Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides

Abstract

The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents in immunotherapy applications. More particularly, the invention provides an immunostimulatory oligonucleotides for use in methods for generating an immune response or for treating a patient in need of immunostimulation. The immunostimulatory oligonucleotides of the invention preferably comprise novel purines. The immunostimulatory oligonucleotides according to the invention further comprise at least two oligonucleotides linked at their 3′ ends, internucleoside linkages or functionalized nucleobase or sugar to a non-nucleotidic linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having an accessible 5′ end.

1 - 30 . (canceled) 
     
     
         31 . A method for therapeutically treating a vertebrate having cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen, such method comprising administering to the patient an immunostimulatory oligonucleotide having a structure from the group of
 5′-TCTGTR′GTTC 1 U 1 -X-U 1 C 1 TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ ID NO: 3-5′);   5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO: 4-3′-X-3′-SEQ ID NO: 4-5′);   5′-CTGTR′GTTCU 1 C 1 -X-C 1 U 1 CTTGR′TGTC-5′ (5′-SEQ ID NO: 5-3′-X-3′-SEQ ID NO: 5-5′);   5′-CTGTR′GTTC 1 U 1 C 1 -X-C 1 U 1 C 1 TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ ID NO: 6-5′);   5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQ ID NO 17-5′);   5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO: 8-3′-X-3′-SEQ ID NO: 8-5′);   5′-TCTGTR′GACA 1 G 1 -X-G 1 A 1 CAGR′TGTCT-5′ (5′-SEQ ID NO: 9-3′-X-3′-SEQ ID NO: 9-5′);   5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO: 10-5′);   5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQ ID NO: 11-5′);   5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO: 12-3′-X-3′-SEQ ID NO: 12-5′);   5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′ (5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO: 13-5′);   5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO: 14-5′); and   5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO: 15-3′-X-3′-SEQ ID NO: 15-5′), wherein R′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; A 1 /C 1 /G 1 /U 1 =2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine and X=glycerol linker.   
     
     
         32 . The method according to  claim 31 , wherein the route of administration is selected from parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash. 
     
     
         33 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GTTC 1 U 1 -X-U 1 C 1 TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ ID NO: 3-5′). 
     
     
         34 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO: 4-3′-X-3′-SEQ ID NO: 4-5′). 
     
     
         35 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-CTGTR′GTTCU 1 C 1 -X-C 1 U 1 CTTGR′TGTC-5′ (5′-SEQ ID NO: 5-3′-X-3′-SEQ ID NO: 5-5′). 
     
     
         36 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-CTGTR′GTTC 1 U 1 C 1 -X-C 1 U 1 C 1 TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ ID NO: 6-5′). 
     
     
         37 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQ ID NO: 17-5′). 
     
     
         38 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO: 8-3′-X-3′-SEQ ID NO: 8-5′). 
     
     
         39 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GACA 1 G 1 -X-G 1 A 1 CAGR′TGTCT-5′ (5′-SEQ ID NO: 9-3′-X-3′-SEQ ID NO: 9-5′). 
     
     
         40 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO: 10-5′). 
     
     
         41 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQ ID NO: 11-5′). 
     
     
         42 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO: 12-3′-X-3′-SEQ ID NO: 12-5′). 
     
     
         43 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′ (5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO: 13-5′). 
     
     
         44 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO: 14-5′). 
     
     
         45 . The method according to  claim 31  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO: 15-3′-X-3′-SEQ ID NO: 15-5′). 
     
     
         46 . A method for preventing cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, skin disorders, allergy, asthma or a disease caused by a pathogen in a vertebrate, such method comprising administering to the vertebrate an immunostimulatory oligonucleotide having a structure from the group of
 5′-TCTGTR′GTTC 1 U 1 -X-U 1 C 1 TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ ID NO: 3-5′);   5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO: 4-3′-X-3′-SEQ ID NO: 4-5′);   5′-CTGTR′GTTCU 1 C 1 -X-C 1 U 1 CTTGR′TGTC-5′ (5′-SEQ ID NO: 5-3′-X-3′-SEQ ID NO: 5-5′);   5′-CTGTR′GTTC 1 U 1 C 1 -X-C 1 U 1 C 1 TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ ID NO: 6-5′);   5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQ ID NO: 17-5′);   5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO: 8-3′-X-3′-SEQ ID NO: 8-5′);   5′-TCTGTR′GACA 1 G 1 -X-G 1 A 1 CAGR′TGTCT-5′ (5′-SEQ ID NO: 9-3′-X-3′-SEQ ID NO: 9-5′);   5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO: 10-5′);   5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQ ID NO: 11-5′);   5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO: 12-3′-X-3′-SEQ ID NO: 12-5′);   5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′ (5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO: 13-5′);   5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO: 14-5′); and   5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO: 15-3′-X-3′-SEQ ID NO: 15-5′), wherein R′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; A 1 /C 1 /G 1 /U 1 =2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine and X=glycerol linker.   
     
     
         47 . The method according to  claim 46 , wherein the route of administration is selected from parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash. 
     
     
         48 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GTTC 1 U 1 -X-U 1 C 1 TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ ID NO: 3-5′). 
     
     
         49 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO: 4-3′-X-3′-SEQ ID NO: 4-5′). 
     
     
         50 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-CTGTR′GTTCU 1 C 1 -X-C 1 U 1 CTTGR′TGTC-5′ (5′-SEQ ID NO: 5-3′-X-3′-SEQ ID NO: 5-5′). 
     
     
         51 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-CTGTR′GTTC 1 U 1 C 1 -X-C 1 U 1 C 1 TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ ID NO: 6-5′). 
     
     
         52 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQ ID NO: 17-5′). 
     
     
         53 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO: 8-3′-X-3′-SEQ ID NO: 8-5′). 
     
     
         54 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCTGTR′GACA 1 G 1 -X-G 1 A 1 CAGR′TGTCT-5′ (5′-SEQ ID NO: 9-3′-X-3′-SEQ ID NO: 9-5′). 
     
     
         55 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO: 10-5′). 
     
     
         56 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQ ID NO: 11-5′). 
     
     
         57 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO: 12-3′-X-3′-SEQ ID NO: 12-5′). 
     
     
         58 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′ (5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO: 13-5′). 
     
     
         59 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO: 14-5′). 
     
     
         60 . The method according to  claim 46  comprising administering an immunostimulatory oligonucleotide having the structure 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO: 15-3′-X-3′-SEQ ID NO: 15-5′). 
     
     
         61 - 63 . (canceled) 
     
     
         64 . The method according to  claim 31 , further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant. 
     
     
         65 . The method according to  claim 46 , further comprising administering an antibody, antisense oligonucleotide, protein, antigen, allergen, chemotherapeutic agent or adjuvant.