Anti-CD40 Antibody Combination Treatment for Cancer

Abstract

This disclosure relates to methods of treating cancer using a combination of an anti-CD40 antibody such as SEA-CD40, and an anti-PD-1 antibody such as pembrolizumab. The treatment can further include a chemotherapy.

Claims

1. A method of treating a pancreatic cancer comprising administering to a patient having the pancreatic cancer: (i) a chemotherapy on day 1, day 8, and day 15 of each 28-day cycle, (ii) a composition comprising an anti-CD40 antibody on day 3 of each 28-day cycle, and (iii) an anti-PD-1 antibody on day 8 of each 42-day cycle; wherein the anti-CD40 antibody: 1) comprises a heavy chain variable region comprising amino acid 1-113 of SEQ ID NO: 1 and a light chain variable region comprising amino acid 1-113 of SEQ ID NO: 2, and a human constant region; wherein the human constant region has an N-glycoside-linked sugar chain at residue N297 according to the EU index; and wherein less than 20% of N-glycoside-linked sugar chains in the composition comprise a fucose residue; and/or 2) is a SEA-CD40 variant; and wherein the anti-PD-1 antibody comprises a light chain comprising CDRs of SEQ ID NOs: 3-5, and a heavy chain comprising CDRs of SEQ ID NOs 8-10.

2. The method of claim 1 wherein less than 10% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

3. The method of claim 1 or 2 wherein less than 5% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

4. The method of any one of claims 1-3 wherein less than 3% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

5. The method of any one of claims 1-4 wherein less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

6. The method of any one of claims 1-5 wherein the anti-CD40 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2.

7. The method of any one of claims 1-6, where in the anti-CD40 antibody is SEA-CD40.

8. The method of any one of claims 1-5 wherein the anti-CD40 antibody is a SEA-CD40 variant.

9. The method of any one of claims 1-8 wherein the light chain of the anti-PD-1 antibody has a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and wherein the heavy chain of the anti-PD-1 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

10. The method of any one of claims 1-9 wherein the light chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 7, and wherein the heavy chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 12.

11. The method of any one of claims 1-10 wherein the anti-PD-1 antibody is pembrolizumab.

12. The method of any one of claims 1-9 wherein the anti-PD-1 antibody is a pembrolizumab variant.

13. The method of any one of claims 1-12 wherein the chemotherapy comprises gemcitabine and/or paclitaxel.

14. The method of claim 13 wherein paclitaxel is nab-paclitaxel.

15. The method of any one of claims 1-14 wherein the anti-CD40 antibody is administered at 10 .Math.g/kg.

16. The method of any one of claims 1-14 wherein the anti-CD40 antibody is administered at 30 .Math.g/kg.

17. The method of any one of claims 1-16 wherein the anti-PD-1 antibody is administered at 400 mg.

18. The method of any one of claims 1-17 wherein the anti-PD-1 antibody is administered intravenously.

19. The method of any one of claims 1-18, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

20. The method of any one of claims 1-19, wherein the anti-CD40 antibody is administered intravenously.

21. The method of any one of claims 1-19, wherein the anti-CD40 antibody is administered subcutaneously.

22. A method of treating a cancer comprising: (i) administering a chemotherapy to a patient having the cancer in a cycle of every 4 weeks, (ii) administering a composition comprising an anti-CD40 antibody to the patient in a cycle of every 4 weeks, and (iii) administering an anti-PD-1 antibody to the patient in a cycle of every 3 weeks or 6 weeks, wherein the chemotherapy is administered on day 1, day 8, day 15 of each 4-week cycle, the anti-CD40 antibody is administered on day 3 of each 4-week cycle, and the anti-PD-1 antibody is administered on day 8 of each of the 3-week cycle or 6-week cycle; wherein the anti-CD40 antibody: 1) comprises a heavy chain variable region comprising amino acid 1-113 of SEQ ID NO: 1 and a light chain variable region comprising amino acid 1-113 of SEQ ID NO: 2, and a human constant region; wherein the human constant region has an N-glycoside-linked sugar chain at residue N297 according to the EU index; and wherein less than 20% of N-glycoside-linked sugar chains in the composition comprise a fucose residue; and/or 2) is a SEA-CD40 variant; and wherein the anti-PD-1 antibody comprises a light chain comprising CDRs of SEQ ID NOs: 3-5, and a heavy chain comprising CDRs of SEQ ID NOs 8-10.

23. The method of claim 22 wherein less than 10% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

24. The method of claim 22 or 23 wherein less than 5% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

25. The method of any one of claims 22-24 wherein less than 3% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

26. The method of any one of claims 22-25 wherein less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

27. The method of any one of claims 22-26 wherein the anti-CD40 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2.

28. The method of any one of claims 22-27, where in the anti-CD40 antibody is SEA-CD40.

29. The method of any one of claims 22-26 wherein the anti-CD40 antibody is a SEA-CD40 variant.

30. The method of any one of claims 22-29 wherein the light chain of the anti-PD-1 antibody has a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and wherein the heavy chain of the anti-PD-1 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

31. The method of any one of claims 22-30 wherein the light chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 7, and wherein the heavy chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 12.

32. The method of any one of claims 22-31 wherein the anti-PD-1 antibody is pembrolizumab.

33. The method of any one of claims 22-30 wherein the anti-PD-1 antibody is a pembrolizumab variant.

34. The method of any one of claims 22-33, wherein the anti-PD-1 antibody is administered in a cycle of every 3 weeks, and the anti-PD-1 antibody is administered on day 8 of each 3-week cycle at a dose of 200 mg.

35. The method of any one of claims 22-33, wherein the anti-PD-1 antibody is administered in a cycle of every 6 weeks, and the anti-PD-1 antibody is administered on day 8 of each 6-week cycle at a dose of 400 mg.

36. The method of any one of claims 22-35 wherein the anti-PD-1 antibody is administered intravenously.

37. A method of treating a cancer comprising: (i) administering an anti-CD40 antibody to a patient having the cancer in a cycle of every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, or every 8 weeks, wherein the cycle comprises a first cycle of administration of the anti-CD40 antibody, (ii) administering an anti-PD-1 antibody to the patient in a cycle of every 3 weeks or every 6 weeks, wherein the cycle comprises a first cycle of administration of the anti-PD-1 antibody, wherein a first administration of the anti-CD40 antibody in the first cycle of administration of the anti-CD40 antibody is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days prior to a first administration of the anti-PD-1 antibody in the first cycle of administration of the anti-PD-1 antibody; wherein the anti-CD40 antibody: 1) comprises a heavy chain variable region comprising amino acid 1-113 of SEQ ID NO: 1 and a light chain variable region comprising amino acid 1-113 of SEQ ID NO: 2, and a human constant region; wherein the human constant region has an N-glycoside-linked sugar chain at residue N297 according to the EU index; and wherein less than 20% of N-glycoside-linked sugar chains in the composition comprise a fucose residue; and/or 2) is a SEA-CD40 variant; and wherein the anti-PD-1 antibody comprises a light chain comprising CDRs of SEQ ID NOs: 3-5, and a heavy chain comprising CDRs of SEQ ID NOs 8-10.

38. The method of claim 37 wherein less than 10% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

39. The method of claim 37 or 38 wherein less than 5% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

40. The method of any one of claims 37-39 wherein less than 3% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

41. The method of any one of claims 37-40 wherein less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

42. The method of any one of claims 37-41 wherein the anti-CD40 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2.

43. The method of any one of claims 37-42, where in the anti-CD40 antibody is SEA-CD40.

44. The method of any one of claims 37-41 wherein the anti-CD40 antibody is a SEA-CD40 variant.

45. The method of any one of claims 37-44 wherein the light chain of the anti-PD-1 antibody has a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and wherein the heavy chain of the anti-PD-1 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

46. The method of any one of claims 37-45 wherein the light chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 7, and wherein the heavy chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 12.

47. The method of any one of claims 37-46 wherein the anti-PD-1 antibody is pembrolizumab.

48. The method of any one of claims 37-45 wherein the anti-PD-1 antibody is a pembrolizumab variant.

49. The method of any one of claims 37-48, wherein the anti-CD40 antibody is administered in a cycle of every 2 weeks, every 4 weeks, every 6 weeks, or every 8 weeks.

50. The method of any one of claims 37-49, wherein the anti-CD40 antibody is administered in a cycle of every 4 weeks or every 8 weeks.

51. The method of any one of claims 37-50, wherein the anti-CD40 antibody is administered in a cycle of every 4 weeks.

52. The method of any one of claims 37-51, wherein the anti-PD-1 antibody is administered in a cycle of every 3 weeks at a dose of 200 mg.

53. The method of any one of claims 37-51, wherein the anti-PD-1 antibody is administered in a cycle of every 6 weeks at a dose of 400 mg.

54. The method of any one of claims 37-53 wherein the anti-PD-1 antibody is administered intravenously.

55. The method of any one of claims 37-54, wherein the first administration of the anti-CD40 antibody in the first cycle is 2 days, 3 days, 4 days, 5 days, or 6 days prior to the first administration of the anti-PD-1 antibody in the first cycle.

56. The method of any one of claims 37-55, wherein the first administration of the anti-CD40 antibody in the first cycle is 3 days, 4 days, or 5 days prior to the first administration of the anti-PD-1 antibody in the first cycle.

57. The method of any one of claims 37-56, wherein the first administration of the anti-CD40 antibody in the first cycle is 5 days prior to the first administration of the anti-PD-1 antibody in the first cycle.

58. The method of any one of claims 37-48, wherein the anti-CD40 antibody and the anti-PD-1 antibody are administered in their first cycles according to a treatment regimen selected from the group consisting of: the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 2; the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 3; the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 4; the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 5; the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 6; the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 7; the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 8; the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 3; the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 4; the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 5; the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 6; the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 7; the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 8; the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 4; the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 5; the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 6; the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 7; the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 8; the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 5; the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 6; the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 7; the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 8; the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 6; the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 7; the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 8; the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 7; the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 8; and the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 8.

59. The method of claim 58, wherein the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 3.

60. The method of claim 58, wherein the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 5.

61. The method of claim 58, wherein the anti-CD40 antibody is first administered on day 1, and the anti-PD-1 antibody is first administered on day 8.

