MODIFIED OLIGORIBONUCLEOTIDE ANALOGS WITH ENHANCED IMMUNOSTIMULATORY ACTIVITY
The invention provides immunostimulatory compositions and methods for their use. In particular, the immunostimulatory compositions of the invention include RNA-like polymers that incorporate an immunostimulatory sequence motif and at least one chemical modification to confer improved stability against nuclease degradation and improved activity. Specific modifications involving phosphate linkages, nucleotide analogs, and combinations thereof are provided. Compositions of the invention optionally include an antigen and can be used to stimulate an immune response. Also provided are compositions and methods useful for treating a subject having an infection, a cancer, an to allergic condition, or asthma. Modified oligoribonucleotide analogs of the invention are believed to stimulate Toll-like receptors TLR7 and TLR8.
1 . An immunostimulatory composition comprising a polymer 4 to 100 units long, wherein each unit comprises a nucleoside or a nucleoside analog, wherein each pair of adjacent units is linked by a covalent linkage, and wherein the composition comprises
(a) an immunostimulatory RNA motif 4 to 8 nucleotides long, and
(b) at least one modified phosphate linkage selected from the group consisting of:
wherein
R1 is hydrogen (H), COOR, OH, C1-C18 alkyl, C 6 H 5 , or (CH 2 ) m —NH—R2, wherein R is H or methyl, butyl, methoxyethyl, pivaloyl oxymethyl, pivaloyl oxybenzyl, or S-pivaloyl thioethyl; R2 is H, C1-C18 alkyl, or C2-C18 acyl; and m is 1 to 17;
X is oxygen (O) or sulfur (S); and
each of Nu and Nu′ independently is a nucleoside or nucleoside analog; with the proviso that if R1 is H, then X is S;
wherein
X is O or S;
X 1 is OH, SH, BH 3 , OR3, or NHR3, wherein R3 is C1-C18 alkyl;
each of X 2 and X 3 independently is O, S, CH 2 , or CF 2 ; and
each of Nu and Nu′ independently is a nucleoside or nucleoside analog; with the proviso that
(a) at least one of X, X 2 , and X 3 is not O or X 1 is not OH,
(b) if X 1 is SH, then at least one of X, X 2 , and X 3 is not O,
(c) if X and X 2 are O and if X 1 is OH, then X 3 is not S and Nu is 3′Nu and Nu′ is 5′Nu′, and
(d) if X 1 is BH 3 , then at least one of X, X 2 , or X 3 is S; and
(iii) any combination of (i) and (ii).
2 . The composition of claim 1 , wherein the immunostimulatory RNA motif has a 15 base sequence selected from
(i) 5′-C/U-U-G/U-U-3′,
(ii) 5′-R-U-R-G-Y-3′,
(iii) 5′-G-U-U-G-B-3′,
(iv) 5′-G-U-G-U-G/U-3′, and
(v) 5′-G/C-U-A/C-G-G-C-A-C-3′,
wherein-C/U- is cytosine (C) or uracil (U), G/U- is guanine (G) or U, R is purine; Y is pyrimidine, B is U, G, or C, G/C is G or C, and A/C is adenine (A) or C.
3 . The composition of claim 1 , wherein the immunostimulatory RNA motif is 5′-C/U-U-G/U-U-3′.
4 . The composition of claim 1 , wherein the immunostimulatory RNA motif is 5’-R-U-R-G-Y-3′.
5 . The composition of claim 1 , wherein the immunostimulatory RNA motif is 5′-G-U-U-G-B-3′.
6 . The composition of claim 1 , wherein the immunostimulatory RNA motif is 5′-G-U-G-U-G/U-3′.
7 . The composition of claim 1 , wherein the immunostimulatory RNA motif is 5 5′-G/C-U-A/C-G-G-C-A-C-3′.
