Patient population suitable for IL23 antagonist therapy

文档序号：1894278 发布日期：2021-11-26 浏览：19次中文

阅读说明：本技术 适合于il23拮抗剂疗法的患者群体 (Patient population suitable for IL23 antagonist therapy ) 是由 Q·周于 2020-03-26 设计创作，主要内容包括：本公开提供了用于通过测量白细胞介素-22结合蛋白的血清水平和/或干扰素-γ的血清水平而选择患有适合于用抗白细胞介素-23疗法治疗的炎症性病症(比如炎症性肠病)的患者亚群或受试者的方法。另外,所述方法可用于鉴定患有适合于用抗IL-23疗法和/或抗IFN-γ疗法治疗的炎症性障碍(比如银屑病、银屑病关节炎、类风湿性关节炎、强直性脊柱炎)的患者亚群。(The present disclosure provides methods for selecting a subpopulation of patients or subjects having an inflammatory disorder (such as inflammatory bowel disease) suitable for treatment with an anti-interleukin-23 therapy by measuring serum levels of interleukin-22 binding protein and/or serum levels of interferon-gamma. In addition, the methods can be used to identify a subpopulation of patients having an inflammatory disorder (such as psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis) suitable for treatment with an anti-IL-23 therapy and/or an anti-IFN- γ therapy.)

1.A method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, comprising:

(a) measuring the serum level of interleukin-22 binding protein (IL-22BP) in the subject;

(b) comparing the serum level of IL-22BP in the subject to the serum level of IL-22BP in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and

2. The method of claim 1, wherein the anti-IL-23 agent is brazimab.

3. The method of claim 1, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

4. The method of claim 3, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

5. The method of claim 1, wherein the inflammatory disorder is refractory to Tumor Necrosis Factor (TNF) therapy.

6. The method of claim 1, wherein the serum level of interleukin-22 binding protein is less than 359 pg/mL.

7. The method of claim 1, further comprising determining that the subject has an inflammatory disorder, wherein the inflammatory disorder is determined by performing a physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner.

8. The method of claim 1, further comprising administering an anti-IL-23 agent in an amount effective to treat the inflammatory disorder.

9. The method of claim 8, wherein the anti-IL-23 agent is brazimab.

10. The method of claim 8, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

11. The method of claim 8, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

12. The method of claim 11, wherein the treatment is administered in the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

13. A method of treating an interleukin-23 (IL-23) -mediated inflammatory disorder in a patient, comprising administering to the patient an effective amount of an anti-IL-23 agent if the patient is determined to have a serum level of IL-22BP that is lower than the level of IL-22BP in a control sample, wherein the control sample is obtained from one or more individuals not suffering from an inflammatory disorder.

14. The method of claim 13, wherein the anti-IL-23 agent is brazimab.

15. The method of claim 13, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

16. The method of claim 15, wherein the inflammatory bowel disease is crohn's disease or ulcerative colitis.

17. The method of claim 13, wherein the inflammatory disorder is refractory to Tumor Necrosis Factor (TNF) therapy.

18. The method of claim 13, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

19. The method of claim 13, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

20. The method of claim 19, wherein the treatment is administered in the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

21. A method of selecting at least one member of a subpopulation of patients having Inflammatory Bowel Disease (IBD) suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subpopulation of patients has IBD that is refractory to treatment with Tumor Necrosis Factor (TNF), the subpopulation of patients has IBD that has not been treated for this purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising:

(a) measuring the serum level of interleukin-22 binding protein (IL-22BP) in a patient with IBD;

(b) comparing the serum IL-22BP level in the IBD patient to the IL-22BP serum level in a control, wherein the IL-22BP serum level in the control is any one of: a serum level of IL-22BP in an individual not suffering from IBD, an average level of IL-22BP in a plurality of individuals not suffering from IBD, or an average level of IL-22BP in a plurality of individuals suffering from IBD; and

(c) selecting the patient as having IBD suitable for treatment with an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in the control.

22. The method of claim 21, wherein the anti-IL-23 agent is brazimab.

23. The method of claim 21, wherein the member of the subpopulation of IBD patients is a crohn's disease patient or an ulcerative colitis patient.

24. The method of claim 21, wherein the serum level of IL-22BP in the control is an average IL-22BP value in a plurality of individuals having IBD.

25. The method of claim 24, wherein the IBD is crohn's disease or ulcerative colitis.

26. The method of claim 21, wherein the patient population having IBD is a patient population having IBD that is refractory to TNF therapy.

27. The method of claim 21, further comprising determining that the subject has inflammatory bowel disease, wherein the inflammatory bowel disease is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner.

28. The method of claim 21, further comprising administering an anti-IL-23 agent in an amount effective to treat inflammatory bowel disease.

29. The method of claim 28, wherein the anti-IL-23 agent is brazimab.

30. The method of claim 28, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

31. The method of claim 28, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

32. The method of claim 31, wherein the treatment is administered in the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

33. A method of treating a patient who is a member of a subpopulation of patients of Inflammatory Bowel Disease (IBD) who are suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subpopulation of patients has IBD that is refractory to treatment with Tumor Necrosis Factor (TNF), the subpopulation of patients has IBD that has not been treated for this purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising: administering an effective amount of an anti-IL-23 agent if the serum level of IL-22BP in the patient is lower than the serum level of IL-22BP in a control, wherein the control is any one of: a serum level of IL-22BP in an individual not suffering from IBD, an average level of IL-22BP in a plurality of individuals not suffering from IBD, or an average level of IL-22BP in a plurality of individuals suffering from IBD.

34. The method of claim 33, wherein the anti-IL-23 agent is brazimab.

35. The method of claim 33, wherein the patient has crohn's disease or ulcerative colitis.

36. The method of claim 33, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

37. The method of claim 33, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

38. The method of claim 37, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

39. A method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent from a population of patients having an inflammatory disorder refractory to tumor necrosis factor treatment, comprising:

(a) measuring the serum level of interleukin-22 binding protein (IL-22BP) in the subject;

(b) comparing the serum IL-22BP level in the subject to an IL-22BP serum level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder refractory to tumor necrosis factor treatment; and

40. The method of claim 39, wherein the anti-IL-23 agent is brazimab.

41. The method of claim 39, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

42. The method of claim 41, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

43. The method of claim 39, wherein the subject selected as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent has a serum level of IL-22BP of less than 359 pg/mL.

44. The method of claim 39, further comprising determining that the subject has an inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy, wherein the inflammatory disorder that is refractory to TNF therapy is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner.

45. The method of claim 39, further comprising administering an anti-IL-23 agent in an amount effective to treat an inflammatory disorder refractory to Tumor Necrosis Factor (TNF) therapy.

46. The method of claim 45, wherein the anti-IL-23 agent is brazimab.

47. The method of claim 45, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

48. The method of claim 45, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

49. The method of claim 48, wherein said treatment is administered at the following amount of said anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

50. A method of treating a subject having an inflammatory disorder refractory to tumor necrosis factor therapy, comprising: administering an effective amount of an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder refractory to tumor necrosis factor treatment.

51. The method of claim 50, wherein the anti-IL-23 agent is brazimab.

52. The method of claim 50, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

53. The method of claim 52, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

54. The method of claim 50, wherein the subject has a serum level of IL-22BP that is less than 359 pg/mL.

55. The method of claim 50, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

56. The method of claim 50, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

57. The method of claim 56, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

58. A method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, comprising:

(a) measuring serum interferon-gamma (IFN- γ) levels in the subject;

(b) comparing the serum IFN- γ level in the subject to the serum IFN- γ level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and

(c) selecting the subject as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent if the serum IFN- γ level in the subject is higher than the serum IFN- γ level in the control.

59. The method of claim 58, wherein the anti-IL-23 agent is brazimab.

60. The method of claim 58, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

61. The method of claim 60, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

62. The method of claim 58, wherein the selected subject has a serum interferon- γ concentration greater than 15 pg/mL.

63. The method of claim 58, wherein the inflammatory disorder is refractory to tumor necrosis factor therapy.

64. The method of claim 58, further comprising determining that the subject has an inflammatory disorder, wherein the inflammatory disorder is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner.

65. The method of claim 58, further comprising administering an anti-IL-23 agent in an amount effective to treat the inflammatory disorder.

66. The method of claim 65, wherein the anti-IL-23 agent is brazimab.

67. The method of claim 65, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

68. The method of claim 65, wherein said treatment is administered at the following amount of said anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

69. The method of claim 68, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

70. A method of treating a subject having an inflammatory disorder, comprising: administering to the subject an effective amount of an anti-IL-23 agent if the serum interferon-gamma level in the subject is greater than the serum interferon-gamma level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder.

71. The method of claim 70, wherein the anti-IL-23 agent is brazimab.

72. The method of claim 70, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

73. The method of claim 72, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

74. The method of claim 70, wherein the inflammatory disorder is refractory to tumor necrosis factor therapy.

75. The method of claim 70, wherein the selected subject has a serum interferon- γ concentration greater than 15 pg/mL.

76. The method of claim 70, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

77. The method of claim 70, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

78. The method of claim 77, wherein said treatment is administered at the following amount of said anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

79. A method of selecting at least one member of a subpopulation of patients having Inflammatory Bowel Disease (IBD) suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subpopulation of patients has IBD that is refractory to treatment with Tumor Necrosis Factor (TNF), the subpopulation of patients has IBD that has not been treated for this purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising:

(a) measuring serum interferon-gamma (IFN- γ) levels in a patient with IBD;

(b) comparing the serum IFN- γ level in the IBD patient to the serum IFN- γ level in a control, wherein the serum IFN- γ level in the control is any one of: a serum IFN- γ level in an individual not suffering from IBD, an average level of IFN- γ in a plurality of individuals not suffering from IBD, or an average level of IFN- γ in a plurality of individuals suffering from IBD; and

(c) selecting the patient as having IBD suitable for treatment with an anti-IL-23 agent if the serum IFN- γ level in the subject is higher than the serum IFN- γ level in the control.

80. The method of claim 79, wherein the anti-IL-23 agent is brazimab.

81. The method of claim 79, wherein the member of the subgroup of patients with IBD is a Crohn's disease patient or an ulcerative colitis patient.

82. The method of claim 79 wherein the serum IFN- γ level in the control is an average of IFN- γ levels in a plurality of individuals having IBD.

83. The method of claim 82, wherein the IBD is Crohn's disease or ulcerative colitis.

84. The method of claim 79, wherein said population of patients having IBD is said population of patients having IBD refractory to TNF therapy.

85. The method of claim 83, wherein the selected subject has a serum interferon- γ concentration greater than 15 pg/mL.

86. The method of claim 79, further comprising determining that the subject has inflammatory bowel disease, wherein the inflammatory bowel disease is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner.

87. The method of claim 79, further comprising administering an anti-IL-23 agent in an amount effective to treat said inflammatory bowel disease.

88. The method of claim 87, wherein the anti-IL-23 agent is brazimab.

89. The method of claim 87, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

90. The method of claim 87, wherein said treatment is administered at the following amount of said anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

91. The method of claim 90, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

92. A method of treating a patient who is a member of a subpopulation of patients having Inflammatory Bowel Disease (IBD) who are suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subpopulation of patients has IBD that is refractory to tumor necrosis factor treatment, the subpopulation of patients has IBD that has not been treated for this purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising: administering an effective amount of an anti-IL-23 agent if the serum interferon-gamma (IFN- γ) level in the patient is higher than the serum IFN- γ level in a control, wherein the control is any one of: serum IFN- γ levels in an individual not suffering from IBD, average levels of IFN- γ in a plurality of individuals not suffering from IBD, or average levels of IFN- γ in a plurality of individuals suffering from IBD.

93. The method of claim 92, wherein the anti-IL-23 agent is brazimab.

94. The method of claim 92, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

95. The method of claim 92 wherein said patient has a serum interferon- γ concentration of greater than 15 pg/mL.

96. The method of claim 92, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

97. The method of claim 92, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

98. The method of claim 97, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

99. A method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent from a population of patients having an inflammatory disorder refractory to treatment with Tumor Necrosis Factor (TNF), comprising:

(a) measuring serum interferon-gamma (IFN- γ) levels in the subject;

(b) comparing the serum IFN- γ level in the subject to the serum IFN- γ level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and

100. The method of claim 99, wherein the anti-IL-23 agent is brazimab.

101. The method of claim 99, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

102. The method of claim 101, wherein the inflammatory bowel disease is crohn's disease or ulcerative colitis.

103. The method of claim 99, wherein the selected subject has a serum interferon- γ concentration greater than 15 pg/mL.

104. The method of claim 99, further comprising determining that the subject has an inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy, wherein the inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner.

105. The method of claim 99, further comprising administering an anti-IL-23 agent in an amount effective to treat an inflammatory disorder refractory to Tumor Necrosis Factor (TNF) therapy.

106. The method of claim 105, wherein the anti-IL-23 agent is brazimab.

107. The method of claim 105, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

108. The method of claim 105, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

109. The method of claim 108, wherein the treatment is administered in the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

110. A method of treating a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subject is a member of a population of patients having an inflammatory disorder refractory to tumor necrosis factor treatment, the method comprising: administering an effective amount of an anti-IL-23 agent if the serum IFN- γ level in the subject is greater than the serum IFN- γ level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder.

111. The method of claim 110, wherein the anti-IL-23 agent is brazimab.

112. The method of claim 110, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

113. The method of claim 112, wherein the inflammatory bowel disease is crohn's disease or ulcerative colitis.

114. The method of claim 110, wherein the subject has a serum interferon- γ concentration greater than 15 pg/mL.

115. The method of claim 110, wherein the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml.

116. The method of claim 110, wherein the treatment is administered in the following amount of the anti-IL-23 agent and at the following interval: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months.

117. The method of claim 116, wherein the treatment is administered at the following amount of the anti-IL-23 agent and at the following interval: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months.

118. A method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, comprising:

(a) measuring the serum level of interleukin-22 binding protein (IL-22BP), interferon-gamma (IFN-gamma), or both IL-22BP and IFN-gamma in the subject;

(b) comparing the serum level of IL-22BP, IFN- γ, or both IL-22BP and IFN- γ in the subject to the serum level of IL-22BP, IFN- γ, or both IL-22BP and IFN- γ in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and

(c) selecting the subject as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in the control, the serum level of IFN- γ in the subject is higher than the serum level of IFN- γ in the control, or the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in the control and the serum level of IFN- γ in the subject is higher than the serum level of IFN- γ in the control.

119. The method of claim 118, wherein the anti-IL-23 agent is brazimab.

120. The method of claim 118, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

121. The method of claim 118, wherein the inflammatory bowel disease is crohn's disease or ulcerative colitis.

122. The method of claim 118, wherein the selected subject has a serum interferon- γ concentration greater than 15 pg/mL.

123. The method of claim 118, further comprising determining that the subject has an inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy, wherein the inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner.

124. The method of claim 118, further comprising administering an anti-IL-23 agent in an amount effective to treat an inflammatory disorder refractory to Tumor Necrosis Factor (TNF) therapy.

125. The method of claim 124, wherein the anti-IL-23 agent is brazimab.

126. The method of claim 14, further comprising administering to the patient an effective amount of an anti-IL-23 agent if the serum IFN- γ level in the patient is greater than the serum IFN- γ level in the control and optionally the IL-22BP serum level in the patient is less than the IL-22BP serum level in the control.

127. The method of claim 126, wherein the anti-IL-23 agent is brazimab.

128. The method of claim 126, wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis.

129. The method of claim 128, wherein the inflammatory bowel disease is crohn's disease or ulcerative colitis.

FIELD

The present disclosure relates generally to the field of selecting a patient subpopulation suitable for treating an inflammatory disorder, and more particularly for treating inflammatory bowel disease.

Background

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family, which is strongly expressed in both Crohn's Disease (CD) and Ulcerative Colitis (UC). Consistent with these observations, IL-22 has been shown to have pro-inflammatory properties, but this cytokine has also been shown to play an organ protective role in the liver and lung. Sonnenberg et al, J. exp. Med. 207:1293-1305 (2010); Cobleigh et al, Am. J. Pathol. 182:21-28 (2013). IL-22 is induced by various environmental and endogenous signals, such as IL-23. Several genome-wide association studies identified IL23R as a susceptibility gene, consistent with the role of the IL-23 pathway in Inflammatory Bowel Disease (IBD). IL-22 is up-regulated in the intestine of IBD patients. IL-22 is generally capable of promoting mucosal healing in the intestine; however, when uncontrolled, it can lead to intestinal morbidity. Therefore, close control of IL-22 activity is essential. IL-22 binding protein (IL-22BP) plays this role in control by specifically binding IL-22 and preventing its binding to membrane-bound IL-22 receptor 1 (IL-22R 1). IL-22 binds to IL-22BP with 20-1000 times greater affinity than the former membrane-bound IL-22R 1. IL-22 and IL-22BP exhibit an inverse expression pattern in a mouse model with tissue damage in the intestine: IL-22BP is most highly expressed in the colon during homeostasis and tissue repair, whereas IL-22 is most highly expressed during peak periods of tissue damage. Thus, careful regulation of IL-22 and IL-22BP may control intestinal homeostasis, but the role of IL-22BP in human IBD is uncertain.

Interferon-gamma (IFN- γ) has been identified as a classical Th1 cytokine that promotes inflammatory responses, but its role in various inflammatory disorders is unclear. Furthermore, IFN- γ is known to play a role in both innate and adaptive immune responses; it also plays a role in defense against microbial infections, including viral and some bacterial and protozoal infections. IFN- γ is stimulated by IL-23, a key mediator of the inflammatory response, and has been identified as a causative factor in inflammatory bowel disease (Ito et al, Clin. and Exper. Immunol. 146:330-338 (2006)). Consistent with these observations, Strober et al reported that IFN- γ and IL-17/IL-22 are important cytokines involved in Inflammatory Bowel Disease (IBD). In addition, aryltuzumab (Fontolizumab), an anti-IFN- γ antibody, was shown to reduce the severity of crohn's disease. Ghosh et al, Gut 55:1071-1073 (2006). Harbour et al, Proc. Natl. Acad. Sci. USA 112:7061-7066 (2015) when analyzing Th 17T cells and showing a subset of Th17 cells to differentiate into Th1 cells (IFN-. gamma.is a defined marker for this Th1 cell) appear to confirm this pro-inflammatory view of IFN-. gamma.. However, Harbour et al also disclose that Sta4 and T-beta are required for the expression of colitis, rather than IFN-gamma, for induction of colitis. This latter finding by Harbour et al is not surprising given the reported anti-inflammatory properties of IFN- γ in early stages of colitis. Sheikh et al, J. Immunol. 184:4069-4073 (2010). Thus, accumulating information about the role of IFN- γ in the inflammatory response establishes that cytokines have significant effects, but the exact nature of the effect has not yet been fully elucidated.

