Activatable antibodies and methods of making and using the same

文档序号：620845 发布日期：2021-05-07 浏览：34次中文

阅读说明：本技术 可活化抗体及其制备和使用方法 (Activatable antibodies and methods of making and using the same ) 是由罗培志杜方勇于 2019-02-02 设计创作，主要内容包括：本文提供了含有编码可活化结合多肽的合成多核苷酸的文库。本文还提供了可活化结合多肽和含有所述可活化结合多肽的多肽文库。本文还提供了制备和使用可活化多肽文库的载体、载体文库、细胞、试剂盒和方法。(Provided herein are libraries containing synthetic polynucleotides encoding activatable binding polypeptides. Also provided herein are activatable binding polypeptides and polypeptide libraries containing the activatable binding polypeptides. Also provided herein are vectors, vector libraries, cells, kits and methods of making and using the activatable polypeptide libraries.)

1. A library comprising polynucleotides, wherein at least one of said polynucleotides encodes a polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

Wherein the FP comprises a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.

2. The library of claim 1, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten unique polypeptides, and each unique polypeptide comprises, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the FP comprises a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.

3. The library of claim 1 or claim 2, wherein each of the polynucleotides in the library encodes a polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.

4. The library of any one of claims 1-3, wherein the FP is formed from a peptide comprising a peptide according to formula (XIV): (NNK)_mTGY(NNK)_nTGY(NNK)_o(SEQ ID NO:87), wherein each N is independently A, G, T or C, wherein each K is independently T or G, and wherein each Y is independently T or C.

5. The library of any one of claims 1-4, wherein each X is not M, W or C.

6. The library of any one of claims 1-5, wherein X of formula (XIII) _mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

7. The library of any one of claims 1-6, wherein X of formula (XIII)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

8. The library of any one of claims 1-7, wherein X of formula (XIII)_oEach X in (a) is independently selected from the group consisting of D, A, Y,S, T, N, I, L, F, V, H and P.

9. The library of any one of claims 1-8, wherein m is 2 or 3-6.

10. The library of any one of claims 1-9, wherein n is 6-8.

11. The library of any one of claims 1-9, wherein n is 6.

12. The library of any one of claims 1-11, wherein o is 1-2.

13. The library of any one of claims 1-11, wherein o is 2.

14. The library of any one of claims 1-13, wherein the FP further comprises an additional amino acid sequence at its N-terminus.

15. The library of claim 14, wherein the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO 16.

16. The library of any one of claims 1-15, wherein the first cleavage site is a protease cleavage site of a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

17. The library of any one of claims 1-16, wherein the CM further comprises a first linker (L) C-terminal to the first cleavage site₁)。

18. The library of claim 17, wherein said L₁Comprising an amino acid sequence selected from the group consisting of SEQ ID NO 17-24.

19. The library of any one of claims 1-18, wherein the CM further comprises a second cleavage site.

20. The library of claim 19, wherein the second cleavage site is at the L₁The C-terminal of (1).

21. The library of claim 19 or claim 20, wherein the second cleavage site is a protease cleavage site of a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

22. The library of any one of claims 19-21, wherein the first cleavage site and the second cleavage site are different.

23. The library of any one of claims 19-22, wherein the CM further comprises a second linker (L) at the C-terminus of the second cleavage site ₂)。

24. The library of claim 23, wherein said L₂Comprising an amino acid sequence selected from the group consisting of SEQ ID NO 17-24.

25. The library of any one of claims 1-24, wherein the polypeptides comprise a polypeptide according to formula (III): EVGSYX₁X₂X₃X₄X₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CX₁₃X₁₄SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:3), wherein X₁Is A, D, I, N, P or Y, X₂Is A, F, N, S or V, X₃Is A, H, L, P, S, V or Y, X₄Is A, H, S or Y, X₅Is A, D, P, S, V or Y, X₆Is A, D, L, S or Y, X₇Is D, P or V, X₈Is A, D, H, P, S or T, X₉Is A, D, F, H, P or Y, X₁₀Is L, P or Y, X₁₁Is F, P or Y, X₁₂Is A, P, S or Y, X₁₃Is A, D, N, S, T or Y, and X₁₄Is A, S or Y.

26. The library of claim 25, wherein each of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence according to formula (III).