8 - 33 . (canceled)
34 . An immunostimulatory composition comprising a polymer 4 to 100 units long, wherein each unit comprises a nucleoside or a nucleoside analog, wherein each pair of adjacent units is linked by a covalent linkage, and wherein the composition comprises
(a) an immunostimulatory RNA motif 4 to 8 nucleotides long, and
(b) at least one nucleotide analog provided as Formula IIIA or Formula IIIB
wherein
R4 is H or OR, wherein R is H or C1-C18 acyl;
B is a nucleobase, a modified nucleobase, or H;
each of X and X 5 independently is O or S; and
X 4 is OH, SH, methyl, or NHR5, wherein R5 is C1-C18 alkyl; and
each dashed line independently represents an optional bond to an adjacent unit, hydrogen, or an organic radical;
with the proviso that at least one of X and X 5 is not O or X 4 is not OH.
35 - 57 . (canceled)
58 . An immunostimulatory composition comprising the immunostimulatory composition of claim 1 , further comprising at least one nucleotide analog provided as Formula IIIA or Formula IIIB
wherein
R4 is H or OR, wherein R is H or C1-C18 acyl;
B is a nucleobase, a modified nucleobase, or H;
each of X and X 5 independently is O or S; and
X 4 is OH, SH, methyl, or NHR5, wherein R5 is C1-C18 alkyl; and
each dashed line independently represents an optional bond to an adjacent unit, hydrogen, or an organic radical;
with the proviso that at least one of X and X 5 is not O or X 4 is not OH.
59 - 87 . (canceled)
88 . A pharmaceutical composition of claim 1 , in association with a delivery vehicle chosen from a cationic lipid, a liposome, a cochleate, a virosome, an immune-stimulating complex (ISCOM), a microparticle, a microsphere, a nanosphere, a unilamellar vesicle (LUV), a multilarnellar vesicle, an oil-in-water emulsion, a water-in oil emulsion, an emulsome, and a polycationic peptide, and, optionally, a pharmaceutically acceptable carrier.
89 . (canceled)
90 . A method of activating an immune cell, the method comprising contacting an 20 immune cell with an effective amount of the composition of claim 1 .
91 . A method of vaccinating a subject, the method comprising administering to the subject an antigen and a composition of claim 1 .
92 - 98 . (canceled)
99 . A method of treating a subject having an infection, the method comprising administering to the subject an effective amount of the composition of claim 1 .
100 . (canceled)
101 . A method of treating a subject having a cancer, the method comprising administering to the subject an effective amount of the composition of claim 1 .
102 . (canceled)
103 . A method of treating a subject having an allergic condition, the method comprising administering to the subject an effective amount of the composition of claim 1 .
104 . (canceled)
105 . A method of treating a subject having asthma, the method comprising administering to the subject an effective amount of the composition of claim 1 .
106 . (canceled)
107 . A method for treating a subject having airway remodeling, the method comprising administering to the subject an effective amount of an immunostimulatory composition of claim 1 .
108 . A method for increasing antibody-dependent cellular cytotoxicity (ADCC), the method comprising administering to a subject in need of increased ADCC an effective amount of an immunostimulatory composition of claim 1 , to increase ADCC.
109 - 110 . (canceled)
111 . A method for enhancing epitope spreading, the method comprising contacting a cell of the immune system with an antigen and subsequently contacting the cell with at least two doses of an immunostimulatory composition of claim 1 .
112 . (canceled)
113 . A method for enhancing epitope spreading in a subject, the method comprising administering to the subject a vaccine comprising an antigen and an adjuvant and subsequently administering to the subject at least two doses of an isolated immunostimulatory composition of claim 1 , in an effective amount to induce multiple epitope-specific immune responses.
114 . (canceled)
115 . A method for enhancing epitope spreading in a subject, the method comprising applying a therapeutic protocol which results in immune system antigen exposure in the subject and subsequently administering at least two doses of an isolated immunostimulatory composition of claim 1 , in an effective amount to induce multiple epitope-specific immune responses.
116 . (canceled)