Immunostimulatory, covalently lipidated oligonucleotides

Abstract

Covalently lipidated oligonucleotides comprising the CpG dinucleotide unit, or an analogue thereof, may be used as immunostimulatory agents to protect against a disease caused by a cancer cell or a pathogen, either alone or in conjunction with immunogens and/or non-immunological agents. Lipidated oligonucleotides with special backbones, lipidated oligonucleotides with fewer than eight nucleotides, and lipidated oligonucleotides comprising a plurality of CpG dinucleotide-containing segments connected by a long internucleoside linkage are of particular interest. These compounds are also novel per se.

1. A method of stimulating the immune system of a subject which comprises administering to the subject an immunologically effective amount of an isolated immunostimulatory molecule which comprises at least one oligonucleotide strand which comprises
 (1) at least one nucleotide sequence comprising a plurality of nucleotides, each nucleotide comprising a nucleobase, and thereby also comprising at least one CxG dinucleotide unit or analogue thereof, 
 in which there are no more than six nucleobases in each oligonucleotide strand, 
 wherein in said analogue, (1) cytosine is replaced with a cytosine analogue which is a pyrimidine other than thymine or uracil, and/or (2) guanine is replaced with a guanine analogue which is a purine other than adenine, and 
 (2) at least one covalently incorporated lipophilic group. 
 
     
     
       2. The method of  claim 1  in which element (1) comprises a CxG dinucleotide unit. 
     
     
       3. The method of  claim 1  in which the CxG dinucleotide unit is a CpG dinucleotide unit. 
     
     
       4. The method of  claim 1  in which there are 2-5 nucleobases on each nucleotide strand. 
     
     
       5. The method of  claim 4  in which at least one lipophilic group is a strongly lipophilic group. 
     
     
       6. The method of  claim 4  in which at least one lipophilic group is a highly lipophilic (Meylan) group. 
     
     
       7. The method of  claim 4  in which at least one lipophilic group has a predicted logP, according to the Meylan algorithm, of at least 4. 
     
     
       8. The method of  claim 4  in which at least one lipophilic group has a predicted logP, according to the Meylan algorithm, of at least 7. 
     
     
       9. The method of  claim 4  in which at least one lipophilic group has a predicted logP, according to the Meylan algorithm, of at least 10. 
     
     
       10. The method of  claim 4  in which at least one lipophilic group is selected from the group consisting of
   —(CH 2 ) 4 NH.CO—(CH 2 ) n CH 3   (a) 
 where n=an integer with values ranging from 6 to 26, 
 
       
         
           
           
               
               
           
         
         where m and n are independent integers with values ranging from 6 to 26, 
         (c) 
         —XR wherein X is —O—, —S—, or —NH— and —R is aliphatic, and 
         (d) 
         —XR wherein X is —O—, —S—, or —NH— and —R is at least partially aromatic. 
       
     
     
       11. The method of  claim 4  where —R is a group selected from the group consisting of one of the following structures: 
       
         
           
           
               
               
           
         
         where m, n, and k are independent integers with values ranging from 3 to 30. 
       
     
     
       12. The method of  claim 4  in which at least one lipophilic group is one of the lipophilic groups depicted below 
       
         
           
           
               
               
           
         
       
     
     
       13. The method of  claim 4  where said molecule comprises at least two lipophilic groups. 
     
     
       14. The method of  claim 12  where said molecule comprises at least two strongly lipophilic groups. 
     
     
       15. The method of  claim 12  where said molecule comprises at least two highly lipophilic (Meylan) groups. 
     
     
       16. The method of  claim 4  in which there are 2-4 nucleobases on each nucleotide strand. 
     
     
       17. The method of  claim 1  in which each of the nucleobases is selected from the group consisting of adenine, guanine, thymine, cytosine, uracil, and hypoxanthine. 
     
     
       18. The method of  claim 1  in which each of the nucleobases is selected from the group consisting of adenine, guanine, thymine, and cytosine. 
     
