Treatment of stage III NSCLC and remission of treatment-related pathological conditions

文档序号：816413 发布日期：2021-03-26 浏览：48次中文

阅读说明：本技术 Iii期nsclc治疗和与治疗相关的病理状况缓解 (Treatment of stage III NSCLC and remission of treatment-related pathological conditions ) 是由 I·度赛特 I·戈棱卡 Y·乌戈梅斯特 A·坎德瓦 O·克里斯顿森 S·厄尔巴瓦博兰燕于 2019-06-12 设计创作，主要内容包括：本公开内容总体上涉及采用双功能融合蛋白的靶向的TGF-β抑制的剂量方案,该剂量方案用于治疗诊断为III期非小细胞肺癌(NSCLC)的未经治疗的对象,和/或缓解与化疗和放疗(cCRT)相关的病理状况的方法。(The present disclosure relates generally to dosage regimens for targeted TGF- β inhibition using bifunctional fusion proteins for use in methods of treating an untreated subject diagnosed with stage III non-small cell lung cancer (NSCLC), and/or alleviating pathological conditions associated with chemotherapy and radiotherapy (crt).)

1. A method of treating an untreated patient diagnosed with stage III non-small cell lung cancer (NSCLC) and at risk of developing a pulmonary pathological condition associated with concomitant chemotherapy and radiation therapy (cCRT), said method comprising a first step of administering to said patient a dose of at least 1200mg of a protein comprising a first polypeptide and a second polypeptide and a concomitant cCRT, and a second step of administering to said patient at least 1200mg of said protein without a concomitant cCRT,

wherein the first polypeptide comprises: (a) at least the heavy chain variable region of an antibody capable of binding to human protein programmed death ligand 1 (PD-L1); and (b) a human transforming growth factor beta receptor II (TGF beta RII) or fragment thereof capable of binding transforming growth factor beta (TGF beta),

wherein the second polypeptide comprises at least the light chain variable region of an antibody that binds PD-L1, and

wherein the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds to PD-L1.

2. The method of claim 1, wherein the method alleviates the pathological condition of the lung associated with crt in the first step.

3. The method of claim 2, wherein the pathological condition is pneumonia and/or pulmonary fibrosis.

4. The method of any one of claims 1-3, wherein the method increases the onset of metastasis and/or the time to distant metastasis of stage III NSCLC in the patient.

5. The method of any one of claims 1-4, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO 3 and the second polypeptide comprises the amino acid sequence of SEQ ID NO 1.

6. The method of any one of claims 1-5, wherein the dose is 1200mg-2400 mg.

7. The method of any one of claims 1-6, wherein the dose is 1800mg-2400 mg.

8. The method of any one of claims 1-7, wherein the dose is 1800 mg.

9. The method of any one of claims 1-7, wherein the dose is 2400 mg.

10. The method of any one of claims 1-6, wherein the dose is administered once every two weeks or once every three weeks.

11. The method of claim 10, wherein the dose is 1200mg administered biweekly.

12. The method of claim 10, wherein the dose is 2400mg administered once every three weeks.

13. The method of claim 10, wherein the dose is 2100mg or 2400mg administered once every three weeks.

14. The method of any one of claims 1-13, wherein the stage III NSCLC exhibits squamous or non-squamous histology.

15. The method of any one of claims 1-14, wherein the stage III NSCLC exhibits PD-L1+ expression.

16. The method of any one of claims 1-14, wherein the stage III NSCLC does not exhibit PD-L1+ expression.

17. The method of any one of claims 1-16, wherein the patient has or does not have an EGFR sensitizing mutation.

18. The method of any one of claims 1-16, wherein the patient has or does not have Anaplastic Lymphoma Kinase (ALK) translocation.

19. The method of any one of claims 1-16, wherein the patient has or does not have ROS1 rearrangement.

20. The method of any one of claims 1-19, wherein the treatment results in remission of disease or improved survival in the patient.

21. The method of claim 20, wherein the disease remission is complete remission, partial remission, or stable disease.

22. The method of claim 21, wherein said survival is Progression Free Survival (PFS).

23. The method of any one of claims 1-22, wherein the chemotherapy comprises administering cisplatin/etoposide, cisplatin/pemetrexed, and/or carboplatin/paclitaxel to the patient.

24. The method of any one of claims 1-23, wherein the chemotherapy comprises cisplatin/pemetrexed and the stage III NSCLC exhibits non-squamous histology.

25. The method of claim 23 or 24, wherein cisplatin is at about 50mg/m²–80mg/m²The dose of (a) is administered intravenously.

26. The method of claim 23 or 24, wherein pemetrexed is at about 500mg/m²The dose of (a) is administered intravenously.

27. The method of claim 23, wherein etoposide is at about 50mg/m²The dose of (a) is administered intravenously.

28. The method of claim 23, wherein paclitaxel is present at about 45mg/m²The dose of (a) is administered intravenously.

29. The method of claim 23, wherein carboplatin is administered intravenously over 30 minutes based on AUC 2.

30. The method of any one of claims 1-29, wherein said radiation therapy comprises a dose of 60-74 Gy.

31. The method of claim 30, wherein the radiation therapy is administered for 6-7 weeks on days 1-5 during the first step.

32. The method of any one of claims 1-31, wherein the protein is administered by intravenous administration.

33. The method of claim 32, wherein the intravenous administration is performed using a pre-filled bag, a pre-filled pen, or a pre-filled syringe comprising a formulation comprising the protein.

34. The method of claim 33, wherein the bag connects a channel comprising a tube and/or a needle.

35. The method of any one of claims 1-34, wherein the second step begins 1-42 days after the end of the first step.

36. The method of claim 35, wherein the second step lasts 12-24 months.

37. A method of ameliorating a pulmonary pathological condition associated with chemotherapy and radiation therapy (cCRT) in an untreated patient diagnosed with stage III non-small cell lung cancer (NSCLC), the method comprising a first step of administering to the patient a dose of at least 1200mg of a protein and concomitant chemotherapy and radiation therapy (cCRT), and a second step of administering to the patient at least 1200mg of the protein without concomitant cCRT, the protein comprising a first polypeptide and a second polypeptide,

wherein the second polypeptide comprises at least the light chain variable region of an antibody that binds PD-L1, and

wherein the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds to PD-L1.

38. The method of claim 37, wherein the pathological condition is pneumonia and/or pulmonary fibrosis.

39. The method of claim 37 or 38, wherein the first polypeptide comprises the amino acid sequence of SEQ ID No. 3 and the second polypeptide comprises the amino acid sequence of SEQ ID No. 1.

40. The method of any one of claims 37-39, wherein the dose is 1200mg-2400 mg.

41. The method of any one of claims 37-40, wherein the dose is 1800mg-2400 mg.

42. The method of any one of claims 37-40, wherein the dose is 1200 mg.

43. The method of any one of claims 37-41, wherein the dose is 2400 mg.

44. The method of any one of claims 37-40, wherein the dose is administered once every two weeks or once every three weeks.

45. The method of claim 44, wherein the dose is 1200mg administered biweekly.

46. The method of claim 44, wherein the dose is 2400mg administered once every three weeks.

47. The method of claim 44, wherein the dose is 2100mg or 2400mg administered once every three weeks.

48. The method of any one of claims 37-47, wherein the stage III NSCLC exhibits squamous or non-squamous histology.

49. The method of any one of claims 37-48, wherein the stage III NSCLC exhibits PD-L1+ expression.

50. The method of any one of claims 37-48, wherein the stage III NSCLC does not exhibit PD-L1+ expression.

51. The method of any one of claims 37-50, wherein the patient has or does not have an EGFR sensitizing mutation.

52. The method of any one of claims 37-50, wherein the patient has or does not have Anaplastic Lymphoma Kinase (ALK) translocation.

53. The method of any one of claims 37-50, wherein the patient has or does not have ROS1 rearrangement.

54. The method of any one of claims 37-53, wherein the treatment results in remission or improved survival of stage III NSCLC disease in the patient.

55. The method of claim 54, wherein the disease remission is complete remission, partial remission, or stable disease.

56. The method of claim 55, wherein said survival is Progression Free Survival (PFS).

57. The method of any one of claims 37-56, wherein the chemotherapy comprises administering cisplatin/etoposide, cisplatin/pemetrexed, and/or carboplatin/paclitaxel to the patient.

58. The method of any one of claims 37-57, wherein the chemotherapy comprises cisplatin/pemetrexed and the stage III NSCLC exhibits non-squamous histology.

59. The method of claim 57 or 58, wherein cisplatin is at about 50mg/m²–80mg/m²The dose of (a) is administered intravenously.

60. The method of claim 57 or 58, wherein pemetrexed is at about 500mg/m²The dose of (a) is administered intravenously.

61. The method of claim 57, wherein etoposide is at about 50mg/m²The dose of (a) is administered intravenously.

62. The method of claim 57, wherein paclitaxel is administered at about 45mg/m²The dose of (a) is administered intravenously.

63. The method of claim 57, wherein carboplatin is administered intravenously over 30 minutes based on AUC 2.

64. The method of any one of claims 37-63, wherein said radiation therapy comprises a dose of 60-74 Gy.

65. The method of claim 64, wherein the radiation therapy is administered for 6-7 weeks on 1-5 days during the first step.

66. The method of any one of claims 37-65, wherein the protein is administered by intravenous administration.

67. The method of claim 66, wherein the intravenous administration is performed using a pre-filled bag, a pre-filled pen, or a pre-filled syringe comprising a formulation comprising the protein.

68. A method as in claim 67, wherein the bag connects a channel comprising a tube and/or a needle.

69. The method of any one of claims 37-68, wherein the second step begins 1-42 days after the end of the first step.

70. The method of claim 69, wherein the second step lasts 12-24 months.

71. The method of any one of claims 1-70, wherein the stage III non-small cell lung cancer (NSCLC) is unresectable.

72. The method of any one of claims 1-22 and 37-56, wherein the chemotherapy is a platinum-based chemotherapy.

73. An anti-PD-L1/TGF β trap protein for use in a method of treating an untreated patient diagnosed with stage III non-small cell lung cancer (NSCLC) and at risk of developing a pulmonary pathological condition associated with concomitant chemotherapy and radiation therapy (cCRT), the method comprising a first step of administering to the patient a dose of at least 1200mg of the protein and concomitant cCRT, and a second step of administering to the patient at least 1200mg of the protein without concomitant cCRT, the protein comprising a first polypeptide and a second polypeptide,

wherein the second polypeptide comprises at least the light chain variable region of an antibody that binds PD-L1, and

wherein the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds to PD-L1.

74. An anti-PD-L1/TGF β trap protein for use in a method of alleviating a pathological condition of the lung associated with chemotherapy and radiation therapy (cCRT) in a treatment-naive patient having stage III non-small cell lung cancer (NSCLC), said method comprising a first step of administering to said patient a dose of at least 1200mg of said protein and concomitant cCRT, and a second step of administering to said patient at least 1200mg of said protein without concomitant cCRT, said protein comprising a first polypeptide and a second polypeptide,

wherein the second polypeptide comprises at least the light chain variable region of an antibody that binds PD-L1, and

wherein the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds to PD-L1.

75. The anti-PD-L1/TGF β trap protein for use according to claim 73 or 74, wherein the method ameliorates the pathological condition in the lung associated with crt in the first step.

76. The anti-PD-L1/TGF β trap protein for use according to claim 75, wherein the pathological condition is pneumonia and/or pulmonary fibrosis.

77. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-76, wherein the method increases the onset of metastasis and/or the time to distant metastasis of stage III NSCLC in the patient.

78. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73 to 77, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO 3 and the second polypeptide comprises the amino acid sequence of SEQ ID NO 1.

79. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73-78, wherein the dose is 1200mg-2400 mg.

80. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73-79, wherein the dose is 1800mg-2400 mg.

81. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73-79, wherein the dose is 1200 mg.

82. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73-80, wherein the dose is 2400 mg.

83. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73-79, wherein the dose is administered once every two weeks or once every three weeks.

84. The anti-PD-L1/TGF β trap protein for use of claim 83, wherein the dose is 1200mg administered biweekly.

85. The anti-PD-L1/TGF β trap protein for use of claim 83, wherein the dose is 2400mg administered once every three weeks.

86. The anti-PD-L1/TGF β trap protein for use according to claim 79, wherein the dose is 2100mg or 2400mg administered once every three weeks.

87. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73-86, wherein the stage III NSCLC exhibits squamous or non-squamous histology.

88. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-87, wherein the stage III NSCLC exhibits PD-L1+ expression.

89. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-87, wherein the stage III NSCLC does not exhibit PD-L1+ expression.

90. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-89, wherein the patient has or does not have an EGFR sensitizing mutation.

91. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-89, wherein the patient has or does not have Anaplastic Lymphoma Kinase (ALK) translocation.

92. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-89, wherein the patient has or does not have a ROS1 rearrangement.

93. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-92, wherein the treatment results in disease remission or improved survival in the patient.

94. The anti-PD-L1/TGF β trap protein for use of claim 93, wherein the remission is complete remission, partial remission, or stable disease.

95. The anti-PD-L1/TGF β trap protein for use of claim 93, wherein the survival is progression-free survival (PFS).

96. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-95, wherein the chemotherapy comprises administration of cisplatin/etoposide, cisplatin/pemetrexed, and/or carboplatin/paclitaxel to the patient.

97. The anti-PD-L1/TGF β trap protein for use of any one of claims 73-95, wherein the chemotherapy comprises cisplatin/pemetrexed and the stage III NSCLC exhibits non-squamous histology.

98. The anti-PD-L1/TGF β trap protein for use of claim 96 or 97, wherein cisplatin is at about 50mg/m²–80mg/m²The dose of (a) is administered intravenously.

99. The anti-PD-L1/TGF β trap protein for use of claim 96 or 97, wherein pemetrexed is at about 500mg/m²The dose of (a) is administered intravenously.

100. The anti-PD-L1/TGF β trap protein for use of claim 96, wherein etoposide is at about 50mg/m²The dose of (a) is administered intravenously.

101. The anti-PD-L1/TGF β trap protein for use of claim 96, wherein paclitaxel is at about 45mg/m²The dose of (a) is administered intravenously.

102. The anti-PD-L1/TGF β trap protein for use of claim 96, wherein carboplatin is administered intravenously over 30 minutes based on AUC 2.

103. The anti-PD-L1/TGF β trap protein for use of any of claims 73-102, wherein the radiation therapy comprises a dose of 60-74 Gy.

104. The anti-PD-L1/TGF β trap protein for use of claim 103, wherein the radiotherapy is administered during the first step on days 1-5 for 6-7 weeks.

105. An anti-PD-L1/TGF β trap protein for use according to any one of claims 73-104, wherein the protein is administered by intravenous administration.

106. The anti-PD-L1/TGF β trap protein for use of claim 105, wherein the intravenous administration is performed using a pre-filled bag, a pre-filled pen, or a pre-filled syringe comprising a formulation comprising the protein.

107. An anti-PD-L1/TGF β trap protein for use according to claim 106, wherein the pocket junction comprises a channel of a tube and/or needle.

108. An anti-PD-L1/TGF β trap protein for use as claimed in any of claims 73-107, wherein the second step commences 1-42 days after the end of the first step.

109. The anti-PD-L1/TGF β trap protein for use of claim 108, wherein the second step lasts 12-24 months.

Technical Field

The present disclosure relates generally to dosage regimens for targeted TGF- β inhibition using bifunctional fusion proteins for use in methods of treating an untreated subject diagnosed with stage III non-small cell lung cancer (NSCLC), and/or alleviating pathological conditions associated with chemotherapy and radiotherapy (crt).

Background

Treatment of locally advanced, unresectable stage III NSCLC with chemotherapy and concurrent radiation therapy (crt) generally fails to control disease progression in NSCLC patients. In addition, radiation therapy causes pathological conditions such as pulmonary fibrosis. Radiation-induced pulmonary fibrosis may occur when lung tissue is irradiated at a dose of ≧ 20Gy within the first 6 months after initiation of treatment.

TGF β is the major profibrotic molecule contributing to the development of pulmonary fibrosis. U.S. patent application publication No. US20150225483a1, which is incorporated herein by reference, describes a bifunctional fusion protein that binds an anti-programmed death ligand 1(PD-L1) antibody and a tumor growth factor beta receptor type II (TGF β RII) extracellular soluble domain as a TGF β neutralizing "sink" as a single molecule. Specifically, the protein is a heterotetramer consisting of two immunoglobulin light chains of anti-PD-L1 and two heavy chains comprising an anti-PD-L1 heavy chain and the extracellular domain of human TGF β RII genetically fused thereto by a flexible glycine-serine linker (see figure 1). This anti-PD-L1/TGF β trap molecule was designed to target two major immunosuppressive mechanisms in the tumor microenvironment. U.S. patent application publication No. US20150225483a1 describes the administration of the trap molecule in a dose based on the weight of the patient.

The present disclosure provides dosage regimens for targeting TGF- β inhibition using anti-PD-L1/TGF β trap molecules for use in methods of treating an untreated subject diagnosed with stage III NSCLC and/or alleviating pathological conditions associated with concurrent crt (e.g., pulmonary fibrosis, pneumonia).

Summary of The Invention

In order to effectively treat patients diagnosed with stage III NSCLC and to cope with acute and long-term symptomatic lung injury due to fibrosis, the present invention provides a treatment for stage III NSCLC while preserving as much normal lung tissue as possible from radiation-induced injury, thereby improving disease prognosis and overall survival of NSCLC patients.

In one aspect, the disclosure provides an anti-PD-L1/TGF β trap with a concomitant crt to target two immunosuppressive pathways simultaneously: PD-L1 and TGF- β, thereby treating stage III NSCLC while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the onset of metastasis and/or the time to distant metastasis of stage III NSCLC in a patient.

The present disclosure provides improved dosing regimens for administering bifunctional proteins targeting PD-L1 and TGF β to treat stage III NSCLC while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the onset of metastasis and/or the time to distant metastasis of stage III NSCLC in a patient. In particular, a weight-independent (BW-independent) dosing regimen and related dosage forms involving the administration of at least 500mg (e.g., 1200mg, 1800mg, 2400mg) of bifunctional protein administered at different dosing frequencies can be used as an anti-tumor and anti-cancer therapy for treating stage III NSCLC while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia) and increasing the onset of metastasis and/or the time to distant metastasis of stage III NSCLC in patients. The BW-independent dosing regimen ensures that all stage III NSCLC patients, regardless of their body weight, have sufficient drug exposure at the tumor site.

The bifunctional proteins of the present disclosure (anti-PD-L1/TGF β trap molecules) comprise a first and a second polypeptide. The first polypeptide comprises: (a) at least the heavy chain variable region of an antibody capable of binding human protein programmed death ligand 1 (PD-L1); and (b) a human transforming growth factor beta receptor II (TGF β RII) or a fragment (e.g., a soluble fragment) thereof capable of binding transforming growth factor beta (TGF β). The second polypeptide comprises: at least a light chain variable region of an antibody capable of binding PD-L1, wherein the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site capable of binding PD-L1 (e.g., any antibody or antibody fragment described herein). Because the bifunctional proteins of the present disclosure are capable of binding to two targets: (1) PD-L1, which is mostly membrane bound, and (2) TGF β, which is a soluble form in blood and stroma, a BW-independent dosing regimen requires a dose that is not only capable of inhibiting PD-L1 at the tumor site, but also is sufficient to inhibit TGF β.

In one aspect, the present disclosure provides dosage regimens for targeted TGF- β inhibition using bifunctional fusion proteins for use in methods of treating an untreated subject diagnosed with stage III non-small cell lung cancer (NSCLC), and/or alleviating pathological conditions associated with chemotherapy and radiotherapy (crt).

In one aspect, the disclosure provides dosage regimens for targeted TGF- β inhibition using bifunctional fusion proteins for use in methods of treating stage III non-small cell lung cancer exhibiting squamous or non-squamous histology, and/or alleviating pathological conditions associated with chemotherapy and radiation therapy (crt). In certain embodiments, stage III NSCLS is unresectable.

In one aspect, the invention provides a method of treating advanced unresectable stage III NSCLC in a patient by administering to the patient an anti-PD-L1/TGF β trap of the invention in combination with crt (e.g., platinum-based chemotherapy), wherein the anti-PD-L1/TGF β trap is administered to the patient after crt. In certain embodiments, the present disclosure provides a method of treating advanced unresectable stage III NSCLC in a patient by: the patient was given an anti-PD-L1/TGF β trap, combined and followed by (in combination with and following) concurrent (current) platinum-based radiotherapy (chemotherapy).

In certain embodiments, crts are administered cisplatin/etoposide, cisplatin/pemetrexed, or carboplatin/paclitaxel concurrently with radiation (e.g., radiation delivered by intensity modulated radiation therapy).

In certain embodiments, the present invention provides a method of treating advanced unresectable stage III NSCLC in a patient having non-squamous histology by: anti-PD-L1/TGF β trap is administered to the patient in combination with crt (e.g., cisplatin/pemetrexed and radiation therapy), followed by administration of anti-PD-L1/TGF β trap to the patient. In certain embodiments, the present disclosure provides a method of treating advanced unresectable stage III NSCLC in a patient by: the patient is administered an anti-PD-L1/TGF β trap that is combined and followed by concurrent (concurrent) cisplatin/pemetrexed and radiation (e.g., radiation delivered by intensity modulated radiotherapy).

The disclosure also includes a method of promoting local subtraction of TGF β. The method comprises administering the above protein, which binds to TGF-beta in solution, binds to PD-L1 on the cell surface, and brings the bound TGF-beta into a cell (e.g., a cancer cell).

The disclosure also includes methods of inhibiting phosphorylation of SMAD3 in a cell (e.g., a cancer cell or an immune cell), the method comprising exposing the cell in a tumor microenvironment to the above-described protein.

Other embodiments and details of the present disclosure will be apparent hereinafter.

Brief description of the drawings

FIG. 1 is a schematic representation of an anti-PD-L1/TGF-beta trap molecule comprising one anti-PD-L1 antibody and two TGF-beta receptor II extracellular domains (ECDs) passing through (Gly₄Ser)₄Gly (SEQ ID NO:11) linker fusion.

FIG. 2 shows a two-step ELISA showing that anti-PD-L1/TGF β trap binds to both PD-L1 and TGF β.

FIG. 3 shows that anti-PD-L1/TGF β trap induces a dramatic increase in IL-2 levels.

FIG. 4A is a graph showing in vivo consumption of TGF-beta 1 in response to anti-PD-L1/TGF-beta trap. The line plots represent the naive, isotype control, and three different doses as shown in the legend. FIG. 4B is a graph showing in vivo consumption of TGF-beta 2 in response to anti-PD-L1/TGF-beta trap. The line plots represent the naive, isotype control, and three different doses as shown in the legend. FIG. 4C is a graph showing in vivo consumption of TGF-beta 3 in response to anti-PD-L1/TGF-beta trap. The line plots represent the naive, isotype control, and three different doses as shown in the legend. FIG. 4D shows that the occupancy of PD-L1 by anti-PD-L1/TGF β trap molecules supports a receptor binding model in the EMT-6 tumor system.

FIG. 5 shows the anti-tumor efficacy of anti-PD-L1/TGF β trap molecule (anti-PD-L1 (mut)/TGF β) in the Detroit562 xenograft model.

FIG. 6A is the whole population C in a median weight mimic population of 68kg for a fixed dose (1200mg) versus a mg/kg body weight dose (17.65mg/kg)_AverageDistribution box plot. FIG. 6B is a boxplot of total population exposure AUC in a median body weight simulated population of 68kg at a fixed dose (1200mg) versus a mg/kg body weight dose (17.65 mg/kg). FIG. 6C is the whole population C in the median weight mimic population of 68kg at a fixed dose (1200mg) versus a mg/kg body weight dose (17.65mg/kg)_GrainDistribution box plot. FIG. 6D is the whole population C in a median weight mimic population of 68kg at a fixed dose (1200mg) versus a mg/kg body weight dose (17.65mg/kg)_{Maximum of}Distribution box plot.

FIG. 6E is the whole population C in a median weight mimic population of 68kg for a fixed dose (500mg) versus a mg/kg body weight dose (7.35mg/kg)_AverageDistribution box plot. FIG. 6F is a boxplot of total population exposure AUC in a median body weight simulated population of 68kg at a fixed dose (500mg) versus a mg/kg body weight dose (7.35 mg/kg). FIG. 6G is the whole population C in the median weight mimic population of 68kg at a fixed dose (500mg) versus a mg/kg body weight dose (7.35mg/kg)_GrainDistribution box plot. FIG. 6H is the whole population C in a median weight mimic population of 68kg for the fixed dose (500mg) versus the mg/kg body weight dose (7.35mg/kg)_{Maximum of}Distribution box plot.

Figures 7A-7C are graphs showing predicted PK and PD-L1 receptor occupancy ("RO") of anti-PD-L1/TGF β trap molecules in doses and regimens associated with tumor arrest in mice. Figure 7A is a graph showing predicted plasma concentrations versus time. Figure 7B is a graph showing predicted PD-L1 RO versus time in PBMC. Figure 7C is a graph showing predicted PD-L1 RO versus time in tumors.

FIG. 8 shows box plots of gene expression signatures associated with fibrosis in control mice (untreated) and mice treated with anti-PD-L1/TGF β trap molecule, radiation, and anti-PD-L1/TGF β trap molecule and radiation.

FIG. 9 shows gene expression profiles (based on RNA sequencing analysis) for Cxcl12, Fap and Cdc6 after treatment of mice with radiation, anti-PD-L1/TGF β trap molecule and concomitant anti-PD-L1/TGF β trap and radiation. "control" represents gene expression in mice that have not been treated.

Figure 10 is a schematic of the treatment protocol described in example 3. Stable disease, partial remission and complete remission are indicated by SD, PR and CR, respectively.

Figure 11 is a schematic of the treatment protocol described in example 4. Stable disease, partial remission and complete remission are indicated by SD, PR and CR, respectively.

Figures 12A-12C are histograms showing that anti-PD-L1/TGF β trap and trap control, but not anti-PD-L1, reduced chemotherapy-induced fibrosis. Figure 12A shows that although anti-PD-L1 antibody did not affect collagen content relative to isotype control, both the trap control and anti-PD-L1/TGF β trap treatment significantly reduced collagen content (total collagen (percentage bitter red (PSR)); PSR staining is a commonly used histological technique that allows visualization of collagen in paraffin-embedded tissue sections; PSR stained collagen appears red under light microscopy)); p-0.0038 and p-0.0019, respectively). Figure 12B shows that anti-PD-L1 antibody did not affect the percentage of alpha SMA relative to isotype control, but both trap control and anti-PD-L1/TGF β trap treatment significantly reduced the percentage of alpha SMA (p 0.0003 and p 0.0013, respectively). Figure 12C is a bar graph showing that anti-PD-L1/TGF β trap decreased the ratio of pSmad2/3 (p 0.0006) relative to isotype control treatment.