Interleukin (IL) -23 is a proinflammatory cytokine involved in the pathogenesis of various inflammatory disorders including, but not limited to, Crohn's Disease (CD), Ulcerative Colitis (UC), psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. IL23 induces T cells to express a variety of inflammatory genes, including IL-17A, IL-17A receptor, TNF- α, and GM-CSF. The main known role of IL-23 is to drive the differentiation of T helper Th17 cells as well as macrophages, Natural Killer (NK) cells, dendritic cells and innate lymphocytes, leading to the upregulation of IL-17, IL-22, TNF- α, GM-CSF and IFN- γ and downregulation of IL-10.

IL-23, a member of the IL-12 cytokine family, is a heterodimeric cytokine that is effective in inducing proinflammatory cytokines. IL-23 is associated with the heterodimeric cytokine interleukin 12 (IL-12), both of which share a common subunit of p 40. In IL-23, a unique p19 subunit is covalently bound to a p40 subunit. In IL-12, the unique subunit is p35 (Oppmann et al, Immunity, 2000, 13: 713-715). IL-23 is expressed by antigen presenting cells (such as dendritic cells and macrophages) in response to activating stimuli (such as CD40 linkages, Toll-like receptor agonists, and pathogens). IL-23 binds to a heterodimeric receptor comprising an IL-12R β 1 subunit (which is shared with the IL-12 receptor) and a unique receptor subunit IL-23R.

IL-23 acts on both activating and memory T cells and promotes the survival and expansion of the T cell subset Th 17. Th17 cells produce proinflammatory cytokines including IL-6, IL-17, TNF α, IL-22, and GM-CSF. IL-23 also acts on natural killer cells, dendritic cells and macrophages to induce expression of proinflammatory cytokines. Unlike IL-23, IL-12 induces differentiation of naive CD4+ T cells into mature IFN- γ producing Th1 effector cells and induces NK and cytotoxic T cell function by stimulating IFN- γ production. Th1 cells driven by IL-12 were previously thought to be a subset of pathogenic T cells in many autoimmune diseases; however, recent animal studies in models of inflammatory bowel disease, psoriasis, inflammatory Arthritis and multiple sclerosis, in which individual contributions of IL-12 and IL-23 were assessed, have firmly established that IL-23 (but not IL-12) is a key driver of autoimmune/inflammatory disease (Ahern et al, Immun. Rev. 2008226: 147-159; Cua et al, Nature 2003421: 744-748; Yago et al, Arthritis Res and ther. 20079 (5): R96). IL-12 is believed to play a key role in the development of protective innate and adaptive immune responses against a number of intracellular pathogens and viruses, as well as in tumor immune surveillance. See Kastelein, et al, Annual Review of Immunology, 2007, 25: 221-42, Liu, et al, Rheumatology, 2007, 46(8): 1266-73, Bowman et al, Current Opinion in efficiencies Diseases, 200619: 245-52, Fieschi and Casanova, Eur. J. immunol. 200333: 1461-4, Meeran et al, mol. Cancer ther. 20065: 825-32, Langwoski et al, Nature 2006442: 461-5. Thus, IL-23 specific inhibition (either by IL-12 or the consensus p40 subunit) is expected to have a higher safety profile than dual inhibition of IL-12 and IL-23.

IL-23 is associated with the heterodimeric cytokine interleukin 12 (IL-12), both of which share a common subunit of p 40. In IL-23, a unique p19 subunit is covalently bound to a p40 subunit. In IL-12, the unique subunit is p35 (Oppmann et al, Immunity, 2000, 13: 713-715). The IL-23 heterodimeric protein is secreted. Like IL-12, IL-23 is expressed by antigen presenting cells (such as dendritic cells and macrophages) in response to activation stimuli (such as CD40 junctions, Toll-like receptor agonists, and pathogens). IL-23 binds to a heterodimeric receptor comprising an IL-12R131 subunit (which is shared with the IL-12 receptor) and a unique receptor subunit IL-23R. The IL-12 receptor is composed of IL-12W and IL-12R 132. IL-23 binds to its heterodimeric receptor and signals STATs 1,3, 4 and 5 by JAK2 and Tyk2 (Parham et al, J. Immunol. 2002, 168: 5699-708). Subunits of the receptor are mainly co-expressed on activated or memory T cells and natural killer cells, and also at lower levels on dendritic cells, monocytes, macrophages, microglia, keratinocytes and synovial fibroblasts. IL-23 and IL-12 act on different T cell subsets and play significantly different roles in vivo.

IL-23 acts on both activating and memory T cells and promotes the survival and expansion of the T cell subset Th 17. Th17 cells produce proinflammatory cytokines including IL-6, IL-17, TNF α, IL-22, and GM-CSF. IL-23 also acts on natural killer cells, dendritic cells and macrophages to induce expression of proinflammatory cytokines. Unlike IL-23, IL-12 induces differentiation of CD4+ T cells into mature IFN- γ producing Th1 effector cells and induces NK and cytotoxic T cell function by stimulating IFN- γ production. Th1 cells driven by IL-12 were previously thought to be a subset of pathogenic T cells in many autoimmune diseases, however, recent animal studies in models of inflammatory bowel disease, psoriasis, inflammatory Arthritis and multiple sclerosis, in which a single contribution of IL-12 relative to IL-23 was assessed, have firmly established that IL-23 (but not IL-12) is a key driver of autoimmune/inflammatory diseases (Ahern et al, Immun. Rev. 2008226: 147-748; Cua et al, Nature 2003421: 744-748; Yago et al, Arthritis Res and Ther. 20079 (5): R96). IL-12 is believed to play a key role in the development of protective innate and adaptive immune responses against a number of intracellular pathogens and viruses, as well as in tumor immune surveillance. See Kastelein, et al, Annual Review of Immunology, 2007, 25: 221-42, Liu, et al, Rheumatology, 2007, 46(8): 1266-73, Bowman et al, Current Opinion in efficiencies Diseases, 200619: 245-52, Fieschi and Casanova, Eur. J. immunol. 200333: 1461-4, Meeran et al, mol. Cancer ther. 20065: 825-32, and Langowski et al, Nature 2006442: 461-5. Thus, IL-23 specific inhibition (either by IL-12 or the consensus p40 subunit) should have potentially higher safety profiles than dual inhibition of IL-12 and IL-23.

Use of antibodies that inhibit human IL-23 (such as at least an antibody that binds to the unique p19 subunit or both p19 and p40 subunits of IL-23) while passing IL-12 through an IL-23 specific antagonist should provide efficacy equal to or greater than that of an IL-12 antagonist or a p40 antagonist without the potential risks associated with IL-12 inhibition. Selection of murine, humanized and phage display antibodies for inhibition of recombinant IL-23 has been described; see, e.g., U.S. Pat. No. 7,491,391, WIPO publication W01999/05280, WO2007/0244846, WO2007/027714, WO2007/076524, WO2007/147019, W02008/103473, WO2008/103432, W02009/043933, and W02009/082624. Fully human therapeutics that are capable of inhibiting native human IL-23 are highly specific for the target, particularly in vivo. Complete inhibition of the target in vivo may result in lower doses of the formulation, less frequent and/or more efficient dosing, which in turn results in reduced cost and increased efficiency.

In view of the continuing prevalence of inflammatory disorders (such as inflammatory bowel disease) in the human population, and in view of the incomplete understanding of inflammation as a biological process, there clearly continues to be a need for: it is useful to identify subjects or patients who are suitable for a particular treatment for such disorders and diseases, particularly when the treatment is effective and cost effective.

SUMMARY

The present disclosure provides effective methods for identifying a patient population or subpopulation suitable for anti-cytokine therapy in the form of an anti-IL-23 agent for treating an inflammatory disorder. The data disclosed herein indicate that subjects with inflammatory disorders exhibiting elevated IFN- γ levels are more likely to be responsive to treatment with an anti-interleukin-23 agent, such as an anti-IL-23 antibody, e.g., Brazikumab (Brazikumab). Accordingly, the present disclosure provides methods for selecting a subject having an inflammatory disorder responsive to treatment with anti-interleukin-23 therapy, such as inflammatory bowel disease, by measuring serum levels of interleukin-22 binding protein and/or serum levels of IFN- γ. In addition, the method can be used to identify a subpopulation of patients having an inflammatory disorder (such as psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis) suitable for treatment with an anti-IL-23 therapy and/or an anti-IFN-gamma therapy.

One aspect of the present disclosure is to provide a method of selecting a subject having an inflammatory disorder amenable to treatment with an anti-interleukin-23 (anti-IL-23) agent, based on the pro-inflammatory properties of IL-22 and not based on its organ protective function, comprising: (a) measuring the serum level of interleukin-22 binding protein (IL-22BP) in the subject; (b) comparing the serum level of IL-22BP in the subject to the serum level of IL-22BP in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and (c) selecting the subject as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in the control. It is apparent from consideration of this aspect of the disclosure that these methods are based in part on the pro-inflammatory properties of IL-22 and its antagonism provided by IL-22BP, and not at all on the organ protective function of IL-22 or any antagonism of that function by IL-22 BP. Therefore, these methods do not prevent the public from using all IL-22 BP. It is also apparent that the methods disclosed herein are based, at least in part, on the role of IFN- γ in immune responses (including autoimmune responses) and not at all on the role of IFN- γ in defending against infection. Thus, these methods do not prevent all public use of IFN- γ.

In some embodiments of this aspect of the disclosure, the anti-IL-23 agent is brakuzumab comprising 6 complementarity determining regions that specifically bind IL-23 (i.e., HCDR1 of SEQ ID NO: 91, HCDR2 of SEQ ID NO: 92, HCDR3 of SEQ ID NO: 93, LCDR1 of SEQ ID NO: 62, LCDR2 of SEQ ID NO: 63, and LCDR3 of SEQ ID NO: 64). In some embodiments, the brakuzumab comprises V of SEQ ID NO 153_HSequence and V of SEQ ID NO 154_LAnd (4) sequencing. In some embodiments, the brakuzumab comprises a fusion with a heavy chain constant region153 of SEQ ID NO_HSequence and V of SEQ ID NO 154 fused to the light chain constant region_LAnd (4) sequencing. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. Some embodiments are provided wherein the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the inflammatory disorder is refractory to Tumor Necrosis Factor (TNF) therapy. In some embodiments, the serum level of interleukin-22 binding protein is less than 359 pg/mL. The present disclosure further contemplates embodiments of the disclosed methods wherein the serum level of IL-22BP in a brekumab-null responder is at least 359 pg/mL or between 359 pg/mL and 6,000 pg/mL. In some embodiments, the IL-22BP level in a brekumab non-responder is 359-5,000 pg/mL, 359-4,000 pg/mL, 359-2,500 pg/mL, 359-1,000 pg/mL, 359-500 pg/mL, 400-6,000 pg/mL, 400-5,000 pg/mL, 400-4,000 pg/mL, 400-2,500 pg/mL, 400-1,000 pg/mL, 400-500 pg/mL, 500-6,000 pg/mL, 500-5,000 pg/mL, 500-4,000 pg/mL, 500-2,500/mL, 500-1,000 pg/mL, 750-6,000 pg/mL, 750,000/750, 5,000 pg/mL, 750,000 pg/mL, 750/750, 750-4,000 pg/mL, 750/mL, 750,000 pg 4,000/mL, 750/000, 750-type 2,500 pg/mL, 750-type 1,000 pg/mL, 1,000-type 6,000 pg/mL, 1,000-type 5,000 pg/mL, 1,000-type 4,000 pg/mL, 1,000-type 2,500 pg/mL, 1,500-type 6,000 pg/mL, 1,500-type 5,000 pg/mL, 1,500-type 4,000 pg/mL, 1,500-type 2,500 pg/mL, 2,000-type 6,000 pg/mL, 2,000-type 5,000 pg/mL, or 2,000-type 2,500 pg/mL. In some embodiments, the method further comprises determining that the subject has an inflammatory disorder, wherein the inflammatory disorder is determined by performing a physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner. In some embodiments, the method further comprises administering an anti-IL-23 agent in an amount effective to treat the inflammatory disorder, such as wherein the anti-IL-23 agent is brauzumab.

In some embodiments of the foregoing aspects of the disclosure, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, an anti-IL 23 agent (e.g., a heterodimer specific anti-IL-23 antibody) is administered to achieve a serum concentration of at least 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, 500 ng/ml, or 990 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months, 299 mg every 4-8 months, 1100 mg every 8-14 months, 300-1100 mg or 1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

A related aspect of the disclosure relates to a method of treating an interleukin-23 (IL-23) -mediated inflammatory disorder in a patient, comprising administering to the patient an effective amount of an anti-IL-23 agent if the patient is determined to have a serum level of IL-22BP that is lower than the level of IL-22BP in a control sample, wherein the control sample is obtained from one or more individuals not suffering from an inflammatory disorder. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the inflammatory disorder is refractory to Tumor Necrosis Factor (TNF) therapy. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

Another aspect of the present disclosure relates to a method of selecting at least one member of a subpopulation of patients having Inflammatory Bowel Disease (IBD) suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, from a population of patients having IBD, wherein the subpopulation of patients has IBD that is refractory to treatment with Tumor Necrosis Factor (TNF), the subpopulation of patients has IBD that has not been treated for the purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising: (a) measuring the serum level of interleukin-22 binding protein (IL-22BP) in a patient with IBD; (b) comparing the serum IL-22BP level in the IBD patient to the IL-22BP serum level in a control, wherein the IL-22BP serum level in the control is any one of: a serum level of IL-22BP in an individual not suffering from IBD, an average level of IL-22BP in a plurality of individuals not suffering from IBD, or an average level of IL-22BP in a plurality of individuals suffering from IBD; and (c) selecting the patient as having IBD responsive to treatment with an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in a control, and optionally, (d) administering to the patient an effective amount of an anti-IL-23 agent. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the member of the subpopulation of IBD patients is a crohn's disease patient or an ulcerative colitis patient. In some embodiments, the serum level of IL-22BP in the control is the average level of IL-22BP in a plurality of individuals having IBD, and some of these embodiments are embodiments in which IBD is crohn's disease or ulcerative colitis. In some embodiments, the population of patients with IBD is a population of patients with IBD that is refractory to TNF therapy. In some embodiments of this aspect of the disclosure, the method further comprises determining that the subject has inflammatory bowel disease, wherein inflammatory bowel disease is determined by performing a physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner. In some embodiments, the method further comprises administering an anti-IL-23 agent in an amount effective to treat inflammatory bowel disease, such as wherein the anti-IL-23 agent is brauzumab. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

In a related aspect, the present disclosure provides a method of treating a patient who is a member of a subpopulation of Inflammatory Bowel Disease (IBD) patients eligible for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subpopulation of patients has IBD that is refractory to Tumor Necrosis Factor (TNF) treatment, the subpopulation of patients has IBD that has not been treated for this purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising: administering an effective amount of an anti-IL-23 agent if the serum level of IL-22BP in the patient is lower than the serum level of IL-22BP in a control, wherein the control is any one of: a serum level of IL-22BP in an individual not suffering from IBD, an average level of IL-22BP in a plurality of individuals not suffering from IBD, or an average level of IL-22BP in a plurality of individuals suffering from IBD. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the patient has crohn's disease or ulcerative colitis. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

Yet another aspect of the present disclosure relates to a method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent from a population of patients having an inflammatory disorder refractory to tumor necrosis factor treatment, comprising: (a) measuring the serum level of interleukin-22 binding protein (IL-22BP) in the subject; (b) comparing the serum IL-22BP level in the subject to the IL-22BP serum level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder refractory to tumor necrosis factor treatment; and (c) selecting the subject as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in the control. In some embodiments, the anti-IL-23 agent is brazimab. Embodiments are also contemplated wherein the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, a subject selected as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent has a serum level of IL-22BP of less than 359 pg/mL. In some embodiments of this aspect of the disclosure, the method further comprises determining that the subject has an inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy, wherein the inflammatory disorder that is refractory to TNF therapy is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner. In some embodiments, the method further comprises administering an anti-IL-23 agent in an amount effective to treat an inflammatory disorder refractory to Tumor Necrosis Factor (TNF) treatment, such as wherein the anti-IL-23 agent is brakumab. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

A related aspect of the disclosure relates to a method of treating a subject having an inflammatory disorder refractory to tumor necrosis factor therapy, comprising: administering an effective amount of an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder refractory to tumor necrosis factor treatment. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the subject has a serum level of IL-22BP that is less than 359 pg/mL. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

Yet another aspect of the present disclosure provides a method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, comprising: (a) measuring serum interferon-gamma (IFN- γ) levels in the subject; (b) comparing the serum IFN- γ level in the subject to the serum IFN- γ level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and (c) selecting the subject as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent if the serum IFN- γ level in the subject is higher than the serum IFN- γ level in the control. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the selected subject has a serum interferon- γ concentration greater than 15 pg/mL. In some embodiments, the inflammatory disorder is refractory to tumor necrosis factor therapy. In some embodiments of this aspect of the disclosure, the method further comprises determining that the subject has an inflammatory disorder, wherein the inflammatory disorder is determined by performing a physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner. In some embodiments, the method further comprises administering an anti-IL-23 agent in an amount effective to treat the inflammatory disorder. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

In a related aspect, the present disclosure provides a method of treating a subject having an inflammatory disorder, comprising: administering to the subject an effective amount of an anti-IL-23 agent if the serum interferon-gamma level in the subject is greater than the serum interferon-gamma level in a control, wherein the control is one or more individuals not suffering from the inflammatory disorder. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the inflammatory disorder is refractory to tumor necrosis factor therapy. In some embodiments, the selected subject has a serum interferon- γ concentration greater than 15 pg/mL. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