27. The library of any one of claims 1-26, wherein the polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID NOs 25-46.

28. The library of any one of claims 1-27, wherein the TBM comprises an antibody light chain variable region.

29. The library of claim 28, wherein the polypeptides further comprise a heavy chain variable region at the C-terminus of the light chain variable region.

30. The library of claim 28, further comprising polynucleotides encoding one or more antibody heavy chain variable regions.

31. The library of any one of claims 1-27, wherein the TBM comprises an antibody heavy chain variable region.

32. The library of claim 31, wherein the polypeptides further comprise a light chain variable region at the C-terminus of the heavy chain variable region.

33. The library of claim 31, further comprising polynucleotides encoding one or more antibody light chain variable regions.

34. The library of any one of claims 1-33, wherein at least one of the polynucleotides encoding the polypeptide is in a vector.

35. The library of claim 34, wherein the vector is an expression vector or a display vector.

36. The library of any one of claims 1-35, wherein at least one of the polynucleotides encoding the polypeptide is in a cell.

37. The library of claim 36, wherein the cells are bacterial cells, yeast cells, insect cells, or mammalian cells.

38. A method of producing an activatable antibody, the method comprising culturing the cell of claim 36 or claim 37 under conditions suitable for production of the activatable antibody.

39. The method of claim 38, further comprising recovering the activatable antibody produced by the cell.

40. The method of claim 39, further comprising testing the activatable antibody for the ability to maintain an activatable phenotype while soluble.

41. A method of screening for activatable antibodies that bind a target using the library of any one of claims 1-37, the method comprising:

a) contacting expression products of the library with the target prior to cleaving the CM;

b) contacting the expression products of the library with the target after cleaving the CM; and

c) isolating one or more of the expression products that bind to the target after cleaving the CM but have reduced binding to the target prior to cleaving the CM.

42. The method of claim 41, wherein an expression product is isolated if its binding affinity after cleaving the CM is at least 2-fold greater than its binding affinity prior to cleaving the CM.

43. The method of claim 41 or 42, wherein the CM comprises at least a first protease cleavage site, the first protease cleavage site being a cleavage site for a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

44. The method of any one of claims 41-43, wherein the target is CTLA4 or CD 137.

45. A polypeptide encoded by one or more polynucleotides from the library of any one of claims 1-37.

46. A kit comprising the library of any one of claims 1-37.

47. A library comprising antigen binding domains, wherein at least one of the antigen binding domains comprises a polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.

48. The library of claim 47, wherein at least two, at least three, at least four, at least five, or at least ten of the antigen binding domains comprise unique polypeptides comprising from N-terminus to C-terminus a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.

49. The library of claim 47 or claim 48, wherein each of the antigen binding domains comprises a unique polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.

50. The library of any one of claims 47-49, wherein the TBM comprises an antibody light chain variable region and the antigen binding domain further comprises an antibody heavy chain variable region.

51. The library of any one of claims 47-49, wherein the TBM comprises an antibody heavy chain variable region and the antigen binding domain further comprises an antibody light chain variable region.

52. The library of any one of claims 47-51, wherein each X is not M, W or C.

53. The library of any one of claims 47-52, X in formula (XIII)_mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

54. The library of any one of claims 47-53, wherein X of formula (XIII)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

55. The library of any one of claims 47-54, wherein X of formula (XIII)_oEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

56. The library of any one of claims 47-55, wherein each of the antigen binding domains is displayed on a cell surface or on a phage.

57. An antibody light chain comprising a polypeptide comprising from N-terminus to C-terminus a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

Wherein the FP comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P;

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region.

58. An antibody comprising a heavy chain and a light chain, wherein the light chain is the light chain of claim 57.

59. An antibody heavy chain comprising, from N-terminus to C-terminus, a polypeptide comprising a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the FP comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1) wherein m is 2 to 10N is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P;

Wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody heavy chain variable region.

60. An antibody comprising a heavy chain and a light chain, wherein the heavy chain is the heavy chain of claim 59.

61. A cell comprising at least one polypeptide displayed on its surface, wherein said at least polypeptide comprises, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the FP comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P;

wherein the CM comprises at least a first cleavage site; and is

Wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.

