Polypeptide-antigen conjugates with non-natural amino acids
Methods for the production of immunogenic compositions containing a non-natural amino acid are disclosed. The non-natural amino acid can be a site for attachment of antigens, such as bacterial capsular polysaccharides, to make immunogenic conjugates. Bio-orthogonal attachment chemistry incorporated into the non-natural amino acids allows for more efficient and potent antigen presentation to the immune system, simplified purification, and more well-defined structure of these semi-synthetic immunogens.
1. A composition comprising at least 21 distinct crosslinked carrier protein-antigen conjugates, wherein each of the distinct crosslinked carrier protein-antigen conjugates comprises:
(i) an antigen, wherein the antigen is a distinct serotype of a capsular polysaccharide antigen from Streptococcus pneumoniae,
wherein there is a distinct crosslinked carrier protein-antigen conjugate for each of the capsular polysaccharide antigens of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F and a distinct crosslinked carrier protein-antigen conjugate for each of at least eight additional capsular polysaccharide antigens of serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F and 38; and
(ii) a carrier protein, wherein the carrier protein comprises a polypeptide comprising at least one T-cell activating epitope and at least 95% sequence identity to SEQ ID NO: 1, wherein a 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF) non-natural amino acid (nnAA) is substituted for a naturally occurring amino acid at residues 34, 213, 245, 265, 386, and 527 numbered according to SEQ ID NO:1,
wherein the capsular polysaccharide antigen is conjugated to the nnAAs in the carrier protein.
2. The composition of claim 1 , wherein the at least 21 distinct crosslinked carder protein-antigen conjugates comprise
(i) at least 21 distinct crosslinked carrier protein-antigen conjugates wherein there is a distinct crosslinked carrier protein-antigen conjugate comprising a capsular polysaccharide for each of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F; or
(ii) at least 24 distinct crosslinked carrier protein-antigen conjugates wherein there is a distinct crosslinked carrier protein-antigen conjugate comprising a capsular polysaccharide for each of Streptococcus pneumonia serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.
3. The composition of claim 2 , wherein the antigen is linked to the carrier protein according to formula XI or XIa:
wherein
R 1 is H, formyl, or at least one amino acid of the carrier protein;
R 2 is OH or at least one amino acid of the carrier protein;
W is C;
y is 1;
n is at least 1; and
X is one monosaccharide within the capsular polysaccharide.
4. The composition of claim 3 , wherein n is 3-16.
5. The composition according to claim 2 , wherein the Streptococcus pneumoniae serotype 20 is serotype 20B.
6. The composition according to claim 5 , wherein the composition additionally comprises a distinct crosslinked carrier protein-antigen conjugate wherein the antigen is a capsular polysaccharide of Streptococcus pneumoniae serotype 6C or 7C.
7. The composition according to claim 2 , wherein the composition additionally comprises a distinct crosslinked carrier protein-antigen conjugate wherein the antigen is a capsular polysaccharide of Streptococcus pneumoniae serotype 6C or 7C.
8. The composition of claim 1 , wherein the overall (weight by weight) ratio of all serotype polysaccharides to carrier protein in the composition is at least 1.1.
9. The composition of claim 1 , wherein the overall (weight by weight) ratio of all serotype polysaccharides to carrier protein in the composition is at least 1.2.
10. The composition of claim 1 , wherein the antigen is linked to the carrier protein according to formula XI or XIa:
wherein
R 1 is H, formyl, or at least one amino acid of the carrier protein;
R 2 is OH or at least one amino acid of the carrier protein;
W is C;
y is 1;
n is at least 1; and
X is one monosaccharide within the capsular polysaccharide.
11. The composition of claim 10 , wherein n is 3-16.
12. The composition of claim 1 , wherein the Streptococcus pneumoniae capsular polysaccharides are conjugated to the nnAAs via a linking moiety, wherein the Streptococcus pneumoniae capsular polysaccharides comprise a cyclooctyne group and are conjugated to the pAMF nnAAs.
13. The composition of claim 12 , wherein the antigen is linked to the carrier protein according to formula XI or Xia:
wherein
R 1 is H, formyl, or at least one amino acid of the carrier protein;
R 2 is OH or at least one amino acid of the carrier protein;
W is C;
y is 1;
n is at least 1; and
X is one monosaccharide within the capsular polysaccharide.
14. The composition of claim 13 , wherein n is 3-16.
15. The composition of claim 1 , wherein the overall (weight by weight) ratio of all serotype polysaccharides to carrier protein in the composition is at least 1.0.
16. The composition of claim 1 , wherein there is less than about 25% (by weight) total free capsular polysaccharide present in the composition.
17. The composition according to claim 1 , wherein the composition additionally comprises a distinct crosslinked carrier protein-antigen conjugate wherein the antigen is a capsular polysaccharide of Streptococcus pneumoniae serotype 6C or 7C.