation, A is a hydrogen bond acceptor or a hydrophilic group; 6-thioguanine and 7-deazaguanine. In some embodiments, A' is selected from the group consisting of hydrogen bond the Sugar moiety S' in (IV) is a naturally occurring Sugar acceptor, hydrophilic group, hydrophobic group, electron 50 moiety, as described above for structure (III). withdrawing group and electron donating group; In a third aspect the invention provides pharmaceutical X is carbon or nitrogen; and compositions. These compositions comprise any one of the S is a pentose or hexose Sugar ring, or a non-naturally compositions disclosed in the first and second of the invention occurring Sugar. either alone or in combination and a pharmaceutically accept In certain embodiments, the Sugar ring is derivatized with 55 able carrier. a phosphate moiety, modified phosphate moiety, or other As used herein, the term “physiologically acceptable' linker moiety suitable for linking the pyrimidine nucleoside refers to a material that does not interfere with the effective to another nucleoside or nucleoside analog. ness of the compositions of the first, second or third aspects of In some embodiments hydrogen bond donors include, the invention and is compatible with a biological system Such without limitation, —NH NH, SH and —OH. Pre 60 as a cell, cell culture, tissue, or organism. In certain embodi ferred hydrogen bond acceptors include, without limitation, ments, the biological system is a living organism, such as a C=O, C=S, and the ring nitrogen atoms of an aromatic vertebrate. heterocycle, e.g., N3 of cytosine. As used herein, the term “carrier encompasses any excipi In some embodiments, the base moiety in (III) is a non ent, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or naturally occurring pyrimidine base. Examples of preferred 65 other material well known in the art for use in pharmaceutical non-naturally occurring pyrimidine bases include, without formulations. It will be understood that the characteristics of limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, the carrier, excipient, or diluent will depend on the route of US 7,427,405 B2 11 12 administration for a particular application. The preparation of therapeutically effective amount of one or more of the thera pharmaceutically acceptable formulations containing these peutic compositions of the invention to an individual as a materials is described in, e.g., Remington's Pharmaceutical single treatment episode. Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Immunostimulatory oligonucleotides were created as immunomers using the following protocols for synthesis. The Easton, Pa., 1990, ISBN: 0-912734-04-3. immunostimulatory oligonucleotides and/or immunomers of Pharmaceutical compositions of the invention may also the invention may conveniently be synthesized using an auto include a cancer vaccine, including a cancer vaccine selected mated synthesizer and phosphoramidite approach as sche from EFG, Anti-idiotypic cancer vaccines, Gp75 antigen, matically depicted in FIGS. 2 and 4. In some embodiments, GMK melanoma vaccine, MGV ganglioside conjugate vac the immunostimulatory oligonucleotides and/or immuno cine, Her2/new, Ovarex R, M-Vax, O-Vax, L-Vax, STn-KHL 10 mers are synthesized by a linear synthesis approach (see FIG. Theratope(R), BLP25 (MUC-1), liposomal idiotypic vaccine, 2). Representative linkers for this synthesis are presented in Melacine R, peptide antigenvaccines, toxinfantigenvaccines, FIG. 3. As used herein, the term “linear synthesis' refers to a MVA-vased vaccine, PACIS, BCG vaccine, TA-HPV, TA synthesis that starts at one end of the immunomer and CTN, DISC-virus and ImmunCyst/TheraCys(R). progresses linearly to the other end. Linear synthesis permits 15 incorporation of either identical or un-identical (in terms of In various embodiments of the invention, the compositions length, base composition and/or chemical modifications of the first, second or third aspects of the invention may be incorporated) monomeric units into the immunostimulatory covalently linked to an antigen or otherwise operatively asso oligonucleotides and/or immunomers. ciated with an antigen. As used herein, the term "operatively An alternative mode of synthesis for immunostimulatory associated with refers to any association that maintains the oligonucleotides and/or immunomers is “parallel synthesis'. activity of both the compositions of the first, second or third in which synthesis proceeds outward from a central linker aspects of the invention and the antigen. Non-limiting moiety (see FIG. 4). Representative linkers for this method of examples of such operative associations include being part of synthesis are presented in FIG. 5. A solid support attached the same liposome or other Such delivery vehicle or reagent. linker can be used for parallel synthesis, as is described in In embodiments wherein the compositions of the first, second 25 U.S. Pat. No. 5,912.332. Alternatively, a universal solid sup or third aspects of the invention are covalently linked to an port, Such as phosphate attached to controlled pore glass antigen, Such covalent linkage is at any position on the com Support, can be used. positions of the first, second or third aspects of the invention Parallel synthesis of immunostimulatory oligonucleotides other than an accessible 5' end of an immunostimulatory and/or immunomers has several advantages over linear Syn oligonucleotide. For example, the antigen may be attached at thesis: (1) parallel synthesis permits the incorporation of an internucleoside linkage or may be attached to the non 30 identical monomeric units; (2) unlike in linear synthesis, both nucleotidic linker. Alternatively, the antigen may itself be the (or all) the monomeric units are synthesized at the same time, non-nucleotidic linker. thereby the number of synthetic steps and the time required In various embodiments of the invention, the compositions for the synthesis is the same as that of a monomeric unit; and of the first, second or third aspects of the invention may (3) the reduction in Synthetic steps improves purity and yield include an oligonucleotide with antisense activity. As used 35 of the final immunomer product. herein, “antisense activity” means that the oligonucleotide, At the end of the synthesis by either linear synthesis or when introduced into a cellorananimal, causes a reduction in parallel synthesis protocols, the immunostimulatory oligo the expression of the gene to which it is complementary. nucleotides or immunomers according to the invention may In various embodiments of the invention, the compositions conveniently be deprotected with concentrated ammonia of the first, second or third aspects of the invention may 40 Solution oras recommended by the phosphoramidite Supplier, include an oligonucleotide sequence that is an aptamer. if a modified nucleoside is incorporated. The product immu Aptamers are nucleic acid molecules that have been selected nostimulatory oligonucleotides and/or immunomer is prefer from random pools based on their ability to bind other mol ably purified by reversed phase HPLC, detritylated, desalted ecules. Aptamers have been selected which bind nucleic and dialyzed. acids, proteins, Small organic compounds, and even entire 45 The compositions disclosed in the first second and third organisms. These novel molecules have many potential uses aspects of the invention can comprise the immunostimulatory in medicine and technology (see, e.g., Burgstaller P. et al. oligonucleotide or immunomer alone or as oligonucleotide? Curr Opin Drug Discov Devel. 