Immunostimulatory nucleic acid molecules

• US 6,239,116 B1
• Filed: 10/30/1997
• Issued: 05/29/2001
• Est. Priority Date: 07/15/1994
• Status: Expired due to Term

• Current Assignee: Health and Human Services Department of United States of America, University of Iowa Research Foundation
• Original Assignee: Coley Pharmaceutical Group, Inc. (Pfizer Inc.), United States Department Of Health And Human Services, University of Iowa Research Foundation
• Inventors: Krieg, Arthur M., Kline, Joel N.
• Primary Examiner(s): Martinell, James
• Application Number:
  US08/960,774
• Time in Patent Office: 1,307 Days
• Field of Search: 514/44, 536/23.1
• US Class Current: 514/44.00A
• CPC Class Codes: A61K 2039/55561 CpG containing adjuvants; Oligonucleotide containing adjuvants
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• CPC Class Codes: A61K 31/4706 4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
• CPC Class Codes: A61K 31/7048 having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin , digitoxin or digoxin
• CPC Class Codes: A61K 31/711 Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/7125 Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/117 : Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 2310/17 : Immunomodulatory nucleic acids
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Nucleic acid sequences containing unmethylated CpG dinucleotides that modulate an immune response including stimulating a Th1 pattern of immune activation, cytokine production, NK lytic activity, and B cell proliferation are disclosed. The sequences are also useful a synthetic adjuvant.

Claims

1. A method for inducing Il-6 in a subject comprising

administering to the subject an effective amount to induce Il-6 in the subject of an immunostimulatory nucleic acid, having a sequence comprising:

2. The method of claim 1, wherein the subject is human.

3. The method of claim 1, wherein the nucleic acid has 8 to 100 nucleotides.

4. The method of claim 1, wherein the nucleic acid backbone includes a phosphate backbone modification on the 5′

inter-nucleotide linkages.

5. The method of claim 1, wherein the nucleic acid backbone includes a phosphate backbone modification on the 3′

inter-nucleotide linkages.

6. The method of claim 1, wherein the nucleic acid includes a phosphate backbone modification.

7. The method of claim 1, wherein X₁X₂ are nucleotides selected from the group consisting of:

GpT, GpG, GpA, ApA, ApT, ApG, GpT, GpA, CpG, TpA, TpT, and TpG; and

X₃X₄ are nucleotides selected from the group consisting of;

TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.

8. The method of claim 1, wherein X₁X₂ are GpA and X₃X₄ are TpT.

9. The method of claim 1, wherein X₁ and X₂ are purines and X₃ and X₄ are pyrimidines.

10. The method of claim 1, wherein X₁X₂ are GpA and X₃ and X₄ are pyrimidines.

11. The method of claim 1, wherein the immunostimulatory nucleic acid is 8 to 40 nucleotides in length.

12. The method of claim 1, wherein the immunostimulatory nucleic acid, has a sequence comprising:

13. The method of claim 1, wherein the immunostimulatory nucleic acid sequence is selected from the group consisting of sequences comprising the following nucleotides:

TCCATGTCGCTCCTGATGCT (SEQ ID NO;

37);

TCCATAACGTTCCTGATGCT (SEQ ID NO;

2);

TCCATGACGATCCTGATGCT (SEQ ID NO;

87);

TCCATGGCGGTCCTGATGCT (SEQ ID NO;

34);

TCCATGTCGGTCCTGATGCT (SEQ ID NO;

28);

TCCATAACGTCCCTGATGCT (SEQ ID NO;

88);

TCCATGTCGTTCCTGATGCT (SEQ ID NO;

38); and

TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO;

46).

14. A method of stimulating natural killer cell lytic activity comprising exposing a natural killer cell to an immunostimulatory nucleic acid to stimulate natural killer cell lytic activity, the immunostimulatory nucleic acid having a sequence comprising:

15. The method of claim 14, wherein the nucleic acid has 8 to 100 nucleotides.

16. The method of claim 14, wherein the nucleic acid backbone includes a phosphate backbone modification on the 5′

inter-nucleotide linkages.

17. The method of claim 14, wherein the nucleic acid backbone includes a phosphate backbone modification on the 3′

inter-nucleotide linkages.

18. The method of claim 14, wherein the nucleic acid includes a phosphate backbone modification.

19. The method of claim 14, wherein X₁X₂ are nucleotides selected from the group consisting of:

GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and

X₃X₄ are nucleotides selected from the group consisting of;

TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.

