Anti-Cancer DNA Vaccine Employing Plasmids Encoding Signal Sequence, Mutant Oncoprotein Antigen, and Heat Shock Protein

Abstract

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;   (b) fused in frame with the first nucleic acid sequence, a second nucleic acid sequence encoding a signal peptide; and   (c) a third nucleic acid sequence that is linked in frame to said first nucleic acid sequence and that encodes an antigenic polypeptide or peptide.   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the antigenic peptide comprises an epitope that binds to a MHC class I protein. 
     
     
         3 . The nucleic acid molecule of  claim 1  wherein the first polypeptide or peptide is Hsp70, an active C-terminal domain thereof, or a functional derivative of Hsp70 or of said C-terminal domain. 
     
     
         4 . The nucleic acid molecule of  claim 1 , wherein the first polypeptide is encoded by SEQ ID NO:9 or a fragment thereof that encodes a functional derivative of said polypeptide or the full length sequence of Hsp70 a set forth in GENBANK Z95324 AL123456 and encoded by nucleotides 10633-12510 of the  Mycobacterium tuberculosis  genome. 
     
     
         5 . The nucleic acid molecule of  claim 1 , wherein the first polypeptide is SEQ ID NO:10 of a functional derivative thereof. 
     
     
         6 . The nucleic acid molecule of  claim 1  wherein the antigen is one which is present on, or/cross-reactive with an epitope of, a pathogenic organism, cell, or virus. 
     
     
         7 . The nucleic acid molecule of  claim 6 , wherein the virus is a human papilloma virus. 
     
     
         8 . The nucleic acid molecule of  claim 7 , wherein the antigen is an E7 polypeptide of HPV-16 having the sequence SEQ ID NO:2, or an antigenic fragment thereof. 
     
     
         9 . The nucleic acid molecule of  claim 8 , wherein the HPV-16 E7 polypeptide is a non-oncogenic mutant or variant of said E7 polypeptide. 
     
     
         10 . The nononcogenic mutant of  claim 9  wherein the sequence of the E7 polypeptide differs from SEQ ID NO:2 by one or more of the following substitutions:
 (a) Cys at position 24 to Gly or Ala 
 (b) Glu at position 26 to Gly or Ala 
 (c) Cys at position 91 to Gly or Ala. 
 
     
     
         11 . The nucleic acid molecule of  claim 7 , wherein the antigen is the E6 polypeptide of HPV-16 having the sequence SEQ ID NO:4 or an antigenic fragment thereof. 
     
     
         12 . The nucleic acid molecule of  claim 11 , wherein the HPV-16 E6 polypeptide is a non-oncogenic mutant or variant of said E6 polypeptide. 
     
     
         13 . The nononcogenic mutant of  claim 12  wherein the sequence of the E6 polypeptide differs from SEQ ID NO:4 by one or more of the following substitutions:
 (a) Cys at position 70 to Gly or Ala 
 (b) Cys at position 113 to Gly or Ala. 
 (c) Ile at position 135 to Thr 
 
     
     
         14 . The nucleic acid molecule of  claim 1  that is characterized as pNG′VL4a-Sig/E7(detox)/HSP70, and has the sequence SEQ ID NO:13. 
     
     
         15 . The nucleic acid molecule of  claim 1  operatively linked to a promoter. 
     
     
         16 . An expression vector comprising the nucleic acid molecule of any of  claims 1 - 13  operatively linked to
 (a) a promoter; and 
 (b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell. 
 
     
     
         17 . An expression vector comprising the nucleic acid molecule of  claim 14 . operatively linked to
 (a) a promoter; and   (b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell.   
     
     
         18 . The expression vector of  claim 16  which is a plasmid. 
     
     
         19 . The expression vector of  claim 18  wherein said plasmid is pNGV4a. 
     
     
         20 . A pharmaceutical composition capable of inducing or enhancing an antigen-specific immune response, comprising:
 (a) pharmaceutically and immunologically acceptable excipient in combination with;   (b) a composition comprising the nucleic acid molecule of any of  claims 1 - 13 .   
     
     
         21 . A pharmaceutical composition capable of inducing or enhancing an antigen-specific immune response, comprising:
 (a) pharmaceutically and immunologically acceptable excipient in combination with;   (b) the nucleic acid molecule of  claim 14 .   
     
     
         22 . A pharmaceutical composition capable of inducing or enhancing an antigen-specific immune response, comprising:
 (a) pharmaceutically and immunologically acceptable excipient in combination with;   (b) the expression vector of  claim 16 .   
     
     
         23 . A pharmaceutical composition capable of inducing or enhancing an antigen-specific immune response, comprising:
 (a) pharmaceutically and immunologically acceptable excipient in combination with;   (b) the expression vector of  claim 19 .   
     
     
         24 . A method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 20 , thereby inducing or enhancing said response. 
     
     
         25 . A method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 21 , thereby inducing or enhancing said response. 
     
     
         26 . A method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 22 , thereby inducing or enhancing said response. 
     
     
         27 . A method of inducing or enhancing an antigen specific immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 23 , thereby inducing or enhancing said response. 
     
     
         28 . The method of  claim 24 , wherein the response is mediated at least in part by CD8 +  cytotoxic T lymphocytes (CTL). 
     
     
         29 . The method of  claim 24  wherein said subject is a human. 
     
     
         30 . The method of  claim 25  wherein said subject is a human. 
     
     
         31 . The method of  claim 26  wherein said subject is a human. 
     
     
         32 . The method of  claim 27  wherein said subject is a human. 
     
     
         33 . The method of  claim 29  wherein said administering is by a intramuscular injection by gene gun administration or by needle-free jet injection. 
     
     
         34 . The method of  claim 30  wherein said administering is by a intramuscular injection by gene gun administration or by needle-free jet injection. 
     
     
         35 . The method of  claim 31  wherein said administering is by a intramuscular injection by gene gun administration or by needle-free jet injection. 
     
     
         36 . The method of  claim 32  wherein said administering is by a intramuscular injection by gene gun administration or by needle-free jet injection. 
     
     
         37 . A method of inhibiting growth or preventing re-growth of a tumor expressing HPV E7 or E6 protein in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition of  claim 20 , wherein said third nucleic acid sequence encodes one or more epitopes of E7 or E6, thereby inhibiting said growth or preventing said re-growth. 
     
     
         38 . A method of inhibiting growth or preventing re-growth of a tumor expressing HPV E7 or E6 protein in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition of  claim 21 , wherein said third nucleic acid sequence encodes one or more epitopes of E7 or E6, thereby inhibiting said growth or preventing said re-growth. 
     
     
         39 . A method of inhibiting growth or preventing re-growth of a tumor expressing HPV E7 or E6 protein in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition of  claim 22 , wherein said third nucleic acid sequence encodes one or more epitopes of E7 or E6, thereby inhibiting said growth or preventing said re-growth. 
     
     
         40 . A method of inhibiting growth or preventing re-growth of a tumor expressing HPV E7 or E6 protein in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition of  claim 23 , wherein said third nucleic acid sequence encodes one or more epitopes of E7 or E6, thereby inhibiting said growth or preventing said re-growth.