5: 690-700 (2002)). immunomer conjugates. An oligonucleotide/immunomer The pharmaceutical compositions of the invention may be conjugate comprises an oligonucleotide or immunomer, as administered by any Suitable route, including, without limi 50 described above, and an antigen conjugated to the oligonucle tation, parenteral, oral, Sublingual, transdermal, topical, intra otide and/or immunomer at a position other than the acces nasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, sible 5' end. In some embodiments, the non-nucleotidic linker by gene gun, dermal patch or in eye drop or mouthwash form. comprises an antigen, which is conjugated to the oligonucle The pharmaceutical compositions can be delivered using otide. In some other embodiments, the antigen is conjugated known procedures at dosages and for periods of time effective to the oligonucleotide at a position other than its 3' end. In obtain the desired effect, e.g. the treatment of cancer, the 55 Some embodiments, the antigen produces a vaccine effect. treatment of infection and the treatment of autoimmune dis The immunostimulatory oligonucleotide or immunomer eases. When administered systemically, the pharmaceutical alone or as oligonucleotidefimmunomer conjugates can be compositions are administered at a Sufficient dosage to attain administered in the methods discussed below. a blood level of the compositions of the first, second and/or The antigen is optionally selected from antigens associated third aspects of the invention from about 0.0001 micromolar 60 with a pathogen, antigens associated with a cancer, antigens to about 10 micromolar. For localized administration, much associated with an auto-immune disorder, and antigens asso lower concentrations than this may be effective, and much ciated with other diseases such as, but not limited to, veteri higher concentrations may be tolerated. In certain embodi nary or pediatric diseases, or wherein the antigen is an aller ments, a total dosage of immunostimulatory oligonucleotide gen. For purposes of the invention, the term “associated with and/or immunomer ranges from about 0.0001 mg per patient 65 means that the antigen is present when the pathogen, cancer, per day to about 200 mg per kg body weight per day. It may be auto-immune disorder, food allergy, skin allergy, respiratory desirable to administer simultaneously, or sequentially a allergy, asthma or other disease is present, but either is not US 7,427,405 B2 13 14 present, or is present in reduced amounts, when the pathogen, receptor (TLR) ligands and cytokines (for example, interleu cancer, auto-immune disorder, food allergy, skin allergy, res kin-1 f; IL-6 and tumor necrosis factor, TNF), which they piratory allergy, or disease is absent. also produce; natural killer (NK) cells and T cells are also The immunomer is covalently linked to the antigen, or it is involved in the pro-inflammatory circuit. After TLR stimula otherwise operatively associated with the antigen. As used tion by bacterial compounds, innate immune cells release a herein, the term “operatively associated with refers to any range of cytokines. Some examples of TLR ligands include, association that maintains the activity of both immunomer but are not limited to, lipoproteins; peptidoglycan, Zymosan and antigen. Nonlimiting examples of Such operative asso (TLR2), double-stranded RNA, polylpolyC (TLR3), ciations include being part of the same liposome or other Such lipopolysaccharide, heat shock proteins, Taxol. R(TLR4), delivery vehicle or reagent. In embodiments wherein the flagellin (TLR5), and imidazoquinolines-R848, residuimod, immunomer is covalently linked to the antigen, Such covalent 10 imiquimod: ssRNA (TLR7/8), beta-lymphocytes (TLR10) linkage preferably is at any position on the immunomer other and uropathogenic E. coli (TLR 11). than an accessible 5' end of an immunostimulatory oligo The methods according to this aspect of the invention are nucleotide. For example, the antigen may be attached at an useful for model studies of the immune system. The methods internucleoside linkage or may be attached to the non-nucleo are also useful for the prophylactic ortherapeutic treatment of tidic linker. Alternatively, the antigen may itself be the non 15 human or animal disease. For example, the methods are use nucleotidic linker. ful for pediatric and Veterinary vaccine applications. In a fourth aspect, the invention provides methods forgen In a fifth aspect, the invention provides methods for thera erating and/or modulating an immune response in a verte peutically treating a vertebrate having a disease or disorder, brate, such methods comprising administering to the verte Such methods comprising administering to the vertebrate an brate an immunomer or immunomer conjugate according to immunomer or immunomer conjugate according to the inven the invention. In some embodiments, the vertebrate is a mam tion. In various embodiments, the disease or disorder to be mal. For purposes of this invention, the term “mammal’ is treated is cancer, an autoimmune disorder, airway inflamma expressly intended to include humans. In certain embodi tion, inflammatory disorders, allergy, asthma or a disease ments, the immunomer or immunomer conjugate is adminis caused by a pathogen. Pathogens include bacteria, parasites, tered to a vertebrate in need of immunostimulation. 25 fungi, viruses, viroids and prions. Administration is carried As used herein, the term “modulating” or “modulate' out as described for the fourth aspect of the invention. means to increase or decrease the immunostimulatory activ For purposes of the invention, the term “allergy” includes, ity of an immunostimulatory nucleic acid relative to that of without limitation, food allergies atopic dermatitis, allergic the parent immunostimulatory nucleic acid. rhinitis (also known as hay fever), allergic conjunctivitis, In the methods according to this aspect of the invention, 30 urticaria (also known as hives), respiratory allergies and aller administration of immunomers can be by any Suitable route, gic reactions to other Substances such as latex, medications including, without limitation, parenteral, oral, Sublingual, and insect stings or problems commonly resulting from aller transdermal, topical, intranasal, intramuscular, intraperi gic rhinitis-sinusitis, otitis media and COPD. The term “air tonal, Subcutaneous, intradermal, aerosol, intraocular, way inflammation' includes, without limitation, asthma. intratracheal, intrarectal, vaginal, by gene gun, dermal patch Specific examples of asthma include, but are not limited to, or in eye drop or mouthwash form. Administration of the 35 allergic asthma, non-allergic asthma, exercised-induced therapeutic compositions of immunomers can be carried out asthma, occupational asthma, and nocturnal asthma. using known procedures at dosages and for periods of time Allergic asthma is characterized by airway obstruction effective to reduce symptoms or Surrogate markers of the associated with allergies and triggered by Substances called disease. When administered systemically, the therapeutic allergens. Triggers of allergic asthma include, but are not composition is preferably administered at a sufficient dosage 40 limited to, airborne pollens, molds, animal