Another aspect of the present disclosure relates to a method of selecting at least one member of a subpopulation of patients having Inflammatory Bowel Disease (IBD) suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, from a population of patients having IBD, wherein the subpopulation of patients has IBD that is refractory to treatment with Tumor Necrosis Factor (TNF), the subpopulation of patients has IBD that has not been treated for the purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising: (a) measuring serum interferon-gamma (IFN- γ) levels in a patient with IBD; (b) comparing the serum IFN- γ level in the IBD patient to the serum IFN- γ level in a control, wherein the serum IFN- γ level in the control is any one of: a serum IFN- γ level in an individual not suffering from IBD, an average level of IFN- γ in a plurality of individuals not suffering from IBD, or an average level of IFN- γ in a plurality of individuals suffering from IBD; and (c) selecting the patient as having IBD suitable for treatment with an anti-IL-23 agent if the serum IFN- γ level in the subject is higher than the serum IFN- γ level in the control. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the member of the subpopulation of IBD patients is a crohn's disease patient or an ulcerative colitis patient. In some embodiments, the serum IFN- γ level in the control is an average of IFN- γ levels in a plurality of individuals having IBD. In some embodiments, the IBD in the control is crohn's disease or ulcerative colitis. In some embodiments, the population of patients with IBD is a population of patients with IBD that is refractory to TNF therapy. In some embodiments, the selected subject has a serum interferon- γ concentration greater than 15 pg/mL. In some embodiments of the methods of this aspect of the disclosure, the method further comprises determining that the subject has inflammatory bowel disease, wherein inflammatory bowel disease is determined by performing a physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner. In some embodiments, the method further comprises administering an anti-IL-23 agent in an amount effective to treat inflammatory bowel disease. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

A related aspect relates to a method of treating a patient who is a member of a subpopulation of Inflammatory Bowel Disease (IBD) patients eligible for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subpopulation of patients has IBD that is refractory to tumor necrosis factor treatment, the subpopulation of patients has IBD that has not been treated for this purpose, and/or the subpopulation of patients is intolerant to treatment with an anti-TNF agent, the method comprising: administering an effective amount of an anti-IL-23 agent if the serum interferon-gamma (IFN- γ) level in the patient is greater than the IFN- γ level in a control, wherein the control is any one of: serum IFN- γ levels in an individual not suffering from IBD, average levels of IFN- γ in a plurality of individuals not suffering from IBD, or average levels of IFN- γ in a plurality of individuals suffering from IBD. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the selected subject has a serum interferon- γ concentration greater than 15 pg/mL. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

Yet another aspect of the present disclosure relates to a method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent from a population of patients having an inflammatory disorder refractory to treatment with Tumor Necrosis Factor (TNF), comprising: (a) measuring serum interferon-gamma (IFN- γ) levels in the subject; (b) comparing the serum IFN- γ level in the subject to the serum IFN- γ level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and (c) selecting the subject as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent if the serum IFN- γ level in the subject is higher than the serum IFN- γ level in the control. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the selected subject has a serum interferon- γ concentration greater than 15 pg/mL. In some embodiments of this aspect of the disclosure, the method further comprises determining that the subject has an inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy, wherein the inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner. In some embodiments, the method further comprises administering an anti-IL-23 agent in an amount effective to treat an inflammatory disorder refractory to Tumor Necrosis Factor (TNF) therapy. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

Another aspect of the disclosure concerns a method of treating a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, wherein the subject is a member of a population of patients having an inflammatory disorder refractory to tumor necrosis factor treatment, the method comprising: administering an effective amount of an anti-IL-23 agent if the serum IFN- γ level in the subject is greater than the serum IFN- γ level in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the selected subject has a serum interferon- γ concentration greater than 15 pg/mL. In some embodiments, the anti-IL-23 agent is administered in an amount sufficient to achieve a serum concentration of 12.5 ng/ml to 1,000 ng/ml. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-54 mg every 0.5-1.5 months, 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months, 300-1100 mg every 8-14 months, or 300-1100 mg every 4-12 months. In some embodiments, the treatment is administered in the following amounts of the anti-IL-23 agent and at the following intervals: 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150 mg every 4-6 months or 700 mg every 4-8 months. In some embodiments, the amount and interval is 21 mg per month, 70 mg per 3 months, 210 mg per 6 months, or 700 mg per 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or at 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month. In some embodiments, the dose of the anti-IL-23 agent administered to the subject is 70 mg, e.g., 70 mg, 140 mg, 219 mg, 420 mg, or 700 mg per dose.

Another aspect of the disclosure relates to a method of selecting a subject having an inflammatory disorder suitable for treatment with an anti-interleukin-23 (anti-IL-23) agent, comprising: (a) measuring serum levels of interleukin-22 binding protein (IL-22BP), interferon-gamma (IFN-gamma), or both IL-22BP and IFN-gamma in the subject; (b) comparing the serum level of IL-22BP, IFN- γ, or both IL-22BP and IFN- γ in the subject to the serum level of IL-22BP, IFN- γ, or both IL-22BP and IFN- γ in a control, wherein the control is one or more individuals not suffering from an inflammatory disorder; and (c) selecting the subject as having an inflammatory disorder suitable for treatment with an anti-IL-23 agent if the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in a control, the serum level of IFN- γ in the subject is higher than the serum level of IFN- γ in a control, or the serum level of IL-22BP in the subject is lower than the serum level of IL-22BP in a control and the serum level of IFN- γ in the subject is higher than the serum level of IFN- γ in a control. In some of these embodiments, the anti-IL-23 agent is brazimab. In some of these embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some of these embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis. In some embodiments, the selected subject has a serum interferon- γ concentration greater than 15 pg/mL. In some of these embodiments, the method further comprises determining that the subject has an inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy, wherein the inflammatory disorder that is refractory to Tumor Necrosis Factor (TNF) therapy is determined by physical examination, by reviewing a medical record of the subject, or by consulting a medical practitioner. In some of these embodiments, the method further comprises administering an anti-IL-23 agent in an amount effective to treat an inflammatory disorder refractory to Tumor Necrosis Factor (TNF) therapy. In some of these embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the method further comprises administering to the patient an effective amount of an anti-IL-23 agent if the serum IFN- γ level in the patient is greater than the serum IFN- γ level in the control and optionally the serum level of IL-22BP in the patient is less than the serum level of IL-22BP in the control. In some embodiments, the anti-IL-23 agent is brazimab. In some embodiments, the inflammatory disorder is inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, or ankylosing spondylitis. In some embodiments, the inflammatory bowel disease is crohn's disease or ulcerative colitis.

Other features and advantages of the present disclosure will be better understood by reference to the following detailed description (including the examples).

Brief Description of Drawings

FIG. 1 raw Optical Density (OD) readings of ELISA plates were performed on an M5 ELISA plate reader (Molecular Devices, Inc., San Jose CA) using Softmax software. Panel a provides the plate layout, panel B provides the raw OD values, and panel C provides the adjusted values for the assay. A1-G1 and A2-G2 are technical duplicates of the standard curve for human IL-22 BP. A3-A11, B3-B11, C3-C11, D3-D11, E3-E11, F3-F11, G3-G10, and H3-H10 are raw OD readings of serum samples from healthy men, healthy women, patients with Crohn's disease, patients with ulcerative colitis, and Medimone phase 2a clinical trial (MEDI2070-1147) placebo group. The raw OD readings were converted to IL-22BP serum concentrations in figures 2-8 below using Softmax software.

FIG. 2. IL-22BP standard curve consists of 7 calibration concentrations in the range of 100-6000 pg/ml. The accuracy (CV%) and accuracy (recovery%) of the ELISA was within the accepted ranges (CV% ≦ 20% and recovery% within the range of 80-120%), indicating the effectiveness of the assay run.

Figure 3 serum levels of IL-22BP from 10 healthy men were calculated based on the standard curve presented in figure 8, with the standard curve data presented in figure 2.

Figure 4 serum levels of IL-22BP from 10 healthy women were calculated based on the standard curve presented in figure 8, with the standard curve data presented in figure 2.

FIG. 5 serum levels of IL-22BP from 19 Crohn's disease patients were calculated based on the standard curve presented in FIG. 8, with the standard curve data presented in FIG. 2.

FIG. 6 serum levels of IL-22BP from 11 patients with ulcerative colitis were calculated based on the standard curve presented in FIG. 8, with the standard curve data presented in FIG. 2.

Figure 7 serum levels of IL-22BP from 20 crohn's disease patients (non-responders to anti-TNF α treatment) in the placebo group serum samples from the medimumne phase 2a trial (MEDI2070-1147) were calculated based on the standard curve presented in figure 8, with the standard curve data presented in figure 2.

FIG. 8 is a graph of the IL-22BP standard generated using Softmax software. IL-22BP concentration is expressed in pg/mL.

Fig. 9 summarizes the results from fig. 3-7. The data show that both the median and mean serum concentrations of IL-22BP in patients with Crohn's Disease (CD) and ulcerative colitis are higher than those in healthy or normal humans (male and female). Furthermore, IL-22BP levels in anti-TNF α non-responsive CD patients are lower than IL-22BP levels in healthy humans. The level of IL-22BP expressed in serum is expected to exhibit a polarization pattern in the brekumab responder versus the non-responder subpopulation, regardless of whether the brekumab responder and non-responder subpopulations are refractory to TNF-a treatment.

FIG. 10 serum IFN- γ levels in anti-TNF- α refractory Crohn's disease patients. Serum samples were obtained from a group of patients treated with brekumab in the Medimmune phase 2a trial (MEDI 2070-1147). Responders to brakumab in clinical trials were marked "0" in the "responders" column of panel a; non-responders to brazimab in clinical trials are identified with a "1" in this column as shown in panel B.

FIG. 11 ELISA results show that both the median and mean serum concentrations of IL-22BP in the Boletumab responder subpopulation (see panel B) were lower in patients with Crohn's disease than in CD patients with Boletumab non-responder (see panel A). Serum samples were obtained from the group of CD patients treated with brekumab in the Melimmune phase 2a trial (MEDI 2070-1147). All patients included in this phase 2a trial were non-responders to anti-TNF α treatment.

Detailed description of the invention

The present disclosure provides methods and materials for selecting a patient population or subpopulation suitable for anti-cytokine therapy, and more specifically anti-IL-23 therapy (including anti-IL-23 immunotherapy) to treat an inflammatory disorder. Based on the experimental data disclosed herein, and contrary to the conventional wisdom that IL-22BP will be elevated in patients who are responsive to anti-IL-23 therapy, there is provided a method of selecting patients suitable for anti-IL-23 therapy to treat an inflammatory disorder, which determines whether a serum sample of the patient shows a reduced level of interleukin-22 binding protein (IL-22BP) and/or an elevated level of interferon-gamma (IFN- γ).

The present disclosure further provides IL-23 antigen binding proteins, including molecules that antagonize IL-23, such as anti-IL-23 antibodies, antibody fragments, and antibody derivatives, e.g., antagonistic anti-IL-23 antibodies, antibody fragments, or antibody derivatives. Also provided are polynucleotides and derivatives and fragments thereof (which comprise a nucleic acid sequence encoding all or a portion of a polypeptide that binds to IL-23, e.g., a polynucleotide encoding all or a portion of an anti-IL-23 antibody, antibody fragment, or antibody derivative), plasmids and vectors comprising such nucleic acids, and cells or cell lines comprising such polynucleotides and/or vectors and plasmids. The provided methods include, for example, methods of making, identifying, or isolating an IL-23 antigen binding protein (such as an anti-IL-23 antibody), methods of determining whether a molecule binds to IL-23, methods of determining whether a molecule antagonizes IL-23, methods of making a composition (such as a pharmaceutical composition) comprising an IL-23 antigen binding protein, and methods for administering an IL-23 antigen binding protein to a subject (e.g., methods for treating a disorder mediated by IL-23 and antagonizing the biological activity of IL-23 in vivo or in vitro).

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used and their techniques related to, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of this disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification, unless otherwise indicated. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990). Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The terms used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as their laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques are available for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

The polynucleotide and protein sequences for the p19 subunit of human IL-23 (SEQ ID NOS: 144 and 145), the consensus p40 subunit (SEQ ID NOS: 146 and 147), the human IL-23 receptor heterodimer subunits IL-12R β 1 (SEQ ID NOS: 150 and 151), and IL-23R (SEQ ID NOS: 148 and 149) are known in the art. See, e.g., GenBank accession No. AB 030000; m65272, NM 005535, NM 144701, as are those from other mammalian species. Recombinant IL-23 and IL-23 receptor proteins (including single chain and Fc proteins) as well as cells expressing the IL-23 receptor have been described or may be obtained from commercial sources (see, e.g., Oppmann et al, Immunity, 2000, 13: 713-715; R & D Systems, Minneapolis, Minnesota; United States Biological, Swampscott, Massachusetts; WIPO publication No. WO 2007/076524). Native human IL-23 can be obtained from human cells (such as dendritic cells) using methods known in the art, including those described herein.

IL-23 is a heterodimeric cytokine comprising a distinct p19 subunit covalently bound to a common p40 subunit. The p19 subunit contains more than 4 up-down motifs of α -helices ("A", "B", "C" and "D") connected by 3 helical inner loops between the A and B helices, between the B and C helices and between the C and D helices, see Oppmann et al, Immunity, 2000, 13: 713-715-andBeyer et al, J Mol Biol, 2008.382 (4): 942-55. The a and D helices of the 4-helix bundle cytokine are believed to be involved in receptor binding. The p40 subunit contains 3 β -pleated sandwich domains D1, D2 and D3 (Lupardus and Garcia, J. mol. biol., 2008, 382: 931-.

The term "polynucleotide" includes both single-and double-stranded nucleic acids, and includes genomic DNA, RNA, mRNA, cDNA, or synthetic sources unrelated to sequences typically found in nature, or some combination thereof. An isolated polynucleotide comprising a particular sequence may comprise, in addition to the particular sequence, a coding sequence for up to 10 or even up to 20 other proteins or portions thereof, or may comprise operably linked regulatory sequences that control expression of the coding region of the nucleic acid sequence and/or may comprise vector sequences. The nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Modifications include base modifications (such as bromouridine and inosine derivatives), ribose modifications (such as 2',3' -dideoxyribose), and internucleotide linkage modifications (such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphorothioate aniline, phosphate aniline, and phosphoramidate).

The term "oligonucleotide" means a polynucleotide comprising 100 or fewer nucleotides. In some embodiments, the oligonucleotide is 10-60 bases in length. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. The oligonucleotides may be single-stranded or double-stranded, for example for use in the construction of mutant genes. The oligonucleotide may be a sense or antisense oligonucleotide. The oligonucleotide may include a detectable label for use in a detection assay, such as a radioactive label, a fluorescent label, a hapten or an antigenic label. Oligonucleotides may be used, for example, as PCR primers, cloning primers, or hybridization probes.

The term "polypeptide" or "protein" means a macromolecule having the amino acid sequence of a native protein (i.e., a protein produced by naturally occurring and non-recombinant cells); or which is produced by genetically engineered or recombinant cells and comprises a molecule having the amino acid sequence of a native protein or a molecule having one or more deletions, insertions and/or substitutions of amino acid residues of a native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" include IL-23 antigen binding proteins (such as antibodies) and sequences having one or more deletions, additions and/or substitutions of amino acid residues to the antigen binding protein sequence. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxy-terminal deletion, and/or an internal deletion as compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. In certain embodiments, fragments are about 5-500 amino acids in length. For example, a fragment can be at least 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of IL-23 antigen binding proteins, such as antibodies, useful fragments include, but are not limited to, one or more CDR regions, a variable domain of a heavy or light chain, a portion of an antibody chain, a portion of a variable region comprising less than 3 CDRs, and the like.

"amino acid" is given its normal meaning in the art. The 20 naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis, 2nd edition, (E.S. Golub and D.R. Gren, eds.), Sinauer Associates: Sunderland, Mass. (1991). Stereoisomers (e.g., D-amino acids) of 20 conventional amino acids, unnatural amino acids (such as α -, α -disubstituted amino acids, N-alkyl amino acids, and other non-conventional amino acids) may also be suitable components of the polypeptide. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid,. epsilon. -N, N, N-trimethyllysine,. epsilon. -N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine and other similar amino and imino acids (e.g., 4-hydroxyproline). In the polypeptide symbols used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, according to standard usage and convention.

The term "isolated protein" refers to a protein, such as an antigen binding protein (an example of which may be an antibody), that is purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. As used herein, "substantially pure" means that the molecular species being described is the predominant species present, i.e., on a molar basis, more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the target species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In certain embodiments, the substantially homogeneous substance has been purified to such an extent that contaminating species cannot be detected in the composition by conventional detection methods, and thus the composition consists of a single detectable macromolecular species.

"variants" of a polypeptide (e.g., an antigen binding protein, such as an antibody) comprise amino acid sequences in which one or more amino acid residues are inserted into, deleted from, and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins. A "derivative" of a polypeptide is a polypeptide that has been chemically modified in some way other than by an insertion, deletion or substitution variant (e.g., via conjugation to another chemical moiety).

The term "naturally-occurring" or "native" as used throughout the specification in connection with biological materials (e.g., polypeptides, nucleic acids, host cells, etc.) refers to materials found in nature, such as native human IL-23. In certain aspects, recombinant antigen binding proteins that bind native IL-23 are provided. In this context, a "recombinant protein" is a protein prepared using recombinant techniques (i.e., by expression of a recombinant nucleic acid as described herein). Methods and techniques for producing recombinant proteins are well known in the art.