62. The cell of claim 61, wherein the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

63. The cell of claim 61 or claim 62, wherein each X is not M, W or C.

64. The method of any one of claims 61-63The cell of formula (I), wherein X of formula (I)_mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

65. The cell of any one of claims 61-64, wherein X of formula (I)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

66. An activatable antibody, comprising:

a first polypeptide comprising, from N-terminus to C-terminus, a Masking Moiety (MM), a Cleavable Moiety (CM), and a target-binding moiety (TBM),

wherein the MM comprises a compound according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the MM inhibits binding of the activatable antibody to human CD137 when the CM is not cleaved;

wherein the CM comprises at least a first cleavage site; and is

Wherein:

a) the TBM comprises an antibody light chain variable region (VL) and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH);

b) the TBM comprises an antibody heavy chain variable region (VH), and the activatable antibody further comprises a second polypeptide comprising an antibody light chain variable region (VL);

c) the TBM comprises, from N-terminus to C-terminus, an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); or

d) The TBM comprises, from N-terminus to C-terminus, an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL); and is

Wherein said activatable antibody binds human CD137 through said VH and said VL when said CM is cleaved.

67. The activatable antibody of claim 66, wherein the TBM comprises an antibody light chain variable region (VL) and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH).

68. The activatable antibody of claim 66 or claim 67, wherein m is 2 or 3-6.

69. The activatable antibody of any one of claims 66-68, wherein n is 6-8.

70. The activatable antibody of claim 69, wherein n is 6.

71. The activatable antibody of any one of claims 66-69, wherein o is 1 or 2.

72. The activatable antibody of any one of claims 66-71, wherein each X is not M, W or C.

73. The activatable antibody of any one of claims 66 to 72, wherein X of formula (I)_mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

74. The activatable antibody of any one of claims 66 to 73, wherein X of formula (I)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

75. The activatable antibody of any one of claims 66-74, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: SEQ ID NO 79-85 and X_mCADAPNHCXX(SEQ ID NO:88)、X_mCHHSPANCXX(SEQ ID NO:89)、X_mCPILRHRCXX(SEQ ID NO:90)、X_mCKWRPSRCXX(SEQ ID NO:91)、X_mCRVLPRRCXX(SEQ ID NO:92)、X_mCLWRHRSCXX (SEQ ID NO:93) and X_mCPRLRRKCXX (SEQ ID NO:94), wherein m is 2-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

76. The activatable antibody of any one of claims 66-75, wherein the MM further comprises an additional amino acid sequence at its N-terminus.

77. The activatable antibody of claim 76, wherein the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO 16.

78. The activatable antibody of any one of claims 66-77, wherein the first cleavage site is a protease cleavage site for a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

79. The activatable antibody of any one of claims 66-78, wherein the CM further comprises a first linker (L) C-terminal to the first cleavage site₁)。

80. The activatable antibody of claim 79, wherein the L ₁Comprises a compound selected fromAmino acid sequence of the group consisting of SEQ ID NO 17-24.

81. The activatable antibody of any one of claims 66-80, wherein the CM further comprises a second cleavage site.

82. The activatable antibody of claim 81, wherein the second cleavage site is at the L₁The C-terminal of (1).

83. The activatable antibody of claim 81 or claim 82, wherein the second cleavage site is a protease cleavage site for a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

84. The activatable antibody of any one of claims 81-83, wherein the first cleavage site and the second cleavage site are different.

85. The activatable antibody of any one of claims 81-84, wherein the CM further comprises a second linker (L) at the C-terminus of the second cleavage site₂)。

86. The activatable antibody of claim 85, wherein the L₂Comprising an amino acid sequence selected from the group consisting of SEQ ID NO 17-24.

87. The activatable antibody of any one of claims 66-86, wherein the CM comprises at least a first protease cleavage site and is cleaved with one or more proteases selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

88. The activatable antibody of any one of claims 66-87, wherein the activatable antibody comprises an antibody according to formula (III): EVGSYX ₁X₂X₃X₄X₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CX₁₃X₁₄SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:3), wherein X₁Is A, D, I, N, P or Y, X₂Is A, F, N, S or V, X₃Is A, H, L, P, S, V or Y, X₄Is A, H, S or Y, X₅Is A, D, P, S, V or Y, X₆Is A, D, L, S or Y, X₇Is D, P or V, X₈Is A, D, H, P, S or T, X₉Is A, D, F, H, P or Y, X₁₀Is L, P or Y, X₁₁Is F, P or Y, X₁₂Is A, P, S or Y, X₁₃Is A, D, N, S, T or Y, and X₁₄Is A, S or Y.