Figure 13A is a scatter plot showing that monotherapy against PD-L1/TGF β trap resulted in a decrease in epithelial-to-mesenchymal transition (EMT) feature score (p <0.0001) relative to isotype control, and that the combination of anti-PD-L1/TGF β trap and radiation therapy significantly down-regulated EMT feature score (p <0.0001) relative to isotype control. Figure 13B is a scatter plot showing that anti-PD-L1/TGF β trap monotherapy also reduced profibrotic gene signature scores, but radiotherapy significantly increased profibrotic gene signature scores relative to isotype controls (p < 0.0001). Furthermore, combining radiation with anti-PD-L1/TGF β trap reduced the profibrotic signature score relative to radiation alone.

Figure 14A depicts a box line plot showing that anti-PD-L1/TGF β trap in combination with radiotherapy significantly reduced ACTA2 expression. Although radiotherapy alone had no significant effect on expression of ACTA2, anti-PD-L1/TGF β trap monotherapy and anti-PD-L1/TGF β trap combination radiotherapy significantly reduced ACTA2 expression in the 4T1 model (p <0.0001 and p ═ 0.0236, respectively).

Fig. 14B depicts a boxplot showing that anti-PD-L1/TGF β trap significantly reduced CTGF expression (P0.0019) relative to isotype control, and as expected, the anti-PD-L1/TGF β trap combination significantly offset the effects of radiation therapy compared to radiation monotherapy despite the radiation therapy increased CTGF (P0.0024).

Fig. 14C depicts a box plot diagram showing that anti-PD-L1/TGF β well significantly reduced FAP expression (P <0.0001) relative to isotype control, and further reduced FAP reduction observed in the case of radiotherapy (P0.0054) by the combination of anti-PD-L1/TGF β well with radiation.

FIG. 15 depicts a box line plot showing the number of α -SMA + pixels determined and normalized to a region of interest (ROI) for a plurality of ROIs per tumor; each symbol represents the positive pixel proportion of a single tumor. P values were determined by one-way analysis of variance. Scale bar, 250 μm.

FIGS. 16A-16D are images showing that anti-PD-L1/TGF β trap treatment reduced alpha-SMA expression in mouse tumors. anti-PD-L1/TGF β trap treatment significantly reduced α -SMA expression (p <0.0001) relative to isotype control (fig. 16A), while radiotherapy significantly increased α -SMA expression (p ═ 0.0002) (fig. 16C). The combination of anti-PD-L1/TGF β trap with radiotherapy significantly reduced α -SMA expression (p ═ 0.0001) relative to single radiotherapy (fig. 16D), suggesting that anti-PD-L1/TGF β trap may reduce radiation-induced cancer-associated fibroblast (CAF) activity.

Figure 17 is a schematic of the treatment protocol described in example 6. Stable disease, partial remission and complete remission are indicated by SD, PR and CR, respectively.

Detailed Description

"TGF-beta RII" or "TGF-beta receptor II" refers to a polypeptide having a wild-type human TGF-beta receptor type 2 isoform A sequence (e.g., the amino acid sequence of NCBI reference sequence (RefSeq) accession number NP-001020018 (SEQ ID NO: 8)), or a polypeptide having a wild-type human TGF-beta receptor type 2 isoform B sequence (e.g., the amino acid sequence of NCBI reference sequence (RefSeq) accession number NP-003233 (SEQ ID NO: 9)), or a sequence that is substantially identical to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO: 9. The tgfbetarii may retain at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95% or 99% of the wild-type sequence tgfbeta binding activity. The expressed TGF-beta RII polypeptide has no signal sequence.

A "fragment of TGF-beta RII capable of binding TGF-beta" refers to any portion of NCBI RefSeq accession No. NP-001020018 (SEQ ID NO:8) or NCBI RefSeq accession No. NP-003233 (SEQ ID NO:9), or a sequence substantially identical to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9, having a length of at least 20 (e.g., at least 30,40,50,60,70,80,90,100,110,120,130,140,150,160,175, or 200) amino acids, and maintaining TGF-beta binding activity (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99%) of at least some wild-type receptors or corresponding wild-type fragments. Typically, such fragments are soluble fragments. One of the exemplary fragments is the extracellular domain of TGF-beta RII having the sequence of SEQ ID NO 10.

By "untreated" is meant a subject or patient who has not received prior systemic treatment for stage III NSCLC since it was diagnosed with the disease. In various embodiments of the disclosure, the untreated patient has not received prior treatment with anti-PD-1, anti-PD-L1, or anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4) antibodies, including ipilimumab, or any other antibody or drug that specifically targets the T cell costimulatory or checkpoint pathway. In various embodiments of the present disclosure, untreated patients are selected for the first line (1L) treatment of the present invention.

"PD-L1 positive" or "PD-L1 +" means ≧ 1% PD-L1 positive tumor cells, as determined, for example, by the Dako IHC 22C3 PharmDx assay or VENTANA PD-L1(SP263) assay.

"PD-L1 high" or "high PD-L1" refers to PD-L1 IHC 73-10 assay (Dako) assay ≧ 80% PD-L1 positive tumor cells, or Dako IHC 22C3 PharmDx assay ≧ 50% Tumor Proportion Score (TPS) (TPS is a term of relevance for IHC 22C3 assays describing the percentage of viable tumor cells with partial or complete membrane staining (e.g., PD-L1 staining)). IHC 73-10 and Dako IHC 22C3 tests similar patient populations were selected at the respective cut-off values. In some embodiments, high expression levels of PD-L1 can also be determined using the VENTANA PD-L1(SP263) assay that is highly correlated with the 22C3 PharmDx assay (see Sughayer et al, appl.

By "substantially identical" is meant that the polypeptide exhibits at least 50%, preferably 60%, 70%, 75% or 80%, more preferably 85%, 90% or 95%, and most preferably 99% amino acid sequence identity to the reference amino acid sequence. The length of the comparison sequences is generally at least 10 amino acids, preferably at least 15 contiguous amino acids, more preferably at least 20, 25, 50, 75, 90,100, 150, 200, 250, 300 or 350 contiguous amino acids, and most preferably the full-length amino acid sequence.

"patient" means a human or non-human animal (e.g., a mammal). "patient," "subject," "patient in need" and "subject in need" are used interchangeably in this disclosure and refer to a living organism suffering from or susceptible to a disease or condition that can be treated by administration using the methods and compositions provided in this disclosure.

The terms "treat," "treatment," or other grammatical equivalents as used in this disclosure include alleviating, ameliorating, improving, or preventing a disease, condition, or symptom, preventing other symptoms, ameliorating, or preventing an underlying metabolic cause of a symptom, inhibiting a disease or condition, e.g., arresting the development of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, relieving a condition caused by a disease or condition, or stopping a symptom of a disease or condition, and are intended to include preventing. The term also includes achieving a therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit refers to eradication or amelioration of the underlying disease being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, whereby an improvement is observed in the patient, although the patient may still be suffering from the underlying disorder.

The term "consolidation" is used in the context of the treatment regimen of the present disclosure, as is commonly understood in the art. For example, according to the U.S. national cancer institute's parlance, the term "consolidation therapy" is a "treatment" administered after the cancer disappears after the initial therapy. Consolidation therapy is used to kill any cancer cells that may remain in the body. It may include radiation therapy, stem cell transplantation or treatment with drugs that kill cancer cells. Also known as intensive therapy and post-remission therapy. "https:// www.cancer.gov/publications/criteria/candidates/cancer-terms/def/association-th copy, last visit on 6/9/2018.

The term "progression-free survival" or PFS is defined as the time from randomized cohort (6 or more weeks after initiation of treatment can occur) to the first documented tumor progression or death without disease progression. The term "overall survival" is defined as the time from random grouping to death of any cause. Researchers evaluated progression-free survival as a pre-defined sensitivity analysis according to RECIST version 1.1.

The terms "alleviate," "alleviate," or "alleviating," and other grammatical equivalents used in this disclosure include alleviating, ameliorating, or preventing a disease, condition, or symptom, preventing other symptoms, ameliorating, or preventing a metabolic cause underlying the symptom, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, alleviating the disease or condition, causing regression of the disease or condition, alleviating the condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include preventing.

By "Cancer" is meant stage III (IIIA, IIIB and/or IIIC) non-small cell lung Cancer (NSCLC) used according to its immediate and ordinary meaning, e.g., as characterized by the National Cancer Institute. Thus, in various embodiments, the cancer has spread to, for example, lymph nodes on the same side of the primary tumor or to lymph nodes on the same opposite side of the breast as the primary tumor.

The term "unresectable" refers to a cancer that cannot be removed by surgery.

The terms "risk", "at risk" and "risk factor" are used herein as is conventionally understood in the art. For example, a risk factor is any attribute, characteristic, or exposure of an individual that increases the likelihood of a disease or injury. In certain embodiments, a human at risk for a disease, disorder or condition refers to exposure of the human to a risk factor that contributes to or increases the probability of the occurrence of the disease, disorder or condition.

Throughout the description and claims of this disclosure, the word "comprise" and other forms of the word, such as "comprises" and "comprising," mean including but not limited to, and are not intended to exclude, for example, other components.

By "co-administration" and "co-administration" is meant that the compositions described herein are administered simultaneously with, immediately prior to, or immediately after the administration of additional therapy. The proteins and compositions of the present disclosure may be administered alone, or may be co-administered to a patient with a second, third, or fourth therapeutic agent. Co-administration is intended to include simultaneous or sequential administration of the protein or composition, either alone or in combination (more than one therapeutic agent).

The terms "a" and "an" are not meant to be limiting. In certain embodiments, the terms "a" and "an" may refer to the plural form. As used throughout this document, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a plurality of such compositions, as well as a single composition.

A "reconstituted" formulation is one prepared by dissolving a lyophilized formulation in an aqueous carrier such that the bifunctional molecule is dissolved in the reconstituted formulation. The reconstituted formulation is suitable for intravenous administration (IV) to a patient in need thereof.

The term "about" refers to any minimal change in the concentration or amount of a drug that does not alter the efficacy of the drug in the preparation of the formulation and in the treatment of a disease or disorder. In embodiments, the term "about" may include ± 15% of a specified numerical value or data point.

In the present disclosure, a range can be expressed as starting from "about" one particular value and/or ending with "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that the disclosure discloses a plurality of values, and that each value is disclosed in the disclosure as "about" that particular value in addition to being disclosed as the value itself. It should also be understood that throughout this application, data is provided in a number of different formats and represents various endpoints and starting points and ranges of any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15, and between 10 and 15 are disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic preparations typically have about 250 to 350mOsmol/KgH₂Osmotic pressure of O. The term "hypertonic" is used to describe formulations with an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using vapor pressure or freezing type osmometers.

The term "buffer" refers to one or more components that are capable of protecting a solution from pH changes when added to an aqueous solution, when added to an acid or base, or when diluted with a solvent. In addition to phosphate buffer, glycinate, carbonate, citrate buffer, etc. may also be used, in which case sodium, potassium or ammonium ions may be used as counter ions.

An "acid" is a substance that generates hydrogen ions in an aqueous solution. "pharmaceutically acceptable acids" include inorganic and organic acids that are non-toxic in their formulated concentrations and manner.

"base" is a substance that generates hydroxide ions in an aqueous solution. "pharmaceutically acceptable bases" include inorganic and organic bases that are non-toxic in the concentrations and manner in which they are formulated.

A "lyoprotectant" is a molecule that, when bound to a protein of interest, prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage.

"preservatives" are substances that reduce the action of bacteria and may optionally be added to the formulations herein. The addition of a preservative may, for example, facilitate the production of a multiple-use (multi-dose) formulation. Examples of useful preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, where the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

A "surfactant" is a surface active molecule containing a hydrophobic moiety (e.g., an alkyl chain) and a hydrophilic moiety (e.g., a carboxyl group and a carboxylate group). Surfactants may be added to the formulations of the present invention. Surfactants suitable for use in the formulations of the present invention include, but are not limited to, polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); sorbitan esters and derivatives; triton (Triton); sodium lauryl sulfate; sodium octyl glucoside; dodecyl-, myristoyl-, linoleyl-or stearyl-sulfobutadiene (sulfobetadine); dodecyl-, myristoyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-or hexadecyl-betaine; lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropylbetaines (e.g., lauramidopropyl); myristoylamidopropyl-, palmitoylamidopropyl-or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-taurate, or disodium methyl oleoyl-taurate; and MONAQUAT^TMSeries (Mona Industries, Inc.), Patterson, N.J.), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., sodium montmorillonite, Inc.), and combinations thereofPluronics, PF68, etc.).

Weight independent dosing regimen

Based on the results of various preclinical and clinical assessments of the molecule, weight-independent dosing regimens have been developed that involve administering at least 500mg of the bifunctional anti-PD-L1/TGF β trap molecule described herein to untreated patients. Two studies investigated the safety, tolerability and pharmacokinetics of the molecule, including the assessment of PD-L1 target occupancy on peripheral blood mononuclear cells obtained from the blood of treated patients, and the measurement of TGF β 1, TGF β 2 and TGF β 3 concentrations. These assessments were based on data from a total of 350 subjects (dose escalation groups of 1, 3, 10 and 20mg/kg in solid tumors, and expansion groups of 3mg/kg, 10mg/kg, 500mg and 1200mg in selected tumor types).

PK/efficacy model (mouse model)

The efficacy of the anti-PD-L1/TGF beta trap molecule in tumor models was also experimentally determined. The efficacy results from EMT-6 xenografts were used to establish a PK/efficacy model. The PK model established in mice was used to simulate anti-PD-L1/TGF β trap plasma exposure for efficacy experimental setup. The estimated parameters are shown in Table 1. The estimated KC50 value was 55.3. mu.g/mL, which represents the mean plasma concentration at which 50% of the maximum anti-tumor activity of the anti-PD-L1/TGF β trap molecule was obtained.

The basic diagnostic map of the model shows no model errors. Model prediction enables the acquisition of tumor volume distribution. The conditionally weighted residuals are typically distributed with 0 means and 1 variance without trends. Tumor Growth Inhibition (TGI) was then simulated using a PK/efficacy model with human predicted concentration-time curves at different doses.

Table 1: mouse PK/efficacy model parameters for anti-PD-L1/TGF beta trap molecules in EMT-6 xenografted mice

Parameter(s)	Estimating	Std	CV％	％IIV
					K_g(h^-1)	0.068	0.0005	0.82	40
K_tr(h^-1)	0.055	0.0024	4.4	76
					KC₅₀(ng/mL)	55324.6	522.3	4.4	232
K_{Maximum of}	2	0.09	1	93
					Base line (mm)³)	88.3	0.87	1	47

Effect analysis based on PD-L1 occupancy (in mouse model)

Using efficacy experiments, the effect in mice was analyzed and sorted by tumor regression or tumor arrest, and PK and PD-L1 Receptor Occupancy (RO) were predicted based on an integrated PK/RO model. This method indicates that anti-PD-L1/TGF β trap molecule plasma concentrations of 40 to 100 μ g/mL are required to achieve tumor regression, which correlates with greater than 95% PD-L1 RO within the tumor. anti-PD-L1/TGF β trap molecule plasma concentrations of 10 to 40 μ g/mL were required to achieve tumor arrest states, correlating to greater than 95% peripheral PD-L1 RO.

Effect analysis and prediction of PK/RO in mice see FIGS. 7A-7C, which summarizes PK/RO/efficacy of anti-PD-L1/TGF β trap molecules in mice. At a plasma concentration of 40 μ g/mL, 95% PD-L1 RO was achieved, with an expected/estimated TGI of only about 65%. Raising the concentration above 40 μ g/mL results in a further enhancement of tumor growth inhibition. At a mean plasma concentration of about 100 μ g/mL, 95% inhibition of tumor growth was achieved.

Based on the population PK model described below, a flat dose of at least 500mg administered once every two weeks is required to maintain an average concentration of about 100 μ g/mL, and a flat dose of about 1200mg administered once every two weeks is required to maintain a C of about 100 μ g/mL_Grain. In certain embodiments, from about 1200mg to about 3000mg (e.g., about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, etc.) of a protein product of the present disclosure (e.g., anti-PD-L1/TGF β trap) is administered to a subject. In some embodiments, about 1200mg of the anti-PD-L1/TGF β trap molecule is administered to the subject once every two weeks. In certain embodiments, the subject is administered about 1800mg of the anti-PD-L1/TGF β trap molecule once every three weeks.

In certain embodiments, about 1200mg to about 3000mg (e.g., about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, etc.) of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID No. 3 and a second polypeptide comprising the amino acid sequence of SEQ ID No. 1 is administered to the subject. In certain embodiments, about 1200mg to about 3000mg (e.g., about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, etc.) of a protein product having a first polypeptide comprising the amino acid sequences SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequences SEQ ID NOs 38, 39, and 40 is administered to a subject.

In certain embodiments, about 1200mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO. 3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO. 1 is administered to a subject biweekly. In certain embodiments, about 1800mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID No. 3 and a second polypeptide comprising the amino acid sequence of SEQ ID No. 1 is administered to a subject once every three weeks. In certain embodiments, about 1200mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40 is administered to a subject once every two weeks. In certain embodiments, about 1800mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40 is administered to a subject once every three weeks.

Establishment of a weight-independent dosing regimen

Based on clinical and preclinical data, a new weight-independent dosing regimen has been developed for the administration of anti-PD-L1/TGF β trap molecules to reduce exposure variability, reduce dosing errors, reduce the time required to perform dose preparation, and reduce drug waste compared to mg/kg doses, thereby facilitating the achievement of good therapeutic results. According to one embodiment, a uniform dose of at least 500mg may be administered regardless of the weight of the patient. According to another embodiment, a uniform dose of at least 1200mg may be administered regardless of the weight of the patient. According to another embodiment, a uniform dose of at least 1,800mg may be administered regardless of the weight of the patient. According to certain embodiments, a uniform dose of at least 2400mg can be administered regardless of the weight of the patient. Typically, these doses will be administered repeatedly, for example once every two weeks or once every three weeks. For example, a uniform dose of 1200mg may be administered once every two weeks, or a uniform dose of 1800mg may be administered once every three weeks, or a uniform dose of 2400mg may be administered once every three weeks.

Pharmacokinetic (PK) analysis sampling in humans

An example of a pharmacokinetic analysis to determine the optimal uniform dose of anti-PD-L1/TGF β trap is provided by the following experiment.

Serum samples for Pharmacokinetic (PK) data analysis were collected at the following time points before the start of the first dose and after the first dose: day 1 immediately after infusion and 4 hours after infusion began; day 2 at least 24 hours after the end of the infusion on day 1; and on days 8 and 15. Samples were collected on days 15, 29, 43 at selected subsequent dosing occasions before dosing, at the end of infusion, and 2 to 8 hours after the end of infusion. For subsequent time points on days 57, 71, and 85, pre-dose samples were collected or scheduled to be collected, followed by PK sampling every 6 weeks for up to 12 weeks, followed by PK sampling every 12 weeks. In the extension phase, sparse PK sampling is performed.

The PK data described above were used to generate a population PK model and possible dosing regimen simulations. A modeling method called Full-plan model (Full-Covariate model) (see Gastongouy, M., Full Covariate model as an Alternative to the method, Relying on Statistical Significance of Inferences about Covariate Effects: methodologies and 42Case study reviews (Full Covariate Models as an Alternative to Methods of optimization and Statistical simulation for information about out Covariate Effects: A Review of Methods and 42Case students), (2011), page 20, abstract 2229) was applied to the population model data obtained from the simulation to obtain parameters with the following characteristics: two-chamber PK model with linear elimination, IIV for CL, V1 and V2, combined sum-proportion residual, full covariate model for CL and V1. The following baseline covariates were included in the final model: age, body weight, gender, race, albumin, CRP, platelet count, eGFR, liver injury, ECOG score, tumor size, tumor type, and prior treatment with biologies. The following pharmacokinetic typical parameter estimates for proteins of the disclosure (e.g., anti-PD-L1/TGF β trap) were obtained: clearance (CL)0.0177L/h (6.2%), central distribution volume (V1)) 3.64L (8.81%), peripheral distribution volume (V2) 0.513L (25.1%), and interchamber clearance (Q) 0.00219L/h (17.8%). The interpatient variation for CL was 22%, V1 was 20%, and V2 was 135%. Body weight is the relevant covariate for CL and V1. To support a uniform dosing regimen, the effect of dosing strategies on contact variation of proteins of the invention (e.g., anti-PD-L1/TGF β trap) was investigated. Specifically, simulations were performed to compare exposure profiles, using a 1200mg once every two weeks unified dosing method, versus a BW adjusted dosing regimen of 17.65mg/kg once every two weeks (corresponding to 1200mg once every two weeks for 68kg subjects) or 15mg/kg once every two weeks (corresponding to 1200mg for 80kg subjects). Further simulations were performed to compare the exposure profiles of the 500mg once every two weeks unitary dose regimen versus the BW adjusted dosing regimen of 7.35mg/kg once every two weeks (corresponding to 500mg once every two weeks for 68kg subjects). Additionally, simulations were performed to evaluate the following once every three weeks unitary dose: 1200mg, 1400mg, 1600mg, 1800mg, 2000mg, 2200mg, 2400mg, 2600mg, 2800mg, 3000 mg.

The following simulation methods were used: using the final PK model variance-covariance matrix, N200 sets of parameter estimates were extracted from the multivariate normal distribution of parameter estimates. For each parameter estimation, 200 IIV estimates were extracted from the $ OMEGA multivariate normal distribution, resulting in a total of 40000(200 × 200) topics. Re-substitution sampling of the original dataset (N-380) generated 40000 sets of matched covariate and steady-state exposure indices (AUC, C) for each dosing regimen_Average，C_GrainAnd C_{Maximum of})。

Simulations show that the difference in BW-based administration for a wide BW range is slightly higher compared to the fixed dose. Figures 6A and 6E show examples of exposure profiles for medium body weights of 68kg, 17.65mg/kg and 1200mg unitary doses or 7.35mg/kg and 500mg unitary doses, respectively. The simulations also show the opposite trend of the exposure distribution of quartile body weight in the patient population: low weight patients had higher exposure with a fixed dose, while high weight patients had higher exposure with a BW adjusted dose.

Establishing an effective dose/dosing regimen for humans: primary dose-response in second-line non-Small cell Lung cancer (2L NSCLC) with anti-PD-L1/TGF β trap administered once every 2 weeks (q2w)

An example of the efficacy of anti-PD-L1/TGF β trap was established by the following clinical study.

Advanced NSCLC patients who were developed after first-line standard therapy (without prior immunotherapy) and without selection of PD-L1 were randomized to receive anti-PD-L1/TGF β trap therapy of the present disclosure at doses of 500mg or 1200mg (n ═ 40 per group), once every two weeks (q2w) until disease progression, unacceptable toxicity or cessation of the trial occurred. The main objective was to evaluate the overall best efficacy (BOR) according to the efficacy evaluation criteria in the "evaluation criteria for solid tumor efficacy, version 1.1" (RECIST v 1.1). Other goals include dose exploration and safety/tolerability assessments. Tumor cell PD-L1 expression levels (Ab clone 73-10(Dako) [ > 80% > 50% with Ab clone 22C3(Dako) ]) were characterized as PD-L1< 1%, > 1% (PD-L1+) or > 80% (PD-L1-high). In 75 patients, the expression of tumor cell PD-L1 was evaluated.

By the cutoff data at the time of analysis, 80 patients received anti-PD-L1/TGF β trap treatment with a median time of 11.9 weeks (range 2-66.1) and a median follow-up time of 51.1 weeks. Ten patients were still receiving treatment. The investigator evaluated a confirmed Overall Remission Rate (ORR) of 23.8% (500mg ORR, 20.0%; 1200mg ORR, 27.5%), with 18 Partial Remissions (PR) observed at both dose levels and 1 Complete Remission (CR) at 1200 mg. As shown in table 2, clinical activity was observed at different PD-L1 expression levels: 1200mg ORR 37.0% in PD-L1+ patients and 85.7% in patients with high PD-L1. The most common treatment-related adverse events (TRAE) are pruritus (20.0%), maculopapular (18.8%) and anorexia (12.5%). 23 patients developed grade 3 TRAE (28.8% of the total), and 2 patients developed grade 4 TRAE. Eight patients (500mg, n-2; 1200mg, n-6) discontinued due to TRAE. No treatment-related deaths occurred.

Table 2: response rates observed in 2L NSCLC patients treated once every 2 weeks with 500mg or 1200mg of anti-PD-L1/TGF beta trap

These results indicate that anti-PD-L1/TGF β trap monotherapy is well tolerated and shows therapeutic efficacy across the PD-L1 subgroup, with an ORR of 1200mg of 37.0% and 85.7% in patients with high PD-L1+ and PD-L1, respectively. In view of the significant improvement in response rates in the case of higher PD-L1 tumor cell expression (e.g., in patients receiving 1200mg therapy), this activity observed as 2L therapy against PD-L1/TGF β trap is expected to transform or promote as first line (1L) therapy in untreated PD-L1-high or PD-L1-independent NSCLC patients.

Establishing dosing regimens with different dosing frequencies

Data protocols with various dosing frequencies have been created to allow for less frequent dosing and/or to allow for the coordination of dosing schedules with concomitant medications. In particular, the foregoing preliminary population PK modeling and simulation methods were used to simulate exposure of various dosing regimens and to make comparisons between regimens based on exposure.

Based on these simulations, for a typical subject, a uniform dose of at least 500mg administered once every two weeks is required to maintain an average concentration of about 100 μ g/mL, and a uniform dose of about 1200mg administered once every two weeks is required to maintain a C of about 100 μ g/mL_Grain。

Based on C_AverageFor the simulations of (1), 1200mg every two weeks is equivalent to 1800mg every three weeks, while for C_GrainEvery two weeks 1200mg corresponds to every three weeks 2800 mg. To pairIn C_AverageFor example, 500mg once every two weeks is equivalent to 750mg once every three weeks; for C_GrainIn other words, 500mg every two weeks is equivalent to 1,167mg every three weeks.