The term "antibody" refers to an intact immunoglobulin of any isotype or fragment thereof that can compete with the intact antibody for specific binding to a target antigen, and includes, for example, chimeric, humanized, fully human, and bispecific antibodies. Antibodies are one type of antigen binding protein. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments and muteins thereof, examples of which are described below. A complete antibody will typically comprise at least two full length heavy chains and two full length light chains, but in some cases may comprise fewer chains, such as antibodies naturally occurring in camelidae that may comprise only heavy chains. Antibodies may be derived from only a single source, or may be "chimeric," i.e., different portions of an antibody may be derived from two different antibodies, as described further below. Antigen binding proteins, antibodies or binding fragments may be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

The term "functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) as used herein is an antigen-binding protein that comprises a portion of the antibody (regardless of how the portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain, but is capable of specifically binding to an antigen. Such fragments are biologically active in that they specifically bind to the target antigen and can compete with other antigen binding proteins (including whole antibodies) for specific binding to a given epitope. In one aspect, such a fragment will retain at least one CDR present in a full-length light or heavy chain, and in some embodiments will comprise a single heavy and/or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including whole antibodies. Fragments include, but are not limited to, immunologically functional fragments such as Fab, Fab ', F (ab')2, Fv, domain antibodies and single chain antibodies, and can be derived from any mammalian source, including, but not limited to, human, mouse, rat, camelid, or rabbit. It is further contemplated that a functional portion (e.g., one or more CDRs) of an antigen binding protein disclosed herein can be covalently bound to a second protein or small molecule to produce a therapeutic agent directed against a specific target in vivo, which has bifunctional therapeutic properties or has an extended serum half-life.

The term "competition," when used in the context of an antigen binding protein (e.g., a neutralizing antigen binding protein or neutralizing antibody), means competition between the antigen binding proteins as determined by an assay in which the antigen binding protein being tested (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., IL-23 protein or fragment thereof). Many types of competitive binding assays can be used, for example: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, Methods in enzymology 92: 242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol. 137: 3614-; use of¹²⁵I-labeled solid phase direct labeling of RIA (see, e.g., Morel et al, 1988, mol. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al, 1990, Virology 176: 546-552); and the direct-labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Generally, such assays involve the use of purified antigens bound to a solid surface or cells carrying any of these: an unlabeled test antigen binding protein and a labeled reference antigen binding protein.

Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of the test antigen binding protein. The antigen binding protein is typically tested for the presence of excess. Antigen binding proteins identified by competition assays (competitive antigen binding proteins) include antigen binding proteins that bind to the same epitope as a reference antigen binding protein and antigen binding proteins that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antigen binding protein to undergo steric hindrance. Typically, when a competing antigen binding protein is present in excess, it will inhibit specific binding of a reference antigen binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an antigen binding protein binds. Epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are generally retained when exposed to denaturing solvents, while epitopes formed from tertiary folds are generally lost when treated with denaturing solvents. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and can have specific three-dimensional structural characteristics and/or specific charge characteristics. Epitopes generally include at least 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 amino acids in a unique spatial conformation. Epitopes can be determined using methods known in the art.

IL-23 antigen binding proteins

"antigen binding protein" as used herein means a protein that specifically binds a particular target antigen; the antigen as provided herein is IL-23, particularly human IL-23, including native human IL-23. An antigen binding protein as provided herein interacts with at least a portion of the unique p19 subunit of IL-23, thereby detectably binding IL-23; but does not have any significant binding to IL-12 (e.g., the p40 and/or p35 subunits of IL-12), thereby "passing IL-12". Thus, the antigen binding proteins provided herein are capable of affecting IL-23 activity without the potential risks associated with inhibiting IL-12 or the p40 subunit shared by IL-12 and IL-23. Antigen binding proteins may affect the ability of IL-23 to interact with its receptor, for example by affecting IL-23 binding to the receptor, such as by interfering with receptor association. In particular, such antigen binding proteins reduce, inhibit, interfere with or modulate, in whole or in part, one or more biological activities of IL-23. Such inhibition or neutralization disrupts the biological response in the presence of the antigen binding protein as compared to the response in the absence of the antigen binding protein, and can be determined using assays known in the art and described herein. The antigen binding proteins provided herein inhibit IL-23-induced pro-inflammatory cytokine production, such as IL-23-induced IL-22 production in whole blood cells and IL-23-induced IFN- γ expression in NK and whole blood cells. The reduction in biological activity can be about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

The antigen binding protein may comprise a moiety that binds to an antigen and optionally a scaffold or framework moiety that enables the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., antigen binding portions of antibodies), antibody derivatives, and antibody analogs. The antigen binding protein may comprise an alternative protein scaffold or an artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of an antigen binding protein, as well as fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., Korndorfer et al, Proteins: Structure, Function, and Bioinformatics, (2003), Vol.53, No. 1: 121-. In addition, peptide antibody mimetics ("PAM") as well as scaffolds based on antibody mimetics that utilize a fibronectin component as a scaffold may be used.

Certain antigen binding proteins described herein are antibodies or are derived from antibodies. Such antigen binding proteins include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, single chain antibodies and fragments thereof, respectively. In some cases, the antigen binding protein is an immunological fragment of an antibody (e.g., Fab ', F (ab')2, or scFv). Various structures are further described and defined herein.

Certain antigen binding proteins provided can comprise one or more CDRs (e.g., 1, 2, 3,4, 5,6, or more CDRs) as described herein. In some cases, the antigen binding protein comprises (a) a polypeptide structure and (b) one or more CDRs inserted and/or attached to the polypeptide structure. The polypeptide structure may take a variety of different forms. For example, it may be or comprise the framework of a naturally occurring antibody or fragment or variant thereof, or may be substantially entirely synthetic. Examples of various polypeptide structures are described further below.

When dissociation equilibrium constant (K)_D) Less than or equal to 10^-8M, an antigen binding protein of the present disclosure is said to "specifically bind" to its target antigen. When K is_DAt least 5x10^-9M, the antigen binding protein binds specifically to the antigen with "high affinity", and when K_DAt least 5x10^-10M, the antigen binding protein specifically binds to the antigen with "very high affinity". In one embodiment, the antigen binding protein will be at 5x10^-12K of M_DBinds to human IL-23, while in yet another embodiment, it will be at 5x10^-13K of M_DAnd (4) combining. In another embodiment of the invention, the antigen binding protein has a size of 5x10^-12KD of M and about 5x10^-61/s K_off. In another embodiment, K_offIs 5x10^-71/s。

Another aspect provides an antigen binding protein having a half-life of at least 1 day in vitro or in vivo (e.g., when administered to a human subject). In one embodiment, the antigen binding protein has a half-life of at least 3 days. In another embodiment, the antibody or portion thereof has a half-life of 4 days or more. In another embodiment, the antibody or portion thereof has a half-life of 8 days or more. In another embodiment, the antibody, or antigen-binding portion thereof, is derivatized or modified such that it has a longer half-life compared to the underivatized or unmodified antibody. In another embodiment, the antigen binding protein contains a point mutation to increase serum half-life, such as described in WIPO publication No. WO 00/09560.

In embodiments in which the antigen binding protein is used for therapeutic applications, the antigen binding protein may reduce, inhibit, interfere with, or modulate one or more biological activities of IL-23, such as inducing the production of proinflammatory cytokines. IL-23 has many different biological effects, which can be measured in many different assays of different cell types; examples of such assays are known and provided herein.

Some of the antigen binding proteins provided have structures that are generally related to naturally occurring antibodies. The building blocks of these antibodies typically comprise one or more tetramers, each of which is composed of two identical polypeptide chain conjugates, although some species of mammal will also produce antibodies with only a single heavy chain. In a typical antibody, each pair or conjugate includes one full length "light" chain (in some embodiments about 25 kDa) and one full length "heavy" chain (in some embodiments about 50-70 kDa). Each individual immunoglobulin chain is composed of several "immunoglobulin domains," each consisting of approximately 90-110 amino acids and exhibiting a characteristic folding pattern. These domains are the basic units that make up antibody polypeptides. The amino-terminal portion of each chain typically includes a variable region responsible for antigen recognition. . The carboxy-terminal portion is evolutionarily more conserved than the other end of the chain, and is referred to as the "constant region" or "C-region". Human light chains are generally classified as kappa and lambda light chains, and each of these chains contains a variable region and a constant domain (CL 1). The z heavy chain is generally classified as a mu, delta, gamma, alpha or epsilon chain, and these chains define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including (but not limited to) IgG1, IgG2, IgG3, and IgG 4. The IgM subtypes include IgM and IgM 2. IgA subtypes include IgA1 and IgA 2. In humans, IgA and IgD isotypes contain 4 heavy chains and 4 light chains; IgG and IgE isotypes contain two heavy chains and two light chains; and IgM isotypes contain 5 heavy chains and 5 light chains. The heavy chain constant region (CH) typically comprises one or more domains that may be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. For example, IgG heavy chains each contain 3 CH regions domains designated CH1, CH2, and CH 3. The provided antibodies can have any of these isotypes and subtypes, e.g., IL-23 antigen binding proteins have the IgG1, IgG2, or IgG4 subtypes. If IgG4 is desired, it may also be desirable to introduce a point mutation at the hinge region (CPSCP- > CPPCP) (as described in Bloom et al, 1997, Protein Science 6: 407) to reduce the tendency to form intra-H chain disulfide bonds, which can lead to heterogeneity in IgG4 antibodies. The subclass switching method can be used to change antibodies provided herein that belong to one type to a different type. See, e.g., Lantto et al, 2002, Methods mol. biol. 178: 303-.

In full-length light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 more amino acids. See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989, New York: Raven Press. The variable regions of each light/heavy chain pair generally form antigen binding sites.

Variable region

The various heavy and light chain variable regions (or domains) provided herein are depicted in tables 1 and 2. Each of these variable regions may be attached to, for example, the heavy and light chain constant regions described above. Further, each of the heavy and light chain sequences so produced may be combined to form a complete antigen binding protein structure.

Providing an antigen binding protein comprising: at least one heavy chain variable region (VH) selected from VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, and VH16 and/or at least one light chain variable region (VL) selected from VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, and VL16, as shown in tables 1 and 2 below.

Each of the heavy chain variable regions listed in table 2 may be combined with any one of the light chain variable regions shown in table 1 to form an antigen binding protein. In some cases, the antigen binding protein includes at least one heavy chain variable region and/or one light chain variable region from those listed in tables 1 and 2. In some cases, the antigen binding protein includes at least two different heavy chain variable regions and/or light chain variable regions from those listed in tables 1 and 2. The various combinations of heavy chain variable regions may be combined with any of the various combinations of light chain variable regions.

In other cases, the antigen binding protein contains two identical light chain variable regions and/or two identical heavy chain variable regions. As one example, the antigen binding protein may be an antibody or immunologically functional fragment comprising two light chain variable regions and two heavy chain variable regions in a combination of light chain variable region pairs and heavy chain variable region pairs as listed in tables 1 and 2. Examples of such antigen binding proteins comprising two identical heavy and light chain variable regions include: antibody A VH14/VL 14; antibody B VH9/VL 9; antibody C VH10/VL 10; antibody D VH15/VL 15; antibody E VH1/VL1, antibody F VH11/VL 11; antibody G VH12/VL 12; antibody H VH13/VL 13; antibody I VH8/VL 8; antibody J VH3/VL 3; antibody K VH7/VL 7; antibody L VH4/VL 4; antibody M VH5/VL5 and antibody N VH6/VL 6.

Some antigen binding proteins provided comprise a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region and/or the light chain variable region comprises an amino acid sequence that differs from a sequence selected from the heavy chain variable region and/or the light chain variable region of tables 1 and 2 only at1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, wherein each such sequence difference is independently a deletion, insertion, or substitution of one amino acid. In some antigen binding proteins, the light and heavy chain variable regions comprise amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences provided in table 1 and table 2. Still other antigen binding proteins (e.g., antibodies or immunologically functional fragments) also include variant heavy chain region forms and/or variant light chain region forms as described herein.

The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more polynucleotides as determined by alignment and comparison of the sequences. "percent identity" means the percentage of residues that are identical between amino acids or nucleotides in the molecules being compared and is calculated based on the smallest molecule size in the molecules being compared.

TABLE 1

Exemplary variant light chain region sequences

TABLE 2

Exemplary variant heavy chain region sequences

For these calculations, the nulls in the alignment (if any) must be resolved by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in: computer Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press, Biocomputing information and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press, Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press, von Heinje, G.1987, Sequence Analysis in Molecular Biology, New York: Academic, Sequence Analysis, Primer Analysis, (Prime, M.M., and device in Molecular Biology, New York: examine, C.G.S., Press, (Prime, M.M.and device, N.J., 1998, sample J.S., S.D.S., 1988, and S.M.D., 1988, and S.M.S. 1988, sample Press, S.S. 3, and S.S.S.S. Press, Inc., S.S. 3, S.S. Appl.

In calculating percent identity, the sequences compared are aligned in a manner that gives the greatest match between the sequences. A Computer program for determining percent identity is the GCG package, which includes GAP (Devereux et al, 1984, Nucl. Acids Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to compare two polypeptides or polynucleotides for which a percentage of sequence identity is to be determined. The sequences are aligned to obtain the best match (the "matching span", as determined by the algorithm) for their corresponding amino acids or nucleotides. Gap opening penalties (calculated as 3x the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix used; "diagonals" are the scores or numbers assigned to each perfect amino acid match by a particular comparison matrix) and gap extension penalties (typically 1/10 times the gap opening penalty), as well as comparison matrices (such as PAM 250 or BLOSUM 62), are used in conjunction with the algorithm. In certain embodiments, the algorithm also uses a standard comparison matrix (for PAM 250 comparison matrix, see Dayhoff et al, 1978, Atlas of Protein Sequence and Structure 5: 345-.

The recommended parameters for determining percent identity of a polypeptide or nucleotide sequence using the GAP program are as follows: the algorithm is as follows: needleman et al, 1970, J. mol. biol.48: 443-; comparing the matrixes: BLOSUM 62 from Henikoff et al, 1992, supra; gap penalties: 12 (but no penalty for end gaps), gap length penalty: 4, similarity threshold: 0. certain alignment schemes for aligning two amino acid sequences can result in matching of only short regions of the two sequences, and this small aligned region can have very high sequence identity, even if there is no significant relationship between the two full-length sequences. Thus, if desired, the alignment method (GAP program) selected can be adjusted to produce an alignment of at least 50 contiguous amino acids across the polypeptide of interest. The heavy and light chain variable regions disclosed herein include consensus sequences derived from the group of related antigen binding proteins. The amino acid sequences of the heavy and light chain variable regions were analyzed for similarity. Set 4, one with kappa light chain variable regions (VH9/VL9, VH10/VL10, VH11/VL11, VH13/VL13, VH14/VL14 and VH15/VL15) and 3 with lambda light chain variable regions: lambda group 1 (VH5/VAS, VH6/VL6 and VH7/VL7), lambda group 2 (VH3/VL3 and VH4/VL4) and lambda group 3 (VH1/VL1 and VH2/VL 2). Representative light chain germline include VK1/A30 and VK 1/L19. Representative light chain lambda germline include VL1/1e, VL3/3p, VL5/5c and VL9/9 a. Representative heavy chain germline includes VH3/3-30, VH3/3-30.3, VH3/3-33, VH3/3-48, VH4/4-31 and VH 4/4-59. As used herein, "consensus sequence" refers to an amino acid sequence having conserved amino acids that are common among many sequences and variable amino acids that vary within a given amino acid sequence. The consensus sequences can be determined using standard phylogenetic analysis of the light and heavy chain variable regions corresponding to the IL-23 antigen binding proteins disclosed herein.

The light chain variable region consensus sequence of the kappa group is DX₁QX₂TQSPSSVSASVGDRVTITCRASQGX₃X₄SX₅WX₆AWYQQKPGX₇APX₈LLIYAASSLQSGVPSRFSGSX0SGTX₁₀FTLTISSLQPX₁₁DFATYX₁₂CQQANSFPFTFGPGTKVDX₁₃K (SEQ ID NO: 30); wherein X₁Is selected from I or S; x₂Is selected from M or L; x₃Selected from G or V and X₄Selected from S, F or I; x₅Selected from S or G; x₆Is selected from F or L; x₇Is selected from K or Q; x₈Selected from K, N or S; x₉Selected from G or V; x₁₀Selected from D or E, X₁₁Selected from E or A; x₁₂Selected from Y or F; and X₁₃Selected from I, V or F.

The light chain variable region consensus sequence of lambda group 1 is QPX₁LTQPPSASASLGASVTLTCTLX₂SGYSDYKVDWYQX₃RPGKGPRFVMRVGTGGX₄VGSKGX₅GIPDRFSVLGSGLNRX₆LTIKNIQEEDESDYHCGADHGSGX₇NFVYVFGTGTKVTVL (SEQ ID NO:61), wherein X₁Selected from V or E; x₂Is selected from N or S; x₃Selected from Q or L and X₄Is selected from I or T; x₅Selected from D or E; x₆Selected from Y or S; and X₇Selected from S or N.

The light chain variable region consensus sequence of lambda group 3 is QSVLTQPPVPSVSGAPGQRVTISCTGTSSSNX₁GAGYDVHWYQQX₂PGTAPKLLIYGSX₃NRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTX₄RLTVL (SEQ ID NO:139), wherein X₁Selected from T or I; x₂Is selected from V or L; x₃Selected from G or N and X₄Is selected from R or K.

The heavy chain variable region of kappa group has the consensus sequence QVQLQEGSGPGLVKPSQTLTCTVSGGSIX₁SGGYYWX₂WIRQHPGKGLEWIGX₃IX₄YSGX₅X₆YYNPSLKSRX₇TX₈SVDTSX₉NQFSLX₁₀LSSVTAADTAVYYCAX₁₁X₁₂RGX₁₃YYGMDVWGQGTTVTVSS (SEQ ID NO:140), wherein X₁Is selected from NOr S; x₂Selected from S or T; x₃Selected from Y or H; x₄Selected from Y or H; x₅Selected from S or N; x₆Selected from S or T; x₇Selected from V or I; x₈Is selected from I or M; x₉Is selected from K or Q; x₁₀Selected from K or S, X₁₁Is selected from R or K; x₁₂Selected from D or N; and X₁₃Selected from H, F or Y.

