Immunostimulatory oligonucleotides

• US 20060211644A1
• Filed: 02/24/2006
• Published: 09/21/2006
• Est. Priority Date: 02/24/2005
• Status: Abandoned Application

• Current Assignee: Coley Pharmaceutical GmbH (Pfizer Inc.), Coley Pharmaceutical Group, Inc. (Pfizer Inc.)
• Original Assignee: Coley Pharmaceutical GmbH (Pfizer Inc.), Coley Pharmaceutical Group, Inc. (Pfizer Inc.)
• Inventors: Krieg, Arthur M., Samulowitz, Ulrike, Vollmer, Jorg
• Application Number:
  US11/361,313
• Publication Number:
  US 20060211644A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 2039/55561 CpG containing adjuvants; Oligonucleotide containing adjuvants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: C12N 15/117 Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 2310/312 Phosphonates
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/334 Modified C
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/336 Modified G
• CPC Class Codes: C12N 2310/351 Conjugate

Abstract

The invention relates to a class of short CpG immunostimulatory oligonucleotides that are useful for stimulating an immune response. Preferably the short oligonucleotides are soft or semi-soft oligonucleotides.

Claims

1. An oligonucleotide of 3 to 24 nucleotides in length comprising at least one YZ dinucleotide with a phosphodiester or phosphodiester-like internucleotide linkage, and at least 4 T nucleotides, wherein Y is a nucleotide comprising a pyrimidine or modified pyrimidine base, wherein Z is a nucleotide comprising a guanine or modified guanine, and wherein the oligonucleotide includes at least one stabilized internucleotide linkage.

2. The oligonucleotide of claim 1, wherein the oligonucleotide includes a TTTT motif.

3. The oligonucleotide of claim 2, wherein the oligonucleotide has only one YZ dinucleotide.

4. The oligonucleotide of claim 3, wherein the oligonucleotide is G*T*C_G*T*T*T*T*G*A*C (SEQ ID NO.:

16) or G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C (SEQ ID NO.;

     11), wherein *refers to the presence of a stabilized internucleotide linkage, and wherein _refers to the presence of a phosphodiester internucleotide linkage.

5. The oligonucleotide of claim 2, wherein the oligonucleotide has only two YZ dinucleotides.

6. The oligonucleotide of claim 5, wherein the oligonucleotide is selected from the group consisting of T*C_G*T*T*T*T*G*A*C_G*T*T (SEQ ID NO.:

3), T*C_G*T*C_G*T*T*T*T*G*A*C (SEQ ID NO.;

     10), G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO.;

     12), G*T*C_G*T*T*T*T*G*A*C_G*T*T (SEQ ID NO.;

     13), T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C (SEQ ID NO.;

     14), and G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C (SEQ ID NO.;

     15), wherein *refers to the presence of a stabilized internucleotide linkage, and wherein _ refers to the presence of a phosphodiester internucleotide linkage.

7. The oligonucleotide of claim 2, wherein the oligonucleotide has only three YZ dinucleotides.

8. The oligonucleotide of claim 7, wherein the oligonucleotide is selected from the group consisting of T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO.:

2), G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO.;

     8), T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C (SEQ ID NO.;

     9), and T*C_G*T*C_G*T*T*T*T*G*A*C (SEQ ID NO.;

     10), wherein *refers to the presence of a stabilized internucleotide linkage, and wherein _refers to the presence of a phosphodiester internucleotide linkage.

9. The oligonucleotide of claim 2, wherein the oligonucleotide has only four YZ dinucleotides.

10. The oligonucleotide of claim 9, wherein the oligonucleotide is selected from the group consisting of T*C_G*T*C_G*T*T*T_T*G*A*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO.:

4), T*C_G*T*C_G*T*T*T_T*G*A*C_G*T*T*T₁₃ T*G*T*C_G*T*T (SEQ ID NO.;

5), T*C_G*T*C_G*T_T*T_T*G_A*C_G*T_T*T_T*G_T*C_G*T*T (SEQ ID NO.;

     6), C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO.;

     17), T*C_I*T*C_I*T*T*T*T*G*A*C_I*T*T*T*T*G*T*C_I*T*T (SEQ ID NO.;

