Therapeutic nuclease-albumin fusions and methods
The invention provides for hybrid nuclease-albumin molecules with increased pharmacokinetic properties. The hybrid nuclease-albumin molecules of the invention have one or more nuclease domains (e.g., an RNase and/or DNase domain) operably coupled to an albumin, or a variant or fragment thereof. The invention also provides methods of treating or preventing a condition associated with an abnormal immune response.
1. A fusion protein comprising:
a mutant human DNase comprising one or more mutations selected from the group consisting of E13R, N74K, A114F and T205K, wherein the mutation numbering corresponds to SEQ ID NO: 66, operably coupled, with or without a linker, to
a human serum albumin or a human serum albumin variant, wherein the human serum albumin variant has greater than 90% sequence identity to the amino acid sequence of human serum albumin set forth in SEQ ID NO: 1, and wherein the human serum albumin or human serum albumin variant is operably coupled, with or without a linker, to
a human RNase; and
wherein the fusion protein has increased serum half-life relative to a fusion protein comprising the human mutant DNase and the human RNase without the human serum albumin or human serum albumin variant.
2. The fusion protein of claim 1 , wherein the mutant human DNase is operably coupled to the N-terminus of the human serum albumin, or human serum albumin variant, and the human RNase is operably coupled to the C-terminus of the human serum albumin, or human serum albumin variant.
3. The fusion protein of claim 2 , wherein the mutant human DNase and human RNase are operably coupled to the N- and C-terminus, respectively, of the human serum albumin, or human serum albumin variant, via a linker.
4. The fusion protein of claim 1 , wherein the human RNase is a wild type human RNase.
5. The fusion protein of claim 3 , wherein the linker is a polypeptide linker.
6. The fusion protein of claim 1 , wherein the fusion protein is selected from the group consisting of: (i) a fusion protein comprising an amino acid sequence set forth in SEQ ID NO: 112-114 or 120-122, or (ii) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 112-114 or 120-122 and having increased serum half-life relative to a fusion protein comprising a human mutant DNase and a human RNase without a human serum albumin or a human serum albumin variant.
7. The fusion protein of claim 1 , wherein the mutant human DNase is operably coupled to the C-terminus of the human serum albumin, or human serum albumin variant, and the human RNase is operably coupled to the N-terminus of the human serum albumin, or human serum albumin variant.
8. The fusion protein of claim 7 , wherein the mutant human DNase and human RNase are operably coupled to the C- and N-terminus, respectively, of the human serum albumin, or human serum albumin variant, via a linker.
9. The fusion protein of claim 1 , wherein the fusion protein comprises a first linker, a second linker, or both, wherein when the fusion protein comprises a first linker, the mutant human DNase is operably coupled to the human serum albumin, or human serum albumin variant, by the first linker, and wherein when the fusion protein comprises a second linker, the human RNase is operably coupled to the human serum albumin, or human serum albumin variant, by the second linker.
10. The fusion protein of claim 9 , wherein the fusion protein comprises a first linker and a second linker.
11. The fusion protein of claim 9 , wherein the first linker, the second linker, or both, comprise a polypeptide linker.
12. The fusion protein of claim 11 , wherein the polypeptide linker of the first linker, the second linker, or both, is a gly-ser linker.
13. The fusion protein of claim 5 , wherein the polypeptide linker is a gly-ser linker.
14. The fusion protein of claim 1 , further comprising a leader sequence.
15. The fusion protein of claim 14 , wherein the leader sequence is a VK3LP peptide, and wherein the leader sequence is coupled to the N-terminus of the mutant human DNase or the N-terminus of the human RNase.
16. The fusion protein of claim 1 , wherein the mutant human DNase comprises the amino acid sequence set forth in SEQ ID NO: 108 or an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 108.
17. The fusion protein of claim 1 , wherein the RNase comprises the amino acid sequence set forth in SEQ ID NO: 75.
18. The fusion protein of claim 1 , wherein the human serum albumin comprises the amino acid sequence set forth in SEQ ID NO: 1.
19. The fusion protein of claim 14 , wherein the leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 86.
20. The fusion protein of claim 1 , wherein the fusion protein is selected from the group consisting of: (i) a fusion protein comprising an amino acid sequence set forth in SEQ ID NO: 113, or (ii) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 113 and having increased serum half-life relative to a fusion protein comprising a human mutant DNase and a human RNase without a human serum albumin or a human serum albumin variant.
21. The fusion protein of claim 1 , wherein the fusion protein is selected from the group consisting of: (i) a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 121, or (ii) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 121 and having increased serum half-life relative to a fusion protein comprising a human mutant DNase and a human RNase without a human serum albumin or a human serum albumin variant.