The common sequence of heavy chain variable regions of lambda set 1 is EVQLVESGGGLVQPGGSLRLSCX₁X₂SGFTFSX₃X₄SMNWVRQAPGKGLEWVSYISSX₅SSTX₆YX₇ADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARRIAAAGX₈X₉X₁₀YYYAX₁₁DVWGQGTTVTVSS (SEQ ID NO: 141); wherein X₁Is selected from A or V; x₂Is selected from A or V; x₃Selected from T or S; x₄Selected from Y or F; x₅Selected from S or R; x₆Is selected from R or I; x₇Selected from H, Y or I; x₈Is selected from P or G; x₉Selected from W or F; x₁₀Selected from G or H and X₁₁Is selected from M or L.

The heavy chain variable region consensus sequence of lambda set 2 is QVQLVESGGGVQPGRSLRLSCAASGFTFSSYX₁MHWVRQAPGKGLEWX₂X₃VISX₄DGSX₅KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERTTLSGSYFDYWGQGTLVTVSS (SEQ ID NO:142), wherein X₁Selected from G or A; x₂Is selected from V or L; x₃Selected from A or S; x₄Selected from F or H; and X₅Is selected from L or I.

The common sequence of heavy chain variable regions of lambda group 3 is QVQLVESGGGVQPGRSLRLSCAASGFTFSSYGMHWVRQACPGKGLEWVAVIYDGSNX₁YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYX₂SSWYPDAFDIWGQGTMVTVSS (SEQ ID NO:143), wherein X₁Selected from E or K and X₂Selected from T or S.

Complementarity determining region

Complementarity determining regions, or "CDRs," are embedded within the framework of the heavy and light chain variable regions, where they constitute the regions responsible for antigen binding and recognition. For example, variable domains of immunoglobulin chains of the same species typically exhibit similar overall structures; comprising relatively conserved Framework Regions (FR) joined by hypervariable CDR regions. The antigen binding protein may have 1, 2, 3,4, 5,6 or more CDRs. For example, the variable regions discussed above typically comprise 3 CDRs. The CDRs from the heavy and light chain variable regions are generally aligned by the framework regions to form a structure that specifically binds on the target antigen (e.g., IL-23). From N-terminus to C-terminus, both naturally occurring light and heavy chain variable regions generally conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. Exemplary CDR and FR regions of the light and heavy chain variable domains are highlighted in tables 1 and 2. It is recognized that the boundaries of the CDR and FR regions may differ from those highlighted. Numbering systems have been designed for assigning numbers to the amino acids occupying positions in each of these domains. These systems can be used to identify the complementarity determining regions and framework regions of a given antigen binding protein. The numbering system is defined in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH publication No. 91-3242, 1991 or Chothia & Lesk, 1987, J. mol. biol. 196: 901-. Other numbering systems for amino acids in immunoglobulin chains include IMGT (International ImMunogeGeneTiCs information System; Lefranc et al, Dev. Comp. lmmunol.2005, 29: 185-; and AHo (Honegger and Pluckthun, J. mol. biol. 2001, 309(3): 657-. The CDRs provided herein can be used not only to define the antigen binding domain of traditional antibody structures, but also to embed into a variety of other polypeptide structures, as described herein.

Antigen binding proteins disclosed herein are polypeptides in which one or more CDRs may be grafted, inserted, embedded, and/or linked. The antigen binding protein may have, for example, one heavy chain CDR1 ("CDRH 1") and/or one heavy chain CDR2 ("CDRH 2") and/or one heavy chain CDR3 ("CDRH 3") and/or one light chain CDR1 ("CDRL 1") and/or one light chain CDR2 ("CDRL 2") and/or one light chain CDR3 ("CDRL 3"). Some antigen binding proteins include both CDRH3 and CDRL 3. Embodiments typically utilize combinations of non-repetitive CDRs, e.g., antigen binding proteins are not typically made with two CDRH2 regions in one variable heavy chain region, etc. An antigen binding protein may comprise one or more amino acid sequences that are identical to the amino acid sequences of one or more CDRs presented in table 3 or differ only at1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently a deletion, insertion or substitution of one amino acid. The CDRs in some antigen binding proteins comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CDR sequences listed in table 3. In some antigen binding proteins, the CDRs are embedded in a "framework" region, which orients the CDRs so that the appropriate antigen binding properties of the CDRs are achieved.

TABLE 3

Exemplary CDRH and CDRL sequences

Provided herein are CDR1 regions comprising: amino acid residues 23-34 of SEQ ID Nos. 7 and 11; amino acid residues 24-34 of SEQ ID NOs 9, 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 23-36 of SEQ ID NOS 1,3 and 4; amino acid residues 31-35 of SEQ ID NOS 31, 33, 34, 38, 40, 44, 52, and 60 and amino acid residues 31-37 of SEQ ID NOS 46, 48, 50, 54, 56, and 58.

Providing a CDR2 region comprising: amino acid residues 50-56 of SEQ ID NOs 9, 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 50-61 of SEQ ID NO 7 and 11; amino acid residues 52-62 of SEQ ID NO 4; amino acid residues 50-65 of SEQ ID NOs 31, 33, 44 and 52; amino acid residues 50-66 of SEQ ID NOS 36, 38, 40, 42 and 60; amino acid residues 52-58 of SEQ ID NO. 1 and 3 and amino acid residues 52-67 of SEQ ID NO. 46, 48, 50, 54, 56 and 58.

Also provided are CDR3 regions comprising: amino acid residues 89-97 of SEQ ID NOS 13, 15, 17, 19, 21, 23, 25, 27 and 29; 1 and 3 amino acid residues 91-101; amino acid residues 94-106 of SEQ ID NOS 7, 9 and 11; amino acid residues 98-107 of SEQ ID NOS: 44 and 52; amino acid residues 97-105 of SEQ ID NO 4; amino acid residues 99-110 of SEQ ID NOS: 34 and 36; 112 of SEQ ID NO: 99 to 112; amino acid residues 99-113 of SEQ ID NOS: 31 and 33; amino acid residues 99-114 of SEQ ID NOs 38, 40 and 42; 46, 48, 54, 56 and 58 of SEQ ID NO, 100-109; and amino acid residue 101-019 of SEQ ID NO: 50.

CDRs disclosed herein include consensus sequences derived from a related set of sequences. As previously described, 4 variable region sequences were identified, 1 κ group and 3 λ groups. CDRL1 consensus sequence from the kappa group consisting of RASQX₁X₂SX₃WX₄A (SEQ ID NO:123) wherein X₁Selected from G or V; x₂Selected from I, F or S; x₃Selected from S or G and X₄Selected from F or L. CDRL1 consensus sequence from Lambda group 1 consists of TLX₁SGYSDYKVD (SEQ ID NO:124), wherein X₁Is selected from N or S. The CDRL1 consensus sequence from lambda group 3 is represented by TGSSSNX₁GAGYDVH (SEQ ID NO:125), wherein X₁Is selected from I or T.

CDRL2 consensus sequence from Lambda group 1 is represented by VGTGGX₁VGSKGX₂(SEQ ID NO: 126) wherein X₁Selected from I or T and X₂Is selected from D or E. CDRL2 consensus sequence from Lambda group 3 consisting of GSX₁NRPS (SEQ ID NO: 127) wherein X₁Is selected from N or G.

CDRL3 consensus sequences include GADHGSGX¹NFVYV (SEQ ID NO:128), where X₁Is S or N.

CDRH1 consensus sequence from the kappa group is represented by SGGYYWX₁(SEQ ID NO:129) wherein X₁Selected from S or T. CDRH from lambda set 1₁The consensus sequence is represented by X₁X₂SMN (SEQ ID NO:131) wherein X₁Selected from S or T and X₂Selected from Y or F. CDRH1 consensus sequence from Lambda group 2 was SYX₁MH (SEQ ID NO:130), wherein X₁Is selected from G or A.

FromThe CDRH2 consensus sequence of the kappa group is represented by X₁IX₂YSGX₃X₄YNPSLKS (SEQ ID NO:132) composition, wherein X₁Selected from Y or H; x₂Selected from Y or H; x₃Selected from S or N; and X₄Selected from T or S. The consensus sequence from lambda set 1 is represented by YISSX₁SSTX₂YX₃ADSVKG (SEQ ID NO:134) with X₁Selected from R or S, X₂Selected from I or R, and X₃Selected from I, H or Y. From lambda group 2 the consensus sequence is represented by VISX₁DGSX₂KYYADSVKG (SEQ ID NO:133), wherein X₁Is F or H and X₂Is L or T. CDRH2 consensus sequence from lambda set 3 was VIWYDGSNX₁YYADSVKG (SEQ ID NO:135), wherein X₁Is selected from K or E.

CDRH3 consensus sequence from the kappa group consisting of X₁RGX₂YYGMDV (SEQID NO:136), wherein X₁Selected from N or D and X₂Selected from H, Y or F. CDRH3 consensus sequence from Lambda group 1 is represented by RIAAAAGX₁X₂X₃YYYAX₄DV (SEQ ID NO:137) in which X₁Is selected from G or P; x₂Selected from F or W; x₃Selected from H or G and X₄Selected from L and M. CDRH3 consensus sequence from lambda set 3 was represented by DRGYX₁SSWYPDAFDI (SEQ ID NO:138), wherein X₁Selected from S or T.

Monoclonal antibodies

Antigen binding proteins provided include monoclonal antibodies that bind to IL-23. Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells harvested from transgenic animals after completion of an immunization program. The spleen cells may be immortalized using any technique known in the art, for example by fusing them with myeloma cells to produce hybridomas. Myeloma cells used for hybridoma-producing fusion procedures preferably do not produce antibodies, have high fusion efficiency, and enzyme deficiency, which renders them incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for mouse fusion include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7, and S194/5XXO Bul; examples of cell lines for rat fusion include R210.RCY3, Y3-Ag 1.2.3, IR983F and 413210. Other cell lines that can be used for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC 729-6.

In some cases, hybridoma cell lines are generated by: immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with an IL-23 immunogen; harvesting spleen cells from the immunized animal; fusing the harvested splenocytes with a myeloma cell line, thereby producing hybridoma cells; hybridoma cell lines are established from the hybridoma cells and identified that produce antibodies that bind to the IL-23 polypeptide while passing IL-12.

Monoclonal antibodies secreted by the hybridoma cell lines can be purified using any technique known in the art. Hybridomas or monoclonal antibodies (mabs) can be further screened to identify mabs with particular properties, such as the ability to inhibit IL-23-induced activity.

Chimeric and humanized antibodies

Chimeric and humanized antibodies based on the foregoing sequences are also provided. Monoclonal antibodies for use as therapeutic agents may be modified in various ways prior to use. One example is a chimeric antibody, which is an antibody consisting of protein segments from different antibodies covalently linked to produce a functional immunoglobulin light or heavy chain or immunologically functional portion thereof. Typically, a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass. For methods related to chimeric antibodies, see, e.g., U.S. Pat. nos. 4,816,567; and Morrison et al, 1985, Proc. Natl. Acad. Sci. USA 81: 6851-6855. CDR grafting is described, for example, in U.S. Pat. nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101.

One useful type of chimeric antibody is a "humanized" antibody. Typically, humanized antibodies are produced from monoclonal antibodies that originally appeared in non-human animals. Certain amino acid residues in the monoclonal antibody (typically from the non-antigen-recognizing portion of the antibody) are modified to be homologous to corresponding residues in a human antibody of the corresponding isotype. Humanization can be performed, for example, by substituting at least a portion of the rodent variable region with the corresponding region of a human antibody using various methods (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,693,762; Jones et al, 1986, Nature321:522-525; Riechmann et al, 1988, Nature 332:323-27; Verhoeyen et al, 1988, Science 239: 1534-1536).

In certain embodiments, constant regions from species other than humans may be used with human variable regions to generate hybrid antibodies.

Fully human antibodies

Fully human antibodies are also provided. Methods are available for making fully human antibodies specific for a given antigen without exposing humans to that antigen ("fully human antibodies"). One particular means provided for carrying out the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which endogenous Ig genes have been inactivated is one means of generating fully human monoclonal antibodies (mabs) in mice, which are animals that can be immunized with any desired antigen. The use of fully human antibodies minimizes immunogenicity and allergic reactions that can sometimes be elicited by administering a mouse or mouse-derived mAb to a human as a therapeutic agent.

Fully human antibodies can be produced by immunizing a transgenic animal (typically a mouse) capable of producing a human antibody repertoire in the absence of endogenous immunoglobulin production. Antigens used for this purpose typically have six or more contiguous amino acids, and are optionally conjugated to a carrier, such as a hapten. See, e.g., Jakobovits et al, 1993, Proc. Natl. Acad. Sci. USA 90:2551 + 2555; Jakobovits et al, 1993, Nature 362:255 + 258; and Bruggermann et al, 1993, Yeast in Immunol 7: 33. In one example of such a method, a transgenic animal is produced by incapacitating an endogenous mouse immunoglobulin locus in which the mouse immunoglobulin heavy and light chains are encoded, and inserting into the mouse genome a large fragment containing human genomic DNA containing the locus encoding the human heavy and light chain proteins. The partially modified animals, which have less than the full complement of the human immunoglobulin locus, are then crossed to obtain animals with all of the desired modifications of the immune system. When administered to an immunogen, these transgenic animals produce antibodies immunospecific for the immunogen but having human rather than murine amino acid sequences (including the variable regions). For further details of this process see, for example, WIPO patent publications WO96/33735 and WO 94/02602. Additional methods related to transgenic mice for the production of human antibodies are described in U.S. patent nos. 5,545,807; 6,713,610, respectively; 6,673,986, respectively; 6,162,963, respectively; 5,545,807, respectively; 6,300,129, respectively; 6,255,458, respectively; 5,877,397, respectively; 5,874,299 and 5,545,806; WIPO patent publications W091/10741, W090/04036 and EP 546073B1 and EP 546073A 1.

The transgenic mice contain human immunoglobulin gene miniloci that encode unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences and targeting mutations that inactivate endogenous mu and kappa chain loci (Lonberg et al, 1994, Nature 368: 856-) -859). Thus, mice display reduced expression of mouse IgM or [ kappa ] in response to immunization, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity human IgG [ kappa ] monoclonal antibodies (Lonberg et al, supra; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci. 764: 536-. The preparation of such mice is described in detail in Taylor et al, 1992, Nucleic Acids Research 20: 6287. about.6295, Chen et al, 1993, International Immunology 5: 647. about.656, Tuailon et al, 1994, J. Immunol 152: 2912. about.2920, Lonberg et al, 1994, Nature 368: 856. about.859, Lonberg, 1994, Handbook of exp. Pharmacology 113:49-101, Taylor et al, 1994, International Immunology 6: 579. about.591, Lonberg and Huszar, 1995, Intern. Rev. immunol. 13:65-93, Harding and Lonberg, 1995, Ann. N.Y. Acerand. 546: 546, Nature 85, Nature 14. about.11. about.t.H.H.H.D.H.H.H.764, 1996. See further U.S. Pat. nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429; and U.S. patent No. 5,545,807; WIPO publication No. WO 93/1227; WO 92/22646; and WO 92/03918. Techniques for the production of human antibodies in these transgenic mice are also disclosed in WIPO publication No. WO 98/24893 and Mendez et al, 1997, Nature Genetics 15: 146-. For example, HCo7 and HCo12 transgenic mouse strains can be used to produce anti-IL-23 antibodies.

Using hybridoma technology, antigen-specific human mabs with the desired specificity can be generated and selected from transgenic mice (such as those described above). Such antibodies can be cloned and expressed using suitable vectors and host cells, or can be harvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage display libraries (such as disclosed in Hoogenboom et al, 1991, J. mol. biol. 227:381; Marks et al, 1991, J. mol. biol. 222: 581; WIPO publication No. WO 99/10494). Phage display technology mimics immunoselection by displaying a pool of antibodies on the surface of filamentous phage, and then selecting the phage by binding it to a selected antigen.

Bispecific or bifunctional antigen binding proteins

A "bispecific", "dual specificity" or "bifunctional" antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites, such as one or more CDRs or one or more variable regions as described above. In some cases, it is an artificial hybrid antibody with two different heavy/light chain pairs and two different binding sites. A multispecific antigen-binding protein or "multispecific antibody" is a protein that targets more than one antigen or epitope. Bispecific antigen binding proteins and antibodies are a multispecific antigen binding protein antibody and may be produced by a variety of methods, including (but not limited to) fusion of hybridomas or attachment of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. exp. Immunol. 79: 315-1553, Kostelny et al, 1992, J. lmmunol. 148: 1547-1553.

Immune fragments

Antigen binding proteins also include immunological fragments of antibodies (e.g., Fab ', F (ab')2, or scFv). A "Fab fragment" comprises one light chain (the light chain variable region (VL) and its corresponding constant domain (CL)) and one heavy chain (the heavy chain variable region (VH) and the first constant domain (CH 1)). The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab ' fragment" contains a portion of one light chain and one heavy chain, the heavy chain portion also containing the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form a F (ab ')2 molecule. Thus, a "F (ab ')2 fragment" consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The "Fv fragment" consists of the variable light and heavy chain regions of a single arm of an antibody. A single chain antibody "scFv" is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain, which forms the antigen binding region. Single chain antibodies are discussed in detail in WIPO publication Nos. WO 88/01649, U.S. Pat. Nos. 4,946,778 and 5,260,203, Bird, 1988, Science 242:423, Huston et al, 1988, Proc. Natl. Acad. Sci. U.S. A.85: 5879, Ward et al, 1989, Nature 334:544, de Graaf et al, 2002, Methods Mol. biol. 178: 379-. The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

Also included are domain antibodies, i.e., immunologically functional immunoglobulin fragments that contain only the heavy chain variable region or the light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to produce a bivalent domain antibody. The two VH regions of the bivalent domain antibody may target the same or different antigens. Diabodies are bivalent antibodies comprising two polypeptide chains, each polypeptide chain comprising a VH and a VL domain connected by a linker, which is too short to allow pairing between the two domains on the same chain, thereby allowing each domain to pair with a complementary domain on the other polypeptide chain (see e.g. Holliger et al, proc. natl. acad. sci. USA 90:6444-48, 1993 and Poljak et al, Structure 2:1121-23, 1994). Similarly, three-chain antibodies and four-chain antibodies are antibodies that comprise 3 and 4 polypeptide chains, respectively, and form 3 and 4 antigen binding sites, respectively, that may be the same or different. Large antibodies (maxibodies) comprise a bivalent scFv covalently attached to the Fc region of IgGi (see, e.g., Fredericks et al, 2004, Protein Engineering, Design & Selection, 17:95-106; Powers et al, 2001, Journal of Immunological Methods, 251: 123-.

