Compositions comprising variant LT-B-R-IG fusion proteins

1. A composition comprising a population of lymphotoxin-β receptor (LT-β-R)-Ig-fusion proteins which comprise a variant LT-β-R extracellular domain of 193 or 194 amino acids in length and a variant Ig portion of 227 amino acids in length, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain, which extracellular domain is set forth in SEQ ID NO:21, and wherein the LT-β-R-Ig-fusion proteins lack N-terminal pyroglutamic acid.

2. The composition of claim 1 , wherein the N-terminal amino acid of the variant LT-β-R-Ig fusion protein is a non-polar amino acid.

3. The composition of claim 2 , wherein the non polar amino acid is either a valine, which corresponds to amino acid six of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21 or an alanine, which corresponds to amino acid five of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21.

4. The composition of claim 1 , wherein the N-terminal amino acid of at least 95% of the LT-β-R-Ig-fusion proteins is either a valine, which corresponds to amino acid six of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21or an alanine, which corresponds to amino acid five of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21.

5. The composition of any one of claim 1 , 3 and 4 , wherein the LT-β-R-Ig fusion proteins are made by expressing a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LT-β-R set forth in SEQ ID NO:4 or SEQ ID NO:23 in a mammalian cell.

6. The composition of claim 5 , wherein variant Ig portion comprises Fc regions of an IgG1 isotype.

7. The composition of claim 5 , wherein the variant Ig portion comprises the amino acid sequence set forth in SEQ ID NO:2.

8. The composition of claim 5 , wherein the Ig portion is non-glycosylated.

9. The composition of claim 5 , wherein the LT-β-R-Ig fusion proteins are made by expressing a nucleic acid molecule encoding the LT-β-R-Ig fusion protein set forth in SEQ ID NO:5 in a mammalian cell.

10. The composition of claim 9 , wherein the nucleic acid molecule comprises the sequence set forth in SEQ ID NO:7.

11. The composition of claim 10 , wherein the step of expressing is done at manufacturing scale.

12. A composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig)-fusion proteins comprising a variant LT-β-R extracellular domain and a variant Ig portion, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain, which extracellular domain is set forth in SEQ ID NO:21, and wherein the variant LT-β-R extracellular domain is aglycosylated.

13. A composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig)-fusion proteins, the fusion proteins comprising a variant LT-β-R extracellular domain of 193 or 194 amino acids in length and a variant Ig portion, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain, which extracellular domain is set forth in SEQ ID NO:21, and wherein the population has reduced N-terminal pyroglutamic acid formation, and reduced C-terminal heterogeneity compared to wild-type LT-β-R-Ig fusion proteins.

14. The composition of claim 13 , wherein at least 90% of the LT-β-R-Ig-fusion proteins comprise a variant LT-β-R extracellular domain as set forth the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 23.

15. The composition of claim 13 , wherein the variant Ig portion comprises a mutation in the hinge region.

16. A pharmaceutical composition comprising a population of lymphotoxin-β receptor (LT-β-R)-Ig-fusion proteins which comprise a variant LT-β-R extracellular domain of 193 or 194 amino acids in length and a variant Ig portion of 227 amino acids in length, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain, which extracellular domain is set forth in SEQ ID NO:21, and wherein the LT-β-R-Ig-fusion proteins lack N-terminal pyroglutamic acid and a pharmaceutically acceptable carrier.

17. The composition of claim 16 , wherein the N-terminal amino acid of the variant LT-β-R-Ig fusion protein is a non-polar amino acid.

18. The composition of claim 17 , wherein the non polar amino acid is either a valine, which corresponds to amino acid six of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21 or an alanine, which corresponds to amino acid five of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21.

19. The composition of claim 16 , wherein the N-terminal amino acid of at least 95% of the LT-β-R-Ig-fusion proteins is either a valine, which corresponds to amino acid six of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:2 or an alanine, which corresponds to amino acid five of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21.

20. The composition of claim 16 , 18 , or 19 , wherein the LT-β-R-Ig fusion proteins are made by expressing a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LT-β-R set forth in SEQ ID NO:4 in a mammalian cell.

21. A method of treating an autoimmune disorder comprising administering the pharmaceutical composition of claim 20 to a subject in need thereof, wherein the autoimmune disorder is rheumatoid arthritis.