TGF beta as cancer target

The present disclosure allows for local reduction of TGF β in a tumor microenvironment by capturing TGF β using soluble cytokine receptors (TGF β RII) tethered to antibody moieties that target cellular immune checkpoint receptors on the outer surface of certain tumor cells or immune cells. An example of an antibody portion of the disclosure is directed against an immune checkpoint protein such as anti-PD-L1. The bifunctional molecules of the present invention, sometimes referred to herein as "antibody-cytokine traps," are truly effective because the anti-receptor antibody is physically linked to the cytokine trap. The resulting advantages (e.g., relative to separate administration of the antibody and the receptor) are due in part to the cytokine being the primary role in the local environment through autocrine and paracrine actions. The antibody moiety directs the cytokine trap to the tumor microenvironment where it may behave most effectively by neutralizing local immunosuppressive autocrine or paracrine effects. Furthermore, when the target of the antibody is internalized upon binding by the antibody, an efficient mechanism for clearance of the cytokine/cytokine receptor complex is thereby provided. The antibody-mediated target internalization of PD-L1 is shown, and the anti-PD-L1/TGF β trap shows similar internalization rates as anti-PD-L1. This is a clear advantage over the use of anti-TGF β antibodies, since anti-TGF β antibodies may not be fully neutralizing in the first place; second, the antibody can act as a carrier to extend the half-life of the cytokine.

Indeed, as described below, treatment with anti-PD-L1/TGF β trap elicits a synergistic anti-tumor effect due to blocking the interaction between PD-L1 on tumor cells and PD-1 on immune cells while neutralizing TGF β in the tumor microenvironment. Without being bound by theory, this is presumably due to the synergistic effect obtained by blocking both major immune escape mechanisms simultaneously, and the TGF β in the tumor microenvironment is depleted by single molecular entities. The depletion is by (1) targeting tumor cells against PD-L1; (2) TGF β autocrine/paracrine in the tumor microenvironment is bound by TGF β traps and (3) the bound TGF β is disrupted by PD-L1 receptor-mediated endocytosis. Also, TGF β RII is fused to the C-terminus of Fc (a crystalline fragment of IgG) several times stronger than TGF β RII-Fc in which TGF β RII is placed at the N-terminus of Fc.

TGF-beta has been a somewhat questionable target in cancer immunotherapy due to its paradoxical role as a cancer "molecular dihedron" ("Jeklyl and Hyde") (Bierie et al, nat. Rev. cancer, 2006; 6: 506-20). Like some other cytokines, TGF β activity is developmentally and background dependent. Indeed, TGF β can act as a tumor promoter or tumor suppressor, affecting tumor development, progression and metastasis. The underlying mechanism of dual TGF-beta action is not clear (Yang et al, Trends Immunol.2010; 31: 220-227). Although Smad-dependent signaling has been hypothesized to mediate growth inhibition of TGF signaling, while Smad-independent signaling pathways may contribute to its tumorigenic effects, there is also data suggesting that Smad-dependent signaling pathways are involved in tumor development (Yang et al, Cancer res.2008; 68: 9107-11).

Both TGF β ligands and receptors are well studied as therapeutic targets. There are three ligand isoforms: TGF β 1,2 and 3, are homodimers. There are three TGF beta receptors (TGF beta R) known as type I, II and III TGF beta R (Lopez-Casillas et al, J Cell biol.1994; 124: 557-68). TGF β RI is a signaling chain and does not bind ligands. TGF β RII binds ligands TGF β 1 and 3 with high affinity, but not TGF β 2. The TGF β RII/TGF β complex recruits TGF β RI to form a signaling complex (Won et al, Cancer Res.1999; 59: 1273-7). Tgfbetariii is a positive regulator of TGF binding to its signaling receptors and binds with high affinity to all 3 TGF isoforms. On the cell surface, the TGF β/TGF β RIII complex binds TGF β RII, and TGF β RI is then recruited to replace TGF β RIII to form a signaling complex.

Although all three different TGF β isoforms signal through the same receptor, they are known to have differential expression patterns and non-overlapping functions in vivo. Mice that have three different TGF-. beta.isoforms knocked out have different phenotypes, suggesting that they have many uncompensated functions (Bujak et al, Cardiovasc Res.2007; 74: 184-95). TGF-beta 1 deficient mice are hematopoietic and angiogenic deficient, TGF-beta 3 deficient mice exhibit defects in pulmonary development and jaw development, and TGF-beta 2 deficient mice exhibit various dysplasias, most notably multiple cardiac malformations (Bartram et al, Circulation, 2001; 103: 2745-52; Yamagishi et al, Ant Rec.2012; 295: 257-67). In addition, TGF also plays an important role in repair of myocardial injury following ischemia and reperfusion injury. In the adult heart, cardiomyocytes secrete TGF β as an autocrine to maintain spontaneous beating rates. Importantly, 70-85% of the TGF β secreted by cardiomyocytes is TGF β 2(Roberts et al, J Clin invest.1992; 90: 2056-62). Although TGF β RI kinase inhibitor treatment caused cardiotoxicity problems, the applicant of the present application found that the anti-PD-L1/TGF β trap was not toxic in monkeys, including cardiotoxicity.

Therapeutic methods of neutralizing TGF β include the use of the extracellular domain of TGF β receptor as a soluble receptor trap and neutralizing antibody. Soluble TGF β 0RIII appears to be an obvious choice in the receptor trap capture method, as it binds all three TGF β ligands. However, the native form of TGF β RIII is the 280-330kD glycosaminoglycan (GAG) -glycoprotein, with an extracellular domain of 762 amino acid residues, a very complex protein for biotherapeutic development. GAG-depleted soluble TGF-. beta.RIII can be produced in insect cells and has been shown to be a potent TGF-. beta.neutralizer (Vilchis-Landeros et al biochem. J., (2001),355: 215). Two independent binding domains of TGF β RIII (endoglin-associated and uromodulin-associated) can be expressed independently, but show 20 to 100-fold lower affinity than soluble TGF β RIII, with greatly reduced neutralization activity (Mendoza et al, biochemistry, 2009; 48: 11755-65). In another aspect, the extracellular domain of TGF-beta RII is only 136 amino acid residues in length and can be produced as a 25-35kD glycoprotein. Recombinant soluble TGF-. beta.RII also showed K at 200pM_DBinding to TGF β 1 is quite similar to the 50pM KD of full-length TGF β RII on cells (Lin et al, J Biol chem.1995; 270: 2747-54). Soluble TGF-. beta.RII-Fc was tested as an anti-Cancer agent and was shown to inhibit the growth of established murine malignant mesothelioma in tumor models (Suzuki et al, Clin Cancer Res.2004; 10: 5907-18). Due to TGF beta RIITGF-beta-RIII binds TGF-beta 1 and 3 with a lower affinity than TGF-beta RII in conjunction with TGF-beta 2, and thus a fusion Protein of the endoglin domain of TGF-beta RIII with the extracellular domain of TGF-beta RII is produced in bacteria, which Protein appears to inhibit signaling by TGF-beta 1 and 2 in cellular experiments, more efficiently than TGF-beta RII or RIII (Verona et al, Protein Eng Des Sel.2008; 21: 463-73).

Another method of neutralizing all three isoforms of TGF-beta ligands is to screen for pan-neutralizing anti-TGF-beta antibodies, or anti-receptor antibodies that block receptor binding to TGF-beta 1,2, and 3. GC1008 is a human antibody specific for all TGF-beta isoforms, and has been entered in phase I/II studies in patients with advanced malignant melanoma or renal cell carcinoma (Morris et al, J Clin Oncol 2008; 26: 9028 (conference abstract)). Although this treatment was found to be safe and well tolerated, only limited clinical efficacy was observed, so it was difficult to explain the importance of anti-TGF β treatment without further characterization of the immunological effects (Flavell et al, Nat Rev immunol.2010; 10: 554-67). TGF β isoform specific antibodies are also entering clinical trials. Metelizumab (Metelimumab), a specific antibody to TGF β 1, has been in phase 2 clinical trials to prevent excessive scarring after glaucoma surgery; in a phase 3 study, the TGF-beta 2-specific antibody, lerdelimumab (lerdelimumab), was found to be safe, but ineffective in improving scarring following ocular surgery (Khaw et al, Ophthalmology 2007; 114: 1822-. anti-TGF-RII antibodies that block receptor binding to all three TGF-beta isoforms, such as anti-human TGF-RII antibody TR1 and anti-mouse TGF-RII antibody MT1, also show some therapeutic efficacy for primary tumor growth and metastasis in mice (Zhong et al, Clin Cancer Res.2010; 16: 1191-. However, in a recent phase I study of antibody TR1(LY3022859), higher doses (uniform doses) of more than 25mg were considered unsafe because of uncontrolled cytokine release despite prophylactic treatment (Tolcher et al, Cancer Chemother Pharmacol 2017; 79: 673-. To date, the vast majority of research on TGF targeted anti-cancer therapies, including TGF signaling small molecule inhibitors that are often quite toxic, is mostly in preclinical stages and has very limited anti-tumor effects (Calone et al, Exp oncol.2012; 34: 9-16; Connolly et al, Int J Biol sci.2012; 8: 964-78).

The antibody-TGF β trap of the present disclosure is a bifunctional protein comprising at least a portion of human TGF β receptor II (TGF β RII) capable of binding TGF β. In some embodiments, the TGF-beta trap polypeptide is a soluble portion of type 2 human TGF-beta receptor isoform A (SEQ ID NO:8) that is capable of binding TGF-beta. In certain embodiments, the TGF-beta trap polypeptide comprises at least amino acids 73-184 of SEQ ID NO 8. In certain embodiments, the TGF-beta trap polypeptide comprises amino acids 24-184 of SEQ ID NO 8. In some embodiments, the TGF-beta trap polypeptide is a soluble portion of type 2 human TGF-beta receptor isoform B (SEQ ID NO:9) that is capable of binding TGF-beta. In certain embodiments, the TGF-beta trap polypeptide comprises at least amino acids 48-159 of SEQ ID NO 9. In certain embodiments, the TGF-beta trap polypeptide comprises amino acids 24-159 of SEQ ID NO 9. In certain embodiments, the TGF-beta trap polypeptide comprises amino acids 24-105 of SEQ ID NO 9.

Mechanism of action

Targeting T cells with therapeutic antibodies to inhibit checkpoints to de-inhibit (dis-inhibition) is an area of intense research (reviewed in pardol, Nat Rev cancer.2012; 12: 253-264). In one embodiment, the antibody moiety or antigen binding fragment thereof targets a T cell on a T cell to inhibit a sentinel receptor protein, such as: CTLA-4, PD-1, BTLA, LAG-3, TIM-3 and LAIR 1. In another approach, the antibody moiety targets counter-receptors (counter-receptors) on antigen presenting cells and tumor cells that select some of these counter-receptors for their own immune escape, such as: PD-L1(B7-H1), B7-DC, HVEM, TIM-4, B7-H3 or B7-H4.

The present disclosure contemplates antibody TGF β traps targeted to T cell inhibition checkpoints by their antibody portions or antigen binding fragments thereof to de-inhibit. To this end, applicants tested the anti-tumor effect of TGF β trap in combination with antibodies targeting multiple T cell inhibitory sentinel receptor proteins (e.g., anti-PD-1, anti-PD-L1, anti-TIM-3, and anti-LAG 3).

The programmed death 1(PD-1)/PD-L1 axis is an important mechanism for tumor immune escape. Long-term antigen-responsive effector T cells exhibit an exhausted phenotype marked by PD-1 expression, in which state tumor cells are involved by upregulation of PD-L1. In addition, in the tumor microenvironment, bone marrow cells, macrophages, parenchymal cells and T cells upregulate PD-L1. Blocking this axis can restore effector function in these T cells. The anti-PD-L1/TGF β trap also binds TGF β (1, 2 and 3 isoforms), an inhibitory cytokine produced by apoptotic neutrophils, myeloid suppressor cells, T cells and tumor cells in the tumor microenvironment. Inhibition of TGF β by soluble TGF β RII reduces malignant mesothelioma in a manner associated with an increase in the anti-tumor effect of CD8+ T cells. Deletion of TGF β 1 produced by activated CD4+ T cells and Treg cells has been shown to inhibit tumor growth and protect mice from spontaneous cancer. Thus, TGF β appears to be important for tumor immune escape.

TGF β has growth inhibitory effects on normal epithelial cells, acts as a regulator of epithelial cell homeostasis, and acts as an anti-neoplastic effect during early cancer development. As tumors progress toward malignancy, the growth inhibitory effect of TGF β on tumors is lost due to mutation or oncogenic reprogramming of one or more TGF β channel signaling components. Once sensitivity to TGF β inhibition is lost, tumors continue to produce high levels of TGF β, thereby promoting tumor growth. TGF β cytokines are overexpressed in a variety of cancer types and associated with tumor staging. TGF β is produced by many types of cells in the tumor microenvironment, including tumor cells themselves, immature myeloid cells, regulatory T cells and stromal fibroblasts; these cells collectively produce a large amount of TGF β reservoir in the extracellular matrix. TGF signaling promotes tumor progression by promoting metastasis, stimulating angiogenesis, and inhibiting innate and adaptive anti-tumor immunity. As a broad immunosuppressive factor, TGF β directly down-regulates effector functions of activated cytotoxic T cells and NK cells and effectively induces differentiation of naive CD4+ T cells into immunosuppressive regulatory T cell (Treg) phenotypes. Additionally, TGF β polarizes macrophages and neutrophils into a wound healing phenotype associated with the production of immunosuppressive cytokines. As a therapeutic strategy, neutralization of TGF β activity has the potential to control tumor growth by restoring effective anti-tumor immunity, blocking metastasis and inhibiting angiogenesis.

Radiation-induced pulmonary fibrosis may occur when lung tissue is irradiated at a dose of ≧ 20Gy within the first 6 months after initiation of treatment. TGF β is the major profibrotic molecule contributing to the development of pulmonary fibrosis. Thus, targeting TGF β during treatment of locally advanced, unresectable stage III NSCLC with crts may help counteract the deleterious effects of crts.

Many cases of pulmonary fibrosis are asymptomatic at onset and early fibrotic changes in the lung tissue are minimally distinguishable from changes in pulmonary inflammation. Symptomatic cases usually involve chronic inflammation, characterized by high levels of circulating platelet-derived and basal fibroblast growth factor expression following initial acute inflammation, fibroblast proliferation and migration, TGF β release and collagen deposition in any histological space of the irradiated lung, including the blood vessels and alveolar spaces. This chronic inflammation of the lungs can lead to ventilation-perfusion mismatch and lead to worsening of lung function (even functional status) as a major symptom. Other symptoms may be similar to acute radiation pneumonitis, including nonproductive cough and dyspnea, although these symptoms are generally more chronic in nature. Due to the time course of pathophysiology, symptoms do not appear until months after radiation treatment and may continue to develop over years after treatment.

Thus, during treatment with concomitant chemotherapy and radiotherapy of patients diagnosed with stage III NSCLC, it is advantageous to retain as much of the normal lung as possible from radiation-induced damage at the beginning of treatment, to avoid acute and long-term symptomatic lung injury, and for effective cancer treatment as well.

The present disclosure provides dosage regimens for targeted TGF- β inhibition using anti-PD-L1/TGF β trap molecules for use in methods of treating an untreated subject diagnosed with stage III NSCLC and/or alleviating pathological conditions associated with concurrent crt (e.g., pulmonary fibrosis). Treatment-ongoing stage III NSCLC was not associated with baseline PD-L1 expression levels. Changes in pulmonary fibrosis from baseline were measured by high resolution CT scan and lung function test.

Concomitant PD-1 and TGF β blockade can reconstitute pro-inflammatory cytokines. anti-PD-L1/TGF β trap includes, for example: the extracellular domain of the human TGF β receptor TGF β RII is covalently linked via a glycine/serine linker to the C-terminus of each heavy chain of the fully human IgG1 anti-PD-L1 antibody. Given the emerging blueprint of the anti-PD-1/PD-L1 class, where the response is clear but has room to increase the magnitude of the effect, it is believed that co-targeting the complementary immunomodulatory step will improve tumor response. A similar TGF-targeting agent, fraysimumab (fresolimumab), is a monoclonal antibody to TGF β 1,2 and 3, showing preliminary evidence of tumor efficacy in a phase I trial on melanoma patients.

The present disclosure provides experiments demonstrating that the TGF β RII portion of the anti-PD-L1/TGF β trap (trap control "anti-PDL-1 (mut)/TGF β trap") elicits anti-tumor activity. For example, after subcutaneous implantation in the Detroit562 human pharyngeal cancer model, the anti-PDL 1(mut)/TGF β trap caused a dose-dependent decrease in tumor volume when administered at doses of 25 μ g, 76 μ g or 228 μ g (FIG. 5).

The present disclosure provides experiments demonstrating that the proteins of the present disclosure bind both PD-L1 and TGF β (fig. 2).

The present disclosure provides experiments demonstrating that proteins of the present disclosure (e.g., anti-PD-L1/TGF β trap) inhibit PD-L1 and TGF β -dependent signaling in vitro. The present disclosure provides experiments demonstrating that the proteins of the present disclosure enhance T cell effector function in vitro by blocking PD-L1-mediated immunosuppression, as measured by an IL-2 induction assay following superantigen stimulation (fig. 3). At about 100ng/ml, the proteins of the present disclosure induced a significant increase in IL-2 levels in vitro (fig. 3).

The present disclosure provides experiments that demonstrate that in vivo, proteins of the present disclosure (e.g., anti-PD-L1/TGF β trap) cause depletion of TGF β in blood. Treatment of EMT-6 breast cancer cells implanted in situ in JH mice with 55 μ g, 164 μ g, or 492 μ g of the proteins of the disclosure effectively and specifically depletes TGF β 1 (fig. 4A), TGF β 2 (fig. 4B), and TGF β 3 (fig. 4C). In addition, the present disclosure provides experiments demonstrating that the proteins of the present disclosure occupy the PD-L1 target, supporting the concept that the proteins of the present disclosure fit the receptor binding model in the EMT-6 tumor system (fig. 4D).

The experiments provided by the present disclosure demonstrate that the proteins of the present disclosure bind efficiently, specifically and simultaneously to PD-L1 and TGF β, have potent anti-tumor activity in a variety of mouse models, inhibit tumor growth and metastasis, and prolong survival (e.g., survival up to and including 6 months, 12 months, 18 months, 22 months, 28 months, 32 months, 38 months, 44 months, 50 months, 56 months, 62 months, 68 months, 74 months, 80 months, 86 months, 92 months, 98 months, 104 months or 110 months), and confer long-term protective anti-tumor immunity. In certain embodiments, the extended lifetime is at least 108 months.

anti-PD-L1 antibody

The anti-PD-L1/TGF β trap molecules of the present disclosure may include any anti-PD-L1 antibody or antigen binding fragment thereof described in the art. anti-PD-L1 antibodies are commercially available, for example, the 29E2A3 antibody (Biolegend, lot 329701). The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody. Antibody fragments include Fab, F (ab') 2, scFv and Fv fragments, as described in more detail below.

Exemplary antibodies can be found in PCT publication WO 2013/079174. These antibodies may comprise a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a) the HVR-H1 sequence is X₁YX₂MX₃(SEQ ID NO:21)；

(b) The HVR-H2 sequence is SIYPSGGX₄TFYADX₅VKG(SEQ ID NO:22)；

And wherein: x₁Is K, R, T, Q, G, A, W, M, I or S; x₂Is V, R, K, L, M or I; x₃Is H, T, N, Q, A, V, Y, W, F or M; x₄Is F or I; x₅Is S or T; x₆Is E or D.

In one embodiment, X₁Is M, I or S; x₂Is R, K, L, M or I; x₃Is F or M; x₄Is F or I; x₅Is S or T; x₆Is E or D.

In another embodiment, X₁Is M, I or S; x₂Is L, M or I; x₃Is F or M; x₄Is I; x₅Is S or T; x₆Is D.

In another embodiment, X₁Is S; x₂Is I; x₃Is M; x₄Is I; x₅Is T; x₆Is D.

In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence located between HVRs, as shown below: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR 4).

In another aspect, the framework sequence is derived from a human consensus framework sequence or a human germline framework sequence.

In another aspect, at least one of the framework sequences is as follows:

HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 24);

HC-FR2 is WVRQAPGKGLEWVS (SEQ ID NO: 25);

HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 26);

HC-FR4 is WGQGTLVTVSS (SEQ ID NO: 27).

In another aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2, and HVR-L3, wherein:

(a) the HVR-L1 sequence is TGTX₇X₈DVGX₉YNYVS(SEQ ID NO:28)；

(b) The HVR-L2 sequence is X₁₀VX₁₁X₁₂RPS(SEQ ID NO:29)；

And wherein: x₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is I, N or S; x₁₂Is D, H or N; x₁₃Is F or Y; x₁₄Is N or S; x₁₅Is R, T or S; x₁₆Is G or S; x₁₇Is I or T.

In another embodiment, X₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is N or S; x₁₂Is N; x₁₃Is F or Y; x₁₄Is S; x₁₅Is S; x₁₆Is G or S; x₁₇Is T.

In another embodiment, X₇Is S; x₈Is S; x₉Is G; x₁₀Is D; x₁₁Is S; x₁₂Is N; x₁₃Is Y; x₁₄Is S; x₁₅Is S; x₁₆Is S; x₁₇Is T.

In another aspect, the light chain further comprises a variable region light chain framework sequence located between the HVRs, as shown below: (LC-FR1MHVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

In another aspect, the light chain framework sequence is derived from a human consensus framework sequence or a human germline framework sequence.

In another aspect, the light chain framework sequence is a lambda light chain sequence.

In another aspect, at least one of the framework sequences is as follows:

LC-FR1 is QSALTQPASVSGSPGQSITISC (SEQ ID NO: 31);

LC-FR2 is WYQQHPGKAPKLMIY (SEQ ID NO: 32);

LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 33);

LC-FR4 is FGTGTKVTVL (SEQ ID NO: 34).

In another embodiment, the present disclosure provides an anti-PD-L1 antibody or antigen-binding fragment comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, and wherein: (i) the HVR-H1 sequence is X₁YX₂MX₃(SEQ ID NO: 21); (ii) the HVR-H2 sequence is SIYPSGGX₄TFYADX₅VKG (SEQ ID NO: 22); (iii) the HVR-H3 sequence is IKLGTVTGVX₆Y (SEQ ID NO:23), and;

(b) the light chain includes HVR-L1, HVR-L2, and HVR-L3, and wherein: (iv) the HVR-L1 sequence is TGTX₇X₈DVGX₉YNYVS (SEQ ID NO: 28); (v) the HVR-L2 sequence is X₁₀VX₁₁X₁₂RPS (SEQ ID NO: 29); (vi) the HVR-L3 sequence is SSX₁₃TX₁₄X₁₅X₁₆X₁₇RV (SEQ ID NO: 30); wherein X is₁Is K, R, T, Q, G, A, W, M, I or S; x₂Is V, R, K, L, M or I; x₃Is H, T, N, Q, A, V, Y, W, F or M; x₄Is F or I; x₅Is S or T; x₆Is E or D; x₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is I, N or S; x₁₂Is D, H or N; x₁₃Is F or Y; x₁₄Is N or S; x₁₅Is R, T or S; x₁₆Is G or S; x₁₇Is I or T.

In one embodiment, X₁Is M, I or S; x₂Is R, K, L, M or I; x₃Is F or M; x₄Is F or I; x₅Is S or T; x₆Is E or D; x₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is N or S; x₁₂Is N; x₁₃Is F or Y; x₁₄Is S; x₁₅Is S; x₁₆Is G or S; x₁₇Is T.

In another embodiment, X₁Is M, I or S; x₂Is L, M or I; x₃Is F or M; x₄Is I; x₅Is S or T; x₆Is D; x₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is N or S; x₁₂Is N; x₁₃Is F or Y; x₁₄Is S; x₁₅Is S; x₁₆Is G or S; x₁₇Is T.

In another embodiment, X₁Is S; x₂Is I; x₃Is M; x₄Is I; x₅Is T; x₆Is D; x₇Is S; x₈Is S; x₉Is G; x₁₀Is D; x₁₁Is S; x₁₂Is N; x₁₃Is Y; x₁₄Is S; x₁₅Is S; x₁₆Is S; x₁₇Is T.

In another aspect, the heavy chain variable region comprises one or more framework sequences located between HVRs as shown below: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and said light chain variable region comprises one or more framework sequences located between HVRs as shown below: (LC-FR1MHVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

In another aspect, the framework sequence is derived from a human consensus framework sequence or a human germline sequence.

In another aspect, one or more of the heavy chain framework sequences are as follows:

HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 24);

HC-FR2 is WVRQAPGKGLEWVS (SEQ ID NO: 25);

HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 26);

HC-FR4 is WGQGTLVTVSS (SEQ ID NO: 27).

In another aspect, the light chain framework sequence is a lambda light chain sequence.

In another aspect, one or more of the light chain framework sequences are as follows:

LC-FR1 is QSALTQPASVSGSPGQSITISC (SEQ ID NO: 31);

LC-FR2 is WYQQHPGKAPKLMIY (SEQ ID NO: 32);

LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 33);

LC-FR4 is FGTGTKVTVL (SEQ ID NO: 34).

In another aspect, the heavy chain variable region polypeptide, antibody or antibody fragment further comprises at least C_H1 domain.

In a more specific aspect, the heavy chain variable region polypeptide, antibody or antibody fragment further comprises C_H1、C_H2 and C_H3 domain.

In another aspect, the variable region light chain, antibody or antibody fragment further comprises C_LA domain.

In another aspect, the antibody isComprising C_H1、C_H2、C_H3 and C_LA domain.

In another more specific aspect, the antibody further comprises a human or murine constant region.

In another aspect, the human constant region is selected from the group consisting of: IgG1, IgG2, IgG2, IgG3, IgG 4.

In a more specific aspect, the human or murine constant region is lgG 1.

In another embodiment, the disclosure includes an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain includes HVR-H1, HVR-H2, and HVR-H3, which have at least 80% overall sequence identity to SYIMM (SEQ ID NO:35), SIYPSGGITFYADTVKG (SEQ ID NO:36), and IKLGTVTTVDY (SEQ ID NO:37), respectively, and

(b) the light chain includes HVR-L1, HVR-L2, and HVR-L3, which have at least 80% overall sequence identity to TGTSSDVGGYNYVS (SEQ ID NO:38), DVSNRPS (SEQ ID NO:39), and SSYTSSSTRV (SEQ ID NO:40), respectively.