Various other forms

Also provided are variant forms of the above-disclosed antigen binding proteins, some of which have one or more conservative amino acid substitutions, for example in one or more of the heavy or light chains, variable regions or CDRs listed in tables 1 and 2. Naturally occurring amino acids can be classified based on common side chain properties: hydrophobicity (norleucine, Met, Ala, Val, Leu, Ile); neutral hydrophilicity (Cys, Ser, Thr, Asn, Gln); acidic (Asp, Glu); basic (His, Lys, Arg); residues affecting the chain orientation (Gly, Pro); and aromatic (Trp, Tyr, Phe).

Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other inverted or inverted forms of amino acid moieties. Such substantial modification of the functional and/or biochemical properties of the antigen binding proteins described herein can be achieved by creating substitutions in the amino acid sequences of the heavy and light chains that differ significantly in their effect of maintaining: (a) the structure of the molecular backbone in the substituted region, for example as a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulkiness of the side chains.

Non-conservative substitutions may involve exchanging a member of one of the above classes for a member from another class. Such substituted residues may be introduced into regions of the antibody homologous to the human antibody, or into non-homologous regions of the molecule.

In making such changes, according to certain embodiments, the hydropathic index (hydropathic index) of the amino acid may be considered. The hydropathic properties of a protein are calculated by assigning a numerical value ("hydropathic index") to each amino acid and then repeatedly averaging these values along the peptide chain. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic/hydrophobic properties in conferring interactive biological function on proteins is understood in the art (see, e.g., Kyte et al, 1982, J. mol. biol. 157: 105-131). It is known that certain amino acids may be substituted for other amino acids having similar hydropathic indices or scores and still retain similar biological activity. Where the alteration is made based on hydropathic index, in certain embodiments, substitutions of amino acids whose hydropathic index is within ± 2 are included. In some aspects, those within ± 1 are included, while in other aspects, those within ± 0.5 are included.

It is also understood in the art that substitution of like amino acids may be effectively made based on hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in an immunological embodiment, as in the case of the present invention. In certain embodiments, the greatest local average hydrophilicity of a protein (as controlled by the hydrophilicity of its adjacent amino acids) is correlated with its immunogenicity and antigen binding or immunogenicity, i.e., with the biological properties of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). Where changes are made based on similar hydrophilicity values, in certain embodiments substitutions of amino acids whose hydrophilicity values are within ± 2 are included, in other embodiments those amino acids within ± 1 are included, and in still other embodiments those amino acids within ± 0.5 are included. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions".

Exemplary conservative amino acid substitutions are described in table 4.

TABLE 4

Conservative amino acid substitutions

The skilled person will be able to determine suitable variants of a polypeptide as described herein using well known techniques. One skilled in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not believed to be important for activity. The skilled artisan will also be able to identify molecular residues and moieties that are conserved among similar polypeptides. In further embodiments, even regions that may be important for biological activity or structure may be conservatively substituted for amino acids without disrupting biological activity or adversely affecting polypeptide structure.

In addition, one skilled in the art can review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In view of this comparison, the importance of amino acid residues in proteins corresponding to those important for activity or structure in similar proteins can be predicted. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the 3-dimensional structure and amino acid sequence associated with this structure in similar polypeptides. In view of this information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make extreme changes to amino acid residues predicted to be on the surface of a protein, as such residues may be involved in important interactions with other molecules. In addition, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using an IL-23 activity assay (see examples below) to generate information about which amino acids can be changed and which cannot be changed. In other words, based on information gathered from such routine experiments, the skilled person can easily determine amino acid positions where further substitutions, alone or in combination with other mutations, should be avoided.

Many scientific publications are devoted to the prediction of secondary structure. See Moult, 1996, curr, Op. in Biotech, 7: 422-. Furthermore, computer programs are currently available to assist in predicting secondary structure. One method of predicting secondary structure is based on homologous modeling. For example, two polypeptides or proteins with sequence identity greater than 30% or similarity greater than 40% typically have similar structural topologies. Recent expansion of protein structure databases (PDBs) has enhanced the predictability of secondary structure, including the number of possible folds within a polypeptide or protein structure. See Holm et al, 1999, nucleic acid. Res. 27: 244-247. It has been suggested (Brenner et al, 1997, Curr. Op. Structure. biol. 7:369-376) that there is a limited number of folds in a given polypeptide or protein and that once a critical number of structures are resolved, the structure prediction will become significantly more accurate.

Additional methods of predicting secondary Structure include "multithreading" (Jones, 1997, curr. Opin. Structure. biol. 7:377-387; Sippl et al, 1996, Structure 4:15-19), "Profile analysis" (Bowie et al, 1991, Science 253:164-170; Gribskov et al, 1990, meth. Enzymol. 183:146-159; Gribov et al, 1987, Proc. Nat. Acad. Sci. 84:4355-4358) and "evolution linkage" (see Holm, Br1999, supra; and enner, 1997, supra).

In some embodiments, amino acid substitutions are made that: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for formation of protein complexes, (4) altered ligand or antigen binding affinity, and/or (4) conferring or modifying other physicochemical or functional properties to such polypeptides, such as maintenance of the structure of the molecular backbone at the substitution region, e.g., as a folded or helical conformation; either maintaining or changing the charge or hydrophobicity of the molecule at the site of interest or maintaining or changing the bulkiness of the side chains.

For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitution may be made in portions of the antibody that are located outside the domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions may be used that do not significantly alter the structural characteristics of the parent sequence (e.g., one or more substituted amino acids that do not disrupt the secondary structure characterizing the parent or native antigen binding protein). Examples of art-recognized secondary and tertiary Structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, Ed.), 1984, W.H. New York: Freeman and Company; Introduction to Protein Structure (Branden and Tooze, eds.), 1991, New York: Garland Publishing; and Thornton et al, 1991, Nature 354: 105.

Additional variants include cysteine variants in which one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, particularly when the antibody (for example) must refold into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native protein, and generally have an even number to minimize interactions due to unpaired cysteines.

The disclosed heavy and light chain variable regions and CDRs can be used to prepare antigen binding proteins comprising antigen binding regions that specifically bind to IL-23 polypeptides. By "antigen-binding region" is meant a protein or portion of a protein that specifically binds a particular antigen, such as a region containing amino acid residues that interact with an antigen and confer on the antigen-binding protein its specificity and affinity for a target antigen. The antigen binding region may include one or more CDRs, and certain antigen binding regions also include one or more "framework" regions. For example, one or more of the CDRs listed in table 3 can be incorporated covalently or non-covalently into a molecule (e.g., a polypeptide) for immunoadhesion. Immunoadhesions can incorporate the CDRs as part of a larger polypeptide chain, can covalently link the CDRs to another polypeptide chain, or can non-covalently incorporate the CDRs. The CDRs enable immunoadhesion to specifically bind to a particular antigen of interest (e.g., an IL-23 polypeptide).

Other antigen binding proteins include mimetics based on the variable regions and CDRs described herein (e.g., "peptide mimetics" or "peptidomimetics"). These analogs can be peptidic, non-peptidic, or a combination of peptidic and non-peptidic regions. Fauchhere, 1986, adv. Drug Res. 15:29, Veber and Freidinger, 1985, TINS p. 392, and Evans et al, 1987, J. Med. chem. 30: 1229. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or prophylactic effects. Such compounds are typically developed by means of computer molecular modeling. Typically, a peptidomimetic is a protein that is structurally similar to an antigen binding protein that exhibits a desired biological activity (such as the ability to bind IL-23), but the peptidomimetic has one or more peptide bonds optionally substituted with a bond selected from, for example: -CH2NH-, -CH2S-, -CH2-CH2-, -CH-CH- (cis and trans), -COCH2-, -CH (OH) CH2-, and-CH 2 SO-. In certain embodiments, systematic substitution of one or more amino acids of the consensus sequence with the same type of D-amino acid (e.g., D-lysine instead of L-lysine) can be used to produce a more stable protein. In addition, restricted peptides comprising a consensus sequence or substantially the same consensus sequence variations can be generated by methods known in the art (Rizo and Gierasch, 1992, Ann. Rev. biochem. 61:387), for example by adding internal cysteine residues capable of forming intramolecular disulfide bonds of cyclized peptides.

Also provided are derivatives of the antigen binding proteins described herein. The derivatized antigen binding protein may comprise any molecule or substance that confers a desired property to the antigen binding protein or fragment, such as increasing half-life in a particular application. The derivatized antigen binding protein can comprise, for example, a detectable (or labeled) moiety (e.g., a radioactive, colorimetric, antigen or enzyme molecule, a detectable bead (such as a magnetic or electron dense (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antigen binding protein for a particular use (e.g., administration to a subject (such as a human subject) or other in vivo or in vitro use). Examples of molecules that can be used to derive antigen binding proteins include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin linked and pegylated derivatives of antigen binding proteins can be prepared using techniques well known in the art. In one embodiment, the antigen binding protein is conjugated or otherwise linked To Transthyretin (TTR) or a TTR variant. TTR or TTR variants can be chemically modified with chemicals selected, for example, from dextran, poly (n-vinyl pyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols, and polyvinyl alcohol.

Other derivatives include covalent or aggregative conjugates of IL-23 antigen binding proteins with other proteins or polypeptides, such as by expression of a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of the IL-23 antigen binding protein. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, such as a yeast alpha-factor leader or peptide (such as an epitope tag). Fusion proteins containing IL-23 antigen binding proteins may comprise peptides (e.g., polyHis) added to facilitate purification or identification of the IL-23 antigen binding protein. IL-23 antigen binding proteins can also be linked to a FLAG peptide, as described by Hopp et al, 1988, Bio/Technology 6: 1204; and U.S. patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that is reversibly bound by a particular monoclonal antibody (mAb), thereby allowing rapid assay and easy purification of expressed recombinant proteins. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

Oligomers containing one or more IL-23 antigen binding proteins are useful as IL-23 antagonists. The oligomers may be in the form of covalently linked or non-covalently linked dimers, trimers or higher oligomers. The use of oligomeric oligomers comprising two or more IL-23 antigen binding proteins is contemplated, one example of which is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, and the like. Also included are oligomers comprising a plurality of IL-23 binding proteins linked via covalent or non-covalent interactions between peptide moieties fused to the IL-23 antigen binding protein. Such peptides may be peptide linkers (spacers) or peptides with properties that promote oligomerization. Among suitable peptide linkers are those described in U.S. patent nos. 4,751,180 and 4,935,233. Leucine zippers and certain antibody-derived polypeptides belong to peptides that promote oligomerization of IL-23 antigen binding proteins attached thereto. Examples of leucine zipper domains suitable for use in generating soluble oligomeric proteins are described in WIPO publication No. WO 94/10308; hoppe et al, 1994, FEBS Letters 344:191, and Fanslow et al, 1994, Semin. Immunol. 6: 267-278. In one method, a recombinant fusion protein comprising an IL-23 antigen-binding protein fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the resulting soluble oligomeric IL-23 antigen-binding protein fragment or derivative is recovered from the culture supernatant.

Such oligomers may comprise 2-4 IL-23 antigen binding proteins. The oligomeric IL-23 antigen binding protein portion can be in any of the above forms, such as variants or fragments. Preferably, the oligomer comprises an IL-23 antigen binding protein having IL-23 binding activity. Oligomers can be prepared using polypeptides derived from immunoglobulins. The preparation of Fusion Proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides, including Fc domains, is described, for example, in Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn et al, 1990, Nature 344:677; and Hollenbaugh et al, 1992, "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, supply 4, pages 10.19.1-10.19.11.

Also included are dimers comprising two fusion proteins produced by fusing an IL-23 antigen binding protein to the Fc region of an antibody. Dimers can be prepared, for example, by inserting the gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with a recombinant expression vector, and allowing the expressed fusion protein to assemble much like an antibody molecule, thereby forming interchain disulfide bonds between the Fc portions to produce the dimer. Such Fc polypeptides include native and mutein forms of polypeptides derived from the Fc region of antibodies. Also included are truncated forms of such polypeptides that contain a hinge region that promotes dimerization. Fusion proteins comprising an Fc portion (and oligomers formed therefrom) offer the advantage of being easily purified by affinity chromatography over protein a or protein G columns. One suitable Fc polypeptide described in WIPO publication No. WO93/10151 and U.S. patent nos. 5,426,048 and 5,262,522 is a single chain polypeptide that extends from the N-terminal hinge region to the natural C-terminus of the Fc region of human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and Baum et al, 1994, EMBO J. 13: 3992-4001. The amino acid sequence of this mutein is identical to the native Fc sequence presented in WIPO publication No. WO93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The muteins exhibit reduced affinity for Fc receptors.

Glycosylation

Antigen binding proteins may have glycosylation patterns that are different or altered from those found in the native species. As is known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, as described below) or the host cell or organism in which the protein is produced. Specific expression systems are described below.

Glycosylation of polypeptides is typically N-linked or O-linked. N-linked means that the carbohydrate moiety is attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to an asparagine side chain linkage. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antigen binding protein may conveniently be accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding one or more serine or threonine residues to the starting sequence or by substituting the starting sequence with one or more serine or threonine residues (for O-linked glycosylation sites). For simplicity, the amino acid sequence of an antigen binding protein can be altered by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on an antigen binding protein is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require the production of proteins in host cells having glycosylation capabilities for N-and O-linked glycosylation. Depending on the coupling mode used, the sugar may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as the sulfhydryl groups of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide groups of glutamine. These methods are described in PCT publication WO 87/05330 and Aplin and Wriston, 1981, CRC Crit. Rev, biochem., pp. 259-propanone 306.

Removal of the carbohydrate moiety present on the starting antigen binding protein may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposing the protein to the compound triflic acid or equivalent compound. This treatment results in the cleavage of most or all of the sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described in Hakimuddin et al, 1987, Arch. biochem. biophysis. 259:52 and Edge et al, 1981, anal. biochem.118: 131. Enzymatic cleavage of the carbohydrate moiety on a polypeptide can be achieved by using a variety of endo-and exoglycosidases, as described by Thotakura et al, 1987, meth. enzymol.138: 350. Glycosylation of potential glycosylation sites can be prevented by the use of the compound tunicamycin as described by Duskin et al, 1982, J. biol. chem. 257: 3105. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

Thus, aspects include glycosylated variants of the antigen binding protein in which the number and/or type of glycosylation sites has been altered as compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the antigen binding protein variant comprises a greater or lesser number of N-linked glycosylation sites than the parent polypeptide. Substitutions that eliminate or alter this sequence will prevent the addition of an N-linked carbohydrate chain present in the parent polypeptide. For example, glycosylation can be reduced by deleting Asn or by substituting Asn with a different amino acid. Antibodies typically have N-linked glycosylation sites in the Fc region.

Labels and effector groups

The antigen binding protein may comprise one or more labels. The term "label" or "labeling group" refers to any detectable label. Generally, labels fall into a variety of categories depending on the assay in which they are to be detected: a) isotopic labeling, which can be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) a redox active moiety; d) an optical dye; enzymatic groups (e.g., horseradish peroxidase, 3-galactosidase, luciferase, alkaline phosphatase); e) a biotinylation group; and f) by secondary reportingA predetermined polypeptide epitope recognized by a molecule (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, etc.). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art. Examples of suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) Fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, 3-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used as appropriate.

The term "effector group" means any group coupled to an antigen binding protein that acts as a cytotoxic agent. Examples of suitable effector groups are radioisotopes or radionuclides (e.g. as³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) In that respect Other suitable groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable groups include calicheamicin, statins, geldanamycin, and maytansine. In some embodiments, the effector group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance.

Polynucleotides encoding IL-23 antigen binding proteins

Also provided are polynucleotides or portions thereof encoding the antigen binding proteins described herein, including polynucleotides encoding one or both strands of an antibody or fragments, derivatives, muteins or variants thereof, polynucleotides encoding a heavy chain variable region or CDR only, polynucleotides sufficient for use as hybridization probes, PCR or sequencing primers for identifying, analyzing, mutating or amplifying the polynucleotides encoding the polypeptides, antisense nucleic acids for inhibiting expression of the polynucleotides, and the complement of the foregoing. The polynucleotide may be of any length. It may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleic acids in length (including all values therebetween), and/or may comprise one or more additional sequences (e.g., regulatory sequences), and/or be part of a larger polynucleotide (e.g., vector). Polynucleotides may be single-stranded or double-stranded and may comprise RNA and/or DNA nucleic acids and artificial variants thereof (e.g., peptide nucleic acids).

Polynucleotides encoding certain antigen binding proteins, or portions thereof (e.g., full length antibody, heavy or light chain, variable domain or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) can be isolated from B cells of mice that have been immunized with IL-23 or an immunogenic fragment thereof. Polynucleotides can be isolated by conventional procedures such as Polymerase Chain Reaction (PCR). Phage display is another example of a known technique by which derivatives of antibodies and other antigen binding proteins can be prepared. In one approach, the polypeptides that are components of the antigen binding protein of interest are expressed in any suitable recombinant expression system and the expressed polypeptides are allowed to assemble to form the antigen binding protein molecule. Phage display has also been used to derive antigen binding proteins with different properties (i.e., different affinities for the antigen to which they bind) via chain shuffling, see Marks et al, 1992, Biotechnology 10: 779.

Because of the degeneracy of the genetic code, each polypeptide sequence described herein is also encoded by a large number of other polynucleotide sequences in addition to those provided. For example, the heavy chain variable domains provided herein can be encoded by the polynucleotide sequences SEQ ID NOs 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or 59. The light chain variable domain may be encoded by the polynucleotide sequence of SEQ ID NO 2,5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28. One of ordinary skill in the art will appreciate that the present disclosure thus provides a sufficient written description and enablement of each degenerate nucleotide sequence encoding each antigen binding protein.