62. The method of claim 58, wherein the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 5.

63. The method of claim 58, wherein the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 8.

64. The method of claim 58, wherein the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 8.

65. A method of treating a cancer comprising: (i) administering a chemotherapy to a patient having the cancer in a cycle of every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks, (ii) administering an anti-CD40 antibody to a patient having the cancer in a cycle of every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, or every 8 weeks, and (iii) administering an anti-PD-1 antibody to the patient in a cycle of every 3 weeks or every 6 weeks, wherein a first administration of the chemotherapy in the first cycle of administration of the chemotherapy is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days prior to a first administration of the anti-CD40 antibody in the first cycle of administration of the anti-CD40 antibody, wherein a first administration of the anti-CD40 antibody in the first cycle of administration of the anti-CD40 antibody is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days prior to the first administration of the anti-PD-1 antibody in the first cycle of administration of the anti-PD-1 antibody; wherein the anti-CD40 antibody: 1) comprises a heavy chain variable region comprising amino acid 1-113 of SEQ ID NO: 1 and a light chain variable region comprising amino acid 1-113 of SEQ ID NO: 2, and a human constant region; wherein the human constant region has an N-glycoside-linked sugar chain at residue N297 according to the EU index; and wherein less than 20% of N-glycoside-linked sugar chains in the composition comprise a fucose residue; and/or 2) is a SEA-CD40 variant; and wherein the anti-PD-1 antibody comprises a light chain comprising CDRs of SEQ ID NOs: 3-5, and a heavy chain comprising CDRs of SEQ ID NOs 8-10.

66. The method of claim 65 wherein less than 10% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

67. The method of claim 65 or 66 wherein less than 5% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

68. The method of any one of claims 65-67 wherein less than 3% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

69. The method of any one of claims 65-68 wherein less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

70. The method of any one of claims 65-69 wherein the anti-CD40 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2.

71. The method of any one of claims 65-70, where in the anti-CD40 antibody is SEA-CD40.

72. The method of any one of claims 65-69 wherein the anti-CD40 antibody is a SEA-CD40 variant.

73. The method of any one of claims 65-72 wherein the light chain of the anti-PD-1 antibody has a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and wherein the heavy chain of the anti-PD-1 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

74. The method of any one of claims 65-73 wherein the light chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 7, and wherein the heavy chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 12.

75. The method of any one of claims 65-74 wherein the anti-PD-1 antibody is pembrolizumab.

76. The method of any one of claims 65-73 wherein the anti-PD-1 antibody is a pembrolizumab variant.

77. The method of any one of claims 65-76, wherein the chemotherapy comprises one or both of gemcitabine and paclitaxel.

78. The method of any one of claims 65-77, wherein the chemotherapy comprises both gemcitabine and paclitaxel.

79. The method of any one of claims 65-78, wherein the chemotherapy consists of gemcitabine and paclitaxel.

80. The method of any one of claims 77-79, wherein paclitaxel is nab-paclitaxel.

81. The method of any one of claims 77-79, wherein paclitaxel is albumin-bound paclitaxel.

82. The method of any one of claims 65-81, wherein the anti-CD40 antibody is administered in a cycle of every 2 weeks, every 4 weeks, every 6 weeks, or every 8 weeks.

83. The method of any one of claims 65-82, wherein the anti-CD40 antibody is administered in a cycle of every 4 weeks or every 8 weeks.

84. The method of any one of claims 65-83, wherein the anti-CD40 antibody is administered in a cycle of every 4 weeks.

85. The method of any one of claims 65-84, wherein the anti-PD-1 antibody is administered in a cycle of every 3 weeks at a dose of 200 mg.

86. The method of any one of claims 65-85, wherein the anti-PD-1 antibody is administered in a cycle of every 6 weeks at a dose of 400 mg.

87. The method of any one of claims 65-86 wherein the anti-PD-1 antibody is administered intravenously.

88. The method of any one of claims 65-87, wherein the first administration of the chemotherapy in the first cycle is 2 days, 3 days, 4 days, 5 days, or 6 days prior to the first administration of the anti-CD40 antibody in the first cycle, and wherein the first administration of the anti-CD40 antibody in the first cycle is 2 days, 3 days, 4 days, 5 days, or 6 days prior to the first administration of the anti-PD-1 antibody in the first cycle.

89. The method of any one of claims 65-88, wherein the first administration of the chemotherapy in the first cycle is 2 days, 3 days, or 4 days prior to the first administration of the anti-CD40 antibody in the first cycle, and wherein the first administration of the anti-CD40 antibody in the first cycle is 3 days, 4 days, or 5 days prior to the first administration of the anti-PD-1 antibody in the first cycle.

90. The method of any one of claims 33-45, where the first administration of the chemotherapy in the first cycle is 2 days prior to the first administration of the anti-CD40 antibody in the first cycle, and wherein the first administration of the anti-CD40 antibody in the first cycle is 5 days prior to the first administration of the anti-PD-1 antibody in the first cycle.

91. The method of any one of claims 65-87, wherein the chemotherapy, the anti-CD40 antibody and the anti-PD-1 antibody are administered in their first cycles according to a treatment regimen selected from the group consisting of: the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 3; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 4; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 5; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 6; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 7; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 8; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 9; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 10; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 12; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 13; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 14; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 2, and the anti-PD-1 antibody is first administered on day 15; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 4; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 5; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 6; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 7; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 8; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 9; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 10; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 12; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 13; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 14; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 15; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 5; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 6; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 7; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 8; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 9; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 10; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 12; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 13; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 14; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 4, and the anti-PD-1 antibody is first administered on day 15; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 6; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 7; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 8; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 9; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 10; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 12; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 13; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 14; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 15; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 7; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 8; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 9; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 10; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 13; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 14; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 6, and the anti-PD-1 antibody is first administered on day 15; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 8; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 9; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 10; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 12; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 13; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 14; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 15; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 9; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 10; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 11; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 12; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 13; the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 14; and the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 15.

92. The method of claim 91 wherein the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 3, and the anti-PD-1 antibody is first administered on day 8.

93. The method of claim 91 wherein the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 5, and the anti-PD-1 antibody is first administered on day 8.

94. The method of claim 91 wherein the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 8.

95. The method of claim 91 wherein the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 7, and the anti-PD-1 antibody is first administered on day 15.

96. The method of claim 91 wherein the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 10, day 11, day 12, or day 15.

97. The method of claim 91 wherein the chemotherapy is first administered on day 1, the anti-CD40 antibody is first administered on day 8, and the anti-PD-1 antibody is first administered on day 15.

98. The method of any one of claims 65-97, wherein the chemotherapy is administered in a cycle of every 4 weeks.

99. The method of any one of claims 65-97, wherein the chemotherapy is administered on day 1, day 5, and day 8 of each cycle.

100. The method of any one of claims 65-99, wherein the anti-CD40 antibody is administered in a cycle of every 4 weeks.

101. The method of any one of claims 22-100, wherein the anti-CD40 antibody is administered at a dose of about 3 .Math.g/kg, about 10 .Math.g/kg, about 30 .Math.g/kg, about 45 .Math.g/kg, or about 60 .Math.g/kg patient body weight.

102. The method of claim 101, wherein the anti-CD40 antibody is administered at a dose of about 10 .Math.g/kg patient body weight.

103. The method of claim 101, wherein the anti-CD40 antibody is administered at a dose of about 30 .Math.g/kg patient body weight.

104. The method of any one of claims 22-103, wherein the cancer is melanoma; breast cancer, metastatic breast cancer; lung cancer, non-small cell lung cancer (NSCLC), or pancreatic cancer.

105. The method of any one of claims 22-104, wherein the cancer is pancreatic cancer.

106. The method of any one of claims 22-105, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC).

107. The method of any one of claims 22-106, wherein the cancer is metastatic pancreatic ductal adenocarcinoma.

Description

DESCRIPTION OF DRAWINGS

[0087] FIG. 1A shows the median tumor volume of mice of a CT26 colon cancer model that were untreated (G1; control); administered with an anti-mPD-1 surrogate antibody (G2; “anti-PD1”); administered with SEA-m1C10 (G3; “SEA-m1C10”); or administered with SEA-m1C10 and the anti-mPD1 surrogate antibody by simultaneous dosing (G4; “SEA-m1C10 + anti-PD1”).

[0088] FIG. 1B shows the median tumor volume of mice of a CT26 colon cancer model that were untreated (G1; control); administered with an anti-mPD-1 surrogate antibody (G2; “anti-PD1”); administered with SEA-m1C10 (G3; “SEA-m1C10”); or administered with SEA-m1C10 and the anti-mPD1 surrogate antibody by staggered dosing (G4; “SEA-m1C10 + anti-PD1”). The time period for SEA-m1C10 dosing and anti-PD1 dosing are labeled by vertical lines on the X-axis.

[0089] FIG. 2A shows the survival curves of mice of a A20 disseminated lymphoma model that were untreated (G1; control); administered with an anti-mPD-1 surrogate antibody (G2; “anti-PD1”); administered with SEA-m1C10 (G3; “SEA-m1C10”); or administered with SEAm1C10 and the anti-mPD1 surrogate antibody by simultaneous dosing (G4; “SEA-m1C10 + anti-PD1”).

[0090] FIG. 2B shows the mean tumor volume of mice of a A20 disseminated lymphoma model that were untreated (G1; control); administered with an anti-mPD-1 surrogate antibody (G2; “anti-PD1”); administered with SEA-m1C10 (G3; “SEA-m1C10”); or administered with SEA-m1C10 and the anti-mPD1 surrogate antibody by staggered dosing (G4; “SEA-m1C10 + anti-PD1”). The time period for SEA-m1C10 dosing and anti-PD1 dosing are labeled by vertical lines on the X-axis.

[0091] FIG. 3A shows the mean tumor volume of mice of a RENCA renal cell carcinoma model that were untreated (G1; control); administered with an anti-mPD-1 surrogate antibody (G2; “anti-PD1”); administered with SEA-m1C10 (G3; “SEA-m1C10”); or administered with SEA-m1C10 and the anti-mPD1 surrogate antibody by simultaneous dosing (G4; “SEA-m1C10 + anti-PD1”).

[0092] FIG. 3B shows the mean tumor volume of mice of a RENCA renal cell carcinoma model that were untreated (G1; control); administered with an anti-mPD-1 surrogate antibody (G2; “anti-PD1”); administered with SEA-m1C10 (G3; “SEA-m1C10”); or administered with SEA-m1C10 and the anti-mPD1 surrogate antibody by staggered dosing (G4; “SEA-m1C10 + anti-PD1”). The time period for SEA-m1C10 dosing and anti-PD1 dosing are labeled by vertical lines on the X-axis.

[0093] FIG. 4 lists sequences discussed in the disclosure. Variable regions are in bold and underlined.

