L-asparaginase variants and fusion proteins with reduced L-glutaminase activity and enhanced stability
Variant Erwinia chrysanthemi L-asparaginases with reduced L-glutaminase activity and enhanced in vivo circulation are described as are fusion proteins containing an L-asparaginase and three tandem soluble domains of TRAIL for use in the treatment of cancers such as acute lymphoblastic leukemia and acute myeloid leukemia.
1. An Erwinia chrysanthemi L-asparaginase (ErA) variant comprising an amino acid substitution at (i) position 31 of SEQ ID NO:1, and (ii) position 63 or 254 of SEQ ID NO:1.
2. The ErA variant of claim 1 , wherein the amino acid substitution at position 31 comprises an isoleucine, valine, leucine, or threonine.
3. The ErA variant of claim 1 , wherein the amino acid substitution at position 63 comprises a glutamine, asparagine, or aspartate.
4. The ErA variant of claim 1 , wherein the amino acid substitution at position 254 comprises an asparagine or glutamine.
5. The ErA variant of claim 1 comprising an amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7.
6. The ErA variant of claim 1 , wherein said ErA variant exhibits an asparaginase reaction rate (k cat ) of at least 75% of wild-type ErA enzyme.
7. The ErA variant of claim 1 , wherein said ErA variant exhibits a Km for L-asparagine of less than 250 μM.
8. The ErA variant of claim 1 , wherein said ErA variant exhibits a glutaminase reaction rate (k cat ) of less than 60% of wild-type ErA enzyme.
9. The ErA variant of claim 1 , wherein said ErA variant exhibits a Km for L-glutamine of greater than 3 mM.
10. The ErA variant of claim 1 , further comprising a histidine tag, a SUMO tag, an albumin-binding domain, or a combination thereof.
11. The ErA variant of claim 1 , wherein said ErA variant is PEGylated.
12. A pharmaceutical composition comprising the ErA variant of claim 1 and a pharmaceutically acceptable excipient.
13. The pharmaceutical composition of claim 12 , further comprising a stable form of TRAIL.
14. The pharmaceutical composition of claim 13 , wherein the stable form of TRAIL comprises the FOLDON sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:31).
15. A method of treating cancer comprising administering to a subject in need of treatment an effective amount of the ErA variant of claim 1 thereby treating the subject's cancer.
16. The method of claim 15 , wherein the cancer is selected from non-Hodgkin's lymphoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.
17. The method of claim 15 , further comprising administering a stable form of TRAIL.
18. The method of claim 17 , wherein the stable form of TRAIL comprises the FOLDON sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:31).
19. A nucleic acid molecule encoding the ErA variant of claim 1 .
20. An expression vector comprising the nucleic acid molecule of claim 19 .
21. A host cell comprising the nucleic acid molecule of claim 19 .
22. An Erwinia chrysanthemi L-asparaginase (ErA) variant comprising an amino acid substitution at one or more of positions 31, 63 and 254 of SEQ ID NO:1, wherein the ErA variant is PEGylated and further comprises a cysteine residue at position 72, 76, 79, 84, 85, 206, 210, 215, 216, 235, 239, 240, 261, 264, 265, 268, 269, 318, 322, or a combination thereof.
23. The ErA variant of claim 22 , wherein the amino acid substitution at position 31 comprises an isoleucine, valine, leucine, or threonine.
24. The ErA variant of claim 22 , wherein the amino acid substitution at position 63 comprises a glutamine, asparagine, or aspartate.
25. The ErA variant of claim 22 , wherein the amino acid substitution at position 254 comprises an asparagine or glutamine.
26. The ErA variant of claim 22 , further comprising a histidine tag, a SUMO tag, an albumin-binding domain, or a combination thereof.
27. A pharmaceutical composition comprising the ErA variant of claim 22 and a pharmaceutically acceptable excipient.
28. The pharmaceutical composition of claim 27 , further comprising a stable form of TRAIL.
29. The pharmaceutical composition of claim 28 , wherein the stable form of TRAIL comprises the FOLDON sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:31).
30. A method of treating cancer comprising administering to a subject in need of treatment an effective amount of the ErA variant of claim 22 thereby treating the subject's cancer.
31. The method of claim 30 , wherein the cancer is selected from non-Hodgkin's lymphoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.
32. The method of claim 30 , further comprising administering a stable form of TRAIL.
33. The method of claim 32 , wherein the stable form of TRAIL comprises the FOLDON sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:31).