22. A pharmaceutical composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig)-fusion proteins, the fusion proteins comprising a variant LT-β-R extracellular domain of 193 or 194 amino acids in length and a variant Ig portion, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain, which extracellular domain is set forth in SEQ ID NO:21, and wherein the population has reduced N-terminal pyroglutamic acid formation and reduced C-terminal heterogeneity compared to wild-type LT-β-R-Ig fusion proteins and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 22 , wherein at least 90% of the LT-β-R-Ig-fusion proteins comprise a variant LT-β-R extracellular domain as set forth the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 23.

24. The pharmaceutical composition of claim 22 , wherein the variant Ig portion comprises a mutation in the hinge region.

25. A pharmaceutical composition, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5.

26. A method of treating an autoimmune disorder comprising administering the pharmaceutical composition of claim 25 to a subject in need thereof, wherein the autoimmune disorder is rheumatoid arthritis.

27. The method of claim 26 , wherein the pharmaceutical composition is administered to the subject at a dose of from about 0.6 to 3 mg/kg biweekly.

28. The method of claim 26 , wherein the pharmaceutical composition is administered subcutaneously.

29. An isolated polypeptide comprising a variant LT-β-R extracellular domain of 193 or 194 amino acids in length and a variant Ig portion of 227 amino acids in length, wherein the polypeptide is missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain, which extracellular domain is set forth in SEQ ID NO:21 and wherein the polypeptide lacks N-terminal pyroglutamic acid.

30. The isolated polypeptide of claim 29 , wherein the N-terminal amino acid is a non-polar amino acid.

31. The isolated polypeptide of claim 30 , wherein the non polar amino acid is either a valine, which corresponds to amino acid six of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21 or an alanine, which corresponds to amino acid five of the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO:21.

32. The isolated polypeptide of claim 29 , which is made by expressing a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LT-β-R set forth in SEQ ID NO:23 SEQ ID NO:4 or in a mammalian cell.

33. An isolated nucleic acid molecule encoding the polypeptide of any one of claims 29 - 32 .

34. The isolated nucleic acid molecule of claim 33 , which nucleic acid molecule comprises of the nucleotide sequence set forth in SEQ ID NO:7.

35. A vector comprising the nucleic acid molecule of claim 34 .

36. An isolated or cultured host cell expressing the vector of claim 35 .

37. The cell of claim 36 , which is a Chinese Hamster Ovary (CHO) cell.

38. A process for making a composition comprising a population of lymphotoxin-β receptor (LT-β-R)-Ig-fusion proteins which comprise a variant LT-β-R extracellular domain and a variant Ig portion, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain set forth in SEQ ID NO:21, the process comprising, expressing a nucleic acid molecule encoding the LT-β-R-Ig fusion protein set forth in SEQ ID NO:8 in a mammalian cell, obtaining the population from the culture supernatant, and, optionally, purifying the supernatant, to thereby obtain a composition comprising a population of lymphotoxin-β receptor (LT-β-R)-Ig-fusion proteins which comprise a variant LT-β-R extracellular domain and a variant Ig portion, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R portion set forth in SEQ ID NO:21.

39. The process of claim 38 , wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:7.

40. The process of claim 38 , wherein the nucleic acid molecule consists of the nucleotide sequence set forth in SEQ ID NO:7.

41. A method of treating rheumatoid arthritis in a human subject, in need thereof, the method comprising administering to the subject a dose of LT-β-R-Ig fusion protein, wherein the dose is sufficient to maintain an average concentration of from about 0.14 ug/ml to about 3.5 ug/ml in the serum of the subject.

42. A method of treating rheumatoid arthritis in a human subject, in need thereof, the method comprising administering to the subject a dose of LT-β-R-Ig fusion protein, wherein the dose is sufficient to maintain an average a minimal average concentration of about 0.6 ug/ml in the serum of the subject.

43. The method of claim 42 , wherein the LT-β-R-Ig fusion protein comprises the amino acid sequence set forth in SEQ ID NO:5.

44. The method of claim 42 , wherein the concentration is achieved by administering LT-β-R-Ig fusion protein at a dose of from about 0.01 to about 5 mg/kg once every 7-60 days.