     
       19. The method of  claim 4  wherein at least one nucleotide comprises a nucleobase-carbohydrate nucleoside. 
     
     
       20. The method of  claim 19  in which the carbohydrate is a monosaccharide. 
     
     
       21. The method of  claim 19  in which the number of carbon atoms in the monosaccharide is 3-8. 
     
     
       22. The method of  claim 21  in which the monosaccharide is a pentose. 
     
     
       23. The method of  claim 20  in which the monosaccharide is an aldose. 
     
     
       24. The method of  claim 20  in which the monosaccharide is cyclized. 
     
     
       25. The method of  claim 19  in which the monosaccharide is a pyranose. 
     
     
       26. The method of  claim 20  in which the monosaccharide is a ribose or a 2-deoxyribose. 
     
     
       27. The method of  claim 4  in which all of the nucleotides of one strand comprise a nucleobase-carbohydrate nucleoside. 
     
     
       28. The method of  claim 4  in which at least one nucleotide further comprises a phosphate. 
     
     
       29. The method of  claim 28  in which at least one nucleotide comprises one and only one phosphate. 
     
     
       30. The method of  claim 29  in which at least one strand is DNA or RNA. 
     
     
       31. The method of  claim 4  in which at least one lipophilic group is covalently incorporated into a free end of at least one strand. 
     
     
       32. The method of  claim 4  in which at least one lipophilic group is covalently incorporated into the 3′ end of at least one strand. 
     
     
       33. The method of  claim 31  in which the lipophilic group is attached to the end through a phosphate group. 
     
     
       34. The method of  claim 4  in which at least one lipophilic group is incorporated into an internucleoside linkage. 
     
     
       35. The method of  claim 4  in which at least one lipophilic group is a substituent of a nucleobase. 
     
     
       36. The method of  claim 15  in which at least part of the oligonucleotide has a backbone which differs from that of DNA and RNA. 
     
     
       37. The method of  claim 36  in which the backbone differs in that the internucleoside linkage is not a phosphate group. 
     
     
       38. The method of  claim 36  in which the backbone differs in that at least one nucleotide is a non-normal nucleotide which does not comprise ribose or 2-deoxyribose. 
     
     
       39. The method of  claim 38  in which the non-normal nucleoside comprises a sugar. 
     
     
       40. The method of  claim 38  in which the non-normal nucleotide does not comprise a sugar. 
     
     
       41. The method of  claim 40  in which the oligonucleotide is at least partially a PNA oligomer. 
     
     
       42. The method of  claim 40  in which at least one non-normal nucleotide comprises a non-normal nucleoside of the form
 nucleobase-O-alkyl 
 where the —O-alkyl is the residue of a polyol, and the alkyl is not more than 6 carbon atoms. 
 
     
     
       43. The method of  claim 42  wherein the polyol is glycerol, and hence the alkyl is 3 carbon atoms. 
     
     
       44. The method of  claim 36  in which at least non-normal nucleoside is bound to a phosphate group. 
     
     
       45. The method of  claim 44  in which there are two adjacent such non-normal nucleosides and the internucleoside linkage between them is a phosphate group. 
     
     
       46. The method of  claim 44  in which there are two adjacent such non-normal nucleosides and the internucleoside linkage between them is
 -phosphate group-linker Z-phosphate group-, 
 where linker Z is aliphatic. 
 
     
     
       47. The method of  claim 46  in which linker Z is of the form -[small alkyl-O] n , where n is 1 to 20, and small alkyl is not more than 6 carbon. 
     
     
       48. The method of  claim 47  in which linker Z is —[CH 2 CH 2 O] n —. 
     
     
       49. The method of  claim 4  in which the dinucleotide unit comprises a non-natural nucleoside, or an internucleoside linkage which is not a phosphate group. 
     
     
       50. The method of  claim 4  in which the CxG dinucleotide unit comprises two non-natural nucleosides and the internucleoside linkage between them is a phosphate group. 
     
     
       51. The method of  claim 49  in which the dinucleotide unit comprises two non-natural nucleosides and the internucleoside linkage between them is
 -phosphate group-linker Z-phosphate group-, 
 where linker Z is aliphatic. 
 
     
     
       52. The method of  claim 51  in which linker Z is —[CH 2 CH 2 O] n — and n is 1 to 20. 
     
     
       53. The method of  claim 49  in which the dinucleotide unit is a PNA oligomer. 
     
     
       54. The method of  claim 49  in which the dinucleotide unit is a GNA oligomer. 
     
     
       55. The method of  claim 4  in which the molecule lacks double stranded structure. 
     
     
       56. The method of  claim 55  in which the molecule has at least some double stranded structure. 
     
     
       57. The method of  claim 3  in which there are no more than four nucleobases in each oligonucleotide strand. 
     
     
       58. The method of  claim 1  in which the molecule further comprises at least one epitope, said epitope being a peptide, carboxydrate, lipid, glycopeptide or glycolipid epitope. 
     