34. A nucleic acid molecule encoding the ErA variant of claim 22 .
35. An expression vector comprising the nucleic acid molecule of claim 34 .
36. A host cell comprising the nucleic acid molecule of claim 34 .
37. An Erwinia chrysanthemi L-asparaginase (ErA) variant comprising an amino acid substitution at one or more of positions 31, 63 and 254 of SEQ ID NO:1, further comprising three tandem soluble domains of TRAIL.
38. The ErA variant of claim 37 , wherein the amino acid substitution at position 31 comprises an isoleucine, valine, leucine, or threonine.
39. The ErA variant of claim 37 , wherein the amino acid substitution at position 63 comprises a glutamine, asparagine, or aspartate.
40. The ErA variant of claim 37 , wherein the amino acid substitution at position 254 comprises an asparagine or glutamine.
41. The ErA variant of claim 37 , wherein the soluble domains of TRAIL comprise residues 115-281 of SEQ ID NO:16.
42. The ErA variant of claim 37 , wherein the three tandem soluble domains of TRAIL are linked to one another via a peptide linking group comprising one to eight amino acid residues.
43. The ErA variant of claim 42 , wherein the amino acid residues are selected from glycine and serine.
44. The ErA variant of claim 37 , wherein the three tandem soluble domains of TRAIL are linked to the ErA variant via a peptide linking group comprising one to twenty amino acid residues.
45. The ErA variant of claim 44 , wherein the amino acid residues are selected from glycine and serine.
46. The ErA variant of claim 37 , further comprising a histidine tag, a SUMO tag, an albumin-binding domain, or a combination thereof.
47. A pharmaceutical composition comprising the ErA variant of claim 37 and a pharmaceutically acceptable excipient.
48. A method of treating cancer comprising administering to a subject in need of treatment an effective amount of the ErA variant of claim 37 thereby treating the subject's cancer.
49. The method of claim 48 , wherein the cancer is selected from non-Hodgkin's lymphoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.
50. A nucleic acid molecule encoding the ErA variant of claim 37 .
51. An expression vector comprising the nucleic acid molecule of claim 50 .
52. A host cell comprising the nucleic acid molecule of claim 50 .
53. A fusion protein comprising an L-asparaginase linked to three tandem soluble domains of TRAIL.
54. The fusion protein of claim 53 , wherein the L-asparaginase is an L-asparaginase from Erwinia chrysanthemi or Escherichia coli.
55. The fusion protein of claim 54 , wherein the Erwinia chrysanthemi L-asparaginase is a variant comprising an amino acid substitution at one or more of positions 31, 63 and 254 of SEQ ID NO:1.
56. The fusion protein of claim 53 , wherein the soluble domains of TRAIL comprise residues 115-281 of SEQ ID NO:16.
57. The fusion protein of claim 53 , wherein the three tandem soluble domains of TRAIL are linked to one another via a peptide linking group comprising one to eight amino acid residues.
58. The fusion protein of claim 57 , wherein the amino acid residues are selected from glycine and serine.
59. The fusion protein of claim 53 , wherein the three tandem soluble domains of TRAIL are linked to the L-asparaginase via a peptide linking group comprising one to twenty amino acid residues.
60. The fusion protein of claim 59 , wherein the amino acid residues are selected from glycine and serine.
61. The fusion protein of claim 53 , further comprising a histidine tag, a SUMO tag, an albumin-binding domain, or a combination thereof.
62. The fusion protein of claim 53 , wherein said fusion protein is PEGylated.
63. The fusion protein of claim 62 , wherein the L-asparaginase is an L-asparaginase from Erwinia chrysanthemi comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, wherein said L-asparaginase has a cysteine residue at position 72, 76, 79, 84, 85, 206, 210, 215, 216, 235, 239, 240, 261, 264, 265, 268, 269, 318, 322, or a combination thereof.
64. A pharmaceutical composition comprising the fusion protein of claim 53 and a pharmaceutically acceptable excipient.
65. A method of treating cancer comprising administering to a subject in need of treatment an effective amount of the fusion protein of claim 53 thereby treating the subject's cancer.
66. The method of claim 65 , wherein the cancer is selected from non-Hodgkin's lymphoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.
67. A nucleic acid molecule encoding the fusion protein of claim 53 .
68. An expression vector comprising the nucleic acid molecule of claim 67 .
69. A host cell comprising the nucleic acid molecule of claim 67 .