DETAILED DESCRIPTION

[0094] This disclosure relates to methods of treating cancer using a combination of an anti-CD40 antibody such as SEA-CD40, and an anti-PD-1 antibody such as pembrolizumab. In one aspect, the disclosure also provides methods of treating cancer using a combination of an anti-CD40 antibody, an anti-PD-1 antibody, and a chemotherapy.

Cd40

[0095] CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is a single chain type I transmembrane protein with an apparent MW of 50 kDa. Its mature polypeptide core consists of 237 amino acids, of which 173 amino acids comprise an extracellular domain (ECD) organized into 4 cysteine-rich repeats that are characteristic of TNF receptor family members. Two potential N-linked glycosylation sites are present in the membrane proximal region of the ECD, while potential O-linked glycosylation sites are absent. A 22 amino acid transmembrane domain connects the ECD with the 42 amino acid cytoplasmic tail of CD40. Sequence motifs involved in CD40-mediated signal transduction have been identified in the CD40 cytoplasmic tail. These motifs interact with cytoplasmic factors called TNF-R-associated factors (TRAFs) to trigger multiple downstream events including activation of MAP kinases and NFκB, which in turn modulate the transcriptional activities of a variety of inflammation-, survival-, and growth-related genes. See, e.g., van Kooten and Banchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al., Immunol. Rev. 229:152-172 (2009).

[0096] Within the hematopoietic system, CD40 can be found on B cells at multiple stages of differentiation, monocytes, macrophages, platelets, follicular dendritic cells, dendritic cells (DC), eosinophils, and activated T cells. In normal non-hematopoietic tissues, CD40 has been detected on renal epithelial cells, keratinocytes, fibroblasts of synovial membrane and dermal origins, and activated endothelium. A soluble version of CD40 is released from CD40-expressing cells, possibly through differential splicing of the primary transcript or limited proteolysis by the metalloproteinase TNFα converting enzyme. Shed CD40 can potentially modify immune responses by interfering with the CD40/CD40L interaction. See, e.g., van Kooten and Banchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al., Immunol. Rev. 229:152-172 (2009).

[0097] The endogenous ligand for CD40 (CD40L) is a type II membrane glycoprotein of 39 kDa also known as CD154. CD40L is a member of the TNF superfamily and is expressed as a trimer on the cell surface. CD40L is transiently expressed on activated CD4+, CD8+, and γδ T cells. CD40L is also detected at variable levels on purified monocytes, activated B cells, epithelial and vascular endothelial cells, smooth muscle cells, and DCs, but the functional relevance of CD40L expression on these cell types has not been clearly defined (van Kooten 2000; Elgueta 2009). However, expression of CD40L on activated platelets has been implicated in the pathogenesis of thrombotic diseases. See, e.g., Ferroni et al., Curr. Med. Chem. 14:2170-2180 (2007).

[0098] The best-characterized function of the CD40/CD40L interaction is its role in contact-dependent reciprocal interaction between antigen-presenting cells and T cells. See, e.g., van Kooten and Banchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al., Immunol. Rev. 229:152-172 (2009). Binding of CD40L on activated T cells to CD40 on antigen-activated B cells not only drives rapid B cell expansion, but also provides an essential signal for B cells to differentiate into either memory B cells or plasma cells. CD40 signaling is responsible for the formation of germinal centers in which B cells undergo affinity maturation and isotype switching to acquire the ability to produce high affinity antibodies of the IgG, IgA, and IgE isotypes. See, e.g., Kehry, J. Immunol. 156:2345-2348 (1996). Thus, individuals with mutations in the CD40L locus that prevent functional CD40/CD40L interaction suffer from the primary immunodeficiency X-linked hyper-IgM syndrome that is characterized by over-representation of circulating IgM and the inability to produce IgG, IgA, and IgE. These patients demonstrate suppressed secondary humoral immune responses, increased susceptibility to recurrent pyrogenic infections, and a higher frequency of carcinomas and lymphomas. Gene knockout experiments in mice to inactivate either CD40 or CD40L locus reproduce the major defects seen in X-linked hyper-IgM patients. These KO mice also show impaired antigen-specific T cell priming, suggesting that the CD40L/CD40 interaction is also a critical factor for mounting cell-mediated immune responses. See, e.g., Elgueta et al., Immunol. Rev. 229:152-172 (2009).

[0099] The immune-stimulatory effects of CD40 ligation by CD40L or anti-CD40 in vivo have correlated with immune responses against syngeneic tumors. See, e.g., French et al., Nat. Med. 5:548-553 (1999). A deficient immune response against tumor cells can result from a combination of factors such as expression of immune checkpoint molecules, such as PD1 or CTLA-4, decreased expression of MHC antigens, poor expression of tumor-associated antigens, appropriate adhesion, or co-stimulatory molecules, and the production of immunosuppressive proteins like TGFβ by the tumor cells. CD40 ligation on antigen presenting and transformed cells results in up-regulation of adhesion proteins (e.g., CD54), co-stimulatory molecules (e.g., CD86) and MHC antigens, as well as inflammatory cytokine secretion, thereby potentially inducing and/or enhancing the antitumor immune response, as well as the immunogenicity of the tumor cells. See, e.g., Gajewski et al., Nat. Immunol. 14:1014-1022 (2013).

[0100] A primary consequence of CD40 cross-linking is DC activation (often termed licensing) and potentiation of myeloid and B cells ability to process and present tumor-associated antigens to T cells. Besides having a direct ability to activate the innate immune response, a unique consequence of CD40 signaling is APC presentation of tumor-derived antigens to CD8+ cytotoxic T cell (CTL) precursors in a process known as ‘cross-priming’. This CD40-dependent activation and differentiation of CTL precursors by mature DCs into tumor-specific effector CTLs can enhance cell-mediated immune responses against tumor cells. See, e.g., Kurts et al., Nat. Rev. Immunol. 10:403-414 (2010).

[0101] Agonistic CD40 mAbs including dacetuzumab, the SEA-CD40 parent molecule (a fucosylated anti-CD40 antibody), have shown encouraging clinical activity in single-agent and combination chemotherapy settings. Dacetuzumab demonstrated some clinical activity in a phase 1 study in NHL and a phase 2 study in diffuse large B-cell lymphoma (DLBCL). See, e.g., Advani et al., J. Clin. Oncol. 27:4371-4377 (2009) and De Vos et al., J. Hematol. Oncol. 7:1-9 (2014). Additionally CP-870,893, a humanized IgG2 agonist antibody to CD40, showed encouraging activity in solid tumor indications when combined with paclitaxel or carboplatin or gemcitabine. In these studies, activation of antigen presenting cells, cytokine production, and generation of antigen-specific T cells were seen. See, e.g., Beatty et al., Clin. Cancer Res. 19:6286-6295 (2013) and Vonderheide et al., Oncoimmunology 2:e23033 (2013)

Anti-CD40 Antibodies

[0102] Anti-CD40 antibodies, e.g., S2C6, have been disclosed in US20170333556A1, which is herein incorporated by reference. The S2C6 antibody is a partial agonist of the CD40 signaling pathway and thus has the following activities: binding to human CD40 protein, binding to cynomolgus CD40 protein, activation of the CD40 signaling pathway, potentiation of the interaction of CD40 with its ligand, CD40L. See, e.g., U.S. Pat. No. 6,946,129, which is herein incorporated by reference.

[0103] Humanized anti-CD40 antibodies, e.g., humanized S2C6 (hS2C6), have been disclosed in US8303955B2 and US8492531B2, both of which are herein incorporated by reference.

[0104] Non-fucosylated anti-CD40 antibodies, e.g., hS2C6 or SEA-CD40 have been disclosed in US20170333556A1. In addition to enhanced binding to Fc receptors, SEA-CD40 also enhances activity of the CD40 pathway, as compared to the parent antibody, dacetuzumab. The SEA-CD40 antibody thus, is administered to patients at lower doses and using different schedules of administration.

[0105] SEA-CD40 exhibits enhanced binding to FcγIII receptors, and enhanced ability to activate the CD40 signaling pathway in immune cells, as described in US20170333556A1. Methods of making the non-fucosylated antibodies including SEA-CD40 are also disclosed in US20170333556A1.

[0106] The amino acid sequences of the heavy chain and light chain for SEA-CD40 are disclosed as SEQ ID NOs: 1 and 2, respectively (See FIG. 4). As disclosed in US20170333556A1, the variable region of the heavy chain is from amino acids 1-113 of SEQ ID NO: 1. The variable region of the light chain is from amino acids 1-113 of SEQ ID NO: 2.

[0107] In some embodiment, a humanized anti-CD40 antibody disclosed herein is useful in the treatment of various disorders associated with the expression of CD40 as described herein. Because SEA-CD40 activates the immune system to respond against tumor-related antigens, its use is not limited to cancers that express CD40. Thus SEA-CD40 can be used to treat both CD40 positive and CD40 negative cancers.

Methods of Making Non-Fucosylated Antibodies

[0108] This disclosure provides compositions and methods for preparing humanized S2C6 antibodies with reduced core fucosylation. As used herein, “core fucosylation” refers to addition of fucose (“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at the reducing terminal of an N-linked glycan.

[0109] Fucosylation of complex N-glycoside-linked sugar chains bound to the Fc region (or domain) of the SEA-CD40 antibody backbone is reduced. As used herein, a “complex N-glycoside-linked sugar chain” is typically bound to asparagine 297 (according to the EU index as set forth in Kabat, “Sequences of Immunological Interest, 5.sup.th Ed., Pub. No. 91-3242, U.S. Dept. Healtth & Human Services, NIH, Bethesda, MD, 1991). As used herein, the complex N-glycoside-linked sugar chain has a biantennary composite sugar chain, mainly having the following structure:

##STR00001##

where ± indicates the sugar molecule can be present or absent, and the numbers indicate the position of linkages between the sugar molecules. In the above structure, the sugar chain terminal which binds to asparagine is called a reducing terminal (at right), and the opposite side is called a non-reducing terminal. Fucose is usually bound to N-acetylglucosamine (“GlcNAc”) of the reducing terminal, typically by an α1,6 bond (the 6-position of GlcNAc is linked to the 1-position of fucose). “Gal” refers to galactose, and “Man” refers to mannose.

[0110] A “complex N-glycoside-linked sugar chain” includes 1) a complex type, in which the non-reducing terminal side of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally has a sialic acid, bisecting N-acetylglucosamine or the like; or 2) a hybrid type, in which the non-reducing terminal side of the core structure has both branches of a high mannose N-glycoside-linked sugar chain and complex N-glycoside-linked sugar chain.