dander, house dust to attain a blood level of immunomer from about 0.0001 mites and cockroach droppings. Non-allergic asthma is micromolar to about 10 micromolar. For localized adminis caused by viral infections, certain medications or irritants tration, much lower concentrations than this may be effective, found in the air, which aggravate the nose and airways. Trig and much higher concentrations may be tolerated. Preferably, gers of non-allergic asthma include, but are not limited to, a total dosage of immunomer ranges from about 0.001 mg per 45 airborne particles (e.g., coal, chalk dust), air pollutants (e.g., patient per day to about 200 mg per kg body weight per day. tobacco Smoke, wood Smoke), strong odors or sprays (e.g., It may be desirable to administer simultaneously, or sequen perfumes, household cleaners, cooking fumes, paints or var tially a therapeutically effective amount of one or more of the nishes), viral infections (e.g., colds, viral pneumonia, sinusi therapeutic compositions of the invention to an individual as tis, nasal polyps), aspirin-sensitivity, and gastroesophageal a single treatment episode. 50 reflux disease (GERD). Exercise-induced asthma (EIA) is Either the immunomer or the vaccine, or both, may option triggered by vigorous physical activity. Symptoms of EIA ally be linked to an immunogenic protein, such as keyhole occur to varying degrees in a majority of asthma Sufferers and limpet hemocyanin (KLH), cholera toxin B subunit, or any are likely to be triggered as a result of breathing cold, dry air other immunogenic carrier protein or nonimmunogenic car while exercising. Triggers of EIA include, but are not limited rier protein. Any of the plethora of adjuvants may be used to, breathing airborne pollens during exercise, breathing air including, without limitation, Freund's complete adjuvant, 55 pollutants during exercise, exercising with viral respiratory Freund's incomplete adjuvant, KLH, monophosphoryl lipid tract infections and exercising in cold, dry air. Occupational A (MPL), alum, and saponins, including QS-21, imiquimod, asthma is directly related to inhaling irritants and other poten R848, or combinations thereof. tially harmful Substances found in the workplace. Triggers of Toll-like receptors (TLRs) function as sensors of infection occupational asthma include, but are not limited to, fumes, and induce the activation of innate and adaptive immune 60 chemicals, gases, resins, metals, dusts, vapors and insecti responses. TLRS recognize a wide variety of ligands, called cides. pathogen-associated molecular patterns (PAMPs). Upon rec As used herein, the term “autoimmune disorder” refers to ognizing conserved pathogen-associated molecular products, disorders in which “self proteins undergo attack by the TLRs activate host defense responses through their intracel immune system. Such term includes autoimmune asthma. lular signaling domain, the Toll/interleukin-1 receptor (TIR) 65 Without wishing to be bound to any particular theory, domain, and the downstream adaptor protein MyD88. Den decreased exposure to bacteria may be partially responsible dritic cells and macrophages normally respond to Toll-like for the increased incidence of severity of, and mortality due US 7,427,405 B2 15 16 to allergic diseases such as asthma, atopic dermatitis, and second and third aspects of the invention. Pathogens include rhinitis in the developed countries. This hypothesis is sup bacteria, parasites, fungi, viruses, Viroids and prions. Admin ported by evidence that bacterial infections or products can istration is carried out as described for the fourth aspect of the inhibit the development of allergic disorders in experimental invention. animal models and clinical studies. Bacterial DNA or syn In any of the methods according to the invention, the thetic oligodeoxynucleotides containing unmethylated CpG immunostimulatory oligonucleotide and/or immunomer or a dinucleotides and/or modified CpG dinucleotides in certain conjugate thereof can be administered in combination with sequence contexts (CpG DNA) potently stimulate innate any other agent useful for treating the disease or condition immune responses and thereby acquired immunity. The that does not diminish the immunostimulatory effect of the immune response to CpG DNA includes activation of innate oligonucleotide or immunomer. For purposes of this aspect of immune cells, proliferation of B cells, induction of Th1 10 the invention, the term “in combination with means in the cytokine secretion, and production of immunoglobulins (Ig). course of treating the same disease in the same patient, and The activation of immune cells by CpG DNA occurs via includes administering the oligonucleotide and/or immuno Toll-like receptor 9 (TLR9), a molecular pattern recognition merandan agent in any order, including simultaneous admin receptor. CpG DNA induce strong Th1-dominant immune istration, as well as any temporally spaced order, for example, responses characterized by secretion of IL-12 and IFN-. 15 from sequentially with one immediately following the other Immunomers (IMO) alone or as allergen conjugates decrease to up to several days apart. Such combination treatment may production of IL-4, IL-5, and IgE and reduce eosinophilia in also include more than a single administration of the immu mouse models of allergic asthma. IMO compounds also nomer, and independently the agent. The administration of effectively reverse established atopic eosinophilic airway dis the oligonucleotide and/or immunomer and agent may be by ease by converting a Th2 response to a Th1 response. the same or different routes. OVA with alum is commonly used to establish a Th2 In any of the methods according to the invention, the agent dominant immune response in various mouse and rat models. useful for treating the disease or condition includes, but is not The Th2 immune response includes increased IL-4, IL-5, and limited to, Vaccines, antigens, antibodies, cytotoxic agents, IL-13 production, elevated serum levels of total and antigen allergens, antibiotics, antisense oligonucleotides, peptides, specific IgE, IgG1, and lower levels of IgG2a. IMO com 25 proteins, gene therapy vectors, DNA vaccines and/or adju pounds prevent and reverse established Th2-dominant vants to enhance the specificity or magnitude of the immune immune responses in mice. The co-administration of IMO response, or co-stimulatory molecules such as cytokines, compounds with OVA/alum to mice reduces IL-4, IL-5, and chemokines, protein ligands, trans-activating factors, pep IL-13 production and induces IFN-Y production in spleen-cell tides and peptides comprising modified amino acids. Addi cultures subjected to antigen re-stimulation. Furthermore, tionally, the agent can include DNA vectors encoding for IMO compounds inhibit antigen-specific and total IgE and 30 antigen or allergen. In these embodiments, the immunomers enhance IgG2a production in these mice. of the invention can variously act as adjuvants and/or produce Injection of OVA/alum and IMO compounds induces a direct immunostimulatory effects. lymphocyte antigen-recall response (Th1-type) in mice char The examples below are intended to further illustrate cer acterized by low levels of Th2-associated cytokines, IgE and tain preferred embodiments of the invention, and are not IgG1, and high levels of Th1-associated cytokines and IgG2a. 35 intended to limit the scope of the invention. Co-administration