22. A composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.
23. A composition comprising the fusion protein of claim 6 and a pharmaceutically acceptable carrier.
24. A composition comprising the fusion protein of claim 20 and a pharmaceutically acceptable carrier.
25. A composition comprising the fusion protein of claim 21 and a pharmaceutically acceptable carrier.
26. The fusion protein of claim 1 , wherein the variant of human serum albumin comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1.
27. The fusion protein of claim 1 , which degrades circulating RNA and/or DNA and RNA and/or DNA in immune complexes, or inhibits interferon-α production, or both.
28. The fusion protein of claim 1 , wherein the variant of the human serum albumin binds FcRn with a higher affinity than that for a corresponding wild type human serum albumin.
29. A fusion protein comprising:
(i) a DNase comprising one or more mutations selected from E13R, N74K, A114F, and T205K, wherein the mutation numbering corresponds to SEQ ID NO: 66, operably coupled, with or without a linker, to
(ii) a human serum albumin, operably coupled, with or without a linker, to
(iii) a human RNase; and
wherein the fusion protein has increased serum half-life relative to a fusion protein comprising the human mutant DNase and the human RNase without the human serum albumin.
30. A fusion protein comprising:
(i) a DNase comprising one or more mutations selected from E13R, N74K, A114F, and T205K, wherein the mutation numbering corresponds to SEQ ID NO: 66, operably coupled, with a first linker, to
(ii) a human serum albumin, operably coupled, with a second linker domain, to
(iii) a human RNase; and
wherein the fusion protein has increased serum half-life relative to a fusion protein comprising the human mutant DNase and the human RNase without the human serum albumin.
31. The fusion protein of claim 30 , wherein the first linker, the second linker, or both, comprise a polypeptide linker.
32. The fusion protein of claim 31 , wherein the polypeptide linker of the first linker, the second linker, or both, is a gly-ser linker.
33. The fusion protein of claim 31 , wherein the polypeptide linker of the first linker and the second linker is a gly-ser linker.
34. The fusion protein of claim 31 , wherein the polypeptide linker of the first linker, the second linker, or both is an NLG linker.
35. The fusion protein of claim 31 , wherein the first linker is an NLG linker, and wherein the second linker is a gly-ser linker.
36. The fusion protein of claim 32 , wherein the gly-ser linker is (Gly 4 Ser) 3 .
37. The fusion protein of claim 33 , wherein the gly-ser linker is (Gly 4 Ser) 3 .
38. The fusion protein of claim 1 , wherein the mutant human DNase comprises the mutations E13R, N74K, A114F, and T205K, wherein the mutation numbering corresponds to SEQ ID NO: 66.
39. The fusion protein of claim 30 , wherein the mutant human DNase comprises the mutations E13R, N74K, A114F, and T205K, wherein the mutation numbering corresponds to SEQ ID NO: 66.
40. The fusion protein of claim 1 , wherein the human serum albumin comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1.
41. The fusion protein of claim 1 , wherein the human serum albumin comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1.
42. The fusion protein of claim 1 , wherein the fusion protein is selected from the group consisting of: (i) a fusion protein comprising an amino acid sequence set forth in SEQ ID NO: 112, or (ii) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 112 and having increased serum half-life relative to a fusion protein comprising a human mutant DNase and a human RNase without a human serum albumin or a human serum albumin variant.
43. The fusion protein of claim 1 , wherein the fusion protein is selected from the group consisting of: (i) a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 120, or (ii) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 120 and having increased serum half-life relative to a fusion protein comprising a human mutant DNase and a human RNase without a human serum albumin or a human serum albumin variant.
44. The fusion protein of claim 1 , wherein the fusion protein is selected from the group consisting of: (i) a fusion protein comprising an amino acid sequence set forth in SEQ ID NO: 114, or (ii) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 114 and having increased serum half-life relative to a fusion protein comprising a human mutant DNase and a human RNase without a human serum albumin or a human serum albumin variant.
45. The fusion protein of claim 1 , wherein the fusion protein is selected from the group consisting of: (i) a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 122, or (ii) a fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 122 and having increased serum half-life relative to a fusion protein comprising a human mutant DNase and a human RNase without a human serum albumin or a human serum albumin variant.
46. A composition comprising the fusion protein of claim 29 and a pharmaceutically acceptable carrier.
47. A composition comprising the fusion protein of claim 30 and a pharmaceutically acceptable carrier.
48. A composition comprising the fusion protein of claim 33 and a pharmaceutically acceptable carrier.
49. A composition comprising the fusion protein of claim 37 and a pharmaceutically acceptable carrier.
50. A composition comprising the fusion protein of claim 39 and a pharmaceutically acceptable carrier.
51. A composition comprising the fusion protein of claim 42 and a pharmaceutically acceptable carrier.
52. A composition comprising the fusion protein of claim 43 and a pharmaceutically acceptable carrier.
53. A composition comprising the fusion protein of claim 44 and a pharmaceutically acceptable carrier.
54. A composition comprising the fusion protein of claim 45 and a pharmaceutically acceptable carrier.