89. The activatable antibody of any one of claims 66-88, wherein the activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 40-46.

90. The activatable antibody of any one of claims 66-89, wherein the VL comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO 68, HVR-L2 comprising the amino acid sequence of SEQ ID NO 69, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 70.

91. The activatable antibody as in any one of claims 66-90, wherein the VL comprises the amino acid sequence of SEQ ID NO 50.

92. The activatable antibody of any one of claims 66-91, wherein the VH comprises HVR-H1 comprising the amino acid sequence of SEQ ID No. 65, HVR-H2 comprising the amino acid sequence of SEQ ID No. 66, and HVR-H3 comprising the amino acid sequence of SEQ ID No. 67.

93. The activatable antibody as in any one of claims 66-92, wherein the VH comprises the amino acid sequence of SEQ ID NO 49.

94. A method of treating or delaying progression of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a polypeptide encoded by one or more polynucleotides from the library of any one of claims 1-37, or the activatable antibody of any one of claims 66-93.

95. The method of claim 94, further comprising administering to the subject an effective amount of at least one additional therapeutic agent.

96. The method of claim 95, wherein the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, and chemotherapy.

97. The method of claim 95 or claim 96, wherein the at least one additional therapeutic agent is selected from the group consisting of: pomalyst, rilimid, lenalidomide, pomalidomide, thalidomide, DNA-alkylated platinum-containing derivative cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD 137 antibody, anti-CTLA 4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD 20 antibody, anti-CD 40 antibody, anti-DR 5 antibody, anti-CD 1d antibody, anti-TIM 3 antibody, anti-SLAMF 7 antibody, anti-KIR receptor antibody, anti-OX 40 antibody, anti-HER 2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pertuzumab, radiotherapy, single dose radiation, fractionated radiation CSF, focal radiation, total organ radiation, IL-12, IFN α, GM-CSF, chimeric antigen receptor, inherited transferred T cells, anti-cancer vaccine, and oncolytic virus.

Technical Field

The present disclosure relates to polynucleotide and polynucleotide libraries useful for screening and/or identifying one or more precision/situation-dependent activatable binding polypeptides (e.g., activatable antibodies), and polypeptide libraries useful for screening and/or identifying precision/situation-dependent activatable binding polypeptides (e.g., activatable antibodies capable of binding CTLA4 or CD137 when in active form), cells, methods, and kits related thereto.

Background

The activatable binding polypeptide exhibits an "activatable" conformation such that the antigen binding portion contained in the activatable binding polypeptide is less prone to bind its target when uncleaved than after cleavage in the presence of one or more specific proteases. Thus, these activatable binding polypeptides provide antigen-specific binding proteins that are only capable of binding their target under certain circumstances (e.g., in a protease-rich tumor microenvironment). Although many interesting activatable binding polypeptides have been developed, the process of developing such proteins is slow, labor intensive and expensive. Accordingly, there is a need for improved methods and products that can be used to identify self-blocking peptides for activatable binding polypeptides.

All references cited herein, including patent applications, patent publications, non-patent documents, and UniProtKB/Swiss-Prot/GenBank accession numbers, are incorporated by reference in their entirety to the same extent as if each individual reference had been specifically and individually indicated to be incorporated by reference.