of IMO compounds with other kinds of antigens, such as S. masoni egg and hen egg lysozyme, also EXAMPLES result in reversal of the Th2-response to a Th1-dominant response in in vitro and in vivo studies. As described herein, Example 1 IMO compounds effectively prevent development of a Th2 40 immune response and allow a strong Th1 response. Oligonucleotide Synthesis, Purification and Thermal While Th2 cytokines trigger an Ig isotype switch towards Melt Profiles production of IgE and IgG1, the Th1 cytokine IFN-Y induces production of IgG2a by B-lymphocytes. Mice injected with CpG oligonucleotides (immunostimulatory oligonucle OVA/alum and IMO compounds produce lower levels of 45 otides/immunomers) were synthesized on a 1 to 2 umole scale IL-4, IL-5, and IL-13 and higher levels of IFN-Y, accompa using B-cyanoethylphosphoramidites on a PerSeptive Bio nied by lower IgE and IgG1 and higher IgG2a levels, than system's 8909 Expedite DNA synthesizer (PerSeptive Bio mice injected with OVA/alum alone. This suggests the exist system, Boston, Mass.). The phosphoramidites of dA, dG, ence of a close link between Th1-cytokine induction and dC, and T were obtained from PE Biosystems (Foster City, immunoglobulin isotype Switch in mice that receive antigen 50 Calif.). As described by Iyer R. P. et al. (J. Am. Chem. Soc. and IMO compounds. 112: 1253-1254 (1990)), an iodine oxidizing agent was used Serum antigen-specific and total IgE levels are signifi to obtain the phosphorothioate backbone modification. All cantly lower in mice receiving OVA/alum and IMO com oligos were deprotected using standard protocols, purified by pounds than in mice receiving OVA/alum alone. In contrast, HPLC, and dialyzed against USP quality sterile water for OVA-specific IgG1 levels are insignificantly changed and irrigation. The oligos were lyophilized and dissolved again in total IgG1 levels are only slightly decreased compared, with 55 distilled water and the concentrations were determined from mice injected with OVA/alum alone (data not shown). The UV absorbance at 260 nm. All oligos were characterized by different response may result from different mechanisms CGE and MALDI-TOF mass spectrometry (Applied Biosys involved in the control of IgE and IgG1 class switch, though tem's Voyager-DETM STR BiospectrometryTM Workstation) both isotypes are influenced by IL-4 and IL-13. For example, for purity and molecular mass, respectively. The purity of IL-6 promotes B lymphocytes to synthesize IgG1 in the pres 60 full-length oligos ranged from 90-96% with the rest being ence of IL-4. shorter by one or two nucleotides (n-1 and n-2) as determined In a sixth aspect the invention provides a method for pre by CGE and/or denaturing PAGE. All oligos contained less venting cancer, an autoimmune disorder, airway inflamma than <0.1 EU/mL of endotoxin as determined by the Limulus tion, inflammatory disorders, skin disorders, allergy, asthma assay (Bio-Whittaker now known as Cambrex Bio Science ora disease caused by a pathogen in a vertebrate. This method 65 Walkersville, Inc., Walkersville, Md.). comprises administering to the vertebrate any one of the Thermal melting studies were carried out in 1 mL solution compositions, alone or in combination, disclosed in the first, of 10 mM disodium hydrogen phosphate, pH 7.2+0.2, con US 7,427,405 B2 17 18 taining 150 mMNaCl, and 2 mM MgCl2. The solutions were were collected for ELISA assays. The experiments were per heated to 95° C. for 10 min and allowed to come to room formed two or three times for each immunomer compound temperature slowly before being stored at 4° C. overnight. and in triplicate for each concentration. The secretion of The final concentration of oligonucleotide strand was 2.0LM. IL-12 and IL-6 was measured by sandwich ELISA as UV thermal melting measurements were performed at 260 described by Bhagat L., et al. (Biochem. Biophys. Res. Com nm on a Perkin-Elmer Lambda 20 Spectrophotometer mun. 300: 853-861 (2003)). The required reagents, including attached to a peltier thermal controller and a personal com cytokine antibodies and standards were purchased from BD puter using 1 cm path length quartz cuvettes at a heating rate Biosciences Pharmingen (San Diego, Calif.). of 0.5°C./min. Melting temperatures (Tm) were taken as the Example 5 temperature of half-dissociation and were obtained from first derivative plots. Each Tm value is an average of two or three 10 independent experiments and the values were within +1.0°C. Mouse Splenomegaly Assay Female BALB/c mice (4-6 weeks, 19-21 gm) were divided Example 2 into groups of three mice. Immunomer compounds were dis 15 solved insterile phosphate buffered saline (PBS) and admin Cell Culture Conditions and Reagents istered subcutaneously (SC) to mice at a dose of 5 mg/kg. The Spleen cells from 4-8 week old BALB/c, C57BL/6 or C3H7 mice were sacrificed after 48 hr and the spleens were har HeJ mice were cultured in RPMI complete medium as vested and weighedas described by Zhao, Q., et al. (Biochem described by Zhao, Q., et at. (Biochem Pharmacol. 51:173 Pharmacol. 51: 173-182 (1996)) and Branda, R. F., et al. 182 (1996)) and Branda, R.F., et at. (Biochem. Pharmacol. (Biochem. Pharmacol. 45: 2037-2043 (1993)). 45: 2037-2043 (1993)). Murine J774 macrophages (Ameri Example 6 can Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209) were cultured in Dulbecco's Activation of the NF-kB Pathway modified Eagles medium supplemented with 10% (v/v) fetal calf serum and antibiotics (100 IU/mL of penicillin G/strep 25 Toll-like receptor 9 (TLR9) has been shown to recognize tomycin). All other culture reagents were purchased from unmethylated CpG-dinucleotides in bacterial, plasmid and Mediatech (Gaithersburg, Md.). synthetic DNAs (Hemmi H., et al. Nature 408: 740–745 (2000)) and activate stress kinase (Yi A. K., et al. J. Immunol. Example 3 161: 4493-4497 (1998)) and NF-kB pathways (Stacey K. J., et al. J. Immunol. 157: 21 16-2122 (1996)). NF-kB activation Spleen Cell Proliferation Assay 30 in J774 cells treated with immunomer compounds was car ried out and analyzed by EMSA as described Yu D., et al. Typically, mouse (Balb-C) spleen cells were cultured with (Biochem. Biophys. Res. Commun. 297: 83-90 (2002)) and immunomer compounds at concentrations of 0.1, 1.0, and Bhagat L., et al. (Biochem. Biophys. Res. Commun. 300: 10.0 ug/ml for 48 h and cell proliferation was determined by 853-861 (2003)). 3H-uridine incorporation, as described by Zhao, Q., et al. 35 Example 7 (Biochem Pharmacol. 51: 173-182 (1996)). Isolation of Human B Cells and Plasmacytoid Example 4 Dendritic Cells (pDCs) Cytokine Induction Assays 40 PBMCs from freshly drawn healthy volunteer blood (CBR Laboratories, Boston, Mass.) were isolated by Ficoll density Mouse spleen or J774 cells were plated in 24-well dishes gradient centrifugation method (Histopaque-1077, Sigma) using 5x10° or 1x10 cells/mL, respectively. The immuno and B cells were isolated from PBMCs by positive selection mer compounds dissolved in TE buffer (10 mM Tris-HCl, pH using the CD19 cell isolation kit (Miltenyi Biotec) according 7.5, 1 mM EDTA) were added to a final concentration of 0.03, 45 to the manufacturer's instructions. Table 1 shows the immu 0.1.0.3, 1.0, 3.0, or 10.0 ug/mL to the cell cultures. The cells nostimulatory activity of immunomer compounds of the were then incubated at 37° C. for 24 hr and the supernatants invention in C57BL/6 Splenocyte Assay.

