Use of specifically engineered enzymes to enhance the efficacy of prodrugs
View Patent ↗The invention provides methods for enhancing efficiency of prodrugs by specifically engineered enzymes with altered or enhanced activity and broader substrate specificity towards nucleoside analogs used in cancer chemotherapy, and delivering the enzymes to specific target cells in a patient. The invention also provides modified deoxycytidine kinase (dCK) mutants with such enhanced activities. Furthermore, the invention provides antibody-conjugated enzymes, pharmaceutical composition and kit containing the same, that can be specifically delivered to tumor cells.
1. A modified human deoxycytidine kinase having amino acid substitutions at amino acid positions 104 and 133, optionally and in addition at amino acid position 74, and optionally and in addition at amino acid position 100, wherein the substitution at amino acid position 104 is glutamine, leucine, or isoleucine, and the substitution at amino acid position 133 is alanine, and wherein the amino acids are identified according to the numbering of wild type human deoxycytidine kinase identified by SEQ ID NO:1.
2. The modified human deoxycytidine kinase of claim 1 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.
3. The modified human deoxycytidine kinase of claim 1 , wherein the substitution at amino acid position 104 is leucine and the substitution at amino acid position 133 is alanine.
4. The modified human deoxycytidine kinase of claim 3 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:10.
5. The modified human deoxycytidine kinase of claim 1 , further comprising a substitution at amino acid position 100.
6. The modified human deoxycytidine kinase of claim 5 wherein the substitution at amino acid position 100 is valine.
7. The modified human deoxycytidine kinase of claim 6 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.
8. The modified human deoxycytidine kinase of claim 6 wherein the substitution at amino acid position 104 is leucine.
9. The modified human deoxycytidine kinase of claim 8 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:9.
10. The modified human deoxycytidine kinase of claim 1 , further comprising a substitution at amino acid position 74, wherein the substitution at amino acid position 74 is glutamic acid.
11. The modified human deoxycytidine kinase of claim 10 wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18.
12. The modified human deoxycytidine kinase of claim 10 wherein the substitution at amino acid position 104 is leucine.
13. The modified human deoxycytidine kinase of claim 12 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:16.
14. The modified human deoxycytidine kinase of claim 10 , further comprising a substitution at amino acid position 100, wherein the substitution at amino acid 100 is valine.
15. The modified human deoxycytidine kinase of claim 14 wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17.
16. The modified human deoxycytidine kinase of claim 14 wherein the substitution at amino acid position 104 is leucine.
17. The modified human deoxycytidine kinase of claim 16 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:15.
18. The modified human deoxycytidine kinase of claim 1 , wherein said modified human deoxycytidine kinase phosphorylates a D-type or L-type thymidine analog.
19. The modified human deoxycytidine kinase of claim 18 , wherein the thymidine analog is (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU).
20. A modified human deoxycytidine kinase having amino acid substitutions at amino acid positions 74, 104 and 133, and optionally and in addition at amino acid position 100, wherein the substitution at amino acid position 74 is glutamic acid, the substitution at amino acid position 104 is methionine, and the substitution at amino acid position 133 is alanine, and wherein the amino acids are identified according to the numbering of wild type human deoxycytidine kinase identified by SEQ ID NO:1.
21. The modified human deoxycytidine kinase of claim 20 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:20.
22. The modified human deoxycytidine kinase of claim 20 , further comprising a substitution at amino acid position 100, wherein the substitution at amino acid position 100 is valine.
23. The modified human deoxycytidine kinase of claim 22 , wherein the modified human deoxycytidine kinase has an amino acid sequence identified by SEQ ID NO:19.
24. The modified human deoxycytidine kinase of claim 20 , wherein said modified human deoxycytidine kinase phosphorylates a D-type or L-type thymidine analog.
25. The modified human deoxycytidine kinase of claim 24 , wherein the thymidine analog is (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU).
26. A conjugate comprising an antibody and a modified human deoxycytidine kinase of claim 1 , wherein the antibody recognizes a cell surface antigen.
27. The conjugate of claim 26 wherein the antibody is Trastuzumab or HuM195.
28. The conjugate of claim 26 wherein the modified human deoxycytidine kinase has the sequence identified by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
29. The conjugate of claim 28 , wherein the modified human deoxycytidine kinase has the sequence identified by SEQ ID NO:15 or SEQ ID NO:16.