[0111] In some embodiments, the “complex N-glycoside-linked sugar chain” includes a complex type in which the non-reducing terminal side of the core structure has zero, one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally further has a structure such as a sialic acid, bisecting N-acetylglucosamine or the like.

[0112] According to the present methods, typically only a minor amount of fucose is incorporated into the complex N-glycoside-linked sugar chain(s) of the SEA-CD40 molecule. For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the antibody has core fucosylation by fucose. In some embodiments, about 2% of the antibody has core fucosylation by fucose.

[0113] In certain embodiments, only a minor amount of a fucose analog (or a metabolite or product of the fucose analog) is incorporated into the complex N-glycoside-linked sugar chain(s). For example, in various embodiments, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the SEA-CD40 antibody has core fucosylation by a fucose analog or a metabolite or product of the fucose analog. In some embodiments, about 2% of the SEA-CD40 antibody has core fucosylation by a fucose analog or a metabolite or product of the fucose analog.

[0114] Methods of making non-fucosylated antibodies by incubating antibody-producing cells with a fucose analogue are described, e.g., in WO/2009/135181. Briefly, cells that have been engineered to express the humanized S2C6 antibody are incubated in the presence of a fucose analogue or an intracellular metabolite or product of the fucose analog. As used herein, an intracellular metabolite can be, for example, a GDP-modified analog or a fully or partially de-esterified analog. A product can be, for example, a fully or partially de-esterified analog. In some embodiments, a fucose analogue can inhibit an enzyme(s) in the fucose salvage pathway. For example, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of fucokinase, or GDP-fucose-pyrophosphorylase. In some embodiments, a fucose analog (or an intracellular metabolite or product of the fucose analog) inhibits fucosyltransferase (preferably a 1,6-fucosyltransferase, e.g., the FUT8 protein). In some embodiments, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of an enzyme in the de novo synthetic pathway for fucose. For example, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of GDP-mannose 4,6-dehydratase or/or GDP-fucose synthetase. In some embodiments, the fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit a fucose transporter (e.g., GDP-fucose transporter).

[0115] In some embodiments, the fucose analogue is 2-flurofucose. Methods of using fucose analogues in growth medium and other fucose analogues are disclosed, e.g., in WO/2009/135181, which is herein incorporated by reference.

[0116] Other methods for engineering cell lines to reduce core fucosylation included gene knock-outs, gene knock-ins and RNA interference (RNAi). In gene knock-outs, the gene encoding FUT8 (alpha 1,6- fucosyltransferase enzyme) is inactivated. FUT8 catalyzes the transfer of a fucosyl residue from GDP-fucose to position 6 of Asn-linked (N-linked) GlcNac of an N-glycan. FUT8 is reported to be the only enzyme responsible for adding fucose to the N-linked biantennary carbohydrate at Asn297. Gene knock-ins add genes encoding enzymes such as GNTIII or a golgi alpha mannosidase II. An increase in the levels of such enzymes in cells diverts monoclonal antibodies from the fucosylation pathway (leading to decreased core fucosylation), and having increased amount of bisecting N-acetylglucosamines. RNAi typically also targets FUT8 gene expression, leading to decreased mRNA transcript levels or knocking out gene expression entirely. Any of these methods can be used to generate a cell line that can produce a non-fucosylated antibody, e.g., an SEA-CD40 antibody.

[0117] Those of skill will recognize that many methods are available to determine the amount of fucosylation on an antibody. Methods include, e.g., LC-MS via PLRP-S chromatography and electrospray ionization quadrupole TOF MS.

[0118] The non-fucosylated antibody, SEA-CD40, when adminstered to a patient induces activation of monocyte maturation into macrophages and induce production of cytokines, including, e.g., interferon-γ (IFN- γ) and chemokine that elicit robust T-cell response to immune system challenges. Unlike fully agoninstic antibodies, such as antibody 24.4.1., SEA-CD40 does not induce production of immune-dampening cytokines, such as interleukin-10 (IL-10). IL-10, in turn, induces activity of T-regulatory cells, which dampen the immune resopnse. Thus, SEA-CD40 is useful for induction of a robust T-cell mediated immune response without promoting activity of T-regulatory cells.

[0119] In some embodiments, the disclosure relates to a composition comprising a non-fucosylated anti-CD40 antibody such as SEA-CD40, wherein the constant region of the antibody such as SEA-CD40 has an N-glycoside-linked sugar chain at residue N297 according to the EU index; and wherein less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 20% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 10% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 5% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 3% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

[0120] In some embodiments, the disclosure relates to treating a cancer by administering a composition comprising a non-fucosylated anti-CD40 antibody such as SEA-CD40, wherein the constant region of the antibody such as SEA-CD40 has an N-glycoside-linked sugar chain at residue N297 according to the EU index; and wherein less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 20% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 10% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 5% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 3% of N-glycoside-linked sugar chains in the composition comprise a fucose residue. In some embodiments, less than 2% of N-glycoside-linked sugar chains in the composition comprise a fucose residue.

[0121] The humanized anti-CD40 antibody or agent is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration (including perfusing or otherwise contacting the graft with the antibody before transplantation). The humanized anti-CD40 antibody or agent can be administered, for example, as an infusion or as a bolus. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the humanized anti-CD40 antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. In one aspect, the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0122] For the prevention or treatment of disease, the appropriate dosage of antibody will depend on a variety of factors such as the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0123] The antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The “therapeutically effective amount” of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat cancers including cancers. Because SEA-CD40 activates the immune system to respond against tumor-related antigens, its use is not limited to cancers that express CD40. Thus SEA-CD40 can be used to treat both CD40 positive and CD40 negative cancers.

[0124] The antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of humanized anti-CD40 antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.

[0125] PD-1 antagonists useful in the treatment method, medicaments and uses of the present invention include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody or a chimeric antibody, and may include a human constant region. In some embodiments the human constant region is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab′-SH, F(ab′).sub.2, scFv and Fv fragments.

[0126] Examples of mAbs that bind to human PD-1, and useful in the treatment method, medicaments and uses of the present invention, are described in US7488802, US7521051, US8008449, US8354509, US8168757, WO2004/004771, WO2004/072286, WO2004/056875, and US2011/0271358. Specific anti-human PD-1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present invention include: pembrolizumab (also known as MK-3475), a humanized IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) and which comprises the heavy and light chain amino acid sequences shown in Table 2; nivolumab (BMS-936558), a human IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013); the humanized antibodies h409A11, h409A16 and h409A17, which are described in WO2008/156712, and AMP-514, which is being developed by MedImmune.

[0127] Examples of mAbs that bind to human PD-L1, and useful in the treatment method, medicaments and uses of the present invention, are described in WO2013/019906, WO2010/077634 A1 andUS8383796. Specific anti-human PD-L1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C and an antibody which comprises the heavy chain and light chain variable regions of SEQ ID NO: 24 and SEQ ID NO: 21, respectively, of WO2013/019906.

[0128] Other PD-1 antagonists useful in the treatment method, medicaments and uses of the present invention include an immunoadhesin that specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1, e.g., a fusion protein containing the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region such as an Fc region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in WO2010/027827 and WO2011/066342. Specific fusion proteins useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present invention include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to human PD-1.

[0129] In some preferred embodiments of the treatment method, medicaments and uses of the present invention, the PD-1 antagonist is a monoclonal antibody, or antigen binding fragment thereof, which comprises: (a) light chain CDRs SEQ ID NOs: 3, 4 and 5 and (b) heavy chain CDRs SEQ ID NOs: 6, 7 and 8.

[0130] In other preferred embodiments of the treatment method, medicaments and uses of the present invention, the PD-1 antagonist is a monoclonal antibody, or antigen binding fragment thereof, which specifically binds to human PD-1 and comprises (a) a heavy chain variable region comprising SEQ ID NO: 11 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO: 6 or a variant thereof. A variant of a heavy chain variable region sequence is identical to the reference sequence except having up to 17 conservative amino acid substitutions in the framework region (i.e., outside of the CDRs), and preferably has less than ten, nine, eight, seven, six or five conservative amino acid substitutions in the framework region. A variant of a light chain variable region sequence is identical to the reference sequence except having up to five conservative amino acid substitutions in the framework region (i.e., outside of the CDRs), and preferably has less than four, three or two conservative amino acid substitution in the framework region.

[0131] In another preferred embodiment of the treatment method, medicaments and uses of the present invention, the PD-1 antagonist is a monoclonal antibody which specifically binds to human PD-1 and comprises (a) a heavy chain comprising SEQ ID NO: 12 and (b) a light chain comprising SEQ ID NO: 7.

[0132] In all of the above treatment method, medicaments and uses, the PD-1 antagonist inhibits the binding of PD-L1 to PD-1, and preferably also inhibits the binding of PD-L2 to PD-1. In some embodiments of the above treatment method, medicaments and uses, the PD-1 antagonist is a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to PD-1 or to PD-L1 and blocks the binding of PD-L1 to PD-1. In one embodiment, the PD-1 antagonist is an anti-PD-1 antibody which comprises a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 12 and SEQ ID NO: 7, respectively.

[0133] In one embodiment, the PD-1 antagonist is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is pembrolizumab. In one embodiment, the anti-PD-1 antibody is a pembrolizumab variant.

[0134] Table 2 below provides a list of the amino acid sequences of exemplary anti-PD-1 mAbs for use in the treatment method, medicaments and uses of the present invention.

TABLE-US-00002 Exemplary PD-1 Antibody Sequences Antibody Feature Amino Acid Sequence SEQ ID NO. Pembrolizumab Light Chain CDR1 RASKGVSTSGYSYLH 3 CDR2 LASYLES 4 CDR3 QHSRDLPLT 5 Variable Region EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWY QQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISS LEPEDFAVYYCQHSRDLPLTFGGGTKVEIK 6 Light Chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWY QQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISS LEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC 7 Pembrolizumab Heavy Chain CDR1 NYYMY 8 CDR2 GINPSNGGTNFNEKFKN 9 CDR3 RDYRFDMGFDY 10 Variable Region QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV RQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSST TTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSS 11 Heavy Chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV RQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSST TTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 12

Dosage and Administration of an anti-CD40 Antibody Such as SEA-CD40 for Treating Cancer

[0135] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0136] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered intravenously. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered subcutaneously. In a further embodiment, an anti-CD40 antibody such as SEA-CD40 is administered subcutaneously at the site of a tumor.

