초록

This invention provides compositions and methods for treating and vaccinating against an Her2/neu antigen-expressing tumor and inducing an immune response against dominant in a non-human animal.

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1. A method of treating a Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering to said non-human animal a recombinant Listeria strain having mutations in the D-alanine racemase (Dal) gene and the D-amino acid transferase (Dat) gene, said

1. A method of treating a Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering to said non-human animal a recombinant Listeria strain having mutations in the D-alanine racemase (Dal) gene and the D-amino acid transferase (Dat) gene, said recombinant Listeria strain comprising a nucleic acid encoding a first and a second open reading frame, wherein said first open reading frame encodes a recombinant polypeptide comprising SEQ ID NO: 2 fused to an additional polypeptide, and wherein said second open reading frame encodes a metabolic enzyme that complements said mutations, wherein said Her-2/neu-expressing tumor growth or cancer is osteosarcoma, and wherein said recombinant Listeria lacks the actA virulence gene. 2. The method of claim 1, wherein said non-human animal is a dog. 3. The method of claim 1, wherein administering said fusion polypeptide to a subject having said osteosarcoma prevents escape mutations within said tumor or cancer. 4. The method of claim 1, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes. 5. The method of claim 1, wherein said nucleic acid molecule is integrated into the Listeria genome. 6. The method of claim 1, wherein said nucleic acid molecule is in a plasmid in said recombinant Listeria strain. 7. The method of claim 6, wherein said plasmid is stably maintained in said recombinant Listeria strain in the absence of antibiotic selection. 8. The method of claim 6, wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria. 9. The method of claim 1, wherein said recombinant Listeria strain is attenuated. 10. The method of claim 1, wherein said additional polypeptide is selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment. 11. The method of claim 1, wherein said metabolic enzyme encoded by said second open reading frame is an amino acid metabolism enzyme. 12. The method of claim 11, wherein said amino acid metabolism enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme. 13. The method of claim 1, wherein said nucleic acid molecule further comprises a third open reading frame. 14. The method of claim 13, wherein said third open reading frame encodes a metabolic enzyme, wherein said metabolic enzyme is a D-amino acid transferase enzyme or an alanine racemase enzyme. 15. The method of claim 1, wherein said recombinant Listeria strain has been passaged through an animal host. 16. The method of claim 1, further comprising an independent adjuvant. 17. The method of claim 16, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide. 18. The method of claim 1, wherein said osteosarcoma cancer is canine osteosarcoma. 19. A method of preventing a Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering to said non-human animal a recombinant Listeria having mutations in the D-alanine racemase (Dal) gene and the D-amino acid transferase (Dat) gene, said recombinant Listeria comprising a nucleic acid encoding a first and a second open reading frame, wherein said first open reading frame encodes a recombinant polypeptide comprising SEQ ID NO: 2 fused to an additional polypeptide, and wherein said second open reading frame encodes a metabolic enzyme that complements said mutations, wherein said Her-2/neu-expressing tumor growth or cancer is osteosarcoma, and wherein said recombinant Listeria lacks the actA virulence gene. 20. The method of claim 19, wherein said non-human animal is a dog. 21. The method of claim 19, wherein administering said fusion polypeptide to a subject having said osteosarcoma prevents escape mutations within said tumor or cancer. 22. The method of claim 19, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes. 23. The method of claim 19, wherein said nucleic acid molecule is integrated into the Listeria genome. 24. The method of claim 19, wherein said nucleic acid molecule is in a plasmid in said recombinant Listeria strain. 25. The method of claim 24, wherein said plasmid is stably maintained in said recombinant Listeria strain in the absence of antibiotic selection. 26. The method of claim 24, wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria. 27. The method of claim 19, wherein said recombinant Listeria strain is attenuated. 28. The method of claim 19, wherein said additional polypeptide is selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment. 29. The method of claim 19, wherein said metabolic enzyme encoded by said second open reading frame is an amino acid metabolism enzyme. 30. The method of claim 29, wherein said amino acid metabolism enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme. 31. The method of claim 19, wherein said nucleic acid molecule further comprises a third open reading frame. 32. The method claim 31, wherein said third open reading frame encodes a metabolic enzyme, wherein said metabolic enzyme is a D-amino acid transferase enzyme or an alanine racemase enzyme. 33. The method of claim 19, wherein said recombinant Listeria strain has been passaged through an animal host. 34. The method of claim 19, further comprising an independent adjuvant. 35. The method of claim 34, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide. 36. The method of claim 19, wherein said osteosarcoma cancer is a canine osteosarcoma. 37. A method of eliciting an enhanced immune response against a Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering to said non-human animal a recombinant Listeria having mutations in the D-alanine racemase (Dal) gene and the D-amino acid transferase (Dat) gene, said recombinant Listeria comprising a nucleic acid encoding a first and a second open reading frame, wherein said first open reading frame encodes a recombinant polypeptide comprising SEQ ID NO: 2 fused to an additional polypeptide, and wherein said second open reading frame encodes a metabolic enzyme that complements said mutations, wherein said Her-2/neu-expressing tumor growth or cancer is osteosarcoma, and wherein said recombinant Listeria lacks the actA virulence gene. 38. The method of claim 37, wherein said non-human animal is a dog. 39. The method of claim 37, wherein administering said fusion polypeptide to a subject having said osteosarcoma prevents escape mutations within said tumor or cancer. 40. The method of claim 37, wherein said Her-2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes. 41. The method of claim 37, wherein said nucleic acid molecule is integrated into the Listeria genome. 42. The method of claim 37, wherein said nucleic acid molecule is in a plasmid in said recombinant Listeria strain. 43. The method of claim 42, wherein said plasmid is stably maintained in said recombinant Listeria strain in the absence of antibiotic selection. 44. The method of claim 42, wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria. 45. The method of claim 37, wherein said recombinant Listeria strain is attenuated. 46. The method of claim 37, wherein said additional polypeptide is selected from the group consisting of: a) non-hemolytic LLO protein or N-terminal fragment, b) a PEST sequence, or c) an ActA fragment. 47. The method of claim 37, wherein said metabolic enzyme encoded by said second open reading frame is an amino acid metabolism enzyme. 48. The method of claim 47, wherein said amino acid metabolism enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme. 49. The method of claim 37, wherein said nucleic acid molecule further comprises a third open reading frame. 50. The method of claim 49, wherein said third open reading frame encodes a metabolic enzyme, wherein said metabolic enzyme is a D-amino acid transferase enzyme or an alanine racemase enzyme. 51. The method of claim 37, wherein said recombinant Listeria strain has been passaged through an animal host. 52. The method of claim 37, further comprising an independent adjuvant. 53. The method of claim 52, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide. 54. The method of claim 37, wherein said osteosarcoma cancer is a canine osteosarcoma. 55. The method of any one of claim 1, 19 or 37, wherein said immune response against said Her-2/neu-expressing tumor comprises an immune response to a subdominant epitope of said Her-2/neu protein. 56. The method of claim 1, wherein said mutations are deletions in the D-alanine racemase and D-amino acid transferase (dal/dat) genes. 57. The method of claim 19, wherein said mutations are deletions in the D-alanine racemase and D-amino acid transferase (dal/dat) genes. 58. The method of claim 37, wherein said mutations are deletions in the D-alanine racemase and D-amino acid transferase (dal/dat) genes. 59. The method of claim 1, wherein said Listeria expresses said recombinant polypeptide. 60. The method of claim 19, wherein said Listeria expresses said recombinant polypeptide. 61. The method of claim 37, wherein said Listeria expresses said recombinant polypeptide.