TABLE 1. Immunomer Structure and C57BL/6 Splenocyte Assay (24 hs)

IL-12 IL-12 IL-12 SEQ Sequences and (pg/ml) (pg/ml) (pg/ml) ID NO Modification (5'-3') 1 lug/ml 1 lug/ml 1 lug/ml 19 5'-TCGAACG2 TTCG-X-GCTTG-CAAGCT-5' 2O 5'-TCGAAC GTTCG-X-GCTTGCAAGCT-5'

21 5'-TCG, AACG, TTCG,-Y-TCTTG, CTGTCT-5' 6396 - 911 and 34 10 61

22 5'-TCG, AACG, TTCG,-Y-TCTTGCTGTCT-5' 7275 749 and 35 77 8O

23 5'-TCG, AACG, TTCG,-Y-TCTTGc, TGTCT-5' 8O34 - 918 and 36 14 136

US 7,427,405 B2 21 22

TABLE 1 - continued Immunomer Structure and C57BL/6 Splenocyte Assay (24 hs)

Media 1O2 12 25 2 Normal phase represents a phosphorothioate linkage; o represents a phosphodiester linkage. l 2'-deoxy-7- deazaguanosine 2 = Arabinoguanosine C l 1 - (2'-deoxy--D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine X = Glycerol Linker Y = C-Linker Example 8 15 1x10° cells/mL, 200 uL/well). The immunomer compounds were added to a final concentration of 0.3, 1.0, 3.0, or 10.0 Human p)C Cultures and IFN-O. and IFN-B ELISA ug/mL to the cell cultures and incubated at 37°C. for 24hr. Supernatants were then harvested and assayed for IFN-C. and pIDCs were isolated from human PBMCs using a BDCA-4 2O IFN-B using ELISA kit (PBL). Tables 2A-2D show an cell isolation kit (Miltenyi Biotec) according to the manufac- average:SD of IFN-C. and IFN-B for immunomer compounds turer's instructions. pl.)C were plated in 96-well plates using according to the invention at a concentration of 10.0 ug/mL.

TABL E 2A Immunomer Structure and Immunostimulatory Activity in Human DC Assay (24 hs)

IFN-O. IFN-O. IFN-O. SEQ Sequences and (pg/ml) (pg/ml) (pg/ml) ID NO Modification (5'-3") 10 ug/ml DN1 10 ug/ml DN3 10 ug/ml DN3

1 5'-TCGAACGTTCG-X-GCTTG CAAGCT-5' 26112 it 25825 96.264 604 416 605

4 O 5'-TCGAACGTTCG-X-GCTTG. CAAGCT-5' 2O34 O 1227O 105804 106 3 O6 688

3 5-TCT CACC TTCT-X-TCTTCCACTCT- 185 31.1 - 1649 O 4. 262

4 5'-TCGAACGTTCG-X-GCTTG-CAAGCT-5'

5 5'-TCGAACGTTCG-X-GCTTG-CAAGCT-5'

media 177 177 O O O O

IFN-O. IFN-O. IFN-O. SEQ Sequences and (pg/ml) (pg/ml) (pg/ml) ID NO Modification (5'-3") 10 ug/ml DN4 10 ug/ml DN5 10 ug/ml DN6

1. 5'-TCGAACGTTCG-X-GCTTG CAAGCT-5' 41718 25O11 it 19608 1015 5 5

4 O 5'-TCGAACGTTCG-X-GCTTG. CAAGCT-5' 49176 14 O14 21988 3O2 1414 1413

3 5-TCT CACC TTCT-X-TCTTCCACTCT- O 197 2O1 O O O

4 5'-TCGAACGTTCG-X-GCTTG-CAAGCT-5'

5 5'-TCGAACGTTCG-X-GCTTG-CAAGCT-5'

media O 2O1 196 O O O

Normal phase represents a phosphorothioate linkage G = 2'-deoxy-7-deazaguanosine G = Arabinoguanosine X = Glycerol linker

US 7,427,405 B2 34

TABLE 2D-continued Immunomer Structure and Immunostimulatory Activity in Human DC Assay (24 hs)

28 5'-CTGTCGTTCTCo-x-oCTCTTGCTGTC-5' 5508 13956 6 OO9 41 O 355 24 O

29 5'-CTGT CoGTTCTC-X-CTCTTGoCTGTC-5' 5599 11824 9977 146 72 O 1379

3O 5'-TCG, TGTCG, TTT-X-TTTG,CTGTG,CT-5' 11946 1.59

31 5'-TG, CTGTG. CTTT-X-TTTCG, TGTCG, T-5' O O

32 5'-TCGAACGTTCG-Y-GACAGCTGTCT-5' 1 OO32 and 38 9

33 5'-TG CAACGCTTGC-Y-GACACGTGTCT-5' 642 O and 39 139

Media 80 101 - O O 7 O Normal phase represents a phosphorothioate linkage; o represents a phosphodiester linkage. G = 2'-deoxy-7-deazaguanosine G = Arabinoguanosine C = 1 - (2'-deoxy--D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine X = Glycerol Linker Y = C3 Linker