[0137] SEA-CD40 is an agonistic antibody and has enhanced binding to Fcγ receptors III and, exhibits enhanced activation of the CD40 signaling pathway. Because of its enhanced activation of the CD40 pathway SEA-CD40 is a potent activator of the immune system. The enhanced activation of the immune system allows SEA-CD40 to be dosed at low levels, as compared to a fucosylated parent antibody.

[0138] As an example, an anti-CD40 antibody such as SEA-CD40 can be administered to patients at levels between about 0.1 to about 70 .Math.g/kg (.Math.g antibody per kilogram patient body weight). Other possible dosage ranges include about 1 .Math.g/kg to about 60 .Math.g/kg, about 10 .Math.g/kg to about 50 .Math.g/kg, and about 20 .Math.g/kg to about 40 .Math.g/kg. Other possible dosage ranges include the following: about 1 .Math.g/kg to about 5 .Math.g/kg, about 5 .Math.g/kg to about 10 .Math.g/kg, about 10 .Math.g/kg to about 15 .Math.g/kg, about 15 .Math.g/kg to about 20 .Math.g/kg, about 20 .Math.g/kg to about 25 .Math.g/kg, about 25 .Math.g/kg to about 30 .Math.g/kg, about 30 .Math.g/kg to about 35 .Math.g/kg, about 35 .Math.g/kg to about 40 .Math.g/kg, about 40 .Math.g/kg to about 45 .Math.g/kg, about 45 .Math.g/kg to about 50 .Math.g/kg, about 50 .Math.g/kg to about 55 .Math.g/kg, and about 55 .Math.g/kg to about 60 .Math.g/kg.

[0139] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered to patients at about 1 .Math.g/kg, about 2 .Math.g/kg, about 3 .Math.g/kg, about 4 .Math.g/kg, about 5 .Math.g/kg, about 6 .Math.g/kg, about 7 .Math.g/kg, about 8 .Math.g/kg, about 9 .Math.g/kg, about 10 .Math.g/kg, about 11 .Math.g/kg, about 12 .Math.g/kg, about 13 .Math.g/kg, about 14 .Math.g/kg, about 15 .Math.g/kg, about 16 .Math.g/kg, about 17 .Math.g/kg, about 18 .Math.g/kg, about 19 .Math.g/kg, about 20 .Math.g/kg, about 21 .Math.g/kg, about 22 .Math.g/kg, about 23 .Math.g/kg, about 24 .Math.g/kg, about 25 .Math.g/kg, about 26 .Math.g/kg, about 27 .Math.g/kg, about 28 .Math.g/kg, about 29 .Math.g/kg, about 30 .Math.g/kg, about 31 .Math.g/kg, about 32 .Math.g/kg, about 33 .Math.g/kg, about 34 .Math.g/kg, about 35 .Math.g/kg, about 36 .Math.g/kg, about 37 .Math.g/kg, about 38 .Math.g/kg, about 39 .Math.g/kg, about 40 .Math.g/kg, about 41 .Math.g/kg, about 42 .Math.g/kg, about 43 .Math.g/kg, about 44 .Math.g/kg, about 45 .Math.g/kg, about 46 .Math.g/kg, about 47 .Math.g/kg, about 48 .Math.g/kg, about 49 .Math.g/kg, about 50 .Math.g/kg, about 51 .Math.g/kg, about 52 .Math.g/kg, about 53 .Math.g/kg, about 54 .Math.g/kg, about 55 .Math.g/kg, about 56 .Math.g/kg, about 57 .Math.g/kg, about 58 .Math.g/kg, about 59 .Math.g/kg, about 60 .Math.g/kg, about 61 .Math.g/kg, about 62 .Math.g/kg, about 63 .Math.g/kg, about 64 .Math.g/kg, about 65 .Math.g/kg, about 66 .Math.g/kg, about 67 .Math.g/kg, about 68 .Math.g/kg, about 69 .Math.g/kg, or about 70 .Math.g/kg. In preferred embodiments, an anti-CD40 antibody such as SEA-CD40 is administered to patients at about 3 .Math.g/kg, about 10 .Math.g/kg, about 30 .Math.g/kg, about 45 .Math.g/kg, or about 60 .Math.g/kg. In a more preferred embodiment, an anti-CD40 antibody such as SEA-CD40 is administered to cancer patients at about 30 .Math.g/kg or about 10 .Math.g/kg. In another more preferred embodiment, an anti-CD40 antibody such as SEA-CD40 is administered to cancer patients at about 10 .Math.g/kg. In yet another more preferred embodiment, an anti-CD40 antibody such as SEA-CD40 is administered to cancer patients at about 30 .Math.g/kg.

[0140] An anti-CD40 antibody such as SEA-CD40 can be administered at different intervals including one week intervals, two weeks intervals, three week intervals, four weeks intervals, five week intervals, six week intervals, seven week intervals, eight week intervals, nine weeks, ten weeks, eleven weeks, twelve weeks, etc. In other words, an anti-CD40 antibody such as SEA-CD40 can be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks etc. In some embodiments, intervals are on a monthly schedule, e.g., one month intervals, two month intervals, or three month intervals. In some embodiments, intervals are based on cycles wherein each cycle can comprise one or more administrations of an anti-CD40 antibody such as SEA-CD40. Exemplary lengths of each cycle include one week, two weeks, three weeks, four weeks, five week, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, and twelve weeks. The lengths of cycles can be different from one cycle to the next. An anti-CD40 antibody such as SEA-CD40 can be administered on any one or more days in each cycle. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on the first day of a cycle. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on the first day of a cycle of three weeks in a treatment period of one cycle, two cycles, three cycles, four cycles, five cycles, six cycles, seven cycles, eight cycles, nine cycles, ten cycles, eleven cycles, twelve cycles, thirteen cycles, fourteen cycles, fifteen cycles, or sixteen cycles.

[0141] An anti-CD40 antibody such as SEA-CD40 can be administered on day 1, day 2, day 3, day 4, day 5, day 6, or day 7 of each 1-week cycle, i.e., an anti-CD40 antibody such as SEA-CD40 is administered every week starting on day 1, day 2, day 3, day 4, day 5, day 6, or day 7 of a treatment regimen. An anti-CD40 antibody such as SEA-CD40 can be administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 of each 2-week cycle, i.e., an anti-CD40 antibody such as SEA-CD40 is administered every two weeks starting on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 of a treatment regimen. An anti-CD40 antibody such as SEA-CD40 can be administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, or day 21 of each 3-week cycle, i.e., an anti-CD40 antibody such as SEA-CD40 is administered every three weeks starting on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, or day 21 of a treatment regimen. An anti-CD40 antibody such as SEA-CD40 can be administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, or day 28 of each 4-week cycle, i.e., an anti-CD40 antibody such as SEA-CD40 is administered every four weeks starting on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, or day 28 of a treatment regimen. An anti-CD40 antibody such as SEA-CD40 can be administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, or day 35 of each 5-week cycle, i.e., an anti-CD40 antibody such as SEA-CD40 is administered every five weeks starting on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, or day 35 of a treatment regimen. An anti-CD40 antibody such as SEA-CD40 can be administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, or day 42 of each 6-week cycle, i.e., an anti-CD40 antibody such as SEA-CD40 is administered every six weeks starting on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, or day 42 of a treatment regimen.

[0142] In this disclosure, administration cycle is described in terms of days or weeks interchangeably as a skilled person would understand. For example, an 1-week administration cycle is the same as a 7-day administration cycle; a 2-week administration cycle is the same as a 14-day administration cycle; a 3-week administration cycle is the same as a 21-week administration cycle; etc.

Dosage and Administration of Pembrolizumab for Treating Cancer in Combination With the Anti-CD40 Antibody

[0143] The pembrolizumab can be administered at 200 mg or 2 mg/kg once every three weeks. In some embodiments, the pembrolizumab is administered at 400 mg once every six weeks.

[0144] Pembrolizumab can be administered at different intervals including three week intervals and six week intervals. In other words, pembrolizumab can be administered every three weeks or every six weeks. In some embodiments, intervals are based on cycles wherein each cycle can comprise one or more administrations of pembrolizumab. Exemplary lengths of each cycle include three weeks and six weeks. The lengths of cycles can be different from one cycle to the next. Pembrolizumab can be administered on any one or more days in each cycle.

[0145] In some embodiments, as an alternative to pembrolizumab, another anti-PD-1 antibody or an anti-PD-L1 antibody is used. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of Nivolumab, Cemiplimab-rwlc, Spartalizumab, AK105, Tislelizumab, Dostarlimab, MEDI0680, Pidilizumab, AMP-224, and SHR-1210. In some embodiments, the anti-PD-1 antibody is Pembrolizumab, Nivolumab, or Cemiplimab-rwlc. In some embodiments, the anti-PDL1 antibody is selected from the group consisting of Atezolizumab, Durvalumab, Avelumab, SHR-1316, MEDI4736, BMS-936559/MDX-1105, MSB0010718C, MPDL3280A, or Envafolimab. In some embodiments, the anti-PDL1 antibody is Atezolizumab, Durvalumab, or Avelumab.

Anti-CD40 Antibody and Pembrolizumab Combination Therapy for Treating Cancer

[0146] An anti-CD40 antibody such as SEA-CD40 can be used in combination with pembrolizumab for treating cancer.

[0147] A treating regime comprising administering an anti-CD40 antibody such as SEA-CD40 and administering pembrolizumab can have different dosing schedules. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on a two-week cycle and pembrolizumab is administered on a three-week cycle. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on a three-week cycle and pembrolizumab is also administered on a three-week cycle. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on a four-week cycle and pembrolizumab is administered on a three-week cycle. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on a two-week cycle and pembrolizumab is administered on a six-week cycle. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on a three-week cycle and pembrolizumab is administered on a six-week cycle. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on a four-week cycle and pembrolizumab is administered on a six-week cycle. In a preferred embodiment, an anti-CD40 antibody such as SEA-CD40 is administered on a four-week cycle and pembrolizumab is administered on a three-week cycle. In another preferred embodiment, an anti-CD40 antibody such as SEA-CD40 is administered on a four-week cycle and pembrolizumab is administered on a six-week cycle.

[0148] In a preferred embodiment, an anti-CD40 antibody such as SEA-CD40 is administered before the administration of pembrolizumab. Giving pembrolizumab after SEA-CD40 may be beneficial, as this timing mitigates the potential for pembrolizumab to bind to immune cells with subsequent immune depletion arising from enhanced clearance of pembrolizumab-bound cells by SEA-CD40. In a combination therapy, the first day of each drug’s first cycle of administration all start on the same day.