Disclosure of Invention

To meet the above and other needs, disclosed herein are, for example, libraries of polynucleotides useful for screening and/or identifying activatable binding polypeptides (i.e., activatable antibodies). The present disclosure is based, at least in part, on the discovery that the polypeptides described herein exhibit significantly improved masking efficiency prior to activation, thereby allowing for better design, screening and/or identification of activatable binding polypeptides (i.e., activatable antibodies) with superior therapeutic indices and safety profiles. The present disclosure is also based, at least in part, on the surprising discovery that polynucleotide libraries described herein can be successfully constructed and screened to identify activatable binding polypeptides (see example 1 and example 2 below). Disclosed herein are precise/situation-dependent activatable binding polypeptides that bind to human CTLA4 (see example 3) or human CD137 (see example 5) when in an active form rather than in an inactive form, i.e., they bind their target (when in an active form) only after cleavage of the Cleavable Moiety (CM) to remove the First Peptide (FP) (i.e., the Masking Moiety (MM) or the self-blocking peptide). The discovered First Peptides (FP) described herein (e.g., masking moieties) are capable of efficiently masking antibody activity and/or reducing or completely inhibiting antigen binding, while in some embodiments, being free of chemically labile residues methionine and/or tryptophan. Furthermore, activatable antibodies identified using the polynucleotide libraries described herein are as efficient as their parent antibodies in treating a variety of cancer types while having significantly reduced cytotoxicity even in susceptible animals (NOD mice, see example 4).

Accordingly, in one aspect, provided herein is a library comprising polynucleotides, wherein at least one of the polynucleotides encodes a polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM) and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, the polynucleotides in the library encodeAt least two, at least three, at least four, at least five, at least ten, at least 50, at least 100, at least 500, at least 1000 unique polypeptides, and each unique polypeptide comprises, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, each of the polynucleotides in the library encodes a polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): x _mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, the FP is formed from a composition comprising a compound according to formula (XIV): (NNK)_mTGY(NNK)_nTGY(NNK)_o(SEQ ID NO:87), wherein each N is independently A, G, T or C, wherein each K is independently T or G, and wherein each Y is independently T or C.

In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W or C. In some embodiments that may be combined with any of the preceding embodiments, X of formula (XIII)_mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In any of the previous embodimentsIn some embodiments of the combinations, X of formula (XIII)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, X of formula (XIII) _oEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, m is 6. In some embodiments that may be combined with any of the preceding embodiments, m is 2 to 5, e.g., 2, 3, 4, or 5. In some embodiments that may be combined with any of the preceding embodiments, n is 6 to 8. In some embodiments that may be combined with any of the preceding embodiments, n is 6. In some embodiments that may be combined with any of the preceding embodiments, o is 1-2. In some embodiments that may be combined with any of the preceding embodiments, o is 2. In some embodiments that may be combined with any of the preceding embodiments, the FP further comprises an additional amino acid sequence at its N-terminus. In some embodiments, the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO 16.

In some embodiments that may be combined with any of the preceding embodiments, the first cleavage site is a protease cleavage site of a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (Tobacco Etch Virus, TEV) protease, plasmin (Thrombin), Thrombin (Thrombin), factor X, PSA, PSMA, cathepsin D (cathepsin D), cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1 (Caspase-1), Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, caspase-12, caspase-13, caspase-14 and TACE. In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a first linker (L) at the C-terminus of the first cleavage site ₁). In some embodiments, L is₁Comprising an amino acid sequence selected from the group consisting of SEQ ID NO 17-24. In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a second cleavage site. In some embodiments, the second cleavage site is at L₁The C-terminal of (1). In some embodiments, the second cleavage site is a protease cleavage site of a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first cleavage site and the second cleavage site are different. In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a second linker (L) at the C-terminus of the second cleavage site ₂). In some embodiments, L is₂Comprising an amino acid sequence selected from the group consisting of SEQ ID NO 17-24. In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a linker at the N-terminus of the first cleavage site.

In some embodiments that may be combined with any of the preceding embodiments, the polypeptide encoded by one or more polynucleotides in the library comprises a polypeptide comprising a sequence according to formula (III): EVGSYX₁X₂X₃X₄X₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CX₁₃X₁₄SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:3), wherein X is a First Peptide (FP) and a Cleavable Moiety (CM) of the amino acid sequence₁Is A, D, I, N, P or Y, X₂Is A, F, N, S or V, X₃Is A, H, L, P, S, V or Y, X₄Is A, H, S or Y, X₅A, D, P,S, V or Y, X₆Is A, D, L, S or Y, X₇Is D, P or V, X₈Is A, D, H, P, S or T, X₉Is A, D, F, H, P or Y, X₁₀Is L, P or Y, X₁₁Is F, P or Y, X₁₂Is A, P, S or Y, X₁₃Is A, D, N, S, T or Y, and X₁₄Is A, S or Y. In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence according to formula (III). In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-46.