Example 9 30 Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers by Cytokine Analysis Ficoll-Paque density gradient centrifugation (Histopaque 1077, Sigma, St. Louis, Mo.). Briefly, heparinized blood was The secretion of IFN-C. in vertebrate cells, preferably layered onto the Histopaque-1077 (equal volume) in a conical BALB/c mouse spleen cells or human PBMC, was measured 35 by sandwich ELISA. The required reagents including cytok centrifuge and centrifuged at 400xg for 30 minutes at room ine antibodies and cytokine Standards were purchased form temperature. The buffy coat, containing the mononuclear PharMingen, San Diego, Calif. ELISA plates (Costar) were cells, was removed carefully and washed twice with isotonic incubated with appropriate antibodies at 5ug/mL in PBSN phosphate buffered saline (PBS) by centrifugation at 250xg buffer (PBS/0.05% sodium azide, pH 9.6) overnight at 4°C. 40 for 10 minutes. The resulting cell pellet was then resuspended and then blocked with PBS/1% BSA at 37°C. for 30 minutes. in RPMI 1640 medium containing L-glutamine (MediaTech, Cell culture Supernatants and cytokine standards were appro Inc., Herndon, Va.) and supplemented with 10% heat inacti priately diluted with PBS/10% FBS, added to the plates in vated FCS and penicillin-streptomycin (100 U/ml). Cells triplicate, and incubated at 25°C. for 2 hours. Plates were Were cultured in 24 well plates for different time periods at overlaid with 1 g/mL appropriate biotinylated antibody and 45 1x10 cells/mi/well in the presence or absence of oligonucle incubated at 25°C. for 1.5 hours. The plates were thenwashed otides. At the end of the incubation period, Supernatants were extensively with PBS-T Buffer (PBS/0.05% Tween 20) and harvested and stored frozen at -70°C. until assayed for vari further incubated at 25°C. for 1.5 hours after adding strepta ous cytokines including IFN-C. (BioSource International) by vidin conjugated peroxidase (Sigma, St. Louis, Mo.). The sandwich ELISA. The results are shown in Table 3A-3D plates were developed with Sure BlueTM (Kirkegaard and 50 Perry) chromogenic reagent and the reaction was terminated below. by adding Stop Solution (Kirkegaard and Perry). The color In all instances, the levels of IFN-C. in the cell culture change was measured on a Ceres 900 HDI Spectrophotom Supernatants was calculated from the standard curve con eter (Bio-Tek Instruments). structed under the same experimental conditions for IFN-C.

TABLE 3A Immunomer Structure and Immunostimulatory Activity in Human PBMC Assay (24 hs)

IFN-O. IFN-O. IFN-O. SEQ Sequences and (pg/ml) (pg/ml) (pg/ml) ID NO Modification (5'-3") 10 ug/ml DN4 10 ug/ml DN5 10 ug/ml DN6

1 5'-TCGAACGTTCG-X-GCTTG CAAGCT-5' 8222 61.14 36O4 44 1. 1.

US 7,427,405 B2 46

TABLE 3D-continued Immunomer Structure and Immunostimulatory Activity in Human PBMC Assay (24 hs) 24 5'-TCG, AACG, TTCG-Y-TCTTCCACTCT-5' 443 678 and 37 29 133

25 5'-TG,CAAG, CTTG,C-Y-TCTTG, CTGTCT-5' 298 and 34 16

26 5'-TG,CAAG, CTTG,C-Y-TCTTCCACTCT-5' 1. 2 and 37

27 5'-TG CAAG. CTTGC-X-CGTTCGAACGT-5'

28 5'-CTGTCGTTCTCo-x-oCTCTTGCTGTC-5' 2 94 512 O

29 5'-CTGT CoGTTCTC-X-CTCTTGoCTGTC-5' 3 61 168 O 18 25

3O 5'-TCG, TGTCG, TTT-X-TTTG,CTGTG,CT-5' 9 9 2

31 5'-TG, CTGTG. CTTT-X-TTTCG, TGTCG, T-5'

32 5'-TCGAACGTTCG-Y-GACAGCTGTCT-5' and 38

33 5'-TG,CAACG, CTTG,C-Y-GACACG, TGTCT-5' and 39

Media O 6 O O Normal phase represents a phosphorothioate linkage; o represents a phosphodiester linkage. G = 2'-deoxy-7-deazaguanosine G = Arabinoguanosine C = 1 - (2'-deoxy--D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine Glycerol Linker Y = C3 Linker

Example 10 briefly descried as follow. The activated culture cells were 40 blocked with 10% Human AB serum (Sigma) in staining Flow Cytometric Analysis buffer (PBS with 1% BSA and 0.1% NaNs) at 4°C. for 1 hour and stained with the antibodies at 4°C. overnight. PBMCs Cell Surface markers of CD69 and CD86 were detected (4x10) were stained with CD69-Fitc and CD86-Fitc. PDCs with a Coulter Epics-XL Flow Cytometer using anti-human (2x10) were stained CD86-Fitc. The cell staining data were CD69-Fitc and CD86-Fitc., which were purchased from BD acquired and analyzed with Coulter System II software (see Pharmingen (San Diego, USA). Staining methods were Tables 4A-4F below).

TABL E 4A

Immunomer Structure and Expression of BC from Human PBMC (2x10 cell/ml) (24 hs)

SEQ Sequences and & CD86 2.& CD86 2.& CD86 ID NO Modification 1 ug/ml DN1 1 lug/ml DN2 1 lug/ml DN3

1 5'-TCGAACGTTCG-X-GCTTG CAAGCT-5' 36.4 17.7 36.4

2 5'-TCGAACGTTCG-X-GCTTG. CAAGCT-5' 27.2 6.3 3 O 4

3 5-TCTCACCTTCT-X-TCT TOCACTCT 15 7.3 11.3

media 1. Of 8 7.6

US 7,427,405 B2 71 72

TABLE 4F-continued Immunomer Structure and Expression of DC from Human PBMC assay (24 hs)

37. and 37

28 5'-CTGTCGTTCTCo-x-oCTCTTGCTGTC-5' 61.1 79.6 58. 29 5'-CTGT CoGTTCTC-X-CTCTTGoCTGTC-5' 68.2 87.3 69.

6O.

44.

