High temperature selection of nucleotide-supported carbohydrate vaccines and resulting glycosylated oligonucleotides
The invention relates to an oligonucleotide including one or more modified nucleoside bases having the structure -B-L-A wherein for each of the modified nucleosides A is independently a monosaccharide or oligosaccharide, L is a linker molecule, and B is independently a pyrimidine or pyridine base linked to the sugar-phosphate backbone of the oligonucleotide; and wherein the oligonucleotide binds specifically to a carbohydrate-binding monoclonal antibody with an affinity of less than 100 nM. Immunogenic conjugates that include the oligonucleotide, and pharmaceutical compositions that include the oligonucleotide or the immunogenic conjugate are also disclosed. Various method of using the oligonucleotides, immunogenic conjugates, and pharmaceutical compositions are disclosed, including inducing an immune response, inhibiting viral or bacterial infection, treating a cancerous condition, and detecting a neutralizing antibody. A method is also disclosed for selecting the oligonucleotides using an alternative Selection of Modified Aptamers (SELMA).
1. An oligonucleotide comprising two to five modified nucleoside bases having the structure:
-B-L-A
wherein for each of the modified nucleosides
A is independently a monosaccharide or oligosaccharide,
L is a linker molecule, and
B is independently a pyrimidine or pyridine base linked to the sugar-phosphate backbone of the oligonucleotide; and
wherein the oligonucleotide binds specifically to a carbohydrate-binding monoclonal antibody with an affinity of less than 100 nM.
2. The oligonucleotide according to claim 1 , wherein the oligonucleotide comprises one or more phosphorothioate-linked nucleotides, or 2′-fluoro-, 2′-amino, 2′-O-methyl-, 5′-iodo-, or 5′-bromo-modified nucleotides.
3. The oligonucleotide according to claim 1 , wherein the oligonucleotide has a length of about 15 to about 100 nucleotides.
4. The oligonucleotide according to claim 1 , wherein the oligonucleotide comprises from two to four of said modified nucleoside bases.
5. The oligonucleotide according to claim 1 , wherein A is an oligosaccharide.
6. The oligonucleotide according to claim 1 , wherein L is a linker molecule that comprises
wherein each of R 1 and R 2 is optionally a direct link or independently selected from the group consisting of a linear or branched C 1 to C 18 hydrocarbon that is saturated or mono- or poly-unsaturated, optionally interrupted by one or more non-adjacent —O—, —C(═O)—, or —NR 4 —; a substituted or unsubstituted C 3 to C 10 cycloalkandiyl, a substituted or unsubstituted aryl diradical; a substituted or unsubstituted heteroaryl diradical; a monosaccharide diradical; or a disaccharide diradical; R 3 is optional and can be —O—, —S—, or —NR 4 —; and R 4 is independently H or a C 1 to C 10 alkyl.
7. The oligonucleotide according to claim 1 , wherein the oligonucleotide binds specifically to the carbohydrate-binding monoclonal antibody with an affinity that is substantially the same as or lower than the affinity of the carbohydrate-binding, neutralizing monoclonal antibody to its naturally occurring binding partner.
8. The oligonucleotide according to claim 1 , wherein the carbohydrate-binding monoclonal antibody is neutralizing against a pathogen.
9. The oligonucleotide according to claim 1 , wherein the carbohydrate-binding monoclonal antibody is cytotoxic against a cancer cell.
10. An oligonucleotide comprising three to five modified nucleoside bases having the structure:
-B-L-A
wherein for each of the modified nucleosides
A is a branched-chain Man 9 oligosaccharide,
L is a linker molecule, and
B is independently a pyrimidine or pyridine base linked to the sugar-phosphate backbone of the oligonucleotide; and
wherein the oligonucleotide binds specifically to HIV neutralizing monoclonal antibody 2G12 with a K d value that is lower than 20 nM.
11. An immunogenic conjugate comprising an oligonucleotide according to claim 1 covalently or non-covalently bound to an immunogenic carrier molecule.
12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an oligonucleotide according to claim 1 .
13. The pharmaceutical composition according to claim 12 further comprising an adjuvant.
14. A method of inducing an immune response in an individual comprising:
administering to an individual an oligonucleotide according to claim 1 , wherein said administering is effective to induce an immune response against the oligonucleotide.
15. An oligonucleotide comprising one or more modified nucleoside bases having the structure:
-B-L-A
wherein for each of the modified nucleosides
A is independently a monosaccharide or oligosaccharide,
L is a linker molecule, and
B is independently a pyrimidine or pyridine base linked to the sugar-phosphate backbone of the oligonucleotide;
wherein the oligonucleotide binds specifically to monoclonal antibody 2G12 with an affinity of less than 100 nM; and
wherein the oligonucleotide comprises the sequence NGNAACCNACGGANA (SEQ ID NO: 103) where N at position 3 is any nucleoside base and N at positions 1, 8, and 14 is one of said modified nucleoside bases.