45. A method of treating rheumatoid arthritis in a human subject, in need thereof, the method comprising administering to the subject a dose of LT-β-R-Ig fusion protein of from about 0.6 to 3 mg/kg not more than twice every 7-30 days.

46. The method of claim 45 , the method comprising administering to the subject a dose of LT-β-R-Ig fusion protein of from about 0.6 to 3 mg/kg once every 7-14 days.

47. The method of claim 45 , wherein administration is once every 14-30 days.

48. The method of claim 45 , wherein administration is once every 28-60 days.

49. The method of claim 45 , wherein administration is once every 7-30 days.

50. A method of treating an autoimmune disorder in a human subject, the method comprising administering to the subject a dose of a pharmaceutical composition comprising a population of LT-β-R-Ig fusion proteins comprising a variant LT-β-R extracellular domain of 193 or 194 amino acids in length, wherein at least 90% of the LT-β-R-Ig-fusion proteins are missing no more than 5 amino acids from the N-terminus of the mature form of the wild type LT-β-R extracellular domain set forth in SEQ ID NO:21 and wherein the dose is sufficient to maintain a minimal average concentration of about 0.6 ug/ml in the serum of the subject, wherein the autoimmune disorder is rheumatoid arthritis.

51. The method of claim 50 , wherein the LT-β-R-Ig fusion protein further comprises a variant Ig portion.

52. The method of claim 50 , wherein the pharmaceutical composition comprises the amino acid sequence set forth in SEQ ID NO:5.

53. The method of claim 52 , wherein administration is twice monthly.

54. The method of claim 52 , wherein administration once monthly.

55. The method of claim 52 , wherein administration is subcutaneous.

56. The method of claim 52 , wherein the dose is about 1 mg/kg.

57. The method of claim 52 , wherein the dose is about 3 mg/kg.

58. The method of claim 52 , wherein the dose is about 1 mg/kg administered about every 7 to 20 days.

59. The method of claim 52 , wherein the dose is about 3 mg/kg administered about every 14 to 30 days.

60. The method of claim 52 , wherein the dose is about 1 mg/kg administered about every 14 days.

61. The method of claim 52 , wherein the subject has been treated with a rheumatoid arthritis drug after being diagnosed with rheumatoid arthritis and prior to administration of the LT-β-R-Ig fusion protein.

62. The method of claim 61 , wherein the rheumatoid arthritis drug is chosen from the group consisting of a DMARD, an NSAID, and a corticosteroid.

63. The method of claim 61 , wherein the human is a DMARD-inadequate responder.

64. The method of claim 61 , wherein the rheumatoid arthritis drug is a TNF inhibitor.

65. The method of claim 61 , wherein the rheumatoid arthritis drug is adalimumab (Humira®), etanercept (Enbrel®), or infliximab (Remicade®).

66. The method of claim 61 , wherein LT-β-R-Ig is administered in combination with the rheumatoid arthritis drug.

67. The method of claim 61 , wherein the human is evaluated to determine if the response to the rheumatoid arthritis drug is inadequate prior to administration of LT-β-R-Ig.

68. The method of claim 67 , wherein the human is determined to have an inadequate response to the rheumatoid arthritis drug, and then the human is administered LT-β-R-Ig.

69. The method of claim 61 , wherein the human is asymptomatic for a first manifestation of rheumatoid arthritis and is symptomatic for a second manifestation of rheumatoid arthritis.

70. The method of claim 61 , wherein LT-β-R-Ig is administered in place of the rheumatoid arthritis drug.

71. The method of claim 61 , wherein administration is in combination with a tumor necrosis factor (TNF) inhibitor.

72. The method of claim 71 , wherein the TNF inhibitor is adalimumab (Humira®), etanercept (Enbrel®), or infliximab (Remicade®).

73. The method of claim 61 , wherein the human is an anti-TNF-inadequate responder.

74. The method of claim 61 , wherein administration is in combination with a non-steroidal anti-inflammatory agent (NSAID), a corticosteroid, or a disease modifying antirheumatic drug (DMARD).

75. The method of claim 61 , wherein administration is in combination with methotrexate.

76. The method of claim 74 , wherein the human is a DMARD-inadequate responder.

77. The isolated polypeptide of claim 29 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.