     18), T* MeC _G*T* MeC _G*T*T*T*T*G*A*MeC_G*T*T*T*T*G*T*MeC_G*T*T (SEQ ID NO.;

     19), T*H_G*T*H_G*T*T*T*T*G*A*H_G*T*T*T*T*G*T*H_G*T*T (SEQ ID NO.;

     20), T*C_—

7*T*C_—

7*T*T*T*T*G*A*C_—

7*T*T*T*T*G*T*C_—

7*T*T (SEQ ID NO.;

     21), and U*C_G*U*C_G*U*U*U*U*G*A*C_G*U*U*U*U*G*U*C_G*U*U (SEQ ID NO.;

     22), wherein *refers to the presence of a stabilized internucleotide linkage, wherein _refers to the presence of a phosphodiester internucleotide linkage, and wherein I is Inosine comprising a Hypoxanthine base;

MeC is 5′

-Methyl-Cytosine, H is 5-Hydroxy-Cytosine, 7 is 7-Deaza-Guanine, and U is Uracil.

11. The oligonucleotide of claim 1, wherein each YZ dinucleotide has a phosphodiester or phosphodiester-like internucleotide linkage.

12. The oligonucleotide of claim 1, wherein Y is a nucleotide comprising an unmethylated cytosine.

13. The oligonucleotide of claim 1, wherein Z is a nucleotide comprising a guanine.

14. The oligonucleotide of claim 1, wherein the phosphodiester-like linkage is boranophosphonate or diastereomerically pure Rp phosphorothioate.

15. The oligonucleotide of claim 1, wherein the stabilized internucleotide linkages are selected from the group consisting of:

phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, and any combination thereof.

16. The oligonucleotide of claim 1, wherein Y is a nucleotide comprising a cytosine or a modified cystosine base selected from the group consisting of 5-methyl cytosine, 5-methyl-isocytosine, 5-hydroxy-cytosine, 5-halogeno cytosine, uracil, N4-ethyl-cytosine, 5-fluoro-uracil, and hydrogen.

17. The oligonucleotide of claim 1, wherein Z is a nucleotide comprising a guanine or a modified guanine base selected from the group consisting of 7-deazaguanine, 7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine, hypoxanthine, 2,6-diaminopurine, 2-aminopurine, purine, 8-substituted guanine such as 8-hydroxyguanine, and 6-thioguanine, 2-aminopurine, and hydrogen.

18. The oligonucleotide of claim 1, wherein the oligonucleotide has a 3′

-3′

linkage with one or two accessible 5′

ends.

19. The oligonucleotide of claim 1, wherein the oligonucleotide has two accessible 5′

ends, each of which are 5′

TCG.

42. A method for treating cancer, comprising administering an oligonucleotide of claim 1, to a subject having cancer in an effective amount to treat the cancer.

44. A method for treating asthma, comprising administering an oligonucleotide of claim 1, to a subject having asthma in an effective amount to treat the asthma.

45. A method for treating infectious disease, comprising administering an oligonucleotide of claim 1, to a subject having or at risk of having infectious disease in an effective amount to treat the infectious disease.

20. An oligonucleotide of 2 to 7 nucleotides in length, wherein the oligonucleotide has at least one YZ dinucleotide with a phosphodiester or phosphodiester-like internucleotide linkage, wherein Y is a a nucleotide comprising a pyrimidine or modified pyrimidine base, wherein Z is a nucleotide comprising a guanine or modified guanine, and wherein the oligonucleotide includes at least one stabilized internucleotide linkage.

21. The oligonucleotide of claim 20, wherein the oligonucleotide has only one YZ dinucleotide.

22. The oligonucleotide of claim 20, wherein the oligonucleotide is selected from the group consisting of T*G*T*C*G*T*T (SEQ ID NO.:

23), T*G*T*C_G*T*T (SEQ ID NO.;

     24), G*T*C*G*T*T (SEQ ID NO.;

     25), G*T*C_G*T*T (SEQ ID NO.;

     26), G*T*C*G*T (SEQ ID NO.;

     27), G*T*C_G*T (SEQ ID NO.;

     28), T*C*G*T*T (SEQ ID NO.;

     29), T*C_G*T*T (SEQ ID NO.;

     30), and C_G (SEQ ID NO.;

     31), wherein * refers to the presence of a stabilized internucleotide linkage, and wherein _refers to the presence of a phosphodiester internucleotide linkage.