One aspect further provides polynucleotides that hybridize to other polynucleotide molecules under specific hybridization conditions. Methods for hybridizing nucleic acids, basic parameters that influence the selection of hybridization conditions, and guidelines for designing suitable conditions are well known in the art. See, e.g., Sambrook, Fritsch, and Maniatis, 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology, 1995, Ausubel et al, eds., John Wiley & Sons, Inc. Moderately stringent hybridization conditions, as defined herein, use a pre-wash solution containing 5x sodium chloride/citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC and a hybridization temperature of 55 ℃ (or other similar hybridization solution, such as a solution containing about 50% formamide, at a hybridization temperature of 42 ℃) and wash conditions of 60 ℃ in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions are performed in 6 XSSC at 45 ℃ followed by one or more washes in 0.1 XSSC, 0.2% SDS at 68 ℃. Furthermore, one skilled in the art can manipulate hybridization and/or wash conditions to increase or decrease the stringency of hybridization such that polynucleotides comprising nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (including all values therebetween) identical to each other generally remain hybridized to each other.

Changes may be introduced into a polynucleotide by mutation, resulting in a change in the amino acid sequence of the polypeptide (e.g., antigen binding protein or antigen binding protein derivative) that it encodes. Mutations can be introduced using any technique known in the art, such as site-directed mutagenesis and random mutagenesis. The mutant polypeptides may be expressed and selected for desired properties. Mutations can be introduced into a polynucleotide without significantly altering the biological activity of the polypeptide that it encodes. Such as substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a polynucleotide that selectively alters the biological activity of the polypeptide encoded thereby. For example, mutations can quantitatively or qualitatively alter biological activities, such as increasing, decreasing, or eliminating activity and altering the antigen specificity of an antigen binding protein.

Another aspect provides polynucleotides suitable for use as primers or hybridization probes for detecting nucleic acid sequences. A polynucleotide may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, e.g., a fragment that can be used as a probe or primer or a fragment encoding an active portion of a polypeptide (e.g., an IL-23 binding portion). Probes based on nucleic acid sequences can be used to detect nucleic acids or similar nucleic acids, such as transcripts encoding polypeptides. The probe may comprise a labelling group, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.

Methods of expressing antigen binding proteins

The antigen binding proteins provided herein can be prepared by any of a variety of conventional techniques. For example, an IL-23 antigen binding protein can be produced by recombinant expression systems using any technique known in the art. See, for example, Monoclonal Antibodies, hybrids: A New Dimension in Biological analytes, Kennet et al (eds.) Plenum Press, New York (1980), and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).

Also provided herein are expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes comprising at least one polynucleotide as described above, and host cells comprising such expression systems or constructs. "vector" as used herein means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) suitable for transferring protein-encoding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors (e.g., recombinant expression vectors). Expression vectors (e.g., recombinant expression vectors) can be used to transform host cells and contain nucleic acid sequences that direct and/or control (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control the transcription, translation, and, if introns are present, RNA splicing of a coding region operably linked thereto. "operably linked" means that the terms so used are in a relationship such that they perform their inherent function. For example, a control sequence (e.g., a promoter) in a vector that is "operably linked" to a protein coding sequence is arranged such that normal activity of the control sequence results in transcription of the protein coding sequence, thereby resulting in recombinant expression of the encoded protein.

Another aspect provides a host cell into which an expression vector (such as a recombinant expression vector) has been introduced. The host cell may be any prokaryotic cell (e.g., E.coli) or eukaryotic cell (e.g., yeast, insect, or mammalian cells (e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells can integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., for resistance to antibiotics) is typically introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced polynucleotide can be identified by methods such as drug selection (e.g., cells that incorporate the selectable marker gene will survive, while other cells die).

The antigen binding protein may be expressed in a hybridoma cell line (e.g. in particular the antibody may be expressed in a hybridoma) or in a cell line other than a hybridoma. Expression constructs encoding antigen binding proteins can be used to transform mammalian, insect or microbial host cells. Transformation can be performed using any known method for introducing a polynucleotide into a host cell, including, for example, packaging the polynucleotide in a virus or phage, and transducing the host cell with the construct by transfection procedures known in the art, such as those described in U.S. Pat. nos. 4,399,216; 4,912,040; 4,740,461; 4,959,455. The optimal transformation procedure to use will depend on the type of host cell being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, mixing of nucleic acids with positively charged lipids, and microinjection of DNA directly into the nucleus.

Recombinant expression constructs generally comprise a polynucleotide encoding a polypeptide. The polypeptide may comprise one or more of: one or more CDRs, such as provided herein; a light chain variable region; a heavy chain variable region; a light chain constant region; heavy chain constant regions (e.g., CH1, CH2, and/or CH 3); and/or another scaffold moiety of an IL-23 antigen binding protein. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In one embodiment, the heavy or light chain constant region is attached to the C-terminus of a heavy or light chain variable region provided herein and ligated into an expression vector. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, thereby allowing amplification and/or expression of the gene to occur). In some embodiments, the vectors used employ protein-fragment complementation assays using protein reporters such as dihydrofolate reductase (see, e.g., U.S. Pat. No. 6,270,964). Suitable expression vectors are available, for example, from Invitrogen Life Technologies (Carlsbad, Calif.) or BD Biosciences (SanJose, Calif.). Other useful vectors for cloning and expressing antibodies and fragments include those described in Bianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84: 439-44. Further suitable expression vectors are described, for example, in Methods enzymol., vol.185 (D. V.Goeddel, ed.), 1990, New York: Academic Press.

In general, an expression vector for any host cell will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences (collectively "flanking sequences") will in certain embodiments generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a polynucleotide encoding the polypeptide to be expressed, and a selectable marker element. The expression vectors provided can be constructed from starting vectors (e.g., commercially available vectors). Such vectors may or may not contain all of the desired flanking sequences. When one or more of the flanking sequences described herein are not already present in the vector, they may be obtained separately and ligated into the vector. Methods for obtaining each flanking sequence are well known to those skilled in the art.

Optionally, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5 'or 3' end of the IL-23 antigen binding protein coding sequence; the oligonucleotide sequence encodes polyhistidine (such as hexa-polyhistidine; SEQ ID NO: 152), or another "tag" such as FLAG, HA (hemagglutinin influenza Virus) or myc, against which there are commercially available antibodies. The tag is typically fused to the polypeptide following expression of the polypeptide and can be used as a means for affinity purification or detection of an IL-23 antigen binding protein from a host cell. Affinity purification can be accomplished, for example, by column chromatography using an antibody against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified IL-23 antigen binding protein by various means, such as cleavage using certain peptidases.

The flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species different from the species or strain of the host cell), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism or any plant, provided that the flanking sequences are functional in and activatable by the host cell machinery.

The flanking sequences useful in the vector may be obtained by any of several methods well known in the art. Generally, flanking sequences useful herein have been previously identified by mapping and/or by restriction endonuclease digestion, and thus may be isolated from an appropriate tissue source using an appropriate restriction endonuclease. In some cases, the entire nucleotide sequence of the flanking sequences may be known. Here, the flanking sequences may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequence is known, it may be obtained using Polymerase Chain Reaction (PCR) and/or by screening a genomic library (such as oligonucleotides and/or fragments of the flanking sequence from the same or another species) with appropriate probes. In the case where the flanking sequences are not known, the DNA segment containing the flanking sequences may be isolated from a larger piece of DNA that may contain, for example, the coding sequence or even another gene or genes. Isolation can be accomplished by restriction endonuclease digestion to generate the appropriate DNA fragments, followed by isolation using agarose gel purification, Qiagen column chromatography (Qiagen, Chatsworth, CA), or other methods known to the skilled artisan. The selection of an appropriate enzyme to achieve this will be readily apparent to those of ordinary skill in the art.

The origin of replication is typically part of those prokaryotic expression vectors that are commercially available, and this origin facilitates the amplification of the vector in the host cell. If the vector selected does not contain an origin of replication site, it can be chemically synthesized based on the known sequence and ligated into the vector. For example, the origin of replication from plasmid pBR322 (New England Biolabs, Beverly, Mass.) is suitable for most gram-negative bacteria and various viral sources (e.g., SV40, polyoma, adenovirus, Vesicular Stomatitis Virus (VSV), or papilloma virus (such as HPV or BPV) can be used to clone vectors in mammalian cells.

Transcription termination sequences are typically located 3' to the end of the polypeptide coding region and serve to terminate transcription. Typically, the transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. Although the sequences are readily cloned from libraries or even purchased commercially as part of a vector, they can also be readily synthesized using methods for nucleic acid synthesis, such as those described herein.

Selectable marker genes encode proteins necessary for survival and growth of host cells grown in selective media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) supplementing the cell for a deficiency in auxotrophy; or (c) provide key nutrients not available from complex or defined media. Specific selectable markers are kanamycin resistance gene, ampicillin resistance gene, and tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

Other alternative genes may be used to amplify the gene to be expressed. Amplification is a process in which genes required for the production of proteins essential for growth or cell survival are repeated in tandem within the chromosomes of successive generation recombinant cells. Examples of suitable selectable markers for use in mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure, wherein only the transformants are uniquely adapted for survival by virtue of the selectable gene present in the vector. Selection pressure is applied by culturing the transformed cells under conditions in which the concentration of the selection agent in the culture medium is continuously increased, resulting in amplification of both the selectable gene and the DNA encoding another gene, such as an antigen binding protein that binds IL-23. As a result, increased amounts of polypeptides, such as antigen binding proteins, are synthesized from the amplified DNA.

The ribosome binding site is usually necessary for the initiation of mRNA translation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is generally located 3 'to the promoter and 5' to the coding sequence for the polypeptide to be expressed.

In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, various pre-or pro-sequences can be manipulated to increase glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered, or the pro sequence added, which can also affect glycosylation. The final protein product may have one or more additional amino acids at the-1 position (relative to the first amino acid of the mature protein) that are concomitantly expressed, which may not be completely removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminus. Alternatively, if the enzyme cuts in such a region within the mature polypeptide, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide.

Expression and cloning will generally comprise a promoter recognized by the host organism and operably linked to a molecule encoding an IL-23 antigen binding protein. A promoter is an untranscribed sequence located upstream (i.e., 5') of the start codon of a structural gene (typically within about 100-1000 bp) that controls transcription of the structural gene. Promoters are routinely classified in one of two categories: inducible promoters and constitutive promoters. Inducible promoters initiate elevated levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or a change in temperature. Constitutive promoters, on the other hand, transcribe a gene to which they are operably linked in concert, i.e., with little or no control over gene expression. Numerous promoters recognized by a variety of potential host cells are well known. A suitable promoter is operably linked to DNA encoding the heavy chain variable region or the light chain variable region of an IL-23 antigen binding protein by removing the promoter from the source DNA by restriction endonuclease digestion and inserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis b virus and simian virus 40 (SV 40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

Other promoters of interest include (but are not limited to): the SV40 early promoter (Benoist and Chambon, 1981, Nature 290: 304-310); the CMV promoter (Thornsen et al, 1984, Proc. Natl. Acad. U.S.A. 81: 659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell 22: 787-797); the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1444-1445); promoter and regulatory sequences from the metallothionein gene (Prinser et al, 1982, Nature 296: 39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al, 1978, Proc. Natl. Acad. Sci. U.S.A. 75: 3727-3731); or the tac promoter (DeBoer et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 21-25). Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been used in transgenic animals: the elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. biol.50:399-409; MacDonald, 1987, Hepatology 7: 425-515); the insulin gene control region which is active in the pancreatic β cell (Hanahan, 1985, Nature 315: 115-122); immunoglobulin gene control regions active in lymphoid cells (Grosschedl et al, 1984, Cell 38: 647-; the mouse mammary tumor virus control region, which is active in testis, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45: 485-; the albumin gene control region which is active in the liver (Pinkert et al, 1987, Genes and Devel. 1: 268-276); the alpha-fetoprotein gene control region that is active in the liver (Krumlauf et al, 1985, Mo/. cell. biol. 5:1639-1648; Hammer et al, 1987, Science 253: 53-58); the α 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel. 1: 161-171); the beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-340; Kollias et al, 1986, Cell 46: 89-94); the myelin basic protein gene control region which is active in oligodendrocytes in the brain (Readhead et al, 1987, Cell 48: 703-712); the myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314: 283-286); and the gonadotropin-releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234: 1372-1378).

Enhancer sequences may be inserted into the vector to increase transcription in higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on a promoter to increase transcription. Enhancers are relatively orientation and position independent, and have been found in both 5 'and 3' positions of the transcriptional unit. Several enhancer sequences are known that can be obtained from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a virus is used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for activating eukaryotic promoters. Although an enhancer may be located 5 ' or 3' to a coding sequence in a vector, it is typically located at a site 5 ' to a promoter. Sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) may be incorporated into the expression vector to facilitate extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cell in which the antibody is produced, and the heterologous signal sequence may replace the native signal sequence. Examples of signal peptides that are functional in a mammalian host cell include the following: the signal sequence of interleukin-7 described in U.S. patent No. 4,965,195; the signal sequence of the interleukin-2 receptor described in Cosman et al, 1984, Nature 312: 768; interleukin-4 receptor signal peptide described in EP patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. patent No. 4,968,607; interleukin-1 receptor signal peptide type II described in EP patent No. 0460846.

After the vector has been constructed, the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression. Transformation of the expression vector for the antigen binding protein into the selected host cell can be accomplished by well known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will depend in part on the type of host cell to be used. These and other suitable methods are well known to the skilled artisan and are set forth, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

The host cell, when cultured under appropriate conditions, synthesizes the protein, which can then be collected from the culture medium (if the host cell secretes it into the culture medium) or directly from the host cell producing it (if it does not secrete). The choice of an appropriate host cell will depend on various factors, such as the desired expression level, the desired or necessary modification of the polypeptide (such as glycosylation or phosphorylation) for activity, and the ease with which folding into a biologically active molecule is facilitated.

Mammalian cell lines useful as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and many other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and constitutively produce an antigen binding protein with IL-23 binding properties. In another embodiment, a cell line from the B cell lineage that does not produce its autoantibodies but has the ability to produce and secrete heterologous antibodies can also be selected.

Use of human IL-23 antigen binding proteins for diagnostic and therapeutic purposes

Antigen binding proteins can be used to detect IL-23 in biological samples and to identify cells or tissues that produce IL-23. An antigen binding protein that specifically binds to IL-23 can be used to diagnose and/or treat IL-23-associated diseases in a patient in need thereof. For example, IL-23 antigen binding proteins can be used in diagnostic assays, such as binding assays that detect and/or quantify IL-23 expressed in blood, serum, cells, or tissues. In addition, IL-23 antigen binding proteins can be used to reduce, inhibit, interfere with, or modulate one or more biological activities of IL-23 in a cell or tissue. Thus, an antigen binding protein that binds to IL-23 can have therapeutic utility in ameliorating IL-23-associated disorders.

Indications of

The present disclosure also relates to the use of IL-23 antigen binding proteins for the prophylactic or therapeutic treatment of medical disorders, such as those disclosed herein. IL-23 antigen binding proteins can be used to treat a variety of disorders in which IL-23 is associated with or plays a role in contributing to an underlying disease or disorder or otherwise contributes to a negative symptom.

Disorders effectively treated by IL-23 antigen binding proteins play a role in inflammatory responses. Such inflammatory disorders include periodontal disease; pulmonary disorders, such as asthma; skin disorders such as psoriasis, atopic dermatitis, contact dermatitis; rheumatic disorders such as rheumatoid arthritis, progressive systemic sclerosis (scleroderma); systemic lupus erythematosus; spinal arthritis including ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis and reactive arthritis. Uveitis is also contemplated, including Vogt-Koyanagi-Harada disease, idiopathic anterior and posterior uveitis, and uveitis associated with spinal arthritis. Also contemplated is the use of an IL-23 antigen binding protein for the treatment of: autoimmune disorders, including multiple sclerosis; autoimmune myocarditis; type 1 diabetes and autoimmune thyroiditis.

Degenerative disorders of the gastrointestinal system may be treated or prevented with an IL-23 antigen binding protein. Such gastrointestinal disorders include inflammatory bowel disease: crohn's disease, ulcerative colitis, and celiac disease.

Also included is the use of IL-23 antigen binding proteins for the treatment of graft versus host disease and complications such as transplant rejection resulting from solid organ transplantation (e.g., heart, liver, skin, kidney, lung or other transplantation, including bone marrow transplantation).

Also provided herein are methods of using IL-23 antigen binding proteins to treat various neoplastic disorders, including various forms of cancer, including colon, gastric, prostate, renal cell, cervical and ovarian cancers, and lung cancers (SCLC and NSCLC). Also included are solid tumors (including sarcomas, osteosarcomas, and carcinomas such as adenocarcinoma and squamous cell carcinoma, esophageal cancer, gastric cancer, gallbladder cancer), leukemias (including acute myelogenous leukemia, chronic myelogenous leukemia, myeloid leukemia, chronic or acute lymphoblastic leukemia, and hairy cell leukemia), and multiple myelomas.

Detection method

The antigen binding proteins can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with IL-23. Examples of methods for detecting the presence of IL-23 include immunoassays, such as enzyme linked immunosorbent assays (ELISA) and Radioimmunoassays (RIA).

For diagnostic applications, the antigen binding protein is typically labeled with a detectable labeling group. Suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g. of³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) Fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used.

Other diagnostic methods for identifying one or more cells that express IL-23 are provided. In a specific embodiment, the antigen binding protein is labeled with a labeling group and the binding of the labeled antigen binding protein to IL-23 is detected. In another embodiment, in vivo detection of antigen binding protein and IL-23 binding. In another embodiment, IL-23 antigen binding proteins are isolated and measured using techniques known in the art. See, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (ed. 1991 and periodical supplement); John E. Coligan, ed., 1993, Current Protocols In Immunology New York: John Wiley & Sons.

Other methods provide for detecting the presence of a test molecule that competes with a provided antigen binding protein for binding to IL-23. One example of such an assay involves detecting the amount of free antigen binding protein in a solution containing an amount of IL-23 in the presence or absence of a test molecule. An increase in the amount of free antigen binding protein (i.e., antigen binding protein that is not bound to IL-23) indicates that the test molecule is capable of competing with the antigen binding protein for IL-23 binding. In one embodiment, the antigen binding protein is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence and absence of the antigen binding protein.

