Anti-cancer DNA vaccine employing plasmids encoding signal sequence, mutant oncoprotein antigen, and heat shock protein
Novel nucleic acid vectors comprising sequences encoding (a) an antigen, (b) a signal peptide, and (c) a heat shock protein, are disclosed, as are methods for using such vectors to induce antigen-specific immune responses and to treat tumors.
1. A nucleic acid molecule encoding a fusion polypeptide useful as a vaccine composition, which molecule comprises:
(a) a first nucleic acid sequence encoding a first polypeptide or peptide that promotes processing via the MHC class I pathway,
wherein the first polypeptide or peptide is encoded by
(i) SEQ ID NO: 9; or
(ii) comprises the amino acid sequence of SEQ ID NO: 10; or
(iii) comprises an active C-terminal domain of the polypeptide or peptide encoded by (i) or comprising (ii);
(b) fused in frame with the first nucleic acid sequence, a second nucleic acid sequence encoding a signal peptide for secreting the fusion polypeptide; and
(c) a third nucleic acid sequence that is linked in frame to said first nucleic acid sequence and that encodes a non-oncogenic mutant of an E7 polypeptide of HPV-16 having the sequence SEQ ID NO: 2, said mutant of SEQ ID NO: 2 comprises a Glycine at positions 24 and 26, wherein positions 24 and 26 correspond to positions 24 and 26 of SEQ ID NO: 2.
2. The nucleic acid molecule of claim 1 , that is characterized as pNGVL4a-Sig/E7 (detox)/HSP70, and has the sequence SEQ ID NO: 13.
3. An expression vector comprising the nucleic acid molecule of claim 1 operatively linked to
(a) a promoter; and
(b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell.
4. The expression vector of claim 3 , which comprises plasmid PNGVL4a.
5. A pharmaceutical composition comprising:
(a) pharmaceutically and immunologically acceptable excipient in combination with;
(b) the nucleic acid molecule of claim 1 .
6. A pharmaceutical composition comprising:
(a) pharmaceutically and immunologically acceptable excipient in combination with;
(b) the nucleic acid molecule of claim 2 .
7. A pharmaceutical composition comprising:
(a) pharmaceutically and immunologically acceptable excipient in combination with;
(b) the expression vector of claim 3 .
8. A pharmaceutical composition immune response, comprising:
(a) pharmaceutically and immunologically acceptable excipient in combination with;
(b) the expression vector of claim 4 .
9. A method of inducing or enhancing a HPV antigen specific immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim 7 , thereby inducing or enhancing said response.
10. A method of inducing or enhancing a HPV antigen specific immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim 8 , thereby inducing or enhancing said response.
11. The method of claim 9 , wherein said subject is a human.
12. The method of claim 10 , wherein said subject is a human.
13. The method of claim 11 , wherein said administering is by an intramuscular injection by gene gun administration or by needle-free jet injection.
14. The method of claim 12 , wherein said administering is by an intramuscular injection by gene gun administration or by needle-free jet injection.
15. The nucleic acid molecule of claim 1 , wherein the mutant of SEQ ID NO: 2 further comprises a Glycine or Alanine substitution at position 91, wherein position 91 corresponds to position 91 of SEQ ID NO: 2.
16. A pharmaceutical composition comprising:
(a) pharmaceutically and immunologically acceptable excipient in combination with;
(b) the nucleic acid molecule of claim 15 .