In a particular aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In another embodiment, the disclosure includes an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, which have at least 80% overall sequence identity to MYMMM (SEQ ID NO:41), SIYPSGGITFYADSVKG (SEQ ID NO:42), and IKLGTVTTVDY (SEQ ID NO:37), respectively, and

(b) the light chain includes HVR-L1, HVR-L2, and HVR-L3, which have at least 80% overall sequence identity to TGTSSDVGAYNYVS (SEQ ID NO:43), DVSNRPS (SEQ ID NO:39), and SSYTSSSTRV (SEQ ID NO:40), respectively.

In a particular aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In another aspect, at least those amino acids highlighted by underlining as shown below remain unchanged compared to the sequences of HVR-H1, HVR-H2, and HVR-H3 in the antibodies or antibody fragments of the present disclosure:

(a) in HVR-H1: sYIMM(SEQ ID NO:35)，

(b) In HVR-H2:SIYPSGGITFYADTVKG(SEQ ID NO:36)，

and wherein at least those amino acids highlighted by underlining as shown below remain unchanged compared to the sequences of HVR-L1, HVR-L2, and HVR-L3:

(a)HVR-L1 TGTSSDVGGYNYVS(SEQ ID NO:38)

(b)HVR-L2 DVSNRPS(SEQ ID NO:39)

(c)HVR-L3 SSYTSSSTRV(SEQ ID NO:40)。

in another aspect, the heavy chain variable region comprises one or more framework sequences located between HVRs as shown below: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and said light chain variable region comprises one or more framework sequences located between HVRs as shown below: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

In another aspect, the framework sequence is derived from a human germline sequence.

In another aspect, one or more of the heavy chain framework sequences are as follows:

HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 24);

HC-FR2 is WVRQAPGKGLEWVS (SEQ ID NO: 25);

HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 26);

HC-FR4 is WGQGTLVTVSS (SEQ ID NO: 27).

In another aspect, the light chain framework sequence is derived from a lambda light chain sequence.

In another aspect, one or more of the light chain framework sequences are as follows:

LC-FR1 is QSALTQPASVSGSPGQSITISC (SEQ ID NO: 31);

LC-FR2 is WYQQHPGKAPKLMIY (SEQ ID NO: 32);

LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 33);

LC-FR4 is FGTGTKVTVL (SEQ ID NO: 34).

In another more specific aspect, the antibody further comprises a human or murine constant region.

In another aspect, the human constant region is selected from the group consisting of: IgG1, IgG2, IgG2, IgG3, IgG 4.

In certain embodiments, the disclosure includes an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMVWRQAPGKGLEWVSSIYPSGGITFYADWKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS (SEQ ID NO:44), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL(SEQ ID NO:45)。

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 44 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 45; the heavy chain sequence has at least 87% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 87% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 44 and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 45; the heavy chain sequence has at least 90% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 90% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 91% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 91% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 92% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 92% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 44 and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 45; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 44 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 45; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 44 and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 45; the heavy chain sequence has at least 97% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 97% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 98% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 98% sequence identity to SEQ ID NO. 45; the heavy chain sequence has at least 99% sequence identity to SEQ ID NO. 44 and the light chain sequence has at least 99% sequence identity to SEQ ID NO. 45; or the heavy chain sequence comprises SEQ ID NO 44 and the light chain sequence comprises SEQ ID NO 45.

In certain embodiments, the present disclosure provides an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLE VWSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAR IKLGTVTTVDYWG QGTLVTVSS (SEQ ID NO:46), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL(SEQ ID NO:47)。

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 46 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 47; the heavy chain sequence has at least 87% sequence identity with SEQ ID NO. 46 and the light chain sequence has at least 87% sequence identity with SEQ ID NO. 47; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 46 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 47; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 46 and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 47; the heavy chain sequence has at least 90% sequence identity to SEQ ID NO. 46 and the light chain sequence has at least 90% sequence identity to SEQ ID NO. 47; the heavy chain sequence has at least 91% sequence identity to SEQ ID NO. 46 and the light chain sequence has at least 91% sequence identity to SEQ ID NO. 47; the heavy chain sequence has at least 92% sequence identity to SEQ ID NO. 46 and the light chain sequence has at least 92% sequence identity to SEQ ID NO. 47; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 46 and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 47; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 46 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 47; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 46 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 47; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 46 and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 47; the heavy chain sequence has at least 97% sequence identity to SEQ ID NO. 46 and the light chain sequence has at least 97% sequence identity to SEQ ID NO. 47; the heavy chain sequence has at least 98% sequence identity with SEQ ID NO. 46 and the light chain sequence has at least 98% sequence identity with SEQ ID NO. 47; the heavy chain sequence has at least 99% sequence identity to SEQ ID NO. 46 and the light chain sequence has at least 99% sequence identity to SEQ ID NO. 47; or the heavy chain sequence comprises SEQ ID NO 46 and the light chain sequence comprises SEQ ID NO 47.

In another embodiment, the antibody binds human, mouse, or cynomolgus PD-L1. In a particular aspect, the antibody is capable of blocking the interaction between human, mouse or cynomolgus PD-L1 and the corresponding human, mouse or cynomolgus PD-1 receptor.

In another embodiment, the antibody is at 5x10^-9M or lower K_DPreferably at 2x10^-9M or lower K_DEven more preferably at 1x10^-9M or lower K_DBinds to human PD-L1.

In another embodiment, the disclosure relates to an anti-PD-L1 antibody or antigen-binding fragment thereof that binds to a functional epitope comprising residues Y56 and D61 of human PD-L1.

In a specific aspect, the functional epitope further comprises E58, E60, Q66, R113, and M115 of human PD-L1.

In a more specific aspect, the antibody binds to a conformational epitope comprising residues 54-66 and 112-122 of human PD-L1.

In certain embodiments, the disclosure relates to an anti-PD-L1 antibody or antigen-binding fragment thereof that cross-competes for binding to PD-L1 with an antibody of the disclosure described herein.

In certain embodiments, the present disclosure includes proteins and polypeptides comprising any of the above anti-PD-L1 antibodies, in combination with at least one pharmaceutically acceptable carrier.

In certain embodiments, the disclosure includes an isolated nucleic acid encoding a polypeptide, or a light chain or heavy chain variable region sequence, of an anti-PD-L1 antibody or antigen-binding fragment thereof described herein. In certain embodiments, the present disclosure provides an isolated nucleic acid encoding a light chain or heavy chain variable region sequence of an anti-PD-L1 antibody, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3 sequences that have at least 80% sequence identity to SYIMM (SEQ ID NO:35), SIYPSGGITFYADTVKG (SEQ ID NO:36), and IKLGTVTTVDY (SEQ ID NO:37), respectively, or

(b) The light chain includes HVR-L1, HVR-L2, and HVR-L3 sequences that have at least 80% sequence identity to TGTSSDVGGYNYVS (SEQ ID NO:38), DVSNRPS (SEQ ID NO:39), and SSYTSSSTRV (SEQ ID NO:40), respectively.

In a particular aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In another aspect, the nucleic acid sequence of the heavy chain is:

(SEQ ID NO:48)

and the nucleic acid sequence of the light chain is:

(SEQ ID NO:49)。

other exemplary anti-PD-L1 antibodies that may be used in an anti-PD-L1/TGF β trap may be found in U.S. patent application publication US 2010/0203056. In one embodiment of the disclosure, the antibody moiety is YW243.55S70. In another embodiment, the antibody moiety is MPDL 3289A.

In certain embodiments, the disclosure includes an anti-PD-L1 antibody portion comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:12), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:13)

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 12 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 13; the heavy chain sequence has at least 87% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 87% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 12 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 90% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 90% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 91% sequence identity to SEQ ID NO. 12 and the light chain sequence has at least 91% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 92% sequence identity to SEQ ID NO. 12 and the light chain sequence has at least 92% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 12 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 97% sequence identity to SEQ ID NO. 12 and the light chain sequence has at least 97% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 98% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 98% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 99% sequence identity with SEQ ID NO. 12 and the light chain sequence has at least 99% sequence identity with SEQ ID NO. 13; or the heavy chain sequence comprises SEQ ID NO 12 and the light chain sequence comprises SEQ ID NO 13.

In certain embodiments, the disclosure includes an anti-PD-L1 antibody portion comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:14), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:13)

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 14 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 13; the heavy chain sequence has at least 87% sequence identity with SEQ ID NO. 14 and the light chain sequence has at least 87% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 14 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 14, and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 90% sequence identity with SEQ ID NO. 14 and the light chain sequence has at least 90% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 91% sequence identity to SEQ ID NO. 14 and the light chain sequence has at least 91% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 92% sequence identity to SEQ ID NO. 14 and the light chain sequence has at least 92% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 14, and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 14 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 14 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 13; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 14, and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 97% sequence identity with SEQ ID NO. 14 and the light chain sequence has at least 97% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 98% sequence identity with SEQ ID NO. 14, and the light chain sequence has at least 98% sequence identity with SEQ ID NO. 13; the heavy chain sequence has at least 99% sequence identity with SEQ ID NO. 14 and the light chain sequence has at least 99% sequence identity with SEQ ID NO. 13; or the heavy chain sequence comprises SEQ ID NO. 14 and the light chain sequence comprises SEQ ID NO. 13.

Other exemplary anti-PD-L1 antibodies that may be used in an anti-PD-L1/TGF β trap may be found in U.S. patent application publication US 2018/0334504.

In certain embodiments, the disclosure includes an anti-PD-L1 antibody portion comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGYISYTGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS (SEQ ID NO:55), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYTFGGGTKVEIK(SEQ ID NO:56)。

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 55 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 56; the heavy chain sequence has at least 87% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 87% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 55 and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 56; the heavy chain sequence has at least 90% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 90% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 91% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 91% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 92% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 92% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 55 and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 56; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 55 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 56; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 55 and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 56; the heavy chain sequence has at least 97% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 97% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 98% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 98% sequence identity to SEQ ID NO. 56; the heavy chain sequence has at least 99% sequence identity to SEQ ID NO. 55 and the light chain sequence has at least 99% sequence identity to SEQ ID NO. 56; or the heavy chain sequence comprises SEQ ID NO. 55 and the light chain sequence comprises SEQ ID NO. 56.

In certain embodiments, the disclosure includes an anti-PD-L1 antibody portion comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS (SEQ ID NO:57), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK(SEQ ID NO:58)。

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 57 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 58; the heavy chain sequence has at least 87% sequence identity with SEQ ID NO. 57 and the light chain sequence has at least 87% sequence identity with SEQ ID NO. 58; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 57 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 58; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 57 and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 58; the heavy chain sequence has at least 90% sequence identity with SEQ ID NO. 57 and the light chain sequence has at least 90% sequence identity with SEQ ID NO. 58; the heavy chain sequence has at least 91% sequence identity to SEQ ID NO. 57 and the light chain sequence has at least 91% sequence identity to SEQ ID NO. 58; the heavy chain sequence has at least 92% sequence identity to SEQ ID NO. 57 and the light chain sequence has at least 92% sequence identity to SEQ ID NO. 58; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 57, and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 58; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 57 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 58; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 57 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 58; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 57, and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 58; the heavy chain sequence has at least 97% sequence identity to SEQ ID NO. 57 and the light chain sequence has at least 97% sequence identity to SEQ ID NO. 58; the heavy chain sequence has at least 98% sequence identity with SEQ ID NO. 57 and the light chain sequence has at least 98% sequence identity with SEQ ID NO. 58; the heavy chain sequence has at least 99% sequence identity with SEQ ID NO. 57 and the light chain sequence has at least 99% sequence identity with SEQ ID NO. 58; or the heavy chain sequence comprises SEQ ID NO 57 and the light chain sequence comprises SEQ ID NO 58.

In certain embodiments, the disclosure includes an anti-PD-L1 antibody portion comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGYISYTGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:59), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:60)。

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 59 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 60; the heavy chain sequence has at least 87% sequence identity with SEQ ID NO. 59 and the light chain sequence has at least 87% sequence identity with SEQ ID NO. 60; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 59 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 60; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 59 and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 60; the heavy chain sequence has at least 90% sequence identity to SEQ ID NO. 59 and the light chain sequence has at least 90% sequence identity to SEQ ID NO. 60; the heavy chain sequence has at least 91% sequence identity to SEQ ID NO. 59 and the light chain sequence has at least 91% sequence identity to SEQ ID NO. 60; the heavy chain sequence has at least 92% sequence identity to SEQ ID NO. 59 and the light chain sequence has at least 92% sequence identity to SEQ ID NO. 60; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 59 and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 60; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 59 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 60; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 59 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 60; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 59 and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 60; the heavy chain sequence has at least 97% sequence identity to SEQ ID NO. 59 and the light chain sequence has at least 97% sequence identity to SEQ ID NO. 60; the heavy chain sequence has at least 98% sequence identity to SEQ ID NO. 59 and the light chain sequence has at least 98% sequence identity to SEQ ID NO. 60; the heavy chain sequence has at least 99% sequence identity with SEQ ID NO. 59 and the light chain sequence has at least 99% sequence identity with SEQ ID NO. 60; or the heavy chain sequence comprises SEQ ID NO 59 and the light chain sequence comprises SEQ ID NO 60.

In certain embodiments, the disclosure includes an anti-PD-L1 antibody portion comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGA (SEQ ID NO:61), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:62)。

in various embodiments, the heavy chain sequence has at least 86% sequence identity to SEQ ID No. 61 and the light chain sequence has at least 86% sequence identity to SEQ ID No. 62; the heavy chain sequence has at least 87% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 87% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 88% sequence identity to SEQ ID NO. 61 and the light chain sequence has at least 88% sequence identity to SEQ ID NO. 62; the heavy chain sequence has at least 89% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 89% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 90% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 90% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 91% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 91% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 92% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 92% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 93% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 93% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 94% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 94% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 95% sequence identity to SEQ ID NO. 61 and the light chain sequence has at least 95% sequence identity to SEQ ID NO. 62; the heavy chain sequence has at least 96% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 96% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 97% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 97% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 98% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 98% sequence identity with SEQ ID NO. 62; the heavy chain sequence has at least 99% sequence identity with SEQ ID NO. 61 and the light chain sequence has at least 99% sequence identity with SEQ ID NO. 62; or the heavy chain sequence comprises SEQ ID NO 61 and the light chain sequence comprises SEQ ID NO 62.

Other exemplary anti-PD-L1 antibodies that may be used in anti-PD-L1/TGF β traps may be found in U.S. patent publication US 7,943,743.

In one of the disclosed embodiments, the anti-PD-L1 antibody is MDX-1105.

In some embodiments, the anti-PD-L1 antibody is MEDI-4736.

Constant region

The proteins and peptides of the present disclosure may include constant regions or constant region fragments, analogs, variants, mutants, or derivatives of immunoglobulins. In certain embodiments, the constant region is derived from a human immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, or other species. In certain embodiments, the constant region comprises a CH2 domain. In other embodiments, the constant region comprises CH2 and CH3 binding domains or comprises the hinge-CH 2-CH 3. Alternatively, the constant region may comprise all or part of the hinge region, the CH2 domain, and/or the CH3 domain.

In one embodiment, the constant region comprises a mutation that reduces affinity for an Fc receptor or reduces Fc effector function. For example, the constant region may comprise a mutation that eliminates a glycosylation site in the IgG heavy chain constant region. In some embodiments, the constant region comprises a mutation, deletion, or insertion at an amino acid position corresponding to Leu234, Leu235, Gly236, Gly237, Asn297, or Pro331 of IgG1 (amino acids numbered according to EU nomenclature). In a specific embodiment, the constant region contains a mutation at the amino acid position corresponding to Asn297 of IgG 1. In another embodiment, the constant region comprises a mutation, deletion or insertion of an amino acid position corresponding to Leu281, Leu282, Gly283, Gly284, Asn344 or Pro378 of IgG 1.

In some embodiments, the constant region comprises a CH2 domain derived from a human IgG2 or IgG4 heavy chain. Preferably, the CH2 domain comprises a mutation that eliminates the glycosylation site in the CH2 domain. In one embodiment, the mutation alters an asparagine within the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence within the CH2 domain of the IgG2 or IgG4 heavy chain. Preferably, the mutation changes asparagine to glutamine. Alternatively, the mutation alters both phenylalanine and asparagine within the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence. In one embodiment, the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence is substituted with a Gln-Ala-Gln-Ser (SEQ ID NO:16) amino acid sequence. The asparagine within the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence corresponds to Asn297 of IgG 1.

In another embodiment, the constant region comprises a CH2 domain and at least a portion of a hinge region. The hinge region may be derived from an immunoglobulin heavy chain such as IgG1, IgG2, IgG3, IgG4, or other species. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable species. More preferably, the hinge region is derived from the heavy chain of human IgG 1. In one embodiment, the cysteine in the IgG1 hinge region Pro-Lys-Ser-Cys-Asp-Lys (SEQ ID NO:17) amino acid sequence is altered. In certain embodiments, the Pro-Lys-Ser-Cys-Asp-Lys (SEQ ID NO:17) amino acid sequence is replaced by a Pro-Lys-Ser-Ser-Asp-Lys (SEQ ID NO:18) amino acid sequence. In certain embodiments, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In certain embodiments, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain, and the hinge region is derived from an altered human IgG1 heavy chain.

Amino acid changes near the junction of the Fc portion and the non-Fc portion significantly increase the serum half-life of the Fc fusion protein (PCT publication WO 01/58957, the disclosure of which is incorporated herein by reference). Thus, the linking region of a protein or polypeptide of the present disclosure may contain alterations relative to the immunoglobulin heavy chain and erythropoietin native sequences, preferably within about 10 amino acids from the point of attachment. These amino acid changes result in increased hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which a C-terminal lysine residue is substituted. Preferably, the C-terminal lysine of the IgG sequence is replaced with a non-lysine amino acid (e.g., alanine or leucine) to further increase serum half-life. In another embodiment, the constant region is derived from an IgG sequence, wherein the Leu-Ser-Leu-Ser (SEQ ID NO:19) amino acid sequence near the C-terminus of the constant region has alterations that eliminate potential conjugative T cell epitopes. For example, in one embodiment, the Leu-Ser-Leu-Ser (SEQ ID NO:19) amino acid sequence is substituted with an Ala-Thr-Ala-Thr (SEQ ID NO:20) amino acid sequence. In other embodiments, amino acids within the Leu-Ser-Leu-Ser (SEQ ID NO:19) segment are replaced with other amino acids such as glycine or proline. Methods for making amino acid substitutions in the Leu-Ser-Leu-Ser (SEQ ID NO:19) segment near the C-terminus of IgG1, IgG2, IgG3, IgG4, or other immunoglobulin molecules are described in detail in U.S. patent publication No. 20030166877, the disclosure of which is incorporated herein by reference.

Suitable hinge regions of the present disclosure may be derived from IgG1, IgG2, IgG3, IgG4, and other immunoglobulin classes. The IgG1 hinge region has three cysteines, two of which are involved in the disulfide bonds between the two heavy chains of immunoglobulins. These cysteines allow efficient and consistent disulfide bond formation between the Fc portions. Thus, one of the hinge regions of the present disclosure is derived from IgG1, e.g., human IgG 1. In a preferred embodiment, the first cysteine in the hinge region of human IgG1 is mutated to another amino acid, preferably serine. The hinge region of the IgG2 isotype has four disulfide bonds, which tend to contribute to oligomerization and possibly incorrect disulfide bonds during secretion of the recombinant system. Suitable hinge regions may be derived from the IgG2 hinge, preferably wherein the first two cysteines are each mutated to other amino acids. It is known that the hinge region of IgG4 is less effective at forming interchain disulfide bonds whereas suitable hinge regions of the present disclosure may be derived from the IgG4 hinge region, preferably containing mutations that enhance the correct formation of disulfide bonds between heavy chain-derived portions (Angal S et al, (1993) mol.

According to the present disclosure, the constant region may comprise CH2 and/or CH3 domains and a hinge region derived from different antibody isotypes, such as a hybrid (hybrid) constant region. For example, in one embodiment, the constant region comprises a CH2 and/or CH3 domain derived from IgG2 or IgG4 and a mutated hinge region derived from IgG 1. Alternatively, mutant hinge regions derived from other IgG subclasses may be employed in the hybrid constant region. For example, a mutated form of the hinge of IgG4 that is effective in forming the disulfide bond between the two double chains may be used. Mutant hinges may also be derived from the IgG2 hinge, in which the first two cysteines are each mutated to other amino acids. The assembly of hybrid constant regions can be found in U.S. patent publication No. 20030044423, the disclosure of which is incorporated herein by reference.

According to the present disclosure, the constant region may comprise one or more of the mutations described herein. The combination of mutations in the Fc portion has additive or synergistic effects on extending serum half-life and increasing potency of the bifunctional molecule in vivo. Thus, in one exemplary embodiment, the constant region may comprise (i) a region derived from an IgG sequence in which the Leu-Ser-Leu-Ser (SEQ ID NO:19) amino acid sequence is replaced with an Ala-Thr-Ala-Thr (SEQ ID NO:20) amino acid sequence; (ii) a C-terminal alanine residue instead of lysine; (iii) CH2 domains and hinge regions derived from different antibody isotypes, such as an IgG2 CH2 domain and an altered IgG1 hinge region; and (iv) a mutation that eliminates the glycosylation site within the IgG 2-derived CH2 domain, such as the Gln-Ala-Gln-Ser (SEQ ID NO:16) amino acid sequence within the IgG 2-derived CH2 domain rather than the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence.

Antibody fragments

The proteins and polypeptides of the present disclosure may also include antigen-binding fragments of antibodies. Exemplary antibody fragments include scFv, Fv, Fab, F (ab')₂And single domain VHH fragments, such as those from camelids.

Single chain antibody fragments, also known as single chain antibodies (scFv), are recombinant polypeptides that typically bind to an antigen or receptor; these fragments comprise at least one antibody variable light chain sequence (V) linked with or without one or more interconnecting linkers_L) Fragment and at least one antibody variable heavy chain amino acid sequence (V)_H) And (3) fragment. Such linkers may be short flexible peptides selected to ensure correct three-dimensional folding after association of the VL and VH domains, thereby retaining the target molecule binding specificity of the whole antibody from which the single chain antibody fragment is derived. In general, V_LOr V_HThe carboxy terminus of the sequence is covalently linked to complementary V through such a peptide linker_LAnd V_HThe amino acid terminus of the sequence. Single chain antibody fragments may be generated by molecular cloning, antibody phage display or similar techniques. These proteins can be produced in eukaryotic cells as well as prokaryotic cells, including bacteria.

Single chain antibody fragments comprise amino acid sequences having at least one of the variable regions or CDRs of intact antibodies described herein, but lacking all or part of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute an integral part of the complete antibody structure. Thus, single chain antibody fragments may overcome some of the problems associated with the use of antibodies comprising part or all of the constant region. For example, single chain antibody fragments tend not to undergo undesirable interactions or other undesirable biological activities between a biomolecule and the heavy chain constant region. Furthermore, single chain antibody fragments are much smaller than intact antibodies and therefore can have higher capillary permeability than intact antibodies, which enables the single chain antibody fragments to more efficiently address and bind to the target antigen binding site. Also, antibody fragments can be produced in prokaryotic cells on a relatively large scale, facilitating their production. Furthermore, the relatively small size of single chain antibody fragments makes them less likely to elicit an immune response in a recipient than intact antibodies.

There may also be antibody fragments having the same or comparable binding characteristics as the intact antibody. Such fragments may contain one or two Fab fragments or F (ab')₂And (3) fragment. Antibody fragments may comprise all six CDRs of the complete antibody, but fragments comprising less than all of these regions, e.g., three, four, or five CDRs, are also functional.

Pharmaceutical composition

The present disclosure also includes pharmaceutical compositions comprising a therapeutically effective amount of a protein described herein. The compositions can be formulated to be suitable for use in a variety of drug delivery systems. The compositions may also contain one or more physiologically acceptable excipients or carriers to make suitable formulations. Suitable formulations for use in the present disclosure can be found in Remington pharmaceutical sciences, 17 th edition, Mark Publishing Company, Iston, Pa. (Mack Publishing Company), 1985. For reviews on drug delivery methods see, for example, Langer (Science 249: 1527) -1533, 1990).

In one aspect, the present disclosure provides an intravenous drug delivery formulation for use in a method of treatment of stage III NSCLC cancer or inhibition of tumor growth in an untreated cancer patient, comprising 500mg to 2400mg of a protein comprising a first polypeptide comprising: (a) at least the heavy chain variable region of an antibody capable of binding human protein programmed death ligand 1 (PD-L1); and (b) a human transforming growth factor beta receptor II (TGF β RII) or fragment thereof capable of binding transforming growth factor beta (TGF β), said second polypeptide comprising: at least the light chain variable region of an antibody capable of binding PD-L1, and the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site capable of binding PD-L1.

In certain embodiments, a protein product of the present disclosure comprises a first polypeptide comprising the amino acid sequence of SEQ ID No. 3 and a second polypeptide comprising the amino acid sequence of SEQ ID No. 1. In certain embodiments, the protein products of the present disclosure include a first polypeptide comprising the amino acid sequences of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequences of SEQ ID NOs 38, 39, and 40.

In certain embodiments of the present disclosure, an intravenous drug delivery formulation for use in a method of treating stage III NSCLC or inhibiting tumor growth in an untreated cancer patient may comprise a dose of about 500mg to about 2400mg (e.g., about 500mg to about 2300mg, about 500mg to about 2200mg, about 500mg to about 2100mg, about 500mg to about 2000mg, about 500mg to about 1900mg, about 500mg to about 1800mg, about 500mg to about 1700mg, about 500mg to about 1600mg, about 500mg to about 1500mg, about 500mg to about 1400mg, about 500mg to about 1300mg, about 500mg to about 1200mg, about 500mg to about 1100mg, about 500mg to about 1000mg, about 500mg to about 900mg, about 500mg to about 800mg, about 500mg to about 700mg, about 500mg to about 600mg, about 600mg to 2400mg, about 700mg to about 2400mg, about 800mg to about 900mg, about 900mg to about 1000mg, about 2400mg to about 1000mg, about 1000mg to 2400mg, about 1300mg to 2400mg, about 1400mg to 2400mg, about 1500mg to 2400mg, about 1600mg to 2400mg, about 1700mg to 2400mg, about 1800mg to 2400mg, about 1900mg to 2400mg, about 2000mg to 2400mg, about 2100mg to 2400mg, about 2200mg to 2400mg, or about 2300mg to 2400mg) of a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap (e.g., a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, an intravenous drug delivery formulation may comprise a 2000mg dose of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, an intravenous drug delivery formulation may comprise a dosage of about 500mg of a protein product of the present disclosure comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID No. 3 and the second polypeptide comprises the amino acid sequence of SEQ ID No. 1. In certain embodiments, an intravenous drug delivery formulation may comprise a 500mg dose of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, an intravenous drug delivery formulation may comprise a protein product of the present disclosure comprising a first polypeptide and a second polypeptide at a dose of about 1,200mg, wherein the first polypeptide comprises the amino acid sequence of SEQ ID No. 3 and the second polypeptide comprises the amino acid sequence of SEQ ID No. 1. In certain embodiments, an intravenous drug delivery formulation may comprise a 1200mg dose of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, an intravenous drug delivery formulation may comprise a dose of about 1800mg of a protein product of the present disclosure comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID No. 3 and the second polypeptide comprises the amino acid sequence of SEQ ID No. 1. In certain embodiments, an intravenous drug delivery formulation may comprise a 1800mg dose of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, an intravenous drug delivery formulation may comprise a 1800mg dose of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40)). In certain embodiments, an intravenous drug delivery formulation may comprise a dosage of about 2400mg of a protein product of the present disclosure comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID No. 3 and the second polypeptide comprises the amino acid sequence of SEQ ID No. 1. In certain embodiments, an intravenous drug delivery formulation may comprise a 2400mg dose of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, an intravenous drug delivery formulation may comprise a 2400mg dose of a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40)).

In certain embodiments, an intravenous drug delivery formulation for use in a method of treatment of stage III NSCLC or tumor growth inhibition in an untreated cancer patient may comprise from about 1200mg to about 3000mg (e.g., from about 1200mg to about 3000mg, from about 1200mg to about 2900mg, from about 1200mg to about 2800mg, from about 1200mg to about 2700mg, from about 1200mg to about 2600mg, from about 1200mg to about 2500mg, from about 1200mg to about 2400mg, from about 1200mg to about 2300mg, from about 1200mg to about 2200mg, from about 1200mg to about 2100mg, from about 1200mg to about 2000mg, from about 1200mg to about 1900mg, from about 1200mg to about 1800mg, from about 1200mg to about 1700mg, from about 1200mg to about 1600mg, from about 1200mg to about 1400mg, from about 1200mg to about 1300mg, from about 1300mg to about 3000mg, from about 1400mg to about 3000mg, from about 1500mg to about 3000mg, from about 1600mg to about 3000mg, from about 3000mg, about 2000mg to about 3000mg, about 2100mg to about 3000mg, about 2200mg to about 3000mg, about 2300mg to about 3000mg, about 2400mg to about 3000mg, about 2500mg to about 3000mg, about 2600mg to about 3000mg, about 2700mg to about 3000mg, about 2800mg to about 3000mg, about 2900mg to about 3000mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, about 2500mg, about 2600mg, about 2700mg, about 2800mg, about 2900mg, or about 3000mg) of a protein product of the present disclosure (e.g., anti-PD-L1/TGF β trap). In certain embodiments, an intravenous drug delivery formulation for use in a method of treatment of stage III NSCLC or tumor growth inhibition in an untreated cancer patient may comprise from about 1200mg to about 3000mg (e.g., from about 1200mg to about 3000mg, from about 1200mg to about 2900mg, from about 1200mg to about 2800mg, from about 1200mg to about 2700mg, from about 1200mg to about 2600mg, from about 1200mg to about 2500mg, from about 1200mg to about 2400mg, from about 1200mg to about 2300mg, from about 1200mg to about 2200mg, from about 1200mg to about 2100mg, from about 1200mg to about 2000mg, from about 1200mg to about 1900mg, from about 1200mg to about 1800mg, from about 1200mg to about 1700mg, from about 1200mg to about 1600mg, from about 1200mg to about 1400mg, from about 1200mg to about 1300mg, from about 1300mg to about 3000mg, from about 1400mg to about 3000mg, from about 1500mg to about 3000mg, from about 1600mg to about 3000mg, from about 3000mg, about 2000mg to about 3000mg, about 2100mg to about 3000mg, about 2200mg to about 3000mg, about 2300mg to about 3000mg, about 2400mg to about 3000mg, about 2500mg to about 3000mg, about 2600mg to about 3000mg, about 2700mg to about 3000mg, about 2800mg to about 3000mg, about 2900mg to about 3000mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, about 2500mg, about 2600mg, about 2700mg, about 2800mg, about 2900mg or about 3000mg) of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1; or a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO 35, 36 and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NO 38, 39 and 40.

In certain embodiments, an intravenous drug delivery formulation for use in a method of treatment of stage III NSCLC or inhibition of tumor growth in a non-treated cancer patient may comprise about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 1075mg, about 1100mg, about 1125mg, about 1150mg, about 1175mg, about 1200mg, about 1225mg, about 1250mg, about 1275mg, about 1300mg, about 1325mg, about 1350mg, about 1375mg, about 1400mg, about 1425mg, about 1450mg, about 5mg, about 1500mg, about 1550mg, about 1600mg, about 1575mg, about 1725mg, about 1650mg, about 1725mg, about 1675mg, about 1850mg, about 1875mg, about 1900mg, about 1925mg, about 1950mg, about 1975mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, or about 2400mg of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap) comprising a first polypeptide having the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide having the amino acid sequence of SEQ ID NOs 38, 39, and 40.

The intravenous drug delivery formulation of the present disclosure for use in a method of treating stage III NSCLC or inhibiting tumor growth in an untreated cancer patient may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a channel that includes a tube and/or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the formulation may be lyophilized, and about 45mg of the lyophilized formulation may be contained in one vial. In certain embodiments, about 40mg to about 100mg of the lyophilized formulation may be contained in one vial. In certain embodiments, freeze-dried preparations from 12, 27 or 45 vials are combined to obtain a therapeutic dose of protein in an intravenous pharmaceutical preparation. In certain embodiments, the formulation may be a liquid formulation of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO. 3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO. 1; or a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO 35, 36 and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NO 38, 39 and 40 and stored from about 250 mg/vial to about 2000 mg/vial (e.g., about 250 mg/vial to about 2000 mg/vial, about 250 mg/vial to about 1900 mg/vial, about 250 mg/vial to about 1800 mg/vial, about 250 mg/vial to about 1700 mg/vial, about 250 mg/vial to about 1600 mg/vial, about 250 mg/vial to about 1500 mg/vial, about 250 mg/vial to about 1400 mg/vial, about 250 mg/vial to about 1300 mg/vial, about 250 mg/vial to about 1200 mg/vial, about 250 mg/vial to about 1100 mg/vial, from about 250 mg/vial to about 1000 mg/vial, from about 250 mg/vial to about 900 mg/vial, from about 250 mg/vial to about 800 mg/vial, from about 250 mg/vial to about 700 mg/vial, from about 250 mg/vial to about 600 mg/vial, from about 250 mg/vial to about 500 mg/vial, from about 250 mg/vial to about 400 mg/vial, from about 250 mg/vial to about 300 mg/vial, from about 300 mg/vial to about 2000 mg/vial, from about 400 mg/vial to about 2000 mg/vial, from about 500 mg/vial to about 2000 mg/vial, from about 600 mg/vial to about 2000 mg/vial, from about 700 mg/vial to about 2000 mg/vial, from about 800 mg/vial to about 2000 mg/vial, from about 900 mg/vial to about 2000 mg/vial, from about 1000 mg/vial to about 2000 mg/vial, from about 1100 mg/vial to about 2000 mg/vial, from about 1200 mg/vial to about 2000 mg/vial, from about 1300 mg/vial to about 2000 mg/vial, from about 1400 mg/hour vial to about 2000 mg/vial, from about 1500 mg/vial to about 2000 mg/vial, from about 1600 mg/vial to about 2000 mg/vial, from about 1700 mg/vial to about 2000 mg/vial, from about 1800 mg/vial to about 2000 mg/vial, or from about 1900 mg/vial to about 2000 mg/vial). In certain embodiments, the formulation may be a liquid formulation and stored at about 600 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 1200 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 1800 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 2,400 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 250 mg/vial.

The present disclosure provides liquid aqueous pharmaceutical formulations comprising a therapeutically effective amount of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap) in a buffered solution to form a formulation for use in a method of treatment of stage III NSCLC or inhibition of tumor growth in an untreated cancer patient.

These compositions for use in methods of treatment of stage III NSCLC or inhibition of tumor growth in untreated cancer patients may be sterilized using conventional sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged as is ("use as-is") type product or lyophilized, the lyophilized formulation being combined with a sterile aqueous carrier prior to administration. The pH of the formulation is generally between 3 and 11, more preferably between 5 and 9 or between 6 and 8, most preferably between 7 and 8, e.g.between 7 and 7.5. The resulting composition in solid form may be packaged in a plurality of single dosage units, each containing a fixed amount of one or more of the agents described above. The composition in solid form can also be packaged in containers to obtain flexible amounts.

In certain embodiments, the present disclosure provides a formulation with extended shelf life for use in a method of treatment of stage III NSCLC or tumor growth inhibition in an untreated cancer patient, the formulation comprising a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap (e.g., a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)) and mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water and sodium hydroxide.

In certain embodiments, the aqueous formulations of the methods of the present disclosure for stage III therapy or tumor growth inhibition in untreated cancer patients are prepared to contain a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., a protein product comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1) or having a first polypeptide comprising the amino acid sequence of SEQ ID NO:35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:38, 39, and 40 in a pH buffered solution the pH of the buffers of the present disclosure can be from about 4 to about 8, e.g., from about 4 to about 8, from about 4.5 to about 8, from about 5 to about 8, from about 5.5 to about 8, from about 6 to about 8, from about 6.5 to about 8, from about 7.5 to about 8, from about 4 to about 7.5, about 4.5 to about 7.5, about 5 to about 7.5, about 5.5 to about 7.5, about 6 to about 7.5, about 6.5 to about 7.5, about 4 to about 7, about 4.5 to about 7, about 5 to about 7, about 5.5 to about 7, about 6 to about 7, about 4 to about 6.5, about 4.5 to about 6.5, about 5 to about 6.5, about 5.5 to about 6.5, about 4 to about 6.0, about 4.5 to about 6.0, about 5 to about 6, or about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Intermediate ranges of the above pH are also part of the present disclosure. For example, a range of values using any combination of the above values as upper and/or lower limits is intended to be included. Examples of buffers to control the pH within this range include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

In certain embodiments, the formulation for use in a method of treatment of cancer stage III NSCLC or tumor growth inhibition in an untreated cancer patient comprises a buffer system comprising citrate and phosphate to maintain a pH in the range of about 4 to about 8. In certain embodiments, the pH range may be from about 4.5 to about 6.0, or from about pH4.8 to about 5.5, or in the pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3mg/ml citric acid (e.g., 1.305mg/ml), about 0.3mg/ml sodium citrate (e.g., 0.305mg/ml), about 1.5mg/ml dibasic sodium phosphate dihydrate (e.g., 1.53mg/ml), about 0.9mg/ml monobasic sodium phosphate dihydrate (e.g., 0.86), and about 6.2mg/ml sodium chloride (e.g., 6.165 mg/ml). In certain embodiments, the buffer system comprises about 1-1.5mg/ml citric acid, about 0.25 to about 0.5mg/ml sodium citrate, about 1.25 to about 1.75mg/ml dibasic sodium phosphate dihydrate, about 0.7 to about 1.1mg/ml monobasic sodium phosphate dihydrate, and 6.0 to 6.4mg/ml sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

Polyols that act as conditioning agents and can stabilize antibodies may also be included in the formulation. The amount of polyol added to the formulation may vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also vary relative to the molecular weight of the polyol. For example, a lower amount of monosaccharide (e.g., mannitol) may be added as compared to a disaccharide (e.g., trehalose). In certain embodiments, the polyol that can be used as a tonicity agent in the formulation is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg/ml. In certain embodiments, the mannitol concentration may be about 7.5 to about 15 mg/ml. In certain embodiments, the mannitol concentration may be about 10 to about 14 mg/ml. In certain embodiments, the mannitol concentration may be about 12 mg/ml. In certain embodiments, the polyol sorbitol may be included in the formulation.

Detergents or surfactants may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and/or minimizes particle formation in the formulation and/or reduces adsorption. In certain embodiments, the formulation may include a surfactant polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or tween 80. Tween 80 is used to denote polyoxyethylene (20) sorbitan monooleate (see Fiedler, encyclopedia of excipients (Lexikon der Hilfsstuffe), edition Cantor Verlag Aulendorf publication, 4 th edition, 1996). In certain embodiments, the formulation may contain from about 0.1mg/mL to about 10mg/mL, or from about 0.5mg/mL to about 5mg/mL of polysorbate 80. In certain embodiments, polysorbate 80 may be added to the formulation at about 0.1%.

Freeze-dried preparation

The lyophilized formulations of the present disclosure for use in methods of treatment of stage III NSCLC or tumor growth inhibition in untreated cancer patients comprise an anti-PD-L1/TGF β trap molecule and a lyoprotectant. The lyoprotectant may be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may further comprise one or more of a buffer, a surfactant, a bulking agent and/or a preservative.

The amount of sucrose or maltose that can be used to stabilize the lyophilized pharmaceutical product can be at least 1:2 protein to sucrose or maltose weight ratio. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1: 5.

In certain embodiments, the pH of the formulation may be set by the addition of a pharmaceutically acceptable acid and/or base prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

Prior to lyophilization, the pH of a solution containing a protein of the present disclosure may be adjusted to between about 6 to about 8. In certain embodiments, the pH of the lyophilized drug product can range from about 7 to about 8.

In certain embodiments, the salt or buffer component may be added in an amount of about 10mM to about 200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) and "alkali-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions may be used as counter ions.

In certain embodiments, a "filler" may be added. A "bulking agent" is a compound that increases the amount of the lyophilized mixture and aids in the physical structure of the lyophilized mass (e.g., aids in producing a substantially uniform lyophilized cake that retains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulation of the present invention may contain such a bulking agent.

Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multiple-use (multi-dose) formulation.

In certain embodiments, a lyophilized pharmaceutical product for use in a method of stage III NSCLC treatment or tumor growth inhibition in an untreated cancer patient may be reconstituted with an aqueous carrier. Aqueous carriers of interest herein are pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) and can be used to prepare liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, ringer's solution, or dextrose solution.

In certain embodiments, the lyophilized pharmaceutical products of the present disclosure are reconstituted with sterile water for injection, USP (swfi) or 0.9% sodium chloride injection, USP. During reconstitution, the lyophilized powder dissolves into a solution.

In certain embodiments, the lyophilized protein products of the present disclosure are dissolved in about 4.5mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

Liquid preparation

In some embodiments, the protein products of the present disclosure are formulated into liquid formulations for use in methods of stage III NSCLC treatment or tumor growth inhibition in untreated cancer patients. The liquid formulation may be present at a concentration of 10mg/mL in USP/Ph Eur type I50R vials, which are sealed with rubber stoppers and sealed with aluminum crimp seals. The stopper may be made of an elastomer conforming to USP and Ph Eur. In certain embodiments, the vial may be filled with about 61.2mL of the protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with a 0.9% saline solution. In certain embodiments, the vial can contain about 61.2mL of a solution of about 20mg/mL to about 50mg/mL (e.g., about 20mg/mL, about 25mg/mL, about 30mg/mL, about 35mg/mL, about 40mg/mL, about 45mg/mL, or about 50mg/mL) of a protein product (e.g., anti-PD-L1/TGF β trap) (e.g., a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1) to allow an extractable volume of 60mL for delivery of about 1200mg to about 3000mg (e.g., about 1200mg to about 3000mg, about 1200mg to about 2900mg, about 1200mg to about 2800mg, about 1200mg to about 2700mg, about 1200mg to about 2600mg, about 1200mg to about 2500mg, about 1200mg to about 2400mg, from about 1200mg to about 2200mg, from about 1200mg to about 2100mg, from about 1200mg to about 2000mg, from about 1200mg to about 1900mg, from about 1200mg to about 1800mg, from about 1200mg to about 1700mg, from about 1200mg to about 1600mg, from about 1200mg to about 1500mg, from about 1200mg to about 1400mg, from about 1200mg to about 1300mg, from about 1300mg to about 3000mg, from about 1400mg to about 3000mg, from about 1500mg to about 3000mg, from about 1600mg to about 3000mg, from about 1700mg to about 3000mg, from about 1800mg to about 3000mg, from about 1900mg to about 3000mg, from about 2000mg to about 2200mg, from about 2100mg to about 3000mg, from about 290mg to about 3000mg, from about 2300mg to about 3000mg, from about 2400mg to about 3000mg, from about 2500mg to about 3000mg, from about 2600mg to about 3000mg, from about 2700mg to about 3000mg, from about 0mg to about 3000mg, from about 1600mg, from about 3000mg to about 3000mg, from about 3000mg to about 3000mg, from about 1200mg, from about, about 2300mg, about 2400mg, about 2500mg, about 2600mg, about 2700mg, about 2800mg, about 2900mg, or about 3000mg) of a protein product (e.g., an anti-PD-L1/TGF β trap, such as a polypeptide having an amino acid sequence comprising SEQ ID NO:3, and a first polypeptide comprising the amino acid sequence of SEQ ID NO:1, or a protein product of a second polypeptide of the amino acid sequence of 1; or a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40) to a subject.

In certain embodiments, the vial may contain about 61.2mL of a solution of about 20mg/mL to about 50mg/mL (e.g., about 20mg/mL, about 25mg/mL, about 30mg/mL, about 35mg/mL, about 40mg/mL, about 45mg/mL, or about 50mg/mL) of a protein product (e.g., an anti-PD-L1/TGF β trap, such as a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:1, or a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40) to allow an extractable volume of 60mL for delivery of about 1200mg to about 3000mg (e.g., about 1200mg to about 3000mg, from about 1200mg to about 2900mg, from about 1200mg to about 2800mg, from about 1200mg to about 2700mg, from about 1200mg to about 2600mg, from about 1200mg to about 2500mg, from about 1200mg to about 2400mg, from about 1200mg to about 2300mg, from about 1200mg to about 2200mg, from about 1200mg to about 2100mg, from about 1200mg to about 2000mg, from about 1200mg to about 1900mg, from about 1200mg to about 1800mg, from about 1200mg to about 1700mg, from about 1200mg to about 1600mg, from about 1200mg to about 1500mg, from about 1200mg to about 1400mg, from about 1200mg to about 1300mg, from about 1300mg to about 3000mg, from about 1400mg to about 3000mg, from about 1500mg to about 3000mg, from about 1600mg to about 3000mg, from about 1700mg to about 3000mg, from about 1800mg to about 3000mg, from about 1900mg to about 3000mg, from about 2000mg to about 3000mg, from about 2100mg to about 3000mg, from about 2700 to about 3000mg, from about 3000mg to about 3000mg, from about 3000mg, about 2900mg to about 3000mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, about 2500mg, about 2600mg, about 2700mg, about 2800mg, about 2900mg, or about 3000mg) of the protein product to an untreated subject.

In certain embodiments, the liquid formulation used in the methods of the present disclosure for treating stage III NSCLC or inhibiting tumor growth in an untreated cancer patient, while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the time to onset of metastasis and/or time to distant metastasis of stage III NSCLC in the patient, may be prepared in a solution at a concentration of 10mg/mL in combination with a stable level of sugar. In certain embodiments, the liquid formulation may be prepared in an aqueous vehicle. In certain embodiments, the stabilizing agent may be added in an amount no greater than that which would result in a viscosity that is unsuitable or undesirable for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may further comprise one or more of a buffer, a surfactant, and a preservative.

In addition to aggregation, deamidation is a common product variant of peptides and proteins, which can occur during fermentation, harvest/cell clarification, purification, drug/drug product storage, and during sample analysis. Deamidation is the loss of NH from proteins₃Forming a hydrolyzable succinimide intermediate. The succinimide intermediate resulted in a 17u mass reduction of the parent peptide. Subsequent hydrolysis resulted in an 18u mass increase. Due to instability under aqueous conditions, it is difficult to isolate the succinimide intermediate. Thus, deamidation is usually measured as 1u mass increase. Deamidation of asparagine to form aspartic acid or isoaspartic acid. Parameters that affect the deamidation rate include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the deamidation rate. Gly and Ser after Asn in the protein sequence lead to easier deamidation.

In certain embodiments, the liquid formulations of the present disclosure for use in methods of stage III NSCLC treatment or tumor growth inhibition in untreated cancer patients may be stored under pH and humidity conditions to prevent deamination of the protein product.

The aqueous vehicles contemplated herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and can be used to prepare liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, ringer's solution, or dextrose solution.

In particular cases, Intravenous (IV) formulations may be the preferred route of administration, for example when a patient receives all drugs via the IV route in a hospital after transplantation. In certain embodiments, the liquid formulation is diluted with a 0.9% sodium chloride solution prior to administration. In certain embodiments, the diluted pharmaceutical product for injection is isotonic and suitable for administration by intravenous infusion.

Methods of treating cancer or inhibiting tumor growth

In one aspect, the present disclosure provides a method of treating stage III NSCLC or inhibiting tumor growth while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia) and increasing the time to onset of metastasis and/or metastasis to distant sites in a patient in an untreated subject in need of treatment, the method comprising administering to the subject a dose of at least 500mg of a protein comprising a first polypeptide and a second polypeptide. The first polypeptide comprises: (a) at least the heavy chain variable region of an antibody capable of binding to human protein programmed death ligand 1 (PD-L1); and (b) a human transforming growth factor beta receptor II (TGF β RII) or fragment thereof capable of binding transforming growth factor beta (TGF β). The second polypeptide includes at least an antibody light chain variable region that binds PD-L1, and the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds PD-L1.

In one aspect, the invention provides a method of treating advanced unresectable stage III non-small cell lung cancer (NSCLC) in a patient by administering to the patient an anti-PD-L1/TGF β trap in combination with a cCRT (e.g., platinum-based chemoradiation), followed by administration to the patient an anti-PD-L1/TGF β trap. In certain embodiments, the present disclosure provides a method of treating advanced unresectable stage III NSCLC in a patient by combining with concurrent platinum-based chemical radiation (crt) and thereafter administering to the patient an anti-PD-L1/TGF β trap.

In certain embodiments, crts are administered concurrently in the form of cisplatin/etoposide, cisplatin/pemetrexed, or carboplatin/paclitaxel with a total radiation dose of 60-66Gy (e.g., 60Gy) delivered by intensity modulated radiation therapy. In certain embodiments, the crt is administered in cisplatin/etoposide form concurrently with a 60-66Gy (e.g., 60Gy) total radiation dose delivered by intensity modulated radiation therapy. In certain embodiments, the crts are administered in the form of carboplatin/paclitaxel concurrently with a total radiation dose of 60-66Gy (e.g., 60Gy) delivered by intensity modulated radiation therapy. In certain embodiments, the crt is administered in cisplatin/pemetrexed form concurrently with a 60-66Gy (e.g., 60Gy) total radiation dose delivered by intensity modulated radiation therapy.

In certain embodiments, the methods of treating stage III NSCLC or inhibiting tumor growth of the present disclosure comprise administering to an untreated subject a protein comprising two peptides, wherein a first polypeptide comprises the amino acid sequence of SEQ ID No. 3 and a second polypeptide comprises the amino acid sequence of SEQ ID No. 1. In certain embodiments, the protein is an anti-PD-L1/TGF β trap molecule.

In some embodiments, a non-treated subject treated according to the methods disclosed herein has not received prior treatment prior to treatment with a bifunctional protein of the present disclosure (anti-PD-1/TGF β trap molecule). In some embodiments, the untreated cancer patient to be treated according to the methods of the present disclosure has or does not have an Epidermal Growth Factor Receptor (EGFR) sensitizing (activating) mutation, an Anaplastic Lymphoma Kinase (ALK) translocation and/or an ROS1 mutation.

In certain embodiments, a method for treating stage III NSCLC or inhibiting tumor growth while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the time to onset of metastasis and/or time to distant metastasis of stage III NSCLC in a patient involves administering a protein (e.g., an anti-PD-L1/TGF β trap (such as a protein product of a first polypeptide having an amino acid sequence comprising SEQ ID NO:3 and a second polypeptide having an amino acid sequence comprising SEQ ID NO: 1; or a protein product of a first polypeptide having an amino acid sequence comprising SEQ ID NOs 35, 36, and 37 and a second polypeptide having an amino acid sequence comprising SEQ ID NOs 38, 39, and 40)) to an untreated subject at a dose of about 1200mg to about 3000mg (e.g., about 1200mg to about 3000mg, about 1200mg to about 2900mg, from about 1200mg to about 2800mg, from about 1200mg to about 2700mg, from about 1200mg to about 2600mg, from about 1200mg to about 2500mg, from about 1200mg to about 2400mg, from about 1200mg to about 2300mg, from about 1200mg to about 2200mg, from about 1200mg to about 2100mg, from about 1200mg to about 2000mg, from about 1200mg to about 1900mg, from about 1200mg to about 1800mg, from about 1200mg to about 1700mg, from about 1200mg to about 1600mg, from about 1200mg to about 1500mg, from about 1200mg to about 1400mg, from about 1200mg to about 1300mg, from about 1300mg to about 3000mg, from about 1400mg to about 3000mg, from about 1500mg to about 3000mg, from about 1600mg to about 1800mg, from about 1700mg to about 3000mg, from about 3000mg to about 3000mg, from about 1900mg to about 3000mg, from about 2000mg to about 3000mg, from about 2100mg to about 3000mg, from about 2300mg to 3000mg, from about 2700mg to about 3000mg, from about 2100mg to about 3000mg, about 2900mg to about 3000mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, about 2500mg, about 2600mg, about 2700mg, about 2800mg, about 2900mg, or about 3000 mg). In certain embodiments, about 1200mg of the anti-PD-L1/TGF β trap molecule is administered biweekly to an untreated stage III NSCLC subject (e.g., a non-resectable stage III NSCLC subject). In certain embodiments, about 1800mg of an anti-PD-L1/TGF β trap molecule is administered to an untreated stage III NSCLC subject (e.g., a non-resectable stage III NSCLC subject) once every three weeks. In certain embodiments, about 1200mg of the protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO. 3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO. 1 is administered biweekly to an untreated subject. In certain embodiments, the untreated stage III NSCLC subject (e.g., a non-resectable stage III NSCLC subject) is administered about 1800mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:1 once every three weeks. In certain embodiments, the untreated stage III NSCLC subject (e.g., a non-resectable stage III NSCLC subject) is administered once every three weeks about 1800mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40. In certain embodiments, about 2400mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO. 3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO. 1 is administered to a subject once every three weeks. In certain embodiments, about 2400mg of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40 is administered to a subject once every three weeks.

In certain embodiments, the dose administered to a non-treated stage III NSCLC subject (e.g., a non-resectable stage III NSCLC subject) may be about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 1075mg, about 1100mg, about 1125mg, about 1150mg, about 1175mg, about 1200mg, about 1225mg, about 1250mg, about 1275mg, about 1300mg, about 1325mg, about 1350mg, about 1375mg, about 1400mg, about 1425mg, about 1575mg, about 1500mg, about 1525mg, about 1600mg, about 1550mg, about 1725mg, about 1575mg, about 16505 mg, about 1850mg, 1875mg, about 1900mg, about 1925mg, about 1950mg, about 1975mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, or about 2400 mg.

In certain embodiments, the dose administered to an untreated stage III NSCLC subject (e.g., a non-resectable stage III NSCLC subject) may be administered once every two weeks. In certain embodiments, the dose administered to a non-treated stage III NSCLC subject (e.g., a non-resectable stage III NSCLC subject) may be administered once every three weeks. In certain embodiments, the protein may be administered intravenously, for example with a pre-filled bag, a pre-filled pen, or a pre-filled syringe. In certain embodiments, the protein is administered intravenously from a 250ml saline bag, and the intravenous infusion may last about 1 hour (e.g., 50 to 80 minutes). In certain embodiments, the bag connects a channel comprising a tube and/or a needle.

In some embodiments, the stage III NSCLC exhibits squamous or non-squamous histology. For example, in one embodiment, the method treats stage III squamous NSCLC. In some embodiments, the method treats non-squamous stage III NSCLC.

In certain embodiments, an untreated subject or patient having stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenously administering at least 500mg (e.g., about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg or more) of an anti-PD-L1/TGF β trap, wherein the anti-PD-L1/TGF β trap comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, an untreated subject or patient having stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenous administration of at least 500mg (e.g., about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg or more) of an anti-PD-L1/TGF β trap, wherein the anti-PD-L1/TGF β trap comprises a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40. In certain embodiments, an untreated subject or patient having stage III NSCLC (such as squamous or non-squamous stage III NSCLC) is treated by intravenous administration of 2400mg of an anti-PD-L1/TGF β trap, wherein the anti-PD-L1/TGF β trap comprises a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40.

In certain embodiments, an untreated subject or patient having stage III NSCLC (e.g., squamous or non-squamous NSCLC) is treated by intravenous administration of anti-TGF L/PD from about 1200mg to about 2400mg (e.g., about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 2200mg, about 1200mg to about 2100mg, about 1200mg to about 2000mg, about 1200mg to about 1900mg, about 1200mg to about 1800mg, about 1200mg to about 1700mg, about 1200mg to about 1600mg, about 1200mg to about 1500mg, about 1200mg to about 1400mg, about 1200mg to about 1300mg, about 1300mg to about 2400mg, about 1400mg to about 2400mg, about 1500mg to about 1800mg, about 1600mg to about 2400mg, about 1700mg to about 2400mg, about 2400mg to about 2400mg, about 1900mg to about 2400mg, about 2000mg to about 2400mg, about 2100mg to about 2100mg, about 2200mg to about 2300mg, or about 1 mg), wherein the anti-PD-L1/TGF β trap comprises a first polypeptide having the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, an untreated subject or patient having stage III NSCLC (e.g., squamous or non-squamous NSCLC) is treated by intravenous administration of anti-TGF L/PD from about 1200mg to about 2400mg (e.g., about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 2200mg, about 1200mg to about 2100mg, about 1200mg to about 2000mg, about 1200mg to about 1900mg, about 1200mg to about 1800mg, about 1200mg to about 1700mg, about 1200mg to about 1600mg, about 1200mg to about 1500mg, about 1200mg to about 1400mg, about 1200mg to about 1300mg, about 1300mg to about 2400mg, about 1400mg to about 2400mg, about 1500mg to about 1800mg, about 1600mg to about 2400mg, about 1700mg to about 2400mg, about 2400mg to about 2400mg, about 1900mg to about 2400mg, about 2000mg to about 2400mg, about 2100mg to about 2100mg, about 2200mg to about 2300mg, or about 1 mg), wherein the anti-PD-L1/TGF β trap comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 35. 36 and 37, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 38. 39 and 40.

In certain embodiments, an untreated subject or patient with stage III NSCLC (e.g., squamous or non-squamous NSCLC) is treated by intravenous administration of a dose of about 1200mg of anti-PD-L1/TGF β trap once every two weeks. In certain embodiments, an untreated subject or patient with stage III NSCLC (e.g., squamous or non-squamous NSCLC) is treated by intravenous administration of a dose of about 1800mg of anti-PD-L1/TGF β trap once every three weeks. In certain embodiments, an untreated subject or patient with stage III NSCLC (e.g., squamous or non-squamous NSCLC) is treated by intravenous administration of an anti-PD-L1/TGF β trap at a dose of about 2400mg once every three weeks. In certain embodiments, an untreated subject or patient with stage III NSCLC (e.g., squamous or non-squamous NSCLC) is treated by intravenous administration of a dose of 2400mg of anti-PD-L1/TGF β trap once every three weeks.

In some embodiments, the stage III NSCLC to be treated is PD-L1 positive. In some embodiments, the stage III NSCLC to be treated is PD-L1 negative. In exemplary embodiments, the stage III NSCLC to be treated exhibits high PD-L1 expression (e.g., "high PD-L1"). In exemplary embodiments, the stage III NSCLC to be treated does not exhibit PD-L1 expression. In an exemplary embodiment, the stage III NSCLC patient to be treated is diagnosed with PD-L1-positive stage III NSCLC. In an exemplary embodiment, the stage III NSCLC patient to be treated is diagnosed with PD-L1 negative stage III NSCLC.

Methods of detecting biomarkers, such as PD-L1, for example, on cancer or tumors are routine in the art and are incorporated herein. Non-limiting examples include immunohistochemistry, immunofluorescence, and fluorescence-activated cell sorting (FACS). In certain embodiments, the level of PD-L1 expression in stage III NSCLC is detected using an anti-PD-L1 antibody. The tissue sample may be formalin fixed, paraffin embedded stage III NSCLC tissue.

In some embodiments, an untreated subject or patient with high PD-L1, stage III NSCLC or without consideration of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenous administration of anti-PD-L1/TGF β trap at a dose of at least 500 mg. In some embodiments, an untreated subject or patient with high PD-L1, stage III NSCLC or without consideration of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenous administration of anti-PD-L1/TGF β trap at a dose of about 1200mg once every two weeks. In some embodiments, an untreated subject or patient with high PD-L1, stage III NSCLC or without consideration of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenous administration of anti-PD-L1/TGF β trap at a dose of about 1800mg once every three weeks. In some embodiments, an untreated subject or patient with high PD-L1, stage III NSCLC or without consideration of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenous administration of the anti-PD-L1/TGF β trap at a dose of about 2400mg once every three weeks.

In certain embodiments, a patient treated with cisplatin/pemetrexed and radiation therapy (crt) in combination with anti-PD-L1/TGF β trap is diagnosed with advanced unresectable stage III NSCLC, which expresses PD-L1. In certain embodiments, a patient treated with cisplatin/pemetrexed and radiation therapy (crt) in combination with anti-PD-L1/TGF β trap is diagnosed with advanced unresectable stage III NSCLC that does not express PD-L1. In certain embodiments, a patient diagnosed with advanced unresectable stage III NSCLC is treated with chemotherapy (e.g., cisplatin/pemetrexed) and radiation therapy (crt) in combination with anti-PD-L1/TGF β trap, regardless of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative).

In some embodiments, a patient diagnosed with advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC) is treated with a combination of chemotherapy (e.g., a combination of cisplatin and etoposide, or a combination of carboplatin and paclitaxel) and radiation therapy (crt) in combination with PD-L1/TGF β trap, independently of PD-L1 expression (stage III NSCLC is both PD-L1 positive or PD-L1 negative), by intravenous administration of at least 500mg of anti-PD-L1/TGF β trap. In some embodiments, a patient diagnosed with advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC) is treated with a combination of chemotherapy (e.g., a combination of cisplatin and etoposide, or a combination of carboplatin and paclitaxel) and radiation therapy (crt) in combination with PD-L1/TGF β trap, independently of PD-L1 expression (both stage III NSCLC are PD-L1 positive or PD-L1 negative), by intravenously administering a dose of about 1200mg of anti-PD-L1/TGF β trap once every two weeks. In some embodiments, a patient diagnosed with advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC) is treated with a combination of chemotherapy (e.g., a combination of cisplatin and etoposide, or a combination of carboplatin and paclitaxel) and radiation therapy (crt) in combination with PD-L1/TGF β trap, independently of PD-L1 expression (both stage III NSCLC are PD-L1 positive or PD-L1 negative), by intravenously administering a dose of about 1800mg or 2400mg of anti-PD-L1/TGF β trap once every three weeks.

In some embodiments, a patient with non-squamous histology diagnosed with advanced unresectable stage III NSCLC is treated with chemotherapy (e.g., a combination of cisplatin and pemetrexed) and radiation therapy (crt) in combination with anti-PD-L1/TGF β trap, independently of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative), by intravenous administration of at least 500mg of anti-PD-L1/TGF β trap. In some embodiments, a patient with non-squamous histology diagnosed with advanced unresectable stage III NSCLC is treated with chemotherapy (e.g., a combination of cisplatin and pemetrexed) and radiation therapy (crt) in combination with anti-PD-L1/TGF β trap, independently of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative), by intravenously administering about 1200mg of anti-PD-L1/TGF β trap once every two weeks. In some embodiments, a patient with non-squamous histology diagnosed with advanced unresectable stage III NSCLC is treated with chemotherapy (e.g., a combination of cisplatin and pemetrexed) and radiation therapy (crt) in combination with anti-PD-L1/TGF β trap, independently of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative), by intravenous administration of about 1800mg or 2400mg of anti-PD-L1/TGF β trap once every three weeks.

In certain embodiments, the present disclosure provides methods of treating advanced unresectable stage III NSCLC in a patient by administering to the patient a combination of anti-PD-L1/TGF β of at least 500mg (e.g., about 500mg, 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, or more) concurrently with and after platinum-based chemical radiation (crt). In certain embodiments, the present disclosure provides methods of treating advanced unresectable stage III NSCLC in a patient by administering to the patient an anti-PD-L1/TGF β trap of about 1200mg concurrently with and following platinum-based chemical radiation (crt). In certain embodiments, the present disclosure provides methods of treating advanced unresectable stage III NSCLC in a patient by administering to the patient about 1800mg of an anti-PD-L1/TGF β trap concurrently with and following platinum-based chemical radiation (crt). In certain embodiments, the present disclosure provides methods of treating advanced unresectable stage III NSCLC in a patient by administering to the patient about 2400mg of an anti-PD-L1/TGF β trap concurrently and after platinum-based chemical radiation (crt).

In some embodiments, the untreated subject or patient to be treated has a mutation selected from the group consisting of an EGFR sensitizing mutation, an ALK translocation, and an ROS1 mutation. For example, in some embodiments, a stage III NSCLC with high PD-L1, or an untreated subject or patient with mutations selected from EGFR-sensitizing mutations, ALK translocations, and ROS1 mutations, unrelated to PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenous administration of at least 500mg (e.g., about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg or more) of an anti-PD-L1/TGF β trap. In some embodiments, an untreated subject or patient with a high PD-L1, stage III NSCLC, or unrelated to PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) having a mutation selected from EGFR sensitizing mutation, ALK translocation and ROS1 mutation is treated by intravenous administration of about 1200mg of anti-PD-L1/TGF β trap once every two weeks. In some embodiments, a untreated subject or patient with a high PD-L1, stage III NSCLC, or unrelated to PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous NSCLC) having a mutation selected from EGFR sensitization, ALK translocation, and ROS1 mutation is treated by intravenous administration of about 1800mg of anti-PD-L1/TGF β trap once every three weeks. In some embodiments, an untreated subject or patient with a high PD-L1, stage III NSCLC, or unrelated to PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous NSCLC) having a mutation selected from EGFR sensitizing mutation, ALK translocation and ROS1 mutation is treated by intravenous administration of about 2400mg of anti-PD-L1/TGF β trap once every three weeks.

In some embodiments, the untreated subject or patient does not have a mutation selected from the group consisting of an EGFR sensitizing mutation, an ALK translocation, an ROS1 mutation, and a BRAF V600E mutation. For example, in some embodiments, a stage III NSCLC with high PD-L1, or an untreated subject or patient not having an EGFR-sensitizing mutation, ALK translocation, ROS1 mutation, and BRAF V600E mutation, regardless of PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) is treated by intravenously administering at least 500mg (e.g., about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg or more) of an anti-PD-L1/TGF β trap. In some embodiments, untreated subjects or patients with high PD-L1, stage III NSCLC, or not associated with PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous stage III NSCLC) that do not have mutations selected from EGFR sensitizing mutation, ALK translocation, ROS1 mutation, and BRAF V600E are treated by intravenous administration of about 1200mg of anti-PD-L1/TGF β trap once every two weeks. In some embodiments, an untreated subject or patient with high PD-L1, stage III NSCLC, or not associated with PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous NSCLC) that does not have mutations selected from EGFR-sensitizing mutations, ALK translocations, ROS1 mutations, and BRAF V600E mutations is treated by intravenous administration of about 1800mg of anti-PD-L1/TGF β trap once every three weeks. In some embodiments, an untreated subject or patient with high PD-L1, stage III NSCLC, or not associated with PD-L1 expression (stage III NSCLC is PD-L1 positive or PD-L1 negative) (e.g., squamous or non-squamous NSCLC) that does not have mutations selected from EGFR-sensitizing mutations, ALK translocations, ROS1 mutations, and BRAF V600E mutations is treated by intravenous administration of about 2400mg of anti-PD-L1/TGF β trap once every three weeks.

In some embodiments, the methods of treatment disclosed herein result in remission or improved survival of the disease in the subject or patient (e.g., up to and including 6 months, 12 months, 18 months, 22 months, 28 months, 32 months, 38 months, 44 months, 50 months, 56 months, 62 months, 68 months, 74 months, 80 months, 86 months, 92 months, 98 months, 104 months, or 110 months). In certain embodiments, the improved survival is at least 108 months. For example, in some embodiments, remission may be complete remission, partial remission, or stable disease. In some embodiments, for example, an improved survival (e.g., a survival up to and including 6 months, 12 months, 18 months, 22 months, 28 months, 32 months, 38 months, 44 months, 50 months, 56 months, 62 months, 68 months, 74 months, 80 months, 86 months, 92 months, 98 months, 104 months, or 110 months) can be a Progression Free Survival (PFS) or an Overall Survival (OS). In certain embodiments, improved PFS and/or OS survival is at least 108 months. In some embodiments, the improvement is determined relative to the period prior to initiation of treatment with an anti-PD-L1/TGF β trap of the invention (e.g., in PFS). Determining disease remission (e.g., complete remission, partial remission, or stable disease) and patient survival (e.g., PFS, overall survival (e.g., up to and including 6 months, 12 months, 18 months, 22 months, 28 months, 32 months, 38 months, 44 months, 50 months, 56 months, 62 months, 68 months, 74 months, 80 months, 86 months, 92 months, 98 months, 104 months, or 110 months)) of cancer or tumor therapy is routine in the art and is contemplated herein. In certain embodiments, the patient survival is at least 108 months. In some embodiments, a patient receiving treatment receives phase contrast enhanced Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) after receiving phase contrast enhanced Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) of the diseased area (e.g., the chest/abdomen and pelvis covering the upper range of pleural access to pubic symphysis), and assesses disease remission according to RECIST 1.1.

Delivery device

In one aspect, the present disclosure provides a drug delivery device for use in a method of stage III NSCLC treatment or tumor growth inhibition in a treatment-naive cancer patient, wherein the device comprises a formulation comprising about 500mg-30000mg of a protein comprising a first polypeptide and a second polypeptide, said first polypeptide comprising: (a) at least the heavy chain variable region of an antibody capable of binding human protein programmed death ligand 1 (PD-L1); and (b) a human transforming growth factor beta receptor II (TGF β RII) or fragment thereof capable of binding transforming growth factor beta (TGF β), said second polypeptide comprising: at least the light chain variable region of an antibody capable of binding PD-L1, and the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site capable of binding PD-L1.

In some embodiments, the device may be a bag, pen or syringe. In certain embodiments, the bag may be connected to a channel that includes a tube and/or a needle.

In certain embodiments, a drug delivery device for use in a method of treating stage III NSCLC or inhibiting tumor growth in an untreated cancer patient while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the time to onset of metastasis and/or metastasis to a distant site in a patient stage III NSCLC may comprise from about 500mg to about 3000mg (e.g., from about 500mg to about 3000mg, from about 500mg to about 2900mg, from about 500mg to about 2800mg, from about 500mg to about 2700mg, from about 500mg to about 2600mg, from about 500mg to about 2500mg, from about 500mg to about 2400mg, from about 500mg to about 2200mg, from about 500mg to about 1600mg, from about 500mg to about 2100mg, from about 500mg to about 2000mg, from about 500mg to about 1900mg, from about 500mg to about 1800mg, from about 500mg to about 1700mg, from about 500mg to about 1600mg, from about 500mg to about 1500mg, from about 1300mg to about 1200mg, about 1300mg to about 3000mg, about 1400mg to about 3000mg, about 1500mg to about 3000mg, about 1600mg to about 3000mg, about 1700mg to about 3000mg, about 1800mg to about 3000mg, about 1900mg to about 3000mg, about 2000mg to about 3000mg, about 2100mg to about 3000mg, about 2200mg to about 3000mg, about 2300mg to about 3000mg, about 2400mg to about 3000mg, about 2500mg to about 3000mg, about 2600mg to about 3000mg, about 2700mg to about 3000mg, about 2800mg to about 3000mg, about 2900mg to about 3000mg, about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 500mg, about 2000mg, about 2100mg, about 2300mg, about 2400mg, about 2500mg, about 2600mg, about 2700mg, about 2800mg, about 3,000mg or about 3000mg of a disclosed anti-TGF-beta-1-beta-alpha-beta-gamma (TGF) trap, which has a sequence comprising SEQ ID NO:3 and a first polypeptide comprising the amino acid sequence of SEQ ID NO: 1; or a protein product of a first polypeptide comprising the amino acid sequences of SEQ ID NOs 35, 36 and 37 and a second polypeptide comprising the amino acid sequences of SEQ ID NOs 38, 39 and 40). In certain embodiments, the drug delivery device may comprise a 1200mg dose of a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1). In certain embodiments, a drug delivery device may comprise a dose of about 500mg of a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1; or a protein product of a first polypeptide comprising the amino acid sequences of SEQ ID NO:35, 36, and 37, and a second polypeptide comprising the amino acid sequences of SEQ ID NO:38, 39, and 40).

In certain embodiments, a drug delivery device may comprise a dose of about 1200mg of a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:1, or a protein product of a first polypeptide comprising the amino acid sequences of SEQ ID NO:35, 36, and 37, and a second polypeptide comprising the amino acid sequences of SEQ ID NO:38, 39, and 40). In certain embodiments, a drug delivery device for use in a method of treatment of stage III NSCLC or tumor growth inhibition in an untreated cancer patient while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the time to onset of metastasis and/or time to distant metastasis of stage III NSCLC in a patient comprises a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:1, or a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40) at a dose of about 1800 mg. In certain embodiments, a drug delivery device for use in a method of treatment of stage III NSCLC or tumor growth inhibition in an untreated cancer patient while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the time to onset of metastasis and/or time to distant metastasis of stage III NSCLC in the patient comprises a dose of about 1200mg, about 1800mg, or 2400mg of a protein having a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1; or a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO 35, 36 and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NO 38, 39 and 40.

In certain embodiments, a drug delivery device for use in a method of treating stage III NSCLC or inhibiting tumor growth in an untreated cancer patient while minimizing the development of pathological conditions associated with concomitant radiotherapy (e.g., pulmonary fibrosis, pneumonia), and increasing the time to onset of metastasis and/or time to distant metastasis of stage III NSCLC in a patient comprises a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap (e.g., a protein product comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1; or a first polypeptide having the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40)) at a dose of about 1,200 mg. In certain embodiments, a drug delivery device for use in a method of stage III NSCLC treatment or tumor growth inhibition in a treatment-naive cancer patient comprises a dose of about 1800mg of a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap, (e.g., a protein product comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1; or having a first polypeptide comprising the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide comprising the amino acid sequence of SEQ ID NOs 38, 39, and 40)). In certain embodiments, a drug delivery device for use in a method of stage III NSCLC treatment or tumor growth inhibition in a treatment-naive cancer patient may comprise about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 1075mg, about 1100mg, about 1125mg, about 1150mg, about 1175mg, about 1200mg, about 1225mg, about 1250mg, about 1275mg, about 1300mg, about 1325mg, about 1350mg, about 1375mg, about 1400mg, about 1425mg, about 1450mg, about 1500mg, about 1800mg, about 1550mg, about 1725mg, about 1575mg, about 1725mg, about 1650mg, about 1825mg, about 1850mg, about 1875mg, about 1900mg, about 1925mg, about 1950mg, about 1975mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, or about 2400mg of a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap, such as a protein product comprising a first polypeptide having the amino acid sequence of SEQ ID NOs 35, 36, and 37 and a second polypeptide having the amino acid sequence of SEQ ID NOs 38, 39, and 40).

Protein production

Antibody-cytokine trap proteins are typically produced by recombinant techniques using mammalian cells containing nucleic acids engineered to express the protein. Although one example of a suitable cell line and protein production method is described in examples 1 and 2 of US20150225483a1, a number of suitable vectors, cell lines and protein production methods exist for the production of antibody-based biopharmaceuticals and may be used to synthesize the antibody-cytokine trap proteins herein.

Treatment indications

The anti-PD-L1/TGF β trap proteins described herein (e.g., including a first polypeptide including the amino acid sequence of SEQ ID NO:3 and a second polypeptide including the amino acid sequence of SEQ ID NO: 1) and intravenous drug delivery formulations and delivery devices of the present disclosure including the anti-PD-L1/TGF β trap proteins may be used to treat stage III NSCLC in an untreated patient or reduce tumor growth in the patient.

A stage III NSCLC or tumor to be treated with an anti-PD-L1/TGF β trap may have increased expression of PD-L1 and/or TGF β in the tumor, a correlation of its expression level with prognosis or disease progression, and be selected for preclinical and clinical experience with respect to the sensitivity of the tumor to treatment targeting PD-L1 and TGF β.

In some embodiments, the untreated cancer patient to be treated according to the methods of the present disclosure has or does not have a mutation selected from: epidermal Growth Factor Receptor (EGFR) sensitizing (activating) mutations, Anaplastic Lymphoma Kinase (ALK) translocation and ROS1 mutations. In some embodiments, patients with untreated cancer (e.g., advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC), PD-L1 positive advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC), or PD-L1 negative advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC)) with high PD-L1 expression to be treated according to the methods of the invention have or do not have an Epidermal Growth Factor Receptor (EGFR) sensitizing (activating) mutation. In some embodiments, a patient with an untreated cancer (e.g., advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC), PD-L1 positive advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC), or PD-L1 negative advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC)) with high PD-L1 expression to be treated according to the methods of the invention has or does not have an Anaplastic Lymphoma Kinase (ALK) translocation. In some embodiments, a patient with an untreated cancer (e.g., advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC), PD-L1-positive advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC), or PD-L1-negative advanced stage III NSCLC (e.g., squamous or non-squamous stage III NSCLC)) with high PD-L1 expression to be treated according to the methods of the invention has or does not have a ROS1 mutation.

Examples

The foregoing is a general description of the present disclosure that will be more readily understood by reference to the following examples, which are intended merely to illustrate certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure in any way.

Example 1: intravenous pharmaceutical formulation packaging

The anti-PD-L1/TGF β trap formulation was prepared as a lyophilized formulation or a liquid formulation. To prepare the lyophilized formulation, the freeze-dried anti-PD-L1/TGF β trap molecules were sterilized and stored in disposable glass vials. Several such glass vials are then packaged in a kit for delivering a specific weight-independent dose to a subject diagnosed with cancer or tumor. The kit contains 12-60 vials, depending on the dosage requirements. Alternatively, the formulation is prepared and packaged as a liquid formulation and stored at 250 mg/bottle to 1000 mg/bottle. For example, the formulation is a liquid formulation and is stored at 600 mg/vial, or at 250 mg/vial. In another example, an anti-PD-L1/TGF β trap molecule is prepared as a 10mg/mL solution and provided in a USP/Ph Eur type I vial with a loading that allows for an extractable volume of 60mL (600mg/60mL), complying with USP and Ph Eur requirements for sealing with rubber stoppers in serological format and with aluminum crimp seals.

A subject diagnosed with stage III NSCLC is administered a formulation comprising 500mg to 2400mg of anti-PD-L1/TGF β trap intravenously. For example, the subject is administered 1200mg of anti-PD-L1/TGF β trap intravenously once every two weeks, or 1800mg of anti-PD-L1/TGF β trap once every three weeks. Intravenous administration was via saline bags. The amount of anti-PD-L1/TGF β trap molecule administered is independent of the subject's body weight.

Example 2: effect of anti-PD-L1/TGF beta trap in alleviating cCRT-induced fibrosis

In this example, experiments were described to evaluate the effect of anti-PD-L1/TGF β trap given in combination with radiation or chemotherapy on the alleviation of pulmonary fibrosis.

Cell line: 4T1 murine breast cancer cells obtained from the American Type Culture Collection (ATCC) were cultured in RPMI1640 medium (Life Technologies) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS). All cells were cultured under sterile conditions and cultured at 37^℃And 5% CO₂And (4) incubating. After passaging, the cells were implanted in vivo and adherent cells were harvested using TrypLE Express (Gibco) or 0.25% trypsin.

Mice: BALB/c was obtained from Charles River Laboratories (Charles River Laboratories). All experimental mice were 6 to 12 week old female mice. Mice were housed in pathogen-free facilities with free access to food and water.

Murine tumor models: 6 days before the start of the treatment, 0.5X 10⁵4T1 cells were inoculated intramuscularly (i.m.) to the thigh of BALB/c mice. Treatment was started after 6 days (day 0) and mice were sacrificed on day 6 (i.e., 12 days after intramuscular injection).

Treatment: for all studies, mice were randomized into treatment groups on the day of treatment initiation (day 0).

anti-PD-L1/TGF β trap and controls: the anti-PD-L1/TGF β trap of the present disclosure is a fully human immunoglobulin 1(IgG1) monoclonal antibody against human PD-L1 (e.g., including a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1) fused to the extracellular domain of human TGF- β receptor II. Isotype control is a mutant form of anti-PD-L1, completely without PD-L1 binding. In tumor-bearing mice, on days 0, 2 and 4, anti-PD-L1/TGF β trap (492 μ g) or isotype control (400 μ g) was formulated for intravenous (i.v.) administration in 0.2mL PBS. Non-tumor bearing BALB/c mice were injected intravenously with anti-PD-L1/TGF β trap (20mg/kg), anti-PD-L1 (16.3mg/kg), trap control (anti-PD-L1 (mut)/TGF- β trap, 20mg/kg) or isotype control (anti-PD-L1 (mut), 16.3 mg/kg).

CCl₄(carbon tetrachloride) -dependent induction of fibrosis: mice were weighed 2 days per week and injected intraperitoneally with 1:3CCl at 1 uL/g using a glass Hamilton syringe and 27G x 1/2 needle₄Olive oil solution.

Radiation: to evaluate the combination of radiation and anti-PD-L1/TGF β trap, mice were randomized into the following treatment groups: isotype control (133, 400 μ g) + vehicle control (0.2mL), radiation (3.6, 7.5, 8 Gy/day), anti-PD-L1/TGF β trap (164, 492 μ g) or anti-PD-L1/TGF β trap + radiation. For radiotherapy, delivery was localized to the tumor-bearing leg of the mouse with a lead-shielded collimator. By timed exposure to cesium 137 gamma irradiators (40Exactor, MDS Nuodian (MDS Nordion), Ottawa, Ontario, Canada). Radiotherapy is performed once a day for four days.

CCl₄Induced liver fibrosis

Histology: the left liver samples were sent to Histotox (Boulder, CO) for processing and staining. Alpha SMA (Abcam, Cat. No. ab124964, 1:200) and bitter red were stained by standard histological methods on 5 μm sections from the upper, middle and lower parts of the medial lobe. To optimize the morphological analysis of the slides, the secondary or background staining was omitted, so only positively stained cells or structures were shown. Primary antibodies were labeled using the Agilent Envision + rabbithrp kit (catalog No. K4011) that included HRP-labeled secondary antibodies that could be subjected to DAB color development.

SMAD/PhosphoSMAD assay: tissue lysates containing 0.02% HALT protease inhibitor cocktail (Thermo) and 1mM EDTA in RIPA buffer (Sigma) were added to frozen liver samples at a weight: volume ratio of 1:2 while thawing. The samples were then homogenized in a tissuelyser (qiagen) using a magnetic bead disruptor for 2 minutes at a frequency of 30/sec. After disruption, the lysate was centrifuged at 12000rpm for 20 minutes at 4 ℃. The supernatant was aliquoted and filtered through a 20 μ M mesh filter plate (EMD Millipore). The final lysates were frozen at-80 ℃ for subsequent analysis or measured directly using SMAD2/3 and phosphoSMAD 2/3ELISA (cell signaling) according to the manufacturer's instructions.

Morphological analysis: slides were digitally scanned using a Hamamatsu Nanozoomer scanner and Digital Pathology software. View software looks at the saved image and reduces it to 1.5% zoom. The final Image was analyzed by thresholding analysis of positively stained cells using Image Pro Premier. The same threshold is applied to all tissues. The images represent the average results.

RNA-seq analysis: RNA was mapped to the genome of Ensembl 75 mice (GRCm38, 2 months 2014), aligned with Bowtie 2 (Langmead and Salzber (2012), nat. methods,9(4): 357-.

The feature score is defined as the average log of all genes in each gene signature₂(fold change). All genes and samples were given a false count of 0.5TPM to determine log₂(TPM) and then from the log of each gene₂-subtracting the median log of each gene in the totality of samples from the TPM₂TPM to calculate the above scores. The characteristic scores for the genome and expression of each gene (log2 fold change) are shown as boxplots, representing the median, 25 th and 75 th percentiles; the dashed line spans from a minimum to a maximum.

α -SMA immunohistochemistry: isolated tumors were fixed in 10% neutral formalin (NBF) for 24 hours at room temperature, dehydrated and embedded in paraffin. The tissue was cut into 5 μm sections and then transferred to positively charged slides. Prior to staining, sections were deparaffinized and rehydrated. Anti- α -SMA immunohistochemistry was performed using an established protocol and a Leica BOND-RX auto stain. Briefly, antigen retrieval was performed using epitope retrieval solution 2(Leica, cat # AR9640) for 20 minutes at 95 ℃. After blocking, sections were incubated with HRP-conjugated 5. mu.g/ml anti-alpha-SMA antibody (clone 1A4, Sigma, Cat. No. SAB420067) for 60 min. Detection was performed using diaminobenzidine substrate (DAB), and sections were counterstained with hematoxylin. After staining was complete, the slides were dehydrated and coverslipped. Stained sections were imaged using a Hamamatsu Nanozoomer microscope.

Images were digitally quantized using Aperio ImageScope software (version 12.3.2.8013). For each tumor, multiple regions of interest (ROIs) (8-11 ROIs) were analyzed. Necrotic areas and tumor margins were excluded from the analysis. The total positive pixels for DAB above background were determined and divided by the total number of pixels in the ROI to obtain the percent positive for each ROI. The percent positive score for each tumor obtained by averaging ROI values was plotted using GraphPad Prism.

Statistical analysis: statistical analysis was performed using GraphPad Prism software version 7.0. For the pSMAD and bitter red analyses, an unpaired two-tailed t-test was used to compare treatment to isotype controls. To assess differences in gene signature scores between treatment groups, one-way analysis of variance (ANOVA) was performed followed by Tukey multiple comparison test.

anti-PD-L1/TGF beta trap and trap controls, but not anti-PD-L1, reduced chemotherapy-induced fibrotic CCl₄Induced hepatic fibrosis in BALB/c mice: to evaluate the anti-fibrotic effect in vivo against PD-L1/TGF beta trap, carbon tetrachloride (CCl) was used₄) Chemotherapy-induced liver fibrosis model. BALB/c mice received CCl twice weekly₄Treatment continued for six weeks while receiving three doses of isotype control, anti-PD-L1, trap control or anti-PD-L1/TGF β trap.

In this experiment, five groups of mice were used: untreated BALB/c mice (Nv) (n-4 mice/group) using CCl₄(1 μ L) treated BALB/c mice (n ═ 8 mice/group) (1 μ L/g, i.p.; 2 days per week for 6 weeks), as well as BALB/c mice receiving treatment with isotype control (16.3mg/kg, i.v.; days 0, 2, 4), anti-PD-L1 (16.3mg/kg, i.v.; days 0, 2, 4), trap control (20mg/kg, i.v.; days 0, 2, 4) or anti-PD-L1/TGF β trap (20mg/kg, iv; days 0, 2, 4).

Mice were harvested after 6 weeks and livers were stained with bitter red or pSMAD 2/3. CCl₄The total collagen content in the liver of isotype control mice was significantly increased as measured by percentage bitter red. anti-PD-L1 antibody did not affect collagen content relative to isotype controls, but both trap control and anti-PD-L1/TGF β trap treatment significantly reduced collagen content (total collagen (percentage bitter red); p 0.0038 and p 0.0019, respectively) (fig. 12A). The percentage of myofibroblast marker, α SMA, in the treated liver samples was similarly unaffected by anti-PD-L1 treatment, but both the well control and anti-PD-L1/TGF β well treatment significantly reduced the percentage of α SMA (p 0.0003 and p 0.0013, respectively) (fig. 12B). Given that phosphorylation of R-Smads (e.g., pSmad2/3) can be induced by TGF- β isoforms 1-3, the ratio of pSmad2/3 to total Smad2/3 in treated liver samples was also determined (the ratio of phosphorylated SMAD2/3 to total SMAD2/3 is expressed as mean. + -. SEM, each point representing one mouse). Treatment was compared to isotype controls using unpaired t-test. Significant differences were indicated by 0.01 and 0.001. anti-PD-L1/TGF β trap was the only treatment capable of reducing the proportion of pSmad2/3 (p 0.0006) relative to isotype control treatment (fig. 12C).

Combination therapy with-PD-L1/TGF beta trap and radiation therapy to reduce EMT and profibrotic gene signature scores

To examine the potential mechanism of action to induce an enhancement of the antitumor activity of the combination therapy, gene expression in 4T1 tumor tissue by targeted RNA sequencing (RNAseq) was profiled.

FIGS. 13A, 13B and 8 show RNAseq analysis from the 4T1 modelThe data of (1). BALB/c mice were inoculated intramuscularly (i.m.) at 0.5X 10⁵4T1 cells (day-6) were treated with isotype control (400 μ g, intravenously; days 0, 2, 4) + vehicle control (0.2mL, oral, twice daily (q.d.), day 0-6), anti-PD-L1/TGF β trap (492 μ g, intravenously (i.v.); days 0, 2, 4), radiation (8 gray (Gy), day 0-3) or anti-PD-L1/TGF β trap + RT (n ═ 10 mice/group). Mice were sacrificed on day 6, tumors were collected and processed to extract RNA. RNAseq was performed using Qiaseq targeted RNA panels and feature scores were defined. The feature scores for EMT and profibrotic genes (defined as the mean log2 fold change between all genes in a feature) are shown in a scatter plot or box plot. The dashed line spans from a minimum to a maximum.

As part of RNA sequencing analysis, genes are divided into functional groups and a "signature" score is determined as a measure of gene expression. anti-PD-L1/TGF β trap monotherapy resulted in a decrease in epithelial-to-mesenchymal transition (EMT) feature score (p <0.0001) relative to isotype control. Although increasing radiation therapy did not significantly affect EMT signature (p >0.05), the combination of anti-PD-L1/TGF β trap and radiation therapy significantly down-regulated EMT signature score (p <0.0001) relative to isotype control (fig. 13A).

anti-PD-L1/TGF β trap monotherapy also reduced profibrotic gene signature scores, but radiotherapy significantly increased profibrotic gene signature scores relative to isotype controls (p < 0.0001). Furthermore, combining radiation with anti-PD-L1/TGF β trap reduced the profibrotic signature score relative to radiation alone (fig. 13B). Following anti-PD-L1/TGF β trap treatment, the radiation-induced fibrosis gene signature score (based on Alsner et al, (2007) Radiotherapy and Oncology,83(3): 261-. Notably, anti-PD-L1/TGF β trap therapy in combination with radiotherapy significantly reduced the characteristic of radiation-induced fibrosis relative to radiation monotherapy (p 0.0365) (fig. 8). For the radiation-induced fibrosis gene signature score, expression levels of Cdc6, Cxcl12 and Fap were measured. At the single gene level, Fap was down-regulated by 27.1% and the p-value was adjusted by limma by 0.0107 (this adjustment is the overall gene measured). Cdc6 and Cxcl12 did not change significantly (fig. 9). In this comparison, Ctgf (the major driver of fibrosis) was also down-regulated by 34.4%, and p is 0.00350.

To further assess the effects of anti-PD-L1/TGF β trap and radiation therapy on EMT and fibrosis, the expression of individual genes associated with fibroblasts and EMT was quantified.

Expression of smooth muscle alpha actin (ACTA2) is restricted to smooth muscle cells, pericytes and myofibroblasts, and is important in myofibroblast function. BALB/c mice were inoculated intramuscularly (i.m.) at 0.5X 10⁵4T1 cells (day-6) were treated with isotype control (400 μ g, intravenous; days 0, 2, 4) + vehicle control (0.2mL, oral, twice daily (q.d.), day 0-6), anti-PD-L1/TGF β trap (492 μ g, intravenous (i.v.); days 0, 2, 4), radiation (8Gy, days 0-3) or anti-PD-L1/TGF β trap + RT (n ═ 10 mice/group). Mice were sacrificed on day 6, tumors were collected and processed to extract RNA. RNAseq was performed using Qiaseq targeted RNA panels and feature scores were defined. Gene expression (log2 fold change) for Acta2, Ctgf and Fap in each treatment is shown in boxed lines. The dashed line spans from a minimum to a maximum.

ACTA2 expression was increased in vitro by TGF-. beta.1 treatment. Although radiotherapy alone had no significant effect on expression of ACTA2, anti-PD-L1/TGF β trap monotherapy and anti-PD-L1/TGF β trap combination radiotherapy significantly reduced ACTA2 expression in the 4T1 model (p <0.0001 and p ═ 0.0236, respectively) (fig. 14A).

Connective Tissue Growth Factor (CTGF) is a secreted protein that is shown to be the core mediator of tissue remodeling and fibrosis. It has even been shown that CTGF inhibition acts to reverse the fibrotic process, while monoclonal antibodies targeting CTGF significantly reduce radiation-induced pulmonary fibrosis in mouse models. The anti-PD-L1/TGF β trap significantly reduced CTGF expression relative to the isotype control (P0.0019), and as expected, the anti-PD-L1/TGF β trap combination significantly offset the effects of radiotherapy (P0.0024) compared to radiation monotherapy despite the radiation treatment increased CTGF (fig. 14B).

Fibroblast Activation Protein (FAP) is highly expressed by cancer-associated fibroblasts (CAF) in more than 90% of human epithelial cancers, where immunosuppression of CAF in TME can be promoted by STAT3 signaling. Relative to isotype controls, anti-PD-L1/TGF β well significantly reduced FAP expression (P <0.0001), and the reduction in FAP observed with radiotherapy (P0.0054) was further reduced by the combination of anti-PD-L1/TGF β well with radiation (fig. 14C).

anti-PD-L1/TGF beta trap treatment reduces alpha-SMA expression in mouse tumors

Increased TGF- β activity induces expression of the CAF marker, smooth muscle actin (α -SMA), which may lead to drug resistance and become a target for Immunotherapy (Calon et al, (2014), Semin. cancer biol.25: 15-22; Kakarla et al, (2012), Immunotherapy,4(11): 1129-. To evaluate the effect of anti-PD-L1/TGF β trap and radiotherapy on α -SMA expression, α -SMA IHC was performed in 4T1 tumor sections. BALB/c mice were inoculated intramuscularly (i.m.) at 0.5X 10⁵4T1 cells (day-6) were treated with isotype control (400 μ g, intravenous; day 0, 2, 4) + vehicle control (0.2mL, oral, twice daily (q.d.), day 0-6), anti-PD-L1/TGF β trap (492 μ g, intravenous; day 0, 2, 4), radiation (8Gy, day 0-3) or anti-PD-L1/TGF β trap + radiation (n ═ 10 mice/group). In the boxplot shown in fig. 15, the number of α SMA + pixels normalized to the region of interest (ROI) was determined and normalized for multiple ROIs per tumor for quantitative considerations; each symbol represents the positive pixel proportion of a single tumor. P values were determined by one-way analysis of variance. Scale bar, 250 μm.

Representative images of anti-a-SMA IHC are shown (fig. 16A-16D). anti-PD-L1/TGF β trap treatment significantly reduced α -SMA expression (p <0.0001) relative to isotype control (fig. 16A), while radiotherapy significantly increased α -SMA expression (p ═ 0.0002) (fig. 16C). The combination of anti-PD-L1/TGF β trap with radiotherapy significantly reduced α -SMA expression (p ═ 0.0001) relative to single radiotherapy (fig. 16D), suggesting that anti-PD-L1/TGF β trap can reduce radiation-induced CAF activity.

Example 3: anti-PD-L1/TGF β trap dosing with concomitant chemotherapy and radiation therapy (cCRT) for untreated group of stage III NSCLC patients-study design 1

Untreated stage III non-small cell lung cancer (NSCLC) patients were treated with anti-PD-L1/TGF β trap in combination with crts, followed by consolidation therapy with anti-PD-L1/TGF β trap (group 1) and compared to patients enrolled in crts, followed by consolidation therapy with anti-PD-L1/TGF β trap (group 2), and patients receiving crts followed by therapy with durvalumab (group 3). In an exemplary embodiment, crts are administered concurrently in the form of cisplatin/etoposide, cisplatin/pemetrexed or carboplatin/paclitaxel with a total radiation dose of 60-66Gy (e.g., 60Gy) delivered by intensity modulated radiation therapy. The chemotherapy regimen is a grading factor.

In an exemplary embodiment, an anti-PD-L1/TGF β trap is administered biweekly to a cancer patient with stage III non-small cell lung cancer (NSCLC) at a BW independent dose of 1200 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In an exemplary embodiment, an anti-PD-L1/TGF β trap is administered to a cancer patient with stage III non-small cell lung cancer (NSCLC) once every three weeks at a BW independent dose of 1800 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In an exemplary embodiment, an anti-PD-L1/TGF β trap is administered to a cancer patient with stage III non-small cell lung cancer (NSCLC) once every three weeks at a BW independent dose of 2400 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In one or more exemplary embodiments, to reduce potential infusion-related reactions, a predose of antihistamine and paracetamol (acetaminophen) (e.g., 25-50mg diphenhydramine and 500-650mg paracetamol [ acetaminophen ] intravenous or oral equivalent) is administered about 30 to 60 minutes prior to each dose of anti-PD-L1/TGF beta trap molecule for the first 2 infusions. If a grade 2 infusion response was seen during the first two infusions, no pre-medication was stopped. Steroids are prohibited for pre-medication.

The inclusion criteria for the patients in this example are as follows. The patients:

- ≧ 18 years old (with informed consent)

Histologically or cytologically, locally advanced unresectable (stage III) NSCLC

At least 3 weeks from the previous thoracotomy (if performed)

Since the diagnosis of stage III NSCLC, it has not been treated by previous systemic therapies, nor has it received any antibodies or drugs targeting T-cell co-regulatory proteins (immune checkpoints), such as anti-PDL 1 or anti-CTLA-4 antibodies

Life expectancy of at least 12 weeks (based on doctor's assessment of prognosis of patient after diagnosis)

Sufficient tumor material (<6 months old) to perform biomarker analysis

-east cooperative tumor cooperative group performance status (ECOG PS) of 0 to 1

Adequate lung function, defined as the expiratory volume in 1 second measured three weeks before the randomized block (FEV)₁) Not less than 1.2 liters or not less than 50% of the expected normal volume

Sufficient hematological function, defined as Absolute Neutrophil Count (ANC) ≥ 1.5X 10⁹/L, platelet count ≥ 100X 10⁹(ii)/L, hemoglobin (Hgb) is not less than 9g/dL

-having sufficient liver function, defined as total bilirubin levels < 1.5x upper normal limit (ULN), aspartate Aminotransferase (AST) levels < 3.0 x ULN, alanine Aminotransferase (ALT) levels < 3.0 x ULN and alkaline phosphatase < 2.5 ULN.

For participants with Cr >1.5 XULN, adequate renal function, defined by creatine ≦ 1.5 XULN or calculated creatinine clearance (CrCl) ≧ 50 mL/min (GFR may also be used)

Sufficient clotting function, defined as the International Normalized Ratio (INR) or Prothrombin Time (PT). ltoreq.1.5 × ULN, unless the test subject is receiving anticoagulant therapy; and activated partial thromboplastin time (aPTT) is less than or equal to 1.5 × ULN unless the test subject is receiving anticoagulant therapy.

Example 4: anti-PD-L1/TGF β trap dosing with concomitant chemotherapy and radiation therapy (cCRT) -study design 2 for untreated group of stage III NSCLC patients

Untreated stage III non-small cell lung cancer (NSCLC) patients were treated with anti-PD-L1/TGF β trap in combination with crts, followed by consolidation treatment with anti-PD-L1/TGF β trap (group 1), and compared with patients receiving 10mg/kg of dutvacizumab every two weeks in combination with crts, followed by 10mg/kg of dutvacizumab every two weeks (group 2), and patients receiving crts alone followed by placebo treatment (group 3). In an exemplary embodiment, crts are administered concurrently in the form of cisplatin/etoposide, cisplatin/pemetrexed or carboplatin/paclitaxel with a total radiation dose of 60-66Gy (e.g., 60Gy) delivered by intensity modulated radiation therapy. The chemotherapy regimen is a grading factor.

In an exemplary embodiment, a 1200mg BW independent dose is administered biweekly to a cancer patient with stage III non-small cell lung cancer (NSCLC). Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In an exemplary embodiment, an anti-PD-L1/TGF β trap is administered to a cancer patient with stage III non-small cell lung cancer (NSCLC) once every three weeks at a BW independent dose of 1800 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In an exemplary embodiment, an anti-PD-L1/TGF β trap is administered to a cancer patient with stage III non-small cell lung cancer (NSCLC) once every three weeks at a BW independent dose of 2400 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In one or more exemplary embodiments, to reduce potential infusion-related reactions, a predose of antihistamine and paracetamol (acetaminophen) (e.g., 25-50mg diphenhydramine and 500-650mg paracetamol [ acetaminophen ] intravenous or oral equivalent) is administered about 30 to 60 minutes prior to each dose of anti-PD-L1/TGF beta trap molecule for the first 2 infusions. If a grade 2 infusion response was seen during the first two infusions, no pre-medication was stopped. Steroids are prohibited for pre-medication.

The inclusion criteria for example 4 are similar to example 2, but can be adjusted at the discretion of the investigator.

Example 5: therapeutic efficacy of anti-PD-L1/TGF beta trap for treatment of stage III NSCLC patients

Progression-free survival (PFS) according to RECIST 1.1 was measured as the primary endpoint of participants receiving anti-PD-L1/TGF β trap in combination with crts as described in examples 3 and 4. The differences in efficacy between groups were studied for each example.

In an exemplary embodiment, during the crt-based induction period, 50mg/m is performed within 60 minutes or according to local standards on days 1,8, 29, 36²The dose of (a) cisplatin is administered intravenously. Etoposide is 50mg/m in 1-5, 29-33 days per day for at least 30 minutes to 60 minutes during crt-based induction²The dose of (a) is administered intravenously.

Standard prescription drugs consisting of H2 blocker, antiemetic, dexamethasone (oral or intravenous) were given according to local regulations. According to local practice, adequate hydration was ensured before and after treatment in cisplatin/etoposide-receiving participants.

In an exemplary embodiment, during the crt-based induction period, 45mg/m is used within 60 minutes or on day 1 of week according to local standards²The dose of (a) was given intravenously. At least 30 minutes prior to paclitaxel treatment, a standard prescription consisting of 25-50mg diphenhydramine, H2 blocker, and dexamethasone (oral or intravenous) is administered according to local standards.

At the discretion of the investigator, for participants who received the carboplatin/paclitaxel regimen and who failed to receive anti-PD-L1/TGF β trap or dovuzumab as a consolidated therapy, 2 additional cycles of carboplatin/paclitaxel (carboplatin AUC 6, paclitaxel 200 mg/m)²Q3W) as a consolidation therapy.

During crt-based induction, carboplatin was administered intravenously within 30 minutes on day 1 of the week according to local standards or based on AUC 2. Following paclitaxel administration, carboplatin will be administered concurrently with the standard antiemetic.

Treatment efficacy can also be measured with three other outcome determining factors. The effectiveness of treatment can be measured by the Objective Remission Rate (ORR), which is the "proportion of patients whose tumor size has been reduced by a predetermined amount and sustained for a minimum period of time" according to the U.S. food and drug administration's doctrine. See FDA 2007. According to the National Cancer Institute (NCI), complete remission is "response to treatment, with all signs of cancer gone". ORR is the first measure of therapeutic efficacy over CR. See Kogan and Haren (2008), Biotech. healthcare,5(1): 22-35. Another measure of treatment efficacy is Overall Survival (OS), i.e. the time from random grouping to plan evaluation, e.g. 57 months. Treatment efficacy may also be measured as duration of efficacy (assessed from CR or Partial Remission (PR) to disease Progression (PD), death or last tumor assessment), which is the time from random grouping to planned assessment, e.g., 57 months.

Chest/abdomen and pelvis contrast enhanced Computed Tomography (CT), which covers the area from above the chest entrance to the pubic symphysis, is the imaging modality of choice for assessing treatment efficacy. Tumor assessment before consolidation therapy should be performed as close as possible to before consolidation therapy starts and within 14 days after CRT-based induction ends. For patients recovering from toxicity associated with crt, the consolidation therapy start time is delayed up to 42 days from the end of crt. Participants were evaluated every 6 weeks for X-ray imaging to assess the response of participants to treatment within 15 months of first dosing and then every 12 weeks.

Other endpoints were studied to further establish therapeutic efficacy. For example, changes in tumor size are assessed by tumor volume analysis compared to baseline, and changes in tumor metabolic volume are measured by PET scanning. Changes in pulmonary fibrosis from baseline were measured by high resolution CT scan and lung function test. Potential predictive biomarkers of clinical response were assessed by examining the type and number of mutations (tumor mutation burden (TMB)) in plasma or tumor tissue and studying the correlation between TMB and clinical outcome.

anti-PD-L1/TGF β trap was considered to have initial clinical activity in untreated stage III NSCLC patients. Patients receiving treatment exhibit remission (e.g., partial remission, complete remission, stable disease) and/or improved survival (e.g., progression-free survival and/or overall survival). It is contemplated that treatment with anti-PD-L1/TGF β trap in combination with concomitant crts followed by anti-PD-L1/TGF β trap consolidation results in better survival of untreated stage III NSCLC patients compared to crts alone or patients treated with crts followed by placebo.

In summary, the anti-PD-L1/TGF β trap and the accompanying crt were found to be an innovative first-class bifunctional fusion protein aimed at targeting two immunosuppressive pathways simultaneously: PD-L1 and TGF- β, thereby treating stage III NSCLC while minimizing the development of fibrosis associated with concomitant radiotherapy and increasing the onset of metastasis and/or the time to distant metastasis of stage III NSCLC in a patient.

Example 6: anti-PD-L1/TGF β trap dosing with concomitant chemotherapy and radiation therapy (cCRT) for a group of advanced unresectable stage III NSCLC patients-study design 3

Advanced unresectable stage III non-small cell lung cancer (NSCLC) patients were treated with anti-PD-L1/TGF β trap in combination with crts (e.g., platinum-based chemotherapy), followed by consolidation therapy with anti-PD-L1/TGF β trap (group 1) and compared to patients treated with crts and placebo matched to anti-PD-L1/TGF β trap, followed by novarum (group 2). A schematic of the treatment protocol is depicted in fig. 17. In an exemplary embodiment, crts are administered concurrently in the form of cisplatin/etoposide, cisplatin/pemetrexed or carboplatin/paclitaxel with a total radiation dose of 60-66Gy (e.g., 60Gy) delivered by intensity modulated radiation therapy. Chemotherapy regimen and/or PD-L1 expression were the grading factors studied.