The treatment method comprises the following steps: pharmaceutical formulation, route of administration

Pharmaceutical compositions are provided comprising a therapeutically effective amount of one or more antigen binding proteins and pharmaceutically acceptable excipients, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Additionally, methods of treating a patient by administering such pharmaceutical compositions are also included. The term "patient" includes human patients. The terms "treat" and "treating" include reducing or preventing at least one symptom or other aspect of the disorder, or reducing the severity of the disease, etc. The term "therapeutically effective amount" or "effective amount" refers to the amount of IL-23 antigen binding protein determined to produce any therapeutic response in a mammal. Such therapeutically effective amounts are readily determined by one of ordinary skill in the art.

For example, therapeutic methods may have a generally beneficial effect on a subject, e.g., they may increase the life expectancy of a subject. Alternatively, the method can, for example, treat, prevent, cure, alleviate, or ameliorate ("treat") a disease, disorder, condition, or discomfort ("condition"). Some embodiments provide a method of treating a disorder in a subject comprising administering to the subject a pharmaceutical composition comprising a specific antibody, wherein the disorder is treatable by reducing the activity (partial or complete) of IL-23 in the subject. Treatment includes both therapeutic administration (i.e., administration when signs and symptoms of the disease or condition are evident) as well as treatment to induce remission and/or maintain remission. Thus, the severity of the disease or condition may be reduced (partially, significantly or completely). Active treatment includes the above-described forms of treatment and prophylactic or maintenance therapy (i.e., administration while the disease or condition is at rest), or may prevent or delay signs and symptoms (delay onset, prolonged remission or rest).

It will be appreciated that the method of treating the diseases described herein will administer an effective amount of an anti-IL-23 antibody. Depending on the indication to be treated, a therapeutically effective amount is sufficient to cause at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more reduction in at least one symptom of the targeted condition relative to an untreated subject.

Antigen binding proteins may constitute viable therapeutic agents without the need to achieve a complete cure or to eradicate every symptom or manifestation of the disease. As recognized in the relevant art, drugs used as therapeutic agents may reduce the severity of a given disease state, but do not require the elimination of every manifestation of the disease to be considered useful therapeutic agents. Similarly, to constitute a viable prophylactic agent, a treatment administered prophylactically need not be completely effective in preventing the onset of the condition. It may be sufficient to simply reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood that the disease will occur or worsen in the subject. Certain methods provided herein comprise administering an IL-23 antagonist (such as an antigen binding protein disclosed herein) to a patient in an amount and for a time sufficient to induce a sustained improvement over baseline that reflects an indicator of the severity of a particular disorder.

As understood in the relevant art, pharmaceutical compositions comprising the molecules of the invention are administered to a patient in a manner appropriate for the indication. The pharmaceutical composition may be administered by any suitable technique, including, but not limited to, parenterally, topically, or by inhalation. If injected, the pharmaceutical composition can be administered, for example, by intraarticular, intravenous, intramuscular, intralesional, intraperitoneal, or subcutaneous routes, by bolus injection or by continuous infusion. Topical administration, for example at the site of disease or injury, is contemplated, as are transdermal delivery and sustained release from the implant. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antagonist in aerosol form, and the like. Other alternatives include eye drops; oral formulations (including pills, syrups, lozenges, or chewing gums); and topical formulations (such as lotions, gels, sprays, and ointments).

The use of antigen binding proteins in ex vivo procedures is also contemplated. For example, the patient's blood or other bodily fluid may be contacted with an antigen binding protein that binds IL-23 ex vivo. The antigen binding protein may be bound to a suitable insoluble matrix or solid support material.

Advantageously, the antigen binding protein is administered in the form of a composition comprising one or more additional components, such as a physiologically acceptable carrier, excipient or diluent. Optionally, the composition further comprises one or more physiologically active agents for use in combination therapy. The pharmaceutical composition may comprise an IL-23 antigen binding protein and one or more agents selected from the group consisting of: buffers, antioxidants (such as ascorbic acid), low molecular weight polypeptides (such as those having less than 10 amino acids), proteins, amino acids, carbohydrates (such as glucose, sucrose or dextrin), chelating agents (such as EDTA), glutathione, stabilizers and excipients. Neutral buffered saline or saline mixed with the same serum albumin are examples of suitable diluents. Preservatives such as benzyl alcohol may also be added according to appropriate industry standards. The composition can be formulated as a lyophilizate using a suitable excipient solution (e.g., sucrose) as a diluent. Suitable components are nontoxic to recipients at the dosages and concentrations employed. Further examples of components useful in Pharmaceutical formulations are presented in any Remington's Pharmaceutical Sciences, including 21 st edition (2005), mack publishing Company, Easton, PA.

The kit used by the practitioner comprises an IL-23 antigen binding protein and a label or other instructions for treating any of the disorders described herein. In one embodiment, the kit comprises a sterile formulation of one or more IL-23 binding antigen binding proteins, which may be in the form of a composition as described above, and may be in one or more vials.

The dosage and frequency of administration may vary depending on factors such as the route of administration, the particular antigen binding protein employed, the nature and severity of the disease being treated, whether the condition is acute or chronic, and the size and general condition of the subject. Appropriate dosages can be determined by procedures known in the relevant art (e.g., clinical trials, which may involve dose escalation studies).

Depending on the factors described above, typical dosages may range from about 0.1 pg/kg up to about 30 mg/kg or more. In particular embodiments, the dose may be in the range of 0.1 pg/kg up to about 30 mg/kg, optionally 1 pg/kg up to about 30 mg/kg, optionally 10 pg/kg up to 20 mg/kg to about 10 mg/kg, optionally about 0.1 mg/kg to 5 mg/kg or optionally about 0.3 mg/kg to 3 mg/kg.

The frequency of administration will depend on the pharmacokinetic parameters of the particular human IL-23 antigen binding protein in the formulation used. Typically, the clinician administers the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implanted device or catheter. Appropriate dosages may be determined by using appropriate dose response data. The IL-23 antigen binding protein of the invention can be administered, e.g., once or more than once, at regular intervals, e.g., over a period of time. In particular embodiments, the IL-23 antigen binding protein is administered over a period of at least one month or more (e.g., 1, 2, or 3 months), or even indefinitely. For the treatment of chronic conditions, long-term treatment is often most effective. However, for the treatment of acute conditions, administration for a shorter period of time (e.g., 1-6 weeks) may be sufficient. Typically, the antigen binding protein is administered until the patient exhibits an improvement in the degree of medical relevance relative to baseline of the one or more selected indicators.

It is contemplated that the IL-23 antigen binding protein is administered to the patient in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the disorder being treated. Various indicators reflecting the extent of the patient's discomfort, disease or condition can be measured to determine whether the amount and time of treatment is sufficient. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestations of the disorder of interest. In one embodiment, an improvement is considered to be sustained if the subject exhibits the improvement at least two moments separated by 2-4 weeks. The degree of improvement is typically determined by a physician, who can make such a determination based on signs, symptoms, biopsies, or other test results, and can also employ questionnaires issued to the subject, such as quality of life questionnaires developed for a given disease.

Treatment of a subject with an IL-23-specific antibody can be administered, e.g., using an assay as described herein to achieve and/or maintain an amount of IL-23-specific antibody per volume of serum and/or a sufficient interval. For example, heterodimer specific antibodies are administered to achieve serum concentrations of 12.5 ng/ml to 1,000 ng/ml. In one embodiment, the heterodimer specific antibody is administered to achieve at least 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, 500 ng/ml, or 990 ng/ml. Those skilled in the art will appreciate that the amounts given herein apply to full length antibodies or immunoglobulin molecules. If an antigen-binding fragment thereof is used, the absolute amount will be different from that given in a manner that can be calculated based on the molecular weight of the fragment relative to the molecular weight of the full-length antibody.

In an exemplary embodiment, 15-54 mg per 0.5-1.5 months; 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8 months; the subject is treated with the IL-23 specific antibody at an amount and interval of 300-1100 mg every 8-14 months or 300-1100 mg every 4-12 months. In some embodiments, the anti-IL-23 antibody is administered at a dose of 15-21 mg every 0.5-1.0 month, 55-70 mg every 1.5-3.0 months, 150-260 mg every 4-6 months, or 300-700 mg every 4-8 months. In some embodiments, the amounts and intervals are selected from: 21 mg per month; 70 mg every 3 months; 210 mg every 6 months; or 700 mg every 6 months. In some embodiments, the anti-IL-23 antibody is administered at 260 mg every 3 months or 700 mg every 3 months. In some embodiments, 260 mg of the antibody is administered every 1 month or 700 mg is administered every 1 month.

The administration and dosage regimen of the anti-IL-23 antibody can be adjusted to provide an effective amount to achieve an optimal therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation.

It is also contemplated that the anti-IL-23 therapies disclosed herein are used with more than one anti-IL-23 therapeutic agent or with other therapies. When multiple therapeutic agents are co-administered, the dosage may be adjusted accordingly, as recognized or known in the relevant art.

Particular embodiments of the methods and compositions of the invention relate to the use of an IL-23 antigen binding protein and one or more additional IL-23 antagonists, e.g., two or more antigen binding proteins of the invention or an antigen binding protein of the invention and one or more additional IL-23 antagonists. Also provided are IL-23 antigen binding proteins administered alone or in combination with other agents for treating a condition in a patient. Examples of such drugs include both protein and non-protein drugs. Such agents include therapeutic moieties that have anti-inflammatory properties (e.g., non-steroidal anti-inflammatory agents, steroids, immunomodulators and/or other cytokine inhibitors, such as those that antagonize, for example, IFN-. gamma., GM-CSF, IL-6, IL-8, IL-17, IL-22 and TNF), or IL-23 antigen binding proteins and one or more other therapies (e.g., surgery, ultrasound or a therapy effective to reduce inflammation). When multiple therapeutic agents are co-administered, the dosage may be adjusted accordingly, as recognized or known in the relevant art. Useful drugs that can be combined with an IL-23 antigen binding protein include those useful for treating, for example, Crohn's disease or ulcerative colitis, such as aminosalicylates (e.g., mesalamine), corticosteroids (including prednisone), antibiotics (such as metronidazole or ciprofloxacin or other antibiotics useful for treating, for example, patients with fistulas), and immunosuppressive agents (such as azathioprine, 6-mercaptopurine, methotrexate, tacrolimus, and cyclosporine. such drugs can be administered orally or by another route (e.g., via suppository or enema). Agents that can be combined with an IL-23 binding protein to treat psoriasis include corticosteroids, calcipotriene and other vitamin D derivatives, avilammic acid and other retinoic acid derivatives, methotrexate, tacrolimus, and cyclosporine. such drugs can be used simultaneously, Continuously, alternately, or according to any other regimen that enables the entire course of therapy to be effective.

In addition to human patients, IL-23 antigen binding proteins may also be used to treat non-human animals, such as domestic pets (dogs, cats, birds, primates, etc.) and domesticated farm animals (horses, cattle, sheep, pigs, birds, etc.). In this case, the appropriate dosage can be determined based on the weight of the animal. For example, dosages of 0.2-1 mg/kg may be used. Alternatively, the dosage is determined on the basis of the surface area of the animal, with exemplary dosages ranging from 0.1 to 20 mg/m²Or more preferably in the range of 5-12 mg/m²Within the range of (1). For small animals, such as dogs or cats, a suitable dose is 0.4 mg/kg. The IL-23 antigen binding protein (preferably constructed from a gene derived from the recipient species) is administered by injection or other suitable route once or more weekly until the condition of the animal is ameliorated, or it can be administered indefinitely.

In one aspect of the invention, the IL-22BP level in a brauzumab non-responder may be at least 359 pg/mL, e.g., 359 pg/mL to 6,000 pg/mL, wherein a subject having a serum level of IL-22BP below this level is responsive to an anti-IL-23 agent for an inflammatory disorder. The level of IL-22BP in the control can be determined based on subjects who do not have this inflammatory disorder, who were previously determined to be responsive to brekumab, or who were previously determined to be non-responsive to brekumab. In some embodiments, the IL-22BP level in a brekumab non-responder is 359-5,000 pg/mL, 359-4,000 pg/mL, 359-2,500 pg/mL, 359-1,000 pg/mL, 359-500 pg/mL, 400-6,000 pg/mL, 400-5,000 pg/mL, 400-4,000 pg/mL, 400-2,500 pg/mL, 400-1,000 pg/mL, 400-500 pg/mL, 500-6,000 pg/mL, 500-5,000 pg/mL, 500-4,000 pg/mL, 500-2,500/mL, 500-1,000 pg/mL, 750-6,000 pg/mL, 750,000/750, 5,000 pg/mL, 750,000 pg/mL, 750/750, 750-4,000 pg/mL, 750/mL, 750,000 pg 4,000/mL, 750/000, 750-2,500 pg/mL, 750-1,000 pg/mL, 1,000-6,000 pg/mL, 1,000-5,000 pg/mL, 1,000-4,000 pg/mL, 1,000-2,500 pg/mL, 1,500-6,000 pg/mL, 1,500-5,000 pg/mL, 1,500-4,000 pg/mL, 1,500-2,500 pg/mL, 2,000-6,000 pg/mL, 2,000-5,000 pg/mL, or 2,000-2,500 pg/mL.

In one aspect of the invention, the level of IFN- γ in a brekumab non-responder is less than 15 pg/mL, wherein a subject with a serum level of IFN- γ of at least 15 pg/mL is responsive to an anti-IL-23 agent for an inflammatory disorder. IFN- γ levels in the control can be determined based on subjects who do not have such an inflammatory disorder, who were previously determined to be responsive to brekumab, or who were previously determined to be non-responsive to brekumab.

The following examples (including experiments performed and results obtained) are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

Examples

Example 1

The data generated from the experiments described in these examples and presented in fig. 1-10 were obtained by Enzyme-Linked ImmunoSorbent assay (ELISA) using the commercial IL-22BP kit of R & D Systems (R & D Systems, inc., Minneapolis, MN). Serum levels of IL-22BP from healthy humans were compared to patients with Crohn's Disease (CD), Ulcerative Colitis (UC) and crohn's disease who was refractory to anti-TNF α therapy. The results show that serum levels of IL-22BP in CD and UC patients are higher than those in healthy humans. However, IL-22BP levels in CD patients refractory to anti-TNF α therapy are lower than IL-22BP levels in healthy humans.

In inflamed intestinal tissue of IBD patients, IL-23-regulated inflammatory cytokines (including IL-22, IL-17a, and IFN- γ) were increased compared to healthy controls. Unexpectedly, the soluble inhibitor of IL-22, IL-22BP, was also increased, indicating that IL-22 activity in the gut was tightly regulated. The data disclosed herein from the phase 2a clinical trial (i.e., the MedI phase 2a clinical trial) indicate that IBD patients, both non-responders to anti-TNF α treatment, have systemic (serum) IL-22BP levels that are lower than those of healthy human controls. See fig. 9 and 11. However, the median IL-22BP serum level in CD patients is generally higher than the median IL-22BP serum level in healthy controls. The MedI 2a phase results show that increased baseline levels of IL-22 in TNF-refractory active CD patients correlate strongly with efficacy of brauzumab treatment. That is, higher baseline levels of IL-22 in these patients may predict a subpopulation of patients most likely to benefit from treatment with brazizumab. Since high IL-22 levels are in this case associated with decreased IL-22BP levels in anti-TNF α non-responders, the present disclosure provides a method of identifying patients or patient subpopulations that are suitable for treatment with an anti-IL-23 agent due to decreased baseline serum levels of IL-22BP, disclosed herein as predictive biomarkers for patients or patient subgroups that are more likely to benefit from treatment with an anti-IL-23 agent than patients that do not exhibit decreased baseline serum levels of IL-22 BP. The subset of patients in figure 11 is identified from a population of patients who failed treatment with an anti-TNF agent (i.e., patients who did not respond to such treatment, patients who were intolerant to such treatment, or untreated IBD patients (i.e., patients who did not receive treatment for IBD)). The data presented in FIGS. 1-8 (as summarized in FIGS. 9 and 11) indicate that the mean serum level of IL-22BP in normal or healthy human males is 680 pg/ml (679.86 pg/ml) and the mean serum level in normal or healthy human females is 440 pg/ml (440.03 pg/ml). Thus, the mean IL-22BP serum level in healthy humans is not higher than 680 pg/ml. In contrast, humans with Crohn's disease exhibit a mean IL-22BP serum level of 2350 pg/ml (2355.78 pg/ml). The summary data in FIG. 9 also indicates that humans with ulcerative colitis have a mean IL-22BP serum level of 895 pg/ml (894.27 pg/ml).

Serum samples from crohn's disease patients refractory to anti-TNF-alpha therapy were also assayed for serum interferon-gamma levels by ELISA. Immunoassays were performed using the Meso Scale Discovery (NSD) 9plex cytokine assay kit (Meso Scale Discovery, Rockville, MD). Serum samples were obtained from crohn's disease patients who participated in the Medimmune phase 2a (MEDI2070-1147) clinical trial. All samples were obtained from patients in the brauzumab treatment group, and all patients provided with samples were non-responders to anti-TNF-a treatment, as described above. The results shown in figure 10 indicate that serum levels of IFN- γ were higher in the brekumab responders than in the non-responders, indicating that serum levels of IFN- γ can be used to identify a subpopulation of CD patients that responded to treatment with brekumab.

Example 2

Retrospective analysis of baseline IFN- γ serum levels showed that elevated IFN- γ levels correlated with a positive response to brekumab treatment in patients who failed, failed to respond, or were unable to tolerate treatment with an anti-TNF agent. See fig. 10. Thus, elevated baseline IFN- γ serum levels may predict or identify a subpopulation of TNF-refractory patients that would benefit from anti-IL-23 therapy.

All publications and patents mentioned in the application are herein incorporated by reference in their entirety or in relevant part as is apparent from the context. Various modifications and alterations of the disclosed subject matter will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. While the present disclosure has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Various modifications of the described modes for making or using the disclosed subject matter which are obvious to those skilled in the relevant fields are intended to be within the scope of the appended claims.

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Patient population suitable for IL23 antagonist therapy

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