     
       59. The method of  claim 58  wherein at least one epitope is a carbohydrate epitope. 
     
     
       60. The method of  claim 58  wherein at least one epitope is a peptide epitope. 
     
     
       61. The method of  claim 58  wherein at least one epitope is a B-cell epitope. 
     
     
       62. The method of  claim 58  wherein at least one epitope is a T-cell epitope. 
     
     
       63. The method of  claim 58  wherein at least one epitope is a MUC1 epitope. 
     
     
       64. The method of  claim 1  in which the oligonucleotide is cyclized, so as to lack a free end, and the lipophilic groups are incorporated elsewhere in the molecule. 
     
     
       65. The method of  claim 1  where said molecule which comprises two or more segments, each segment consisting of nucleosides joined to each other by short internucleoside linkages, each segment being joined to at least one other segment by a long internucleoside linkage,
 at least two of said segments each comprising at least one CxG dinucleotide unit or analogue thereof. 
 
     
     
       66. The method of  claim 65  in which said segments are connected by said internucleoside linkages to form one or more linear chains. 
     
     
       67. The method of  claim 65  in which two or more segments are cyclized by two or more internucleoside linkages. 
     
     
       68. The method of  claim 1  where said nucleotide sequence comprises at least one pair of adjacent thymine nucleobases which are dimerized to form a thymine dimer. 
     
     
       69. The method of  claim 4  in which at least one lipophilic group is covalently incorporated into the 5′ end of an oligonucleotide strand. 
     
     
       70. The method of  claim 4  in which the molecule does not have cytotoxic activity against cancer cells. 
     
     
       71. The method of  claim 4  in which the subject is not suffering from a cancer. 
     
     
       72. The method of  claim 4  in which the subject is not being medicated with any other cancer preventative. 
     
     
       73. The method of  claim 4  in which the molecule potentiates the specific innate immune response to a pathogen or cancer already present in the subject. 
     
     
       74. The method of  claim 4  which further comprises administering a pharmaceutical composition comprising an immunogen to the subject, said molecule potentiating the specific elicited immune response to said immunogen. 
     
     
       75. The method of  claim 74  in which the molecule and the immunogen are administered simultaneously. 
     
     
       76. The method of  claim 75  in which the molecule and the immunogen are administered in the same composition. 
     
     
       77. The method of  claim 74  in which the immunostimulatory oligonucleotide molecule is also an immunogen which elicits a specific immune response protective against said pathogen or cancer. 
     
     
       78. The method of  claim 1  where none of the internucleoside linkages is selected from the group consisting of poly(N-viny 1 ), poly(methacryloxyethyl), poly(methacrylamide), and poly(etheylenimine) 
     
     
       79. The method of  claim 1  wherein the nucleotide sequence consists of two nucleotides. 
     
     
       80. The method of  claim 1  wherein the nucleotide sequence consists of three nucleotides. 
     
     
       81. The method of  claim 1  wherein the nucleotide sequence consists of four nucleotides. 
     
     
       82. The method of  claim 1  wherein the nucleotide sequence consists of five nucleotides. 
     
     
       83. The method of  claim 1  wherein the nucleotide sequence consists of six nucleotides. 
     
     
       84. The method of  claim 1  wherein the nucleotide sequence consists of seven nucleotides. 
     
     
       85. A method of stimulating the immune system of a subject which comprises administering to the subject an immunologically effective amount of an immunostimulatory molecule which comprises at least one oligonucleotide strand which comprises
 (1) at least one nucleotide sequence comprising a plurality of nucleotides, each nucleotide comprising a nucleobase, and thereby also comprising at least one CxG dinucleotide unit or analogue thereof, 
 in which there are no more than six nucleobases in each oligonucleotide strand, 
 wherein in said analogue, (1) cytosine is replaced with a cytosine analogue which is a pyrimidine other than thymine or uracil, and/or (2) guanine is replaced with a guanine analogue which is a purine other than adenine, and 
 (2) at least one covalently incorporated lipophilic group. 
 
     
     
       86. The method of  claim 1  wherein the molecule is immunostimulatory in the absence of lipofectin. 
     
     
       87. The method of  claim 1  wherein the stimulation of the immune system comprises stimulation of T-cell proliferation. 
     
     
       88. The method of  claim 76  in which the immunostimulatory molecule and the immunogen are separate molecules. 
     
     
       89. The method of  claim 4  in which the molecule further comprises at least one epitope, said epitope being a peptide, carbohydrate, lipid, glycopeptide or glycolipid epitope.