20. The method of claim 14, wherein X₁X₂ are GpA and X₃X₄ are TpT.

21. The method of claim 14, wherein X₁ and X₂ are purines and X₃ and X₄ are pyrimidines.

22. The method of claim 14, wherein X₁X₂ are GpA and X₃ and X₄ are pyrimidines.

23. The method of claim 14, wherein the immunostimulatory nucleic acid is 8 to 40 nucleotides in length.

24. The method of claim 15, wherein the immunostimulatory nucleic acid, has a sequence comprising:

25. The method of claim 14, wherein the immunostimulatory nucleic acid sequence is selected from the group consisting of sequences comprising the following nucleotides:

TCGTCGTTGTCGTTGTCGTT (SEQ ID NO;

47);

TCCATGACGGTCCTGATGCT (SEQ ID NO;

35);

TCCATGACGATCCTGATGCT (SEQ ID NO;

87);

TCCATGACGCTCCTGATGCT (SEQ ID NO;

89);

TCCATGACGTTCCTGATGCT (SEQ ID NO;

7);

TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO;

46);

TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO;

49);

GCGTGCGTTGTCGTTGTCGTT (SEQ ID NO;

56);

TGTCGTTTGTCGTTTGTCGTT(SEQ ID NO;

48);

TGTCGTTGTCGTTGTCGTT (SEQ ID NO;

50); and

TCGTCGTCGTCGTT (SEQ ID NO;

51).

26. A method for inducing interferon-gamma in a subject to treat an immune system deficiency, comprising:

administering to a subject having an immune system deficiency an effective amount to induce interferon-gamma production in the subject of an immunostimulatory nucleic acid, having a sequence comprising;

27. The method of claim 26, wherein the nucleic acid has 8 to 100 nucleotides.

28. The method of claim 26, wherein the nucleic acid backbone includes a phosphate backbone modification on the 5′

inter-nucleotide linkages.

29. The method of claim 26, wherein the nucleic acid backbone includes a phosphate backbone modification on the 3′

inter-nucleotide linkages.

30. The method of claim 26, wherein the nucleic acid includes a phosphates backbone modification.

31. The method of claim 26, wherein X₁X₂ are nucleotides selected from the group consisting of:

GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and

X₃X₄ are nucleotides selected from the group consisting of;

TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.

32. The method of claim 26, wherein X₁X₂ are GpA and X₃X₄ are TpT.

33. The method of claim 26, wherein X₁ and X₂ are purines and X₃ and X₄ are pyrimidines.

34. The method of claim 26, wherein X₁X₂ are GpA and X₃ and X₄ are pyrimidines.

35. The method of claim 26, wherein the immunostimulatory nucleic acid is 8 to 40 nucleotides in length.

36. The method of claim 26, wherein the immunostimulatory nucleic acid, has a sequence comprising:

37. A method for inducing Il-12 in a subject comprising:

administering to the subject an effective amount to induce Il-12 in the subject, of an immunostimulatory nucleic acid having a sequence comprising;

38. The method of claim 37, wherein the subject is human.

39. The method of claim 37, wherein the immunostimulatory nucleic acid sequence is selected from the group consisting of sequences comprising the following nucleotides:

TCCATGTCGCTCCTGATGCT (SEQ ID NO;

37);

TCCATGACGATCCTGATGCT (SEQ ID NO;

87);

TCCATGGCGGTCCTGATGCT (SEQ ID NO;

34);

TCCATGTCGGTCCTGATGCT (SEQ ID NO;

28);

TCCATAACGTCCCTGATGCT (SEQ ID NO;

88);

TCCATGTCGTTCCTGATGCT (SEQ ID NO;

38); and

TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO;

46).

40. The method of claim 37, wherein the nucleic acid has 8 to 100 nucleotides.

41. The method of claim 37, wherein the nucleic acid backbone includes a phosphate backbone modification on the 5′

inter-nucleotide linkages.

42. The method of claim 37, wherein the nucleic acid backbone includes a phosphate backbone modification on the 3′

inter-nucleotide linkages.

43. The method of claim 37, wherein the nucleic acid includes a phosphate backbone modification.

44. The method of claim 37, wherein X₁X₂ are nucleotides selected from the group consisting of:

GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and

X₃X₄ are nucleotides selected from the group consisting of;

TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.

45. The method of claim 37, wherein X₁X₂ are GpA and X₃X₄ are TpT.

46. The method of claim 37, wherein X₁ and X₂ are purines and X₃ and X₄ are pyrimidines.

47. The method of claim 37, wherein X₁X₂ are GpA and X₃ and X₄ are pyrimidines.

48. The method of claim 37, wherein the immunostimulatory nucleic acid is 8 to 40 nucleotides in length.

49. The method of claim 37, wherein the immunostimulatory nucleic acid, has a sequence comprising: