Tumor specific delivery of therapeutic agents via liposomase
Clostridium novyi is an obligate anaerobe that can infect hypoxic regions within experimental tumors. We found that mice bearing large, established tumors were often cured when treated with C. novyi plus a single dose of liposomal doxorubicin. The secreted factor responsible for this phenomenon was identified and, surprisingly, proved to be a member of the lipase family. The gene encoding this protein, called liposomase, has the potential to be incorporated into diverse therapeutic methods to deliver specifically a variety of chemotherapeutic agents to tumors.
1. A composition comprising:
toxin-defective Clostridium novyi spores; and
a liposome comprising an anti-tumor drug or biological agent.
2. A composition comprising:
a toxin-defective Clostridium novyi liposomase according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif at residues 160-164; and
a liposome comprising an anti-tumor drug or biological agent.
3. The composition of claim 1 or 2 wherein the composition is formed in vivo in a non-human mammal.
4. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is doxorubicin.
5. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is irinotecan.
6. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is an antibody.
7. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is a polynucleotide.
8. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is a polynucleotide in a viral vector.
9. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is a polynucleotide in a non-viral vector.
10. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is a protein.
11. The composition of claim 1 or 2 wherein the anti-tumor drug or biological agent is a cytokine.
12. A kit comprising:
a toxin-defective Clostridium novyi liposomase according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif at residues 160-164; and
a liposome comprising an anti-tumor drug or biological agent.
13. A kit comprising:
toxin-defective Clostridium novyi spores; and
a liposome comprising an anti-tumor drug or biological agent.
14. The kit of claim 12 or 13 wherein the anti-tumor drug or biological agent is doxorubicin.
15. The kit of claim 12 or 13 wherein the anti-tumor drug or biological agent is irinotecan.
16. The kit of claim 12 or 13 wherein the anti-tumor drug or biological agent is an antibody.
17. The kit of claim 12 or 13 wherein the anti-tumor drug or biological agent is a polynucleotide.
18. The kit of claim 12 or 13 wherein the anti-tumor drug or biological agent is a polynucleotide in a viral vector.
19. The kit of claim 12 or 13 wherein the anti-tumor drug or biological agent is a polynucleotide in a non-viral vector.
20. A method of treating a tumor bearing mammal, comprising:
administering to the tumor bearing mammal a first agent which is toxin-defective Clostridium novyi spores and a second agent which is a liposome comprising an anti-tumor drug or biological agent, whereby the tumor regresses or its growth is slowed or arrested.
21. A method of treating a tumor bearing mammal, comprising:
administering to the tumor bearing mammal a first agent which is a toxin-defective Clostridium novyi liposomase according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif at residues 160-164 and a second agent which is a liposome comprising an anti-tumor drug or biological agent, whereby the tumor regresses or its growth is slowed or arrested.
22. The method of claim 20 or 21 wherein the first agent and the second agent are administered sequentially.
23. The method of claim 20 or 21 wherein the first agent and the second agent are administered simultaneously.
24. The method of claim 20 or 21 wherein the anti-tumor drug or biological agent is doxorubicin.
25. The method of claim 20 or 21 wherein the anti-tumor drug or biological agent is irinotecan.
26. The method of claim 20 or 21 wherein the anti-tumor drug or biological agent is an antibody.
27. The method of claim 20 or 21 wherein the anti-tumor drug or biological agent is a polynucleotide.
28. The method of claim 20 or 21 wherein the anti-tumor drug or biological agent is a polynucleotide in a viral vector.
29. The method of claim 20 or 21 wherein the anti-tumor drug or biological agent is a polynucleotide in a non-viral vector.
30. A composition comprising an isolated and purified toxin-defective Clostridium novyi liposomase protein according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif at residues 160-164.
31. A conjugate protein comprising:
a toxin-defective Clostridium novyi liposomase protein according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif at residues 160-164; and
a polypeptide ligand which binds to a receptor on a tumor cell.
32. The conjugate protein of claim 31 wherein the polypeptide ligand is a variable region of a heavy or light chain of an antibody.
33. The conjugate protein of claim 31 further comprising a linker peptide between the liposomase protein and the polypeptide ligand.
34. The conjugate protein of claim 31 wherein the polypeptide ligand comprises a variable region of a heavy chain and a variable region of a light chain of an antibody.
35. The conjugate protein of claim 34 which comprises a linker peptide between the variable region of the heavy chain and the variable region of the light chain.
36. The conjugate protein of claim 31 which is post-translationally conjugated.
37. The conjugate protein of claim 31 which is translated as a single polypeptide chain.
38. A polynucleotide which encodes the conjugate protein of claim 37 .
39. A composition comprising an isolated and purified polynucleotide encoding a toxin-defective Clostridium novyi liposomase protein according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif at residues 160-164.
40. The composition of claim 39 wherein the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 2.
41. The composition of claim 39 wherein the polynucleotide comprises a vector.
42. The composition of claim 39 wherein the polynucleotide is in a viral vector.
43. The composition of claim 39 wherein the polynucleotide comprises a non-viral vector.
44. The composition of claim 39 wherein the polynucleotide comprises a promoter which is at least two-fold more transcriptionally active in a tumor than in a normal tissue.
45. The method of claim 20 wherein the tumor bearing mammal is a human.
46. The method of claim 20 wherein the tumor bearing mammal is a pet.
47. The method of claim 20 wherein the tumor bearing mammal is an agricultural animal.
48. The method of claim 20 wherein the tumor bearing mammal is a laboratory animal.
49. The method of claim 46 wherein the pet is a dog.
50. The method of claim 1 wherein the anti-tumor drug or biological agent is a topoisomerase inhibitor.
51. The method of claim 1 wherein the anti-tumor drug or biological agent is a DNA synthesis inhibitor.