[0149] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 2 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 3 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 4 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 5 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 6 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 7 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 1 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0150] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 3 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 4 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 5 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 6 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 7 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 2 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0151] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 4 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 5 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 6 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 7 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0152] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 5 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 6 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 7 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 4 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0153] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 6 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 7 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 5 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0154] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 7 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 6 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0155] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 7 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 7 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 7 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 7 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 7 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 7 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 7 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0156] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 8 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 9 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 8 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 8 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 8 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 8 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 8 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0157] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 9 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 10 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 9 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 9 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 9 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 9 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0158] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 10 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 11 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 10 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 10 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 10 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0159] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 11 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 12 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 11 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 11 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0160] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 12 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 13 of the first cycle of pembrolizumab. In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 12 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0161] In some embodiments, an anti-CD40 antibody such as SEA-CD40 is administered on day 13 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 14 of the first cycle of pembrolizumab.

[0162] In some embodiments, the first administration of an anti-CD40 antibody such as SEA-CD40 in the first cycle is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days prior to the first administration of pembrolizumab in the first cycle. In some embodiments, the first administration of pembrolizumab is 1 day, days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after the first administration of an anti-CD40 antibody such as SEA-CD40 in the first cycle.

[0163] In some embodiments, each dose of the anti-PD-1 antibody is administered at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 days after a dose of the anti-CD40 antibody. In some embodiments, the anti-PD-1 antibody and the anti-CD40 antibody are not administered on the same day. In some embodiments, the interval between the administration of the anti-PD-1 antibody and the administration of the anti-CD40 antibody is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours, or at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0164] In some embodiments, pembrolizumab is administered in a cycle of about every 2-4 weeks (e.g., about every 2 weeks, about every 3 weeks, or about every 4 weeks). In some embodiments, pembrolizumab is administered in a cycle of every 14 days, every 15 days, every 16 days, every 17 days, every 18 days, every 19 days, every 20 days, every 21 days, every 22 days, every 23 days, every 24 days, every 25 days, every 26 days, every 27 days, or every 28 days. In some embodiments, pembrolizumab is administered in a cycle of about every 5-7 weeks (e.g., about every 5 weeks, about every 6 weeks, or about every 7 weeks). In some embodiments, pembrolizumab is administered in a cycle of every 35 days, every 36 days, every 37 days, every 38 days, every 39 days, every 40 days, every 41 days, every 42 days, every 43 days, every 44 days, every 45 days, every 46 days, every 47 days, every 48 days, or every 49 days.

[0165] In some embodiments, pembrolizumab is administered every 3 weeks at a dose of about 200 mg. In some embodiments, pembrolizumab is administered every 3 weeks at a dose of 200 mg. In some embodiments, pembrolizumab is administered every3 at a dose of about 2 mg/kg. In some embodiments, pembrolizumab is administered every 6 weeks at a dose of 400 mg.

Combination Therapy With anti-CD40 Antibody and Chemotherapy

[0166] The combination therapy of an anti-CD40 antibody such as SEA-CD40 can be combined with chemotherapy. In some embodiments, the combination therapy of an anti-CD40 antibody such as SEA-CD40 and pembrolizumab can be further combined with chemotherapy.

[0167] In most humans, millions of cells die via apoptosis and are removed without generating an immune response. However, after treatment with some chemotherapeutic agents, immune cells have been observed to infiltrate tumors. Thus, some tumor cells killed by chemotherapeutic agents act as vaccines and raise a tumor-specific immune response. This phenomenon is referred to as immunogenic cell death (ICD). See, e.g., Kroemer et al., Annu. Rev. Immunol., 31:51-72 (2013). The ability of a chemotherapeutic agent to induce ICD can be determined experimentally. Two criteria must be met. First, injection of an immunocompetent mouse with cancer cells that have been treated in vitro with a chemotherapeutic agent must elicit a protective immune response that is specific for tumor antigens, in the absence of adjuvant. Second, ICD occurring in vivo, e.g., a mouse syngeneic model with treatment using a potential ICD-inducing chemotherapeutic agent, must drive an immune response in the tumor that is dependent on the immune system.

[0168] Chemotherapeutic agents that induce ICD include, e.g., anthracyclines, anti-EGFR antibodies, bortezomib, cyclophosphamide, gemcitabine, irradiation of the tumor, and oxaliplatin. A combination of an anti-CD40 antibody such as SEA-CD40 and pembrolizumab can be used in combination with any of these chemotherapy agents to generate an enhanced immune response and treat cancer in a patient. In some embodiments, the combination of an anti-CD40 antibody such as SEA-CD40 and pembrolizumab is used in combination with one or more of gemcitabine, dacarbazine, temozolomide, paclitaxel, albumin-bound paclitaxel (nab-paclitaxel), or carboplatin. ABRAXANE® is a brand name of paclitaxel containing albumin-bound paclitaxel. In some embodiments, the combination of an anti-CD40 antibody such as SEA-CD40 and pembrolizumab is used in combination with both the chemotherapeutic agent gemcitabine and paclitaxel/nab-paclitaxel.

[0169] In some embodiments, the combination therapy includes an anti-CD40 antibody such as SEA-CD40, pembrolizumab, and chemotherapy. In some embodiments, chemotherapy used in the combination includes gemcitabine or paclitaxel. In some embodiments, chemotherapy used in the combination includes both gemcitabine and paclitaxel. In some embodiments, paclitaxel is nab-paclitaxel, e.g., ABRAXANE®.

[0170] Chemotherapy used in the combination can be administered in cycles. In some embodiments, the cycle is 1 week, i.e., chemotherapy is administered every week. In some embodiments, the cycle is 2 weeks, i.e., chemotherapy is administered every 2 weeks. In some embodiments, the cycle is 3 weeks, i.e., chemotherapy is administered every 3 weeks. In some embodiments, the cycle is 4 weeks, i.e., chemotherapy is administered every 4 weeks. In some embodiments, the cycle is 5 weeks, i.e., chemotherapy is administered every 5 weeks. In some embodiments, the cycle is 6 weeks, i.e., chemotherapy is administered every 6 weeks. In some embodiments, the cycle is 7 weeks, i.e., chemotherapy is administered every 7 weeks. In some embodiments, the cycle is 8 weeks, i.e., chemotherapy is administered every 8 weeks. In each cycle, chemotherapy can be administered one or more times.

[0171] In some embodiments, chemotherapy used in the combination is administered in a 4 week cycle, i.e., chemotherapy is administered every 4 weeks, wherein chemotherapy is administered three times in each cycle. In some embodiments, chemotherapy administered in the combination is administered on days 1, 8, and 15 in each cycle.

[0172] In some embodiments, chemotherapy used in the combination is administered in a cycle of about every 3-5 weeks (e.g., about every 3 weeks, about every 4 weeks, or about every 5 weeks). In some embodiments, chemotherapy used in the combination is administered in a cycle of every 21 days, every 22 days, every 23 days, every 24 days, every 25 days, every 26 days, every 27 days, every 28 days, every 29 days, every 30 days, every 31 days, every 32 days, every 33 days, every 34 days, or every 35 days.

[0173] In some embodiments, chemotherapy used in the combination includes gemcitabine (e.g., INFUGEM™) and/or paclitaxel (e.g., ABRAXANE®). In some embodiments, gemcitabine is administered 1 time, 2 times, 3 times, 4 times, or 5 times in each cycle. In some embodiments, gemcitabine is administered on days 1, 8, and 15 in each cycle (e.g., a 28-day cycle). In some embodiments, gemcitabine is administered on days 1 and 8 of each cycle (e.g., a 21-day cycle). In some embodiments, gemcitabine is administered at a dose of about 800-1500 mg/m.sup.2 (e.g., about 800 mg/m.sup.2, about 850 mg/m.sup.2, about 900 mg/m.sup.2, about 950 mg/m.sup.2, about 1000 mg/m.sup.2, about 1050 mg/m.sup.2, about 1100 mg/m.sup.2, about 1150 mg/m.sup.2, about 1200 mg/m.sup.2, about 1250 mg/m.sup.2, about 1300 mg/m.sup.2, about 1350 mg/m.sup.2, about 1400 mg/m.sup.2, about 1450 mg/m.sup.2, or about 1500 mg/m.sup.2), e.g., over about 20-60 minutes (e.g., about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes). In some embodiments, paclitaxel is administered 1 time, 2 times, 3 times, 4 times, or 5 times in each cycle. In some embodiments, paclitaxel is administered on days 1, 8, and 15 of each cycle (e.g., a 21-day cycle, or a 28-day cycle). In some embodiments, paclitaxel is administered at a dose of about 50-300 mg/m.sup.2 (e.g., about 50 mg/m.sup.2, about 60 mg/m.sup.2, about 70 mg/m.sup.2, about 80 mg/m.sup.2, about 90 mg/m.sup.2, about 100 mg/m.sup.2, about 110 mg/m.sup.2, about 120 mg/m.sup.2, about 125 mg/m.sup.2, about 130 mg/m.sup.2, about 140 mg/m.sup.2, about 150 mg/m.sup.2, about 160 mg/m.sup.2, about 170 mg/m.sup.2, about 180 mg/m.sup.2, about 190 mg/m.sup.2, about 200 mg/m.sup.2, about 210 mg/m.sup.2, about 220 mg/m.sup.2, about 230 mg/m.sup.2, about 240 mg//m.sup.2, about 250 mg/m.sup.2, about 260 mg/m.sup.2, about 270 mg/m.sup.2, about 280 mg/m.sup.2, about 290 mg/m.sup.2, or about 300 mg/m.sup.2), e.g., over about 20-60 minutes (e.g., about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes).

[0174] In a preferred embodiment, the first administration of the first cycle of chemotherapy is given prior to the administration of an anti-CD40 antibody such as SEA-CD40 to allow for antigen release to occur from tumor cells as a consequence of chemotherapy. In some embodiments, the chemotherapy is given 1 day prior to the administration of an anti-CD40 antibody such as SEA-CD40. In some embodiments, the chemotherapy is given 2 days prior to the administration of an anti-CD40 antibody such as SEA-CD40. In some embodiments, the chemotherapy is given 3 days prior to the administration of an anti-CD40 antibody such as SEA-CD40. This timing is anticipated to enhance the potential for an anti-CD40 antibody such as SEA-CD40 to lead to an anti-tumor immune response. Specifically, an anti-CD40 antibody such as SEA-CD40 can stimulate antigen update and presentation--and thus is expected to be most effective in the setting of increased levels of circulating antigen. Additionally, waiting 1-3 days after chemotherapy before administering an anti-CD40 antibody such as SEA-CD40 may mitigate the potential for synergistic toxicity.

