Compositions and methods for prevention of escape mutation in the treatment of Her2/neu over-expressing tumors

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.