30. A pharmaceutical composition comprising the conjugate of claim 26 , and a pharmaceutically acceptable carrier.
31. A kit for inhibiting or reducing proliferation of tumor cells comprising the conjugate of claim 26 in at least one container.
32. The kit of claim 31 further comprising a prodrug.
33. The kit of claim 32 , wherein the prodrug is a D-type or L-type thymidine analog.
34. The kit of claim 33 wherein the thymidine analog is (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU).
35. A conjugate comprising an antibody and a modified human deoxycytidine kinase of claim 20 , wherein the antibody recognizes a cell surface antigen.
36. The conjugate of claim 35 wherein the antibody is Trastuzumab or HuM195.
37. The conjugate of claim 35 wherein the modified human deoxycytidine kinase has the sequence identified by SEQ ID NO:19 or SEQ ID NO:20.
38. The conjugate of claim 37 , wherein the modified human deoxycytidine kinase has the sequence identified by SEQ ID NO:20.
39. A pharmaceutical composition comprising the conjugate of claim 35 , and a pharmaceutically acceptable carrier.
40. A kit for inhibiting or reducing proliferation of tumor cells comprising the conjugate of claim 35 and at least one container.
41. The kit of claim 40 further comprising a prodrug.
42. The kit of claim 41 , wherein the prodrug is a D-type or L-type thymidine analog.
43. The kit of claim 42 wherein the thymidine analog is (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU).
44. An isolated polynucleotide comprising a nucleotide sequence encoding the modified human deoxycytidine kinase of claim 1 .
45. The isolated polynucleotide of claim 44 wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
46. An expression vector comprising the isolated polynucleotide of claim 44 wherein the expression vector is capable of expressing the modified human deoxycytidine kinase.
47. A host cell comprising the expression vector of claim 46 .
48. A method for producing a modified human deoxycytidine kinase polypeptide comprising steps of:
a. culturing the host cell of claim 47 under conditions effective to allow expression of the modified human deoxycytidine kinase polypeptide; and
b. recovering the modified human deoxycytidine kinase polypeptide from the cell culture.
49. An isolated polynucleotide comprising a nucleotide sequence encoding the modified human deoxycytidine kinase of claim 20 .
50. The isolated polynucleotide of claim 49 wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:19 or SEQ ID NO:20.
51. An expression vector comprising the isolated polynucleotide of claim 49 wherein the expression vector is capable of expressing the modified human deoxycytidine kinase.
52. A host cell comprising the expression vector of claim 51 .
53. A method for producing a modified human deoxycytidine kinase polypeptide comprising steps of:
a. culturing the host cell of claim 52 under conditions effective to allow the expression of the modified human deoxycytidine kinase polypeptide; and
b. recovering the modified human deoxycytidine kinase polypeptide from the cell culture.
54. A method of reducing or inhibiting proliferating of a tumor cell, comprising the step of contacting the tumor cell with a prodrug and the conjugate of claim 26 , wherein the conjugate comprises an antibody and a modified human deoxycytidine kinase that converts the prodrug to a therapeutically active drug.
55. The method of claim 54 , wherein the antibody is Trastuzumab or HuM195.
56. The method of claim 54 , wherein the prodrug is a D-type or L-type thymidine analog.
57. The method of claim 56 , wherein the thymidine analog is (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU).
58. The method of claims 54 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
59. The method of claim 58 wherein the modified human deoxycytidine kinase has an amino acid sequence identified by SEQ ID NO:15.
60. The method of claim 54 wherein the tumor cell is a breast tumor cell or a leukemia cell.
61. A method of reducing or inhibiting proliferating of a tumor cell, comprising a step of contacting the tumor cell with a prodrug and the conjugate of claim 35 , wherein the conjugate comprises an antibody and a modified human deoxycytidine kinase that converts the prodrug to a therapeutically active drug.
62. The method of claim 61 , wherein the antibody is Trastuzumab or HuM195.
63. The method of claim 61 , wherein the prodrug is a D-type or L-type thymidine analog.
64. The method of claim 63 , wherein the thymidine analog is (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU).
65. The method of claims 61 , wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:19 or SEQ ID NO:20.
66. The method of claim 65 wherein the modified human deoxycytidine kinase has the amino acid sequence identified by SEQ ID NO:20.
67. The method of claim 61 wherein the tumor cell is a breast tumor cell or a leukemia cell.