[0175] In some embodiments, each dose of the anti-CD40 antibody is administered at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 days after a dose of the chemotherapy. In some embodiments, the chemotherapy and the anti-CD40 antibody are not administered on the same day. In some embodiments, the interval between the administration of the chemotherapy and the administration of the anti-CD40 antibody is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours, or at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0176] In some embodiments, the chemotherapy is administered on day 1 of the first cycle of the chemotherapy, an anti-CD40 antibody such as SEA-CD40 is administered on day 3 of the first cycle of the anti-CD40 antibody, and pembrolizumab is administered on day 8 of the first cycle of pembrolizumab, wherein day 1 of the chemotherapy cycle, the anti-CD40 antibody cycle, and the pembrolizumab cycle starts on the same day.

[0177] In a preferred embodiment, the combination therapy includes a chemotherapy administered on day 1, day 8, and day 16 of a 28 day cycle, an anti-CD40 antibody such as SEA-CD40 administered on day 3 of a 28 day cycle, and pembrolizumab administered on day 8 of a 42 day cycle. In some embodiments, the combination therapy includes a chemotherapy administered on day 1, day 8, and day 16 of a 28 day cycle, an anti-CD40 antibody such as SEA-CD40 administered on day 3 of a 28 day cycle, and pembrolizumab administered on day 8 of a 21 day cycle. In a preferred embodiment, the combination therapy includes a chemotherapy administered on day 1, day 8, and day 15 of a 28 day cycle, an anti-CD40 antibody such as SEA-CD40 administered on day 3 of a 28 day cycle, and pembrolizumab administered on day 8 of a 42 day cycle. In some embodiments, the combination therapy includes a chemotherapy administered on day 1, day 8, and day 15 of a 28 day cycle, an anti-CD40 antibody such as SEA-CD40 administered on day 3 of a 28 day cycle, and pembrolizumab administered on day 8 of a 21 day cycle. In some embodiments, the chemotherapy includes both gemcitabine and paclitaxel. In some embodiments, paclitaxel is nab-paclitaxel, e.g., ABRAXANE®.

[0178] In one aspect, the disclosure relates to treating a pancreatic cancer with a combination of chemotherapy, pembrolizumab, and SEA-CD40, wherein the chemotherapy is administered on days 1, 8, and 15 of each 28-day cycle, wherein SEA-CD40 is administered on day 3 of each 28-day cycle, and wherein pembrolizumab is administered on day 8 of each 42-day cycle. In some embodiments, the chemotherapy consists of gemcitabine and nab-paclitaxel (ABRAXANE®). In some embodiments, SEA-CD40 is administered intravenously. In some embodiments, SEA-CD40 is administered subcutaneously. In some embodiments, pembrolizumab is administered at 400 mg. In some embodiments, pembrolizumab is administered at 200 mg. In some embodiments, SEA-CD40 is administered at 10 .Math.g/kg. In some embodiments, SEA-CD40 is administered at 30 .Math.g/kg. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the pancreatic cancer is metastatic pancreatic ductal adenocarcinoma (PDAC).

[0179] In another aspect, this disclosure relates to treating a pancreatic cancer with a combination of chemotherapy, an anti-PD-1 antibody, and SEA-CD40, wherein the chemotherapy is administered on days 1, 8, and 15 of each each 28-day cycle, wherein SEA-CD40 is administered on day 3 of each 28-day cycle, and wherein the anti-PD-1 antibody is administered on day 8 of each 42-day cycle. In some embodiments, the chemotherapy consists of gemcitabine and nab-paclitaxel (ABRAXANE®). In some embodiments, SEA-CD40 is administered intravenously. In some embodiments, SEA-CD40 is administered subcutaneously. In some embodiments, SEA-CD40 is administered at 10 .Math.g/kg. In some embodiments, SEA-CD40 is administered at 30 .Math.g/kg. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the pancreatic cancer is metastatic pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, pembrolizumab is administered at 400 mg. In some embodiments, pembrolizumab is administered at 200 mg.

Cancer

[0180] A combination therapy of an anti-CD40 antibody such as SEA-CD40, pembrolizumab, and chemotherapy can be used for treating various types of cancer including, e.g., a solid tumor or a blood cancer. In some embodiments, the cancer is melanoma, breast cancer, including metastatic breast cancer, lung cancer, including non-small cell lung cancer, pancreatic cancer, lymphoma; colorectal cancer; or renal cancer. In some embodiments, the cancer is a melanoma; a breast cancer, including metastatic breast cancer; a lung cancer, including a non-small cell lung cancer; or pancreatic cancer. In some embodiments, the pancreatic cancer is a pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the PDAC is metastatic.

[0181] Pancreatic cancer has one of the highest mortality rates among all cancers and is the fourth most common cause of adult cancer death in the United States with an estimated 42,470 cases per year. See Nieto et al., The Oncologist, 13:562-576 (2008); and Cancer Facts and Figures, American Cancer Society (2009). It accounts for about 3% of all newly diagnosed cancers in the United States each year. However, almost double that number cancer patients, about 6%, die from pancreatic cancer. See Cancer Facts and Figures, American Cancer Society (2009). The high mortality rate from pancreatic cancer is a result of the high incidence of metastatic disease at the time of diagnosis. As a result, only 5%-15% of patients are candidates present with tumors are amenable to resection. See Nieto et al, The Oncologist, 13:562-576 (2008).

[0182] In a preferred embodiment, the combination therapy of an anti-CD40 antibody such as SEA-CD40, pembrolizumab, and chemotherapy can be used for treating pancreatic cancer. In some embodiments, the pancreatic cancer is metastatic pancreatic ductal adenocarcinoma (PDAC).

[0183] In some embodiments, the combination therapy of an anti-CD40 antibody such as SEA-CD40, pembrolizumab, and chemotherapy is used to treat tumors that are known to be immune responsive, particularly if the cancer expresses low levels of CD40 or does not detectably express CD40. Immune responsive cancers include, e.g., melanoma; bladder cancer; lung cancer, e.g., small cell lung cancer and non-small cell lung cancer; ovarian cancer; kidney cancer; pancreatic cancer; breast cancer; cervical cancer; head and neck cancer, prostate cancer; glioblastoma; non-hodgkin lymphoma; chronic lymphocytic leukemia; hepatocellular carcinoma; and multiple myeloma.

[0184] In some embodiments, the combination therapy of an anti-CD40 antibody such as SEA-CD40, pembrolizumab, and chemotherapy is used to treat solid tumors. In a further embodiment, SEA-CD40 is used to treat blood cancers, e.g., lymphoma, including non-Hodgkin lymphoma and Hodgkin lymphoma; chronic lymphocytic leukemia; or multiple myeloma.

[0185] The present disclosure also provides methods of manufacturing the combination therapies for various uses as described herein. The combination therapy can be included in a container, pack, kit, or dispenser together with instructions for administration.

[0186] Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

EXAMPLES

[0187] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Treating Cancer Patients With a Combination of SEA-CD40, Pembrolizumab, and Chemotherapy

[0188] Treatment of pancreatic cancer is being assessed with the combination of chemotherapy, SEA-CD40, and pembrolizumab. Chemotherapy is given on day 1 to stimulate antigen release, followed by SEA-CD40 on day 3. Waiting 1-2 days after chemotherapy before giving SEA-CD40 allows for antigen release to occur from tumor cells as a consequence of chemotherapy. This timing is anticipated to enhance the potential for SEA-CD40 to lead to an anti-tumor immune response. Specifically, SEA-CD40 can stimulate antigen update and presentation--and thus is expected to be most effective in the setting of increased levels of circulating antigen. Additionally, waiting 1-2 days after chemotherapy before administering SEA-CD40 may mitigate the potential for synergistic toxicity. Pembrolizumab is given on day 8. Giving pembrolizumab after SEA-CD40 may be beneficial, as this timing mitigates the potential for pembrolizumab to bind to immune cells with subsequent immune depletion arising from enhanced clearance of pembrolizumab-bound cells by SEA-CD40. SEA-CD40 is dosed at a level that results in significant immune stimulation in humans (e.g. 10 .Math.g/kg or 30 .Math.g/kg).

[0189] Background: SEA-CD40 is an investigational non-fucosylated, humanized 1gG1 monoclonal antibody directed against CD40, a co-stimulatory receptor expressed on antigen-presenting cells (APCs). Activation of CD40 on APCs upregulates cytokine production and co-stimulatory receptors, enhancing tumor antigen presentation to T cells. Preclinical data indicate that treatment of pancreatic ductal adenocarcinoma (PDAC) with chemotherapy in conjunction with a CD40 agonist could enhance antigen presentation and initiate an antitumor immune response (Byrne KT and Vonderheide RH, Cell Rep 2016;15, 2719-32). An ongoing Phase 1 study (SGNS40-001) is evaluating SEA-CD40 as monotherapy and in combination with pembrolizumab in patients with advanced solid or hematologic malignancies. A new cohort is enrolling to evaluate the combination of SEA-CD40, gemcitabine, nab-paclitaxel, and pembrolizumab in metastatic PDAC. ABRAXANEⓇ is a brand name of paclitaxel containing albumin-bound paclitaxel.

[0190] Methods: The cohort consists of patients with metastatic PDAC who have had no prior therapy for metastatic disease. Patients must be 18 years old or older, with (neo)adjuvant therapy completed > 4 months prior to enrollment, ECOG (Eastern Cooperative Oncology Group) status less than or equals to 1, adequate renal, hepatic, and hematologic function, and measurable disease per RECIST v 1.1 criteria. A standard regimen of gemcitabine and nab-paclitaxel on Days 1, 8, and 15 of each 28-day cycle is administered with SEA-CD40 intravenously (IV) on Day 3. Pembrolizumab is administered every 42 days starting on Day 8. The primary objective is to evaluate the antitumor activity of the administration regimen, and the secondary objectives are to assess the safety and tolerability of SEA-CD40 and pembrolizumab by pharmacokinetic analysis. Efficacy endpoints are confirmed according to RECIST (Response Evaluation Criteria in Solid Tumors) ORR (objective response rate/overall response rate) per investigator (primary), disease control rate (response or stable disease ~16 weeks), duration of response, PFS (progression-free survival), and OS (objective response/overall response). Disease is assessed every 8 weeks using RECIST (Response Evaluation Criteria in Solid Tumors) and immune-based RECIST (iRECIST). Treatment continues until occurrence of unacceptable toxicity, progressive disease per iRECIST, consent withdrawal, or study closure. Assessment of dose-limiting toxicity will occur initially in groups of 6 patients to identify the recommended phase 2 dose of SEA-CD40 for the cohort. Table 3 below illustrates days of administration of chemotherapy, SEA-CD40, and pembrolizumab for the initial 84 days (12 weeks; 3 cycles of 28-day cycle for chemotherapy and SEA-CD40; 2 cycles of 42-day cycle for pembrolizumab). The days of administration of the following days after day 84 follow the same scheme.