In an exemplary embodiment, anti-PD-L1/TGF β trap is administered biweekly to a cancer patient with stage III non-small cell lung cancer (NSCLC) at a BW independent dose of 1200 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In an exemplary embodiment, an anti-PD-L1/TGF β trap is administered to a cancer patient with stage III non-small cell lung cancer (NSCLC) once every three weeks at a BW independent dose of 1800 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In an exemplary embodiment, an anti-PD-L1/TGF β trap is administered to a cancer patient with stage III non-small cell lung cancer (NSCLC) once every three weeks at a BW independent dose of 2400 mg. Intravenous administration is for about 1 hour (-10 min/+ 20 min, e.g., 50 min to 80 min). In one or more exemplary embodiments, to reduce potential infusion-related reactions, a predose of antihistamine and paracetamol (acetaminophen) (e.g., 25-50mg diphenhydramine and 500-650mg paracetamol [ acetaminophen ] intravenous or oral equivalent) is administered about 30 to 60 minutes prior to each dose of anti-PD-L1/TGF beta trap molecule for the first 2 infusions. If a grade 2 infusion response was seen during the first two infusions, no pre-medication was stopped. Steroids are prohibited for pre-medication.

In an exemplary embodiment, patients with advanced unresectable stage III NSCLC infused 1200mg of anti-PD-L1/TGF β trap intravenously within 1 hour every two weeks until unacceptable toxicity occurred during crts, had confirmed disease progression and was as long as one year after crts. In an exemplary embodiment, 4 doses (e.g., 1200mg each) of anti-PD-L1/TGF β trap are administered during the induction period associated with crt. In one or more exemplary embodiments, 26 doses (e.g., 1200mg each) of anti-PD-L1/TGF β trap are administered during consolidation.

In an exemplary embodiment, etoposide is at 50mg/m²Or intravenously administered according to local standards on days 1-5 and days 29-33 for at least 30 minutes to 60 minutes per day during crts. In an exemplary embodiment, etoposide is at 500mg/m²Or intravenously according to local standards over 10 minutes on days 1, 22 and 43 during a crt. In an exemplary embodiment, carboplatin is administered intravenously over 30 minutes based on area under the curve (AUC)2 on days 1,8, 15, 22, 29, 36, and 43 during crts. In an exemplary embodiment, cisplatin is at 45mg/m²Or intravenously administered according to local standards on days 1,8, 15, 22, 29, 36, and 43 during crts over 60 minutes. At least 30 minutes prior to paclitaxel treatment, a standard prescription consisting of 25-50mg diphenhydramine, H2 blocker, and dexamethasone (oral or intravenous) is administered according to local standards.

At one isIn an exemplary embodiment, during crts-based induction, 75mg/m is performed within 60 minutes or on days 1, 22, 43 according to local standards²The dose of (a) cisplatin is administered intravenously. Etoposide 500mg/m²Or intravenously according to local standards over 10 minutes on days 1, 22 and 43 during a crt.

In group 2, patients with advanced unresectable stage III NSCLC were infused intravenously every 2 weeks with placebo matched to anti-PD-L1/TGF β trap until acceptable toxicity during crts, confirming disease progression. Dolvauzumab was administered at a dose of 10mg/kg once every two weeks over a period of 1 hour until acceptable toxicity during cCRT, confirming disease progression and up to 1 year post-cCRT. In one or more exemplary embodiments, 26 doses (e.g., 10mg/kg each) of dulvacizumab are administered during consolidation.

In an exemplary embodiment, to alleviate potential infusion-related reactions, a predose of antihistamine and paracetamol (acetaminophen) (e.g., 25-50mg diphenhydramine and 500-650mg paracetamol [ acetaminophen ] intravenous or oral equivalent) is administered about 30 to 60 minutes prior to administration of each dose of anti-PD-L1/TGF β trap molecule for the first 2 infusions. If a grade 2 infusion response was seen during the first two infusions, no pre-medication was stopped. Steroids are prohibited for pre-medication.

In one exemplary embodiment, a standard prescription consisting of an H2 blocker, an antiemetic, dexamethasone (oral or intravenous) is administered, according to local regulations. According to local practice, adequate hydration was ensured before and after treatment in cisplatin/etoposide-receiving participants.

The inclusion criteria for the patients in this example are as follows. The patients:

- ≧ 18 years old (with informed consent)

Histologically confirmed NSCLC, manifested as locally advanced stage III, unresectable disease (International Association for Manual of Lung cancer staging in thoracic tumors)

Tumor-bearing patients without Epidermal Growth Factor (EGFR) sensitive (activating) mutations, without Anaplastic Lymphoma Kinase (ALK) translocations, without ROS1 rearrangements were included

Sufficient hematological function, defined as Absolute Neutrophil Count (ANC) ≥ 1.5X 10⁹/L, platelet count ≥ 100X 10⁹(ii)/L, and hemoglobin is not less than 9g/dL

For participants with Cr >1.5 × ULN, sufficient renal function, as defined by creatine ≦ 1.5 × ULN or calculated creatinine clearance (CrCl) ≧ 50 mL/min (GFR may also be used)

Use of contraceptives (male and female) in accordance with the regulations of the local contraceptive method

Eastern cooperative tumor cooperative group (ECOG PS) performance status 0-1

Patients may be excluded from the study by any prior systemic cytotoxic chemotherapy or any antibody or drug targeting T cell co-regulatory proteins directed against their NSCLC.

Example 7: therapeutic efficacy of treatment of advanced unresectable stage III NSCLC patients as described in example 6

Progression-free survival (PFS) according to RECIST 1.1 was measured as the primary endpoint of participants receiving anti-PD-L1/TGF β trap in combination with crts, followed by anti-PD-L1/TGF β trap treatment, as described in example 6. The differences in efficacy between groups were studied for each example.

Treatment efficacy may also be measured with additional outcome determinants. One measure of treatment efficacy is Overall Survival (OS), i.e., time from random grouping to planned assessment, e.g., 59 months. The best overall efficacy (BOR), which is recorded from the start of study treatment to disease progression/recurrence, can also be used to further determine treatment efficacy. Other measures of treatment efficacy are by assessing PD-L1 expression at baseline. Another secondary endpoint is safety. Other endpoints were studied to further establish therapeutic efficacy. For example, changes in tumor size are assessed by tumor volume analysis compared to baseline, and changes in tumor metabolic volume are measured by PET scanning. Changes in pulmonary fibrosis from baseline were measured by high resolution CT scan and lung function test.

Chest/abdomen and pelvis contrast enhanced Computed Tomography (CT), which covers the area from above the chest entrance to the pubic symphysis, is the imaging modality of choice for assessing treatment efficacy. Participants underwent radiographic examinations every 8 weeks to assess participants' response to study intervention as long as 24 months after first dose, unless disease progression or withdrawal from study was preceded by a first-onset. Follow-up scans were performed every 8-12 weeks until progression, initiation of new treatment or death.

Potential predictive biomarkers of clinical efficacy can be assessed by examining the type and number of mutations (tumor mutation burden (TMB)) in plasma or tumor tissue and studying the correlation between TMB and clinical outcome.

Other exploratory endpoints were studied to further establish therapeutic efficacy. For example, based on immune-related efficacy assessment criteria (irRECIST), circulating tumor dna (ctdna) levels, immune-related optimal overall efficacy (irBOR) and immune-related changes in progression-free survival (irPFS) in solid tumors.

anti-PD-L1/TGF β trap was considered to have initial clinical activity in patients with untreated advanced unresectable stage III NSCLC. Patients receiving treatment exhibit remission (e.g., partial remission, complete remission, stable disease) and/or improved survival (e.g., progression-free survival and/or overall survival). It is contemplated that treatment with anti-PD-L1/TGF β trap in combination with concomitant crts followed by anti-PD-L1/TGF β trap consolidation results in better survival of untreated stage III NSCLC patients compared to patients treated with crts and placebo matched to anti-PD-L1/TGF β trap followed by doxoruzumab.

In an exemplary embodiment, PD-L1 expression is determined by FDA approved testing (e.g., (tumor proportion score (TPS) or VENTANA PD-L1(SP263) assay)). In an exemplary embodiment, an anti-PD-L1 antibody is used to determine PD-L1 protein expression in formalin-fixed, paraffin-embedded tissues. In an exemplary embodiment, patients were recruited independent of PD-L1 expression and retrospectively graded for PD-L1 expression by SP263 assay. In one exemplary embodiment, PD-L1 data (retrospective and prospective) were considered sensitivity analyses in primary efficacy analyses (graded log rank test, PD-L1 graded Cox model, PD-L1 adjusted Cox model) for assessing therapeutic efficacy with respect to PFS and OS.

In an exemplary embodiment, a chemotherapeutic regimen (e.g., cisplatin/pemetrexed) is used as a staging factor in the study. In an exemplary embodiment, a patient diagnosed with stage III NSCLC (e.g., squamous or non-squamous) is treated by intravenous administration of cisplatin/etoposide or carboplatin/paclitaxel in combination with anti-PD-L1/TGF β trap, followed by treatment with anti-PD-L1/TGF β trap. In an exemplary embodiment, a stage III NSCLC patient diagnosed with non-squamous histology is treated by intravenous administration of cisplatin/etoposide in combination with anti-PD-L1/TGF β trap, followed by treatment with anti-PD-L1/TGF β trap.

In summary, it was found that the anti-PD-L1/TGF β trap and the accompanying crt are innovative first-class bifunctional fusion proteins aimed at targeting two immunosuppressive pathways simultaneously: PD-L1 and TGF- β, thereby treating stage III NSCLC while minimizing the development of fibrosis associated with concomitant radiotherapy and increasing the onset of metastasis and/or the time to distant metastasis of stage III NSCLC in a patient.

Sequence of

SEQ ID NO:1

Peptide sequence of secreted anti-PD-L1 lambda light chain

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:2

Secreted peptide sequence against the H chain of PDL1

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:3

Secreted anti-PDL 1/TGF beta trap H chain peptide sequence

SEQ ID NO:4

DNA sequence of anti-PD-L1 lambda light chain from translation initiation codon to translation termination codon (leader sequence before VL is signal peptide from urokinase plasminogen activator)

atgagggccctgctggctagactgctgctgtgcgtgctggtcgtgtccgacagcaagggcCAGTCCGCCCTGACCCAGCCTGCCTCCGTGTCTGGCTCCCCTGGCCAGTCCATCACCATCAGCTGCACCGGCACCTCCAGCGACGTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGTCCAACCGGCCCTCCGGCGTGTCCAACAGATTCTCCGGCTCCAAGTCCGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCAGAGGACGAGGCCGACTACTACTGCTCCTCCTACACCTCCTCCAGCACCAGAGTGTTCGGCACCGGCACAAAAGTGACCGTGCTGggccagcccaaggccaacccaaccgtgacactgttccccccatcctccgaggaactgcaggccaacaaggccaccctggtctgcctgatctcagatttctatccaggcgccgtgaccgtggcctggaaggctgatggctccccagtgaaggccggcgtggaaaccaccaagccctccaagcagtccaacaacaaatacgccgcctcctcctacctgtccctgacccccgagcagtggaagtcccaccggtcctacagctgccaggtcacacacgagggctccaccgtggaaaagaccgtcgcccccaccgagtgctcaTGA

SEQ ID NO:5

DNA sequence from translation initiation codon to translation termination codon (mVK SP leader: lowercase underlined; VH: uppercase; IgG1m3 containing the K to A mutation: lowercase; (G4S) x4-G (SEQ ID NO:11) linker: bold uppercase; TGF. beta. RII: bold underlined lowercase; two termination codons: bold underlined uppercase)

SEQ ID NO:6

Polypeptide sequence of secreted anti-PD-L1 (mut)/TGF beta trap lambda light chain with mutations A31G, D52E, R99Y

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:7

Polypeptide sequence of secreted anti-PD-L1 (mut)/TGF beta trap heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:8

Human TGF-. beta.RII isoform A precursor polypeptide (NCBI RefSeq accession No.: NP-001020018)

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

SEQ ID NO:9

Human TGF-. beta.RII isoform B precursor polypeptide (NCBI RefSeq accession No.: NP-003233)

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

SEQ ID NO:10

Human TGF-beta RII isoform B ectodomain polypeptides

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:11

(Gly₄Ser)₄Gly linker

GGGGSGGGGSGGGGSGGGGSG

SEQ ID NO:12

Polypeptide sequence of heavy chain variable region of secreted anti-PD-L1 antibody MPDL3289A

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

SEQ ID NO:13

Polypeptide sequence of light chain variable region of secreted anti-PD-L1 antibody MPDL3289A

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR

SEQ ID NO:14

Polypeptide sequence of YW243.55S70 heavy chain variable region of secreted anti-PD-L1 antibody

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA

SEQ ID NO:50

Truncated human TGF-beta-RII isoform B extracellular domain polypeptides

GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:51

Truncated human TGF-beta-RII isoform B extracellular domain polypeptides

VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:52

Truncated human TGF-beta-RII isoform B ectodomain polypeptides

VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:53

Truncated human TGF-beta-RII isoform B ectodomain polypeptides

LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:54

Mutant human TGF-beta RII isoform B ectodomain polypeptides

VTDNAGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:55

Polypeptide sequence of heavy chain variable region of anti-PD-L1 antibody

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGYISYTGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS

SEQ ID NO:56

Polypeptide sequence of anti-PD-L1 antibody light chain variable region

DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYTFGGGTKVEIK

SEQ ID NO:57

Polypeptide sequence of heavy chain variable region of anti-PD-L1 antibody

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

SEQ ID NO:58

Polypeptide sequence of anti-PD-L1 antibody light chain variable region

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

SEQ ID NO:59

Polypeptide sequence of anti-PD-L1 antibody heavy chain

SEQ ID NO:60

Polypeptide sequence of anti-PD-L1 antibody light chain

SEQ ID NO:61

Polypeptide sequence of anti-PD-L1 antibody heavy chain

SEQ ID NO:62

Polypeptide sequence of anti-PD-L1 antibody light chain

Is incorporated by reference

The entire disclosure of each patent document and scientific article referred to herein is incorporated by reference for all purposes.

Equivalent forms

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the disclosure described herein. The various structural elements and the various method steps described in the different embodiments may be arranged in any combination and all such variations are to be considered in the manner of this disclosure. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Sequence listing

<110> Merck patent Co., Ltd (MERCK PATENT GMBH)

<120> treatment of stage III NSCLC and remission of pathological conditions associated with the treatment

<130> EMD-010WO

<150> 62/684,385

<151> 2018-06-13

<150> 62/800,808

<151> 2019-02-04

<150> 62/855,170

<151> 2019-05-31

<160> 62

<170> PatentIn version 3.5

<210> 1

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 1

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 2

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 2

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 3

<211> 607

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 3

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val

465 470 475 480

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

485 490 495

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

500 505 510

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

515 520 525

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

530 535 540

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

545 550 555 560

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

565 570 575

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

580 585 590

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

595 600 605

<210> 4

<211> 711

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of polynucleotides

<400> 4

atgagggccc tgctggctag actgctgctg tgcgtgctgg tcgtgtccga cagcaagggc 60

cagtccgccc tgacccagcc tgcctccgtg tctggctccc ctggccagtc catcaccatc 120

agctgcaccg gcacctccag cgacgtgggc ggctacaact acgtgtcctg gtatcagcag 180

caccccggca aggcccccaa gctgatgatc tacgacgtgt ccaaccggcc ctccggcgtg 240

tccaacagat tctccggctc caagtccggc aacaccgcct ccctgaccat cagcggactg 300

caggcagagg acgaggccga ctactactgc tcctcctaca cctcctccag caccagagtg 360

ttcggcaccg gcacaaaagt gaccgtgctg ggccagccca aggccaaccc aaccgtgaca 420

ctgttccccc catcctccga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 480

tcagatttct atccaggcgc cgtgaccgtg gcctggaagg ctgatggctc cccagtgaag 540

gccggcgtgg aaaccaccaa gccctccaag cagtccaaca acaaatacgc cgcctcctcc 600

tacctgtccc tgacccccga gcagtggaag tcccaccggt cctacagctg ccaggtcaca 660

cacgagggct ccaccgtgga aaagaccgtc gcccccaccg agtgctcatg a 711

<210> 5

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of polynucleotides

<400> 5

atggaaacag acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg ctccacaggc 60

gaggtgcagc tgctggaatc cggcggagga ctggtgcagc ctggcggctc cctgagactg 120

tcttgcgccg cctccggctt caccttctcc agctacatca tgatgtgggt gcgacaggcc 180

cctggcaagg gcctggaatg ggtgtcctcc atctacccct ccggcggcat caccttctac 240

gccgacaccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 300

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc ccggatcaag 360

ctgggcaccg tgaccaccgt ggactactgg ggccagggca ccctggtgac agtgtcctcc 420

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 720

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1140

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc cccgggtgct ggcggcggag gaagcggagg aggtggcagc 1440

ggtggcggtg gctccggcgg aggtggctcc ggaatccctc cccacgtgca gaagtccgtg 1500

aacaacgaca tgatcgtgac cgacaacaac ggcgccgtga agttccctca gctgtgcaag 1560

ttctgcgacg tgaggttcag cacctgcgac aaccagaagt cctgcatgag caactgcagc 1620

atcacaagca tctgcgagaa gccccaggag gtgtgtgtgg ccgtgtggag gaagaacgac 1680

gaaaacatca ccctcgagac cgtgtgccat gaccccaagc tgccctacca cgacttcatc 1740

ctggaagacg ccgcctcccc caagtgcatc atgaaggaga agaagaagcc cggcgagacc 1800

ttcttcatgt gcagctgcag cagcgacgag tgcaatgaca acatcatctt tagcgaggag 1860

tacaacacca gcaaccccga ctgataa 1887

<210> 6

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 6

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 7

<211> 607

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 7

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr

20 25 30

Met Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val

465 470 475 480

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

485 490 495

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

500 505 510

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

515 520 525

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

530 535 540

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

545 550 555 560

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

565 570 575

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

580 585 590

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

595 600 605

<210> 8

<211> 592

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 8

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp

20 25 30

Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn

35 40 45

Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr

50 55 60

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

65 70 75 80

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

85 90 95

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

100 105 110

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

115 120 125

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

130 135 140

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

145 150 155 160

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

165 170 175

Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln Val

180 185 190

Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile

195 200 205

Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser Ser

210 215 220

Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu His

225 230 235 240

Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr Cys

245 250 255

Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu Asp

260 265 270

Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys Leu

275 280 285

Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile Phe

290 295 300

Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser

305 310 315 320

Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala Glu

325 330 335

Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala Phe

340 345 350

His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile Ser

355 360 365

Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile Ala

370 375 380

His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro Ile

385 390 395 400

Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu

405 410 415

Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr

420 425 430

Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg

435 440 445

Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val

450 455 460

Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu Trp

465 470 475 480

Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr Glu

485 490 495

Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser Met

500 505 510

Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser Phe

515 520 525

Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu

530 535 540

Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala

545 550 555 560

Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser

565 570 575

Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr Lys

580 585 590

<210> 9

<211> 567

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 9

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val

20 25 30

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

35 40 45

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

50 55 60

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

65 70 75 80

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

85 90 95

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

100 105 110

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

115 120 125

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

130 135 140

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu

145 150 155 160

Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu

165 170 175

Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn

180 185 190

Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys

195 200 205

Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg

210 215 220

Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu

225 230 235 240

Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala

245 250 255

Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu

260 265 270

Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys

275 280 285

Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile

290 295 300

Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln

305 310 315 320

Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr

325 330 335

Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser

340 345 350

Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys

355 360 365

Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn

370 375 380

Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu

385 390 395 400

Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser

405 410 415

Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser

420 425 430

Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr

435 440 445

Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val

450 455 460

Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu

465 470 475 480

His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly

485 490 495

Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met

500 505 510

Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg

515 520 525

Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu

530 535 540

Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp

545 550 555 560

Gly Ser Leu Asn Thr Thr Lys

565

<210> 10

<211> 136

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 10

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 11

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 11

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly

<210> 12

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 13

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 14

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 15

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 15

Gln Phe Asn Ser

<210> 16

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 16

Gln Ala Gln Ser

<210> 17

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 17

Pro Lys Ser Cys Asp Lys

1 5

<210> 18

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 18

Pro Lys Ser Ser Asp Lys

1 5

<210> 19

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 19

Leu Ser Leu Ser

<210> 20

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 20

Ala Thr Ala Thr

<210> 21

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Lys, Arg, Thr, Gln, Gly, Ala, Trp, Met, Ile or Ser

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Val, Arg, Lys, Leu, Met or Ile

<220>

<221> MOD_RES

<222> (5)..(5)

<223> His, Thr, Asn, Gln, Ala, Val, Tyr, Trp, Phe or Met

<400> 21

Xaa Tyr Xaa Met Xaa

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Phe or Ile

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Ser or Thr

<400> 22

Ser Ile Tyr Pro Ser Gly Gly Xaa Thr Phe Tyr Ala Asp Xaa Val Lys

1 5 10 15

Gly

<210> 23

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Glu or Asp

<400> 23

Ile Lys Leu Gly Thr Val Thr Thr Val Xaa Tyr

1 5 10

<210> 24

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 24

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 25

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 25

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 26

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 26

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 27

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 27

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 28

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (4)..(4)

<223> Asn or Ser

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Thr, Arg or Ser

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Ala or Gly

<400> 28

Thr Gly Thr Xaa Xaa Asp Val Gly Xaa Tyr Asn Tyr Val Ser

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Glu or Asp

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Ile, Asn or Ser

<220>

<221> MOD_RES

<222> (4)..(4)

<223> Asp, His or Asn

<400> 29

Xaa Val Xaa Xaa Arg Pro Ser

1 5

<210> 30

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Phe or Tyr

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Asn or Ser

<220>

<221> MOD_RES

<222> (6)..(6)

<223> Arg, Thr or Ser

<220>

<221> MOD_RES

<222> (7)..(7)

<223> Gly or Ser

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Ile or Thr

<400> 30

Ser Ser Xaa Thr Xaa Xaa Xaa Xaa Arg Val

1 5 10

<210> 31

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 31

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys

<210> 32

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 32

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr

1 5 10 15

<210> 33

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 33

Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

1 5 10 15

Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

20 25 30

<210> 34

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 34

Phe Gly Thr Gly Thr Lys Val Thr Val Leu

1 5 10

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 35

Ser Tyr Ile Met Met

1 5

<210> 36

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 36

Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val Lys

1 5 10 15

Gly

<210> 37

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 37

Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr

1 5 10

<210> 38

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 38

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 39

Asp Val Ser Asn Arg Pro Ser

1 5

<210> 40

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 40

Ser Ser Tyr Thr Ser Ser Ser Thr Arg Val

1 5 10

<210> 41

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 41

Met Tyr Met Met Met

1 5

<210> 42

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 42

Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 43

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 43

Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr Asn Tyr Val Ser

1 5 10

<210> 44

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 44

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Trp Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 45

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 45

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 46

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 46

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr

20 25 30

Met Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Val Trp

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 47

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 48

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polynucleotides from human Fab libraries

<400> 48

atggagttgc ctgttaggct gttggtgctg atgttctgga ttcctgctag ctccagcgag 60

gtgcagctgc tggaatccgg cggaggactg gtgcagcctg gcggctccct gagactgtct 120

tgcgccgcct ccggcttcac cttctccagc tacatcatga tgtgggtgcg acaggcccct 180

ggcaagggcc tggaatgggt gtcctccatc tacccctccg gcggcatcac cttctacgcc 240

gacaccgtga agggccggtt caccatctcc cgggacaact ccaagaacac cctgtacctg 300

cagatgaact ccctgcgggc cgaggacacc gccgtgtact actgcgcccg gatcaagctg 360

ggcaccgtga ccaccgtgga ctactggggc cagggcaccc tggtgacagt gtcctccgcc 420

tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcacg ggatgagctg 1140

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260

gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1320

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380

aagagcctct ccctgtcccc gggtaaa 1407

<210> 49

<211> 705

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polynucleotides from human Fab libraries

<400> 49

atggagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cttaagccag 60

tccgccctga cccagcctgc ctccgtgtct ggctcccctg gccagtccat caccatcagc 120

tgcaccggca cctccagcga cgtgggcggc tacaactacg tgtcctggta tcagcagcac 180

cccggcaagg cccccaagct gatgatctac gacgtgtcca accggccctc cggcgtgtcc 240

aacagattct ccggctccaa gtccggcaac accgcctccc tgaccatcag cggactgcag 300

gcagaggacg aggccgacta ctactgctcc tcctacacct cctccagcac cagagtgttc 360

ggcaccggca caaaagtgac cgtgctgggc cagcccaagg ccaacccaac cgtgacactg 420

ttccccccat cctccgagga actgcaggcc aacaaggcca ccctggtctg cctgatctca 480

gatttctatc caggcgccgt gaccgtggcc tggaaggctg atggctcccc agtgaaggcc 540

ggcgtggaaa ccaccaagcc ctccaagcag tccaacaaca aatacgccgc ctcctcctac 600

ctgtccctga cccccgagca gtggaagtcc caccggtcct acagctgcca ggtcacacac 660

gagggctcca ccgtggaaaa gaccgtcgcc cccaccgagt gctca 705

<210> 50

<211> 117

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 50

Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe

1 5 10 15

Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr

20 25 30

Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys

35 40 45

Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu

50 55 60

Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile

65 70 75 80

Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys

85 90 95

Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn

100 105 110

Thr Ser Asn Pro Asp

115

<210> 51

<211> 115

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 51

Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr

1 5 10 15

Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile

20 25 30

Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp

35 40 45

Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr

50 55 60

His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys

65 70 75 80

Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser

85 90 95

Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser

100 105 110

Asn Pro Asp

115

<210> 52

<211> 122

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 52

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

1 5 10 15

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

20 25 30

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

35 40 45

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

50 55 60

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

65 70 75 80

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

85 90 95

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

100 105 110

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

115 120

<210> 53

<211> 110

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 53

Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys

1 5 10 15

Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln

20 25 30

Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu

35 40 45

Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu

50 55 60

Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro

65 70 75 80

Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp

85 90 95

Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

100 105 110

<210> 54

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 54

Val Thr Asp Asn Ala Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

1 5 10 15

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

20 25 30

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

35 40 45

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

50 55 60

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

65 70 75 80

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

85 90 95

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

100 105 110

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

115 120

<210> 55

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 55

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 56

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 56

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr His

20 25 30

Ser Asn Gln Lys His Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 57

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 57

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 58

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 58

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 59

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 59

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 60

<211> 220

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 60

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr His

20 25 30

Ser Asn Gln Lys His Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 61

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala

435 440 445

<210> 62

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 62

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

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