23. The oligonucleotide of claim 20, wherein Y is an unmethylated C.

24. The oligonucleotide of claim 20, wherein Z is a nucleotide comprising a guanine.

25. The oligonucleotide of claim 20, wherein the stabilized internucleotide linkage is phosphorothioate.

26. The oligonucleotide of claim 20, wherein Y is a nucleotide comprising a cytosine or a modified cystosine base selected from the group consisting of 5-methyl cytosine, 5-methyl-isocytosine, 5-hydroxy-cytosine, 5-halogeno cytosine, uracil, N4-ethyl-cytosine, 5-fluoro-uracil, and hydrogen.

27. The oligonucleotide of claim 20, wherein Z is a nucleotide comprising a guanine or a modified guanine base selected from the group consisting of 7-deazaguanine, 7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine, hypoxanthine, 2,6-diaminopurine, 2-aminopurine, purine, 8-substituted guanine such as 8-hydroxyguanine, and 6-thioguanine, 2-aminopurine, and hydrogen.

28. The oligonucleotide of claim 20, wherein the oligonucleotide has a 3′

-3′

linkage with one or two accessible 5′

ends.

29. The oligonucleotide of claim 20, wherein the oligonucleotide has two accessible 5′

ends, each of which are 5′

TCG.

30. An oligonucleotide of 7 nucleotides in length, wherein the oligonucleotide has at least one CG dinucleotide, wherein the oligonucleotide includes at least one stabilized internucleotide linkage.

31. The oligonucleotide of claim 30, wherein the all of the internucleotide linkages are phosphorothioate linkages.

32. An oligonucleotide of 5 to 7 nucleotides in length, wherein the oligonucleotide comprises GTCGT or TCGTT, and wherein the oligonucleotide includes at least one stabilized internucleotide linkage.

33. The oligonucleotide of claim 32, wherein the all of the internucleotide linkages are phosphorothioate linkages.

34. An oligonucleotide comprising at least one YZ dinucleotide with a linkage that is an ethylphosphate or methylphosphonate, wherein Y is a nucleotide comprising a pyrimidine or modified pyrimidine base, wherein Z is a nucleotide comprising a guanine or modified guanine.

35. The oligonucleotide of claim 34, wherein the oligonucleotide has a length of 4-100 nucleotides.

36. An oligonucleotide comprising at least one YZ dinucleotide with a phosphodiester or phosphodiester-like internucleotide linkage, and wherein Y is a nucleotide comprising a pyrimidine or modified pyrimidine base, wherein Z is a nucleotide comprising a guanine or modified guanine, and wherein the oligonucleotide contains an aminohexylgroup at the 3′

end of the oligonucleotide.

37. An oligonucleotide comprising at least one YZ dinucleotide with a phosphodiester or phosphodiester-like internucleotide linkage, and wherein Y is a nucleotide comprising a pyrimidine or modified pyrimidine base, wherein Z is a nucleotide comprising a guanine or modified guanine, and wherein the oligonucleotide contains an aminohexylgroup at the 5′

end of the oligonucleotide.

38. An oligonucleotide comprising at least one YZ dinucleotide with a phosphodiester or phosphodiester-like internucleotide linkage, and wherein Y is a nucleotide comprising a pyrimidine or modified pyrimidine base, wherein Z is a nucleotide comprising a guanine or modified guanine, and wherein the oligonucleotide contains an aminohexylgroup at the 5′

and 3′

ends of the oligonucleotide.

39. The oligonucleotide of claim 38, wherein the oligonucleotide includes at least one stabilized internucleotide linkage.

40. The oligonucleotide of claim 38, wherein the oligonucleotide has a length of 4-100 nucleotides.

41. The oligonucleotide of claim 38, wherein Y is a nucleotide comprising an unmethylated cytosine.

43. A method for treating allergy, comprising administering an oligonucleotide of claim 41, to a subject having or at risk of having an allergy in an effective amount to treat the allergy.