TABLE-US-00003 Chemotherapy (gemcitabine and nab-paclitaxel); 28 day-cycle SEA-CD40; 28 day-cycle Pembrolizumab; 42 day-cycle Day 1 1st cycle 1st administration Day 3 1st cycle administration Day 8 1st cycle 2nd administration 1st cycle administration Day 15 1st cycle 3rd administration Day 29 2nd cycle 1st administration Day 31 2nd cycle administration Day 36 2nd cycle 2nd administration Day 43 2nd cycle 3rd administration Day 50 2nd cycle administration Day 57 3rd cycle 1st administration Day 59 3rd cycle administration Day 64 3rd cycle 2nd administration Day 71 3rd cycle 3rd administration

Example 2: Murine Tumor Models With Concomitant or Staggered Dosing of a SEA-CD40 Surrogate and/or an Anti-mPD-1 Surrogate Antibody

[0191] Mouse models have been proven to be very useful in assessing efficacy and mechanisms of cancer therapeutics. Study of SEA-CD40 in murine cancer models has been difficult because SEA-CD40 does not recognize murine CD40. Therefore, to assess the activity of the non-fucosylated anti-CD40 antibodies, syngeneic murine tumor models were developed. The murine functional equivalents of human IgG1 and human FcγRIII/CD16 are murine IgG2a and murine FcyRIV, respectively, and binding of murine IgG2a to murine FcyRIV mediates antibody-dependent cellular cytotoxicity (ADCC). See, e.g., Bruhns, Blood 119:5640-5649 (2012) and Nimmeriahn et al., Immunity 23:41-51 (2005). The rat antibody 1C10 was used to generate a surrogate of SEA-CD40. See, e.g., Heath et al., Eur. J. Immunol. 24:1828-1834 (1994). Briefly, the VL and VH gene fragments of a rat monoclonal antibody can recognize murine CD40. The 1C10 antibody were cloned in-frame 5′ to murine Ckappa and murine IgG2a CH1-CH2-CH3 fragments, respectively. Expression of the resulting genes in CHO cells generated a chimeric 1C10 antibody with rat VL and VH domains and murine light and heavy chain domains of the IgG2a isotype (mIgG2a 1C10). mIgG2a 1C10 was expressed in the presence of 2-fluorofucose in the CHO cell growth medium using the methods described in U.S. Pat.Application Publication No. US 2017/0333556 A1, to generate a non-fucosylated form of mIgG2a 1C10 (mIgG2a SEA 1C10, or SEA-m1C10).

[0192] The single agent activity of SEA-m1C10 or an anti-mPD-1 surrogate antibody (“anti-PD1”), and a combination thereof, were investigated in solid and syngeneic tumor models. Based on SEA-CD40′s mechanism (e.g., enhanced activation of antigen presenting cells, and subsequent induction of an amplified anti-tumor T cell response), SEA-m1C10 was administered prior to the initial treatment with the anti-mPD-1 surrogate antibody.

[0193] Stock solutions of the antibodies were diluted to the appropriate concentration and then injected into animals in a volume of 100 .Math.l. Final dosages were 1 mg/kg for SEA-m1C10, and 1 mg/kg for the anti-mPD-1 surrogate antibody. Tumor length, tumor width, and mouse weight were measured throughout the experimental period, and tumor volume was calculated. Euthanasia was performed when tumor volume of a mouse reached 1000 mm.sup.3.

CT26 Colon Cancer Model

[0194] The combinatorial activity of the SEA-m1C10 antibody and the anti-mPD-1 surrogate antibody was tested in a CT26 colon cancer model, which is responsive to anti-mPD-1 surrogate antibody treatment. BALB/c mice were implanted with the CT26 syngeneic tumor cell line subcutaneously in the flank of mice on day 0. When the mean tumor size (measured using the formula: Volume (mm.sup.3) = 0.5 * Length * Width.sup.2, wherein length is the longer dimension and width is the shorter dimension) reached 100 mm.sup.3, mice were randomly placed into a control group G1 and three treatment groups G2-G4 (5 mice per group).

[0195] In one experiment, the treatment group mice were administered intraperitoneally with either a single agent (the anti-mPD-1 surrogate antibody (G2) or SEA-m1C10 (G3)), or a combination thereof (G4) on the same day. The administration frequency was once every three days for a total of three treatments. The control group mice (G1) were untreated. Median tumor volume of the mice are shown in FIG. 1A.

[0196] Alternatively, mice in treatment groups G3 and G4 were administered with 3 doses of SEA-m1C10 three days apart (e.g., within the period from day 9 to day 15). On the last day of SEA-m1C10 treatment (e.g., on day 15), the first dose of the anti-mPD-1 surrogate antibody was administered to the G2 and G4 group mice, which then received 2 additional doses three days apart (e.g., within the period from day 15 to day 21). The control group mice (G1) were untreated. Median tumor volume of the mice are shown in FIG. 1B.

[0197] The results showed that SEA-m1C10 did not exhibit any anti-tumor effect when administered alone. As shown in FIG. 1A, no combinatorial activity, or even an antagonistic activity, was observed when SEA-m1C10 and the anti-mPD-1 surrogate antibody were administered concomitantly. However, the anti-tumor activity was enhanced when SEA-m1C10 was administered in a staggered manner with the anti-mPD-1 surrogate antibody (FIG. 1B). The above results indicate that the timing of administration of these agents is important to achieve a therapeutic benefit. In addition, the results are in line with the proposed mechanism of action of SEA-CD40.

A20 Disseminated Lymphoma Model

[0198] The combinatorial activity of the SEA-m1C10 antibody and the anti-mPD-1 surrogate antibody was also tested in an A20 lymphoma model. BALB/c mice were injected intravenously with A20 cells which established a disseminated lymphoma in about 2-4 weeks. A20 disseminated lymphoma model was initiated in immune-competent female BALB/c mice by injecting 1 × 10.sup.6 A20 cells per mouse intravenously (IV). Mice were randomly placed into a control group G1 and three treatment groups G2-G4 (6 mice per group).

[0199] In one experiment, the treatment group mice were administered with antibodies at 3 mg/kg intraperitoneally (i.p.) on a q3dx3 schedule (once every three days for a total of three treatments) starting on day 7 post tumor cell inoculation. The control group mice were untreated. All mice were monitored for weight loss and symptoms of tumor burden, e.g., ascites in the peritoneum. The tumor burden was further verified after sacrificing the mice. FIG. 2A shows the survival curves of the control group mice (G1), mice treated with the anti-mPD-1 surrogate antibody (G2), SEA-m1C10 (G3), and a combination thereof on the same days (G4).

[0200] In a different experiment, an A20 lymphoma model was established via subcutaneous methodology. Specifically, tumors were allowed to grow to about 100 mm.sup.3 and mice in treatment groups G3 and G4 were administered with 3 doses of SEA-m1C10 three days apart (e.g., within the period from day 4 to day 10). On the last day of SEA-m1C10 treatment, the first dose of the anti-mPD1 surrogate antibody was administered (e.g., on day 10) to the G2 and G4 group mice, which then received 2 additional doses three days apart (e.g., within the period from day 10 to day 16). The control group mice (G1) were untreated. Mean tumor volume of the mice are shown in FIG. 2B.

[0201] As shown in FIG. 2A, mice treated with SEA-m1C10 (G3) significantly extended animal survival, as compared to that of the control group mice (G1). Further, concomitant administration of SEA-m1C10 and the anti-mPD1 surrogate antibody in a disseminated model of A20 did not exhibit any anti-tumor activity and even appeared antagonistic when administered together. In contrast, as shown in FIG. 2B, the anti-tumor activity was enhanced when SEAm1C10 was administered in a staggered manner with the anti-mPD-1 surrogate antibody. For example, while both the anti-mPD-1 surrogate antibody (G2) and SEA-m1C10 (G3) delayed tumor progression with 4/6 and ⅚ animals achieving a complete response (CR), respectively, the combinatorial activity of these two agents was striking, driving complete tumor regression in 6/6 animals (G4).

RENCA Renal Cell Carcinoma Model

[0202] The combinatorial activity of the SEA-m1C10 antibody with the anti-mPD1 surrogate antibody was also tested in a subcutaneous RENCA renal cell syngeneic model. BALB/c mice were implanted with the RENCA syngeneic tumor cell line subcutaneously in the flank of mice on day 0. When the mean tumor size (measured using the formula: Volume (mm.sup.3) = 0.5 * Length * Width.sup.2, wherein length is the longer dimension and width is the shorter dimension) reached 100 mm.sup.3, mice were randomly placed into a control group G1 and three treatment groups G2-G4 (5 mice per group).

[0203] In one experiment, the treatment group mice were administered intraperitoneally with either a single agent (the anti-mPD-1 surrogate antibody (G2) or SEA-m1C10 (G3)), or a combination thereof (G4) on the same day. The administration frequency was once every three days for a total of three treatments. The control group mice (G1) were untreated. Mean tumor volume of the mice are shown in FIG. 3A.

[0204] Alternatively, mice in treatment groups G3 and G4 were administered with 3 doses of SEA-m1C10 three days apart (e.g., within the period from day 5 to day 11). On the last day of SEA-m1C10 treatment (e.g., on day 11), the first dose of the anti-mPD-1 surrogate antibody was administered to the G2 and G4 group mice, which then received 2 additional doses three days apart (e.g., within the period from day 9 to day 15). The control group mice (G1) were untreated. Mean tumor volume of the mice are shown in FIG. 3B.

[0205] As shown in FIG. 3A, the results showed that concomitant administration of SEAm1C10 and the anti-mPD-1 surrogate antibody did not exhibit any anti-tumor activity and even appeared antagonistic when administered together. As shown in FIG. 3B, while both SEAm1C10 and the anti-mPD-1 surrogate antibody delayed tumor progression, the combinatorial activity of these two agents administered in a staggered manner improved anti-tumor activity and resulted in tumor growth delay.

OTHER EMBODIMENTS

[0206] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.