32 5'-TCGAACGTTCG-Y-GACAGCTGTCT-5' 65. and 38

33 5'-TG CAACGCTTGC-Y-GACACG, TGTCT-5' 49. and 39

Media 69. 6 58.3 35. 8 Normal phase represents a phosphorothioate linkage; o represents a phosphodiester linkage. G = 2'-deoxy-7-deazaguanosine G = Arabinoguanosine C = 1 - (2'-deoxy--D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine X = Glycerol Linker Y = C3 Linker

Example 11 30 pounds of the invention for 16 hr, then pulsed with 0.75 uCi of H-thymidine and harvested 8 h later. The incorporation of B Cell Proliferation Assay radioactivity was measured using liquid scintillation counter. A total of 1x10 B cells/200 ul were stimulated with 0.3, Table 5 shows an average:SD of B cell proliferation at a final 1.0, 3.0, or 10.0 ug/mL concentrations of immunomer com concentration of 1.0 g/mL.

TABLE 5 Immunomer Structure and Immunostimulatory Activity in Human B-Cell Proliferation Assay (24 hs) HT HT 3HT SEQ Sequences and (cpm) (cpm) (cpm) ID NO Modification (5'-3') 1 ug/ml DN4 1 lug/ml DN51 ug/ml DN6

19 CCG-X-GCTTG-CAAGCT-5' CCG-X-GCTTGCAAGCT-5'

21 CCG-Y-TCTTG,CTGTCT-5 31127 17626 and 34 68OO 2809

22 CCG-Y-TCTTGCTGTCT-5' 333 68 17131 and 35 1364 1366

23 CCG-Y-TCTTGc, TGTCT-5 3O845 13826 and 36 2514 2331

24 CCG-Y-TCTTCCACTCT-5 34 Off 8073 and 37 36.36 583

25 and 34

26 5'-TG CAAGCT) GI C-Y-TCTTCCACTCT-5 and 37

27 5'-TG CAAGCT)

28 US 7,427,405 B2 73 74

TABLE 5 - continued Immunomer Structure and Immunostimulatory Activity in Human B-Cell Proliferation Assay (24 hs)

32 5'-TCGAACGTTCG-Y-GACAGCTGTCT-5' and 38

33 5'-TG CAACGCTTGC-Y-GACACG, TGTCT-5' and 39

Media 646 457 236 121 HT HT HT SEQ Sequences and (cpm) (cpm) (cpm) ID NO Modification (5'-3") 1 ug/ml DN7 1 lug/ml DN8 1 lug/ml DN9 19 5'-TCGAACGTTCG-X-GCTTG-CAAGCT-5'

21 5'-TCG, AACG, TTCG-Y-TCTTG,CTGTCT-5' 37731 and 34 29 O1

22 5'-TCG, AACG, TTCG,-Y-TCTTGCTGTCT-5' 384 OS and 35 8056

23 5'-TCG, AACG, TTCG-Y-TCTTGc, TGTCT-5' 347 O2 and 36 61.96

24 5'-TCG, AACGTTCG-Y-TCTTCCACTCT-5' 23O3O and 37 1941

25 5'-TG,CAAG, CTTG,C-Y-TCTTG,CTGTCT-5' and 34

26 5'-TG,CAAG, CTTG,C-Y-TCTTCCACTCT-5'

28 5'-CTGTCGTTCTCo-x-oCTCTTGCTGTC-5'

32 5'-TCGAACGTTCG-Y-GACAGCTGTCT-5' and 38

33 5'-TG CAACGCTTGC-Y-GACACG, TGTCT-5' and 39

21 5'-TG CAACGC-Y-GACACGTGTCT-5' and 34

Media 658 2O5 Normal phase represents a phosphorothioate linkage; o represents a phosphodi ester linkage. G = 2'-deoxy-7- deazaguanosine = Arabinoguanosine g = 1 - (2'-deoxy-3-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine X = Glycerol Linker Y = C3 Linker

While the foregoing invention has been described in some 65 disclosure that various changes in form and detail can be detail for purposes of clarity and understanding, it will be made without departing from the true scope of the invention appreciated by one skilled in the art from a reading of this and appended claims. US 7,427,405 B2 75 76

SEQUENCE LISTING

NUMBER OF SEO ID NOS: 41

SEQ ID NO 1 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 1 tona act to in 11

SEQ ID NO 2 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 2 tcnaacntitc g 11

SEQ ID NO 3 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 3 t ct cacct to t 11

SEQ ID NO 4 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: Arabinoguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: Arabinoguanosine US 7,427,405 B2 77 78

- Continued

&220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (11) <223> OTHER INFORMATION: Arabinoguanosine <4 OO SEQUENCE: 4 tona act to in 11

<210 SEQ ID NO 5 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (3) <223> OTHER INFORMATION: Arabinoguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (7) <223> OTHER INFORMATION: Arabinoguanosine

<4 OO SEQUENCE: 5 tcnaacntitc g 11

<210 SEQ ID NO 6 <211 LENGTH: 21 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (3) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (10) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (14) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 6 t cnt cnaacn titcnagatga t 21

<210 SEQ ID NO 7 <211 LENGTH: 21 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (3) <223> OTHER INFORMATION: Arabinoguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: Arabinoguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (10) <223> OTHER INFORMATION: Arabinoguanosine US 7,427,405 B2 79 80

- Continued

FEATURE: NAMEAKEY: modified base LOCATION: (14) <223> OTHER INFORMATION: Arabinoguanosine SEQUENCE: 7 t cnt cnaacn titcnagatga t

SEQ ID NO 8 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxyinosine FEATURE: NAMEAKEY: modified base LOCATION: (6) OTHER INFORMATION: 2'-deoxyinosine FEATURE: NAMEAKEY: modified base LOCATION: (10) OTHER INFORMATION: 2'-deoxyinosine FEATURE: NAMEAKEY: modified base LOCATION: (14) OTHER INFORMATION: 2'-deoxyinosine

SEQUENCE: 8 t cnt cnaacn titcnagatga t

SEO ID NO 9 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (2) OTHER INFORMATION: 1- (2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine FEATURE: NAMEAKEY: modified base LOCATION: (5) OTHER INFORMATION: 1- (2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine FEATURE: NAMEAKEY: modified base LOCATION: (9) OTHER INFORMATION: 1- (2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine FEATURE: NAMEAKEY: modified base LOCATION: (13) OTHER INFORMATION: 1- (2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine

SEQUENCE: 9 tngtingaang ttngagatga t

SEQ ID NO 10 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide US 7,427,405 B2 81 82

- Continued

FEATURE: NAMEAKEY: modified base LOCATION: (2) OTHER INFORMATION: Arabinocytidine FEATURE: NAMEAKEY: modified base LOCATION: (5) OTHER INFORMATION: Arabinocytidine FEATURE: NAMEAKEY: modified base LOCATION: (9) OTHER INFORMATION: Arabinocytidine FEATURE: NAMEAKEY: modified base LOCATION: (13) OTHER INFORMATION: Arabinocytidine

SEQUENCE: 1.O tngtingaang ttngagatga t 21

SEQ ID NO 11 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (2) OTHER INFORMATION: 2'-deoxy-5-hydroxycytidine FEATURE: NAMEAKEY: modified base LOCATION: (5) OTHER INFORMATION: 2'-deoxy-5-hydroxycytidine FEATURE: NAMEAKEY: modified base LOCATION: (9) OTHER INFORMATION: 2'-deoxy-5-hydroxycytidine FEATURE: NAMEAKEY: modified base LOCATION: (13) OTHER INFORMATION: 2'-deoxy-5-hydroxycytidine

SEQUENCE: 11 tngtingaang ttngagatga t 21

SEQ ID NO 12 LENGTH: 10 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 12 tona act to 10

SEQ ID NO 13 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide US 7,427,405 B2 83 84

- Continued

FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 13 tonttcaac in 11

SEQ ID NO 14 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

SEQUENCE: 14 tcca acct tc g 11

SEO ID NO 15 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (6) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 15 tonttcaac in 11

SEQ ID NO 16 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: Arabinoguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: Arabinoguanosine

SEQUENCE: 16 tona act to t 11

SEO ID NO 17 LENGTH: 11 US 7,427,405 B2 85 86

- Continued

TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: Arabinoguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 17 tona act to in 11

SEQ ID NO 18 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAME/KEY: modified base LOCATION: (6) OTHER INFORMATION: 1- (2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 18 tcnaangttc n 11

SEQ ID NO 19 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: Arabinoguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 19 tona act to in 11

SEQ ID NO 2 O LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: US 7,427,405 B2 87 88

- Continued OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (6) OTHER INFORMATION: 1- (2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (11) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 2O tcnaangttc n 11

<210 SEQ ID NO 21 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base <222> LOCATION: (11) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 21 tona act to in 11

<210 SEQ ID NO 22 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (3) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (7) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 22 tona act to in 11

SEQ ID NO 23 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: US 7,427,405 B2 89 90

- Continued NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 23 tona act to in 11

SEQ ID NO 24 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 24 tona act to in 11

SEO ID NO 25 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (2) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (6) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (10) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 25 tnca actt. C 11

SEQ ID NO 26 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (2) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: US 7,427,405 B2 91

- Continued <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (10) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 26 tnca actt. C 11

<210 SEQ ID NO 27 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (2) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (10) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 27 tnca actt. C 11

<210 SEQ ID NO 28 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: Arabinoguanosine

<4 OO SEQUENCE: 28 citgtcnttct c 11

<210 SEQ ID NO 29 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: Arabinoguanosine <4 OO SEQUENCE: 29 citgtcnttct c 11

<210 SEQ ID NO 3 O <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide US 7,427,405 B2 93 94

- Continued

FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (8) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 3 O tontgtcntt t 11

SEQ ID NO 31 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (2) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 31 tnctgtinctt t 11

SEQ ID NO 32 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (3) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

SEQUENCE: 32 tona act to in 11

SEQ ID NO 33 LENGTH: 12 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide FEATURE: NAMEAKEY: modified base LOCATION: (2) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (7) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine FEATURE: NAMEAKEY: modified base LOCATION: (11) OTHER INFORMATION: 2'-deoxy-7- deazaguanosine US 7,427,405 B2 95 96

- Continued

<4 OO SEQUENCE: 33 tncaacnctt C 12

<210 SEQ ID NO 34 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (7) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 34 totgtcnttct 11

<210 SEQ ID NO 35 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (7) <223> OTHER INFORMATION: Arabinoguanosine <4 Oos SEQUENCE: 35 totgtcnttct 11

<210 SEQ ID NO 36 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: 1- (2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7- deaza- 8-methylpurine

<4 OO SEQUENCE: 36 tctgtingttct 11

<210 SEQ ID NO 37 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO SEQUENCE: 37 t ct cacct to t 11

<210 SEQ ID NO 38 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: US 7,427,405 B2 97 98

- Continued <221 NAMEAKEY: modified base <222> LOCATION: (7) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 38 tctgtc.naca g 11

<210 SEQ ID NO 39 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (6) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 39 tctgtncaca g 11

<210 SEQ ID NO 4 O <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (3) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (7) <223> OTHER INFORMATION: 2'-deoxy-7- deazaguanosine

<4 OO SEQUENCE: 40 tona act to t 11

<210 SEQ ID NO 41 <211 LENGTH: 11 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO SEQUENCE: 41 tcgaacgttc g 11

What is claimed is: transdermal, topical, intranasal, aerosol, intraocular, intratra 1. An immunostimulatory oligonucleotide having the cheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash. structure 5'-TCGAACGTTCG-X-GCTTG,CAAGCT 55 5'; wherein X is a glycerol linker and G is 2'-deoxy-7-dea 5. The oligonucleotide according to claim 1, further com Zaguanosine. prising an antibody, antisense oligonucleotide, protein, anti 2. A composition comprising the oligonucleotide accord gen, allergen, chemotherapeutic agent or adjuvant. ing to claim 1 and a physiologically acceptable carrier. 6. The composition according to claim 2, further compris 3. A method for generating an immune response in a ver 60 ing an antibody, antisense oligonucleotide, protein, antigen, tebrate, the method comprising administering to the verte allergen, chemotherapeutic agent or adjuvant. brate an immunostimulatory oligonucleotide having the 7. The method according to claim 3, further comprising structure 5'-TCGAACGTTCG-X-GCTTG,CAAGCT administering an antibody, antisense oligonucleotide, pro 5'; wherein X is a glycerol linker and G is 2'-deoxy-7-dea tein, antigen, allergen, chemotherapeutic agent or adjuvant. Zaguanosine. 65 4. The method according to claim 3, wherein the route of administration is selected from parenteral, oral, Sublingual,