In some embodiments that may be combined with any of the preceding embodiments, the TBM comprises an antibody light chain variable region. In some embodiments, the TBM further comprises a heavy chain variable region at the C-terminus of the light chain variable region. In some embodiments, the library further comprises polynucleotides encoding one or more antibody heavy chain variable regions. In some embodiments, the heavy chain variable region and the light chain variable region form an antigen binding site capable of binding a target in the absence of a Masking Moiety (MM).

In some embodiments that may be combined with any of the preceding embodiments, the TBM comprises an antibody heavy chain variable region. In some embodiments, the TBM further comprises a light chain variable region at the C-terminus of the heavy chain variable region. In some embodiments, the library further comprises polynucleotides encoding one or more antibody light chain variable regions. In some embodiments, the heavy chain variable region and the light chain variable region form an antigen binding site capable of binding a target in the absence of a Masking Moiety (MM).

In some embodiments that may be combined with any of the preceding embodiments, at least one of the encoded polypeptides in the polynucleotide is in a vector. In some embodiments, the vector is an expression vector or a display vector. In some embodiments that may be combined with any of the preceding embodiments, at least one of the encoded polypeptides in the polynucleotide is in a cell. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

Other aspects of the disclosure relate to a method of producing an activatable antibody, the method comprising culturing any of the cells described herein under conditions suitable for production of the activatable antibody. In some embodiments, the method further comprises recovering the activatable antibody produced by the cell. In some embodiments, the method further comprises testing the ability of the activatable antibody to maintain an activatable phenotype while soluble.

Other aspects of the disclosure relate to a method of screening for an activatable antibody that binds to a target using any of the libraries described herein, the method comprising the steps of: a) contacting the expression products of the library with the target prior to cleaving the CM, b) contacting the expression products of the library with the target after cleaving the CM, and c) isolating one or more of the expression products that bind to the target after cleaving the CM, but not the target prior to cleaving the CM. Also provided herein is a method of screening for activatable antibodies that bind a target using any of the libraries described herein, the method comprising the steps of: a) contacting the expression products of the library with the target prior to cleaving the CM, b) contacting the expression products of the library with the target after cleaving the CM, and c) isolating one or more of the expression products that bind the target after cleaving the CM but have a reduced binding affinity for the target prior to cleaving the CM as compared to the binding affinity after cleaving the CM. In some embodiments, the K of a product is expressed if it is prior to cleaving the CM _DIs K of the expression product after cleavage of CM_DAt least 2-fold (e.g., at least 5-fold, at least 10-fold, at least 15-fold, or higher), then isolating the expression product. In some embodiments, the CM comprises at least a first protease cleavage site that is a cleavage site for a protease selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4Caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14 and TACE. In some embodiments, the target is CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, and/or B7-H4. In some embodiments, the target is CTLA4 or CD 137.

Other aspects of the disclosure relate to a polypeptide encoded by one or more polynucleotides of any of the libraries described herein, or a library having polypeptides encoded by one or more polynucleotides of any of the libraries described herein.

Other aspects of the disclosure relate to a kit comprising any of the libraries described herein.

Other aspects of the present disclosure relate to a library comprising antigen binding domains, wherein at least one of the antigen binding domains comprises a polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, at least two, at least three, at least four, at least five, at least ten, at least 50, at least 100, at least 1000 of the antigen binding domains comprise a unique polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): x _mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, IK, L, M, N, P, Q, R, S, T, V, W and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, each of the antigen binding domains comprises a unique polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, the TBM comprises an antibody light chain variable region and the antigen binding domain further comprises an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, the TBM comprises an antibody heavy chain variable region and the antigen binding domain further comprises an antibody light chain variable region. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W or C. In some embodiments that may be combined with any of the preceding embodiments, X in formula (XIII) _mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, X of formula (XIII)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, X of formula (XIII)_oEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, each of the antigen binding domains in the library is displayed on a phage or cell (e.g., a yeast cell).

The disclosure of whichOthers aspect relates to an antibody light chain comprising a polypeptide comprising from N-terminus to C-terminus a First Peptide (FP), a Cleavable Moiety (CM) and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region. Other aspects of the disclosure relate to an antibody comprising a heavy chain and a light chain, wherein the light chain is any antibody light chain described herein. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W or C. In some embodiments that may be combined with any of the preceding embodiments, X in formula (XIII) _mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

Other aspects of the present disclosure relate to an antibody heavy chain comprising a polypeptide comprising, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody heavy chain variable region. Other aspects of the disclosure relate to an antibody comprising a heavy chain and a light chain, wherein the heavy chain is any antibody heavy chain described herein. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W or C. In a manner similar to any of the previous embodimentsIn some embodiments, X in formula (XIII) _mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

Other aspects of the present disclosure relate to a cell comprising at least one polypeptide displayed on its surface, wherein the at least polypeptide comprises, from N-terminus to C-terminus, a First Peptide (FP), a Cleavable Moiety (CM) and a target-binding moiety (TBM), wherein the FP comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W or C. In some embodiments that may be combined with any of the preceding embodiments, X of formula (I) _mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, X of formula (I)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

Other aspects of the disclosure relate to an activatable antibody comprising: a polypeptide comprising, from N-terminus to C-terminus, a Masking Moiety (MM), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the MM comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,s, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein when the CM is not cleaved, the MM inhibits binding of the activatable antibody to human CTLA 4; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region (VL) and/or an antibody heavy chain variable region (VH); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 through the VH and the VL. In some embodiments, the TBM comprises a VL and the activatable antibody further comprises a VH. In some embodiments, the TBM comprises a VH, and the activatable antibody further comprises a VL. In some embodiments, the TBM comprises, from N-terminus to C-terminus, VH and VL or VL and VH. In some embodiments, the CM comprises at least a first protease cleavage site and is cleaved with one or more proteases selected from the group consisting of: urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W or C. In some embodiments that may be combined with any of the preceding embodiments, X of formula (I) _mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, X of formula (I)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 72-78. Can be combined with any oneIn some embodiments of the foregoing combination of embodiments, the VL comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:62, HVR-L2 comprising the amino acid sequence of SEQ ID NO:63, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments that may be combined with any of the preceding embodiments, the VL comprises the amino acid sequence of SEQ ID No. 48. In some embodiments that may be combined with any of the preceding embodiments, the VH comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:59, HVR-H2 comprising the amino acid sequence of SEQ ID NO:60, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments that may be combined with any of the preceding embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, m is 3 to 10.

Other aspects of the disclosure relate to an activatable antibody comprising: a polypeptide comprising, from N-terminus to C-terminus, a Masking Moiety (MM), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the MM comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the MM inhibits binding of the activatable antibody to human CD137 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region (VL) and/or an antibody heavy chain variable region (VH); and wherein when said CM is cleaved, said activatable antibody binds human CD137 via said VH and said VL. In some embodiments, the TBM comprises a VL and the activatable antibody further comprises a VH. In some embodiments, the TBM comprises a VH, and the activatable antibody further comprises a VL. In some embodiments, the TBM comprises, from N-terminus to C-terminus, VH and VL or VL and VH. In some embodiments, the CM comprises at least a first protease cleavage site and is cleaved with one or more proteases selected from the group consisting of: urokinase type fibre Prolysin activating factor (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W or C. In some embodiments that may be combined with any of the preceding embodiments, X of formula (I)_mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, X of formula (I)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments that may be combined with any of the preceding embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-85 and 88-94. In some embodiments that may be combined with any of the preceding embodiments, the VL comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:68, HVR-L2 comprising the amino acid sequence of SEQ ID NO:69, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments that may be combined with any of the preceding embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments that may be combined with any of the preceding embodiments, the VH comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:65, HVR-H2 comprising the amino acid sequence of SEQ ID NO:66, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments that may be combined with any of the preceding embodiments, the VH comprises the amino acid sequence of SEQ ID No. 49. In some embodiments, m is 3 to 10.

Other aspects of the disclosure relate to a polynucleotide encoding any of the activatable antibodies described herein. In other aspects, the disclosure relates to a vector comprising any of the polynucleotides described herein (e.g., polynucleotides encoding activatable antibodies). In some embodiments, the vector is an expression vector and/or a display vector. In other aspects, the disclosure relates to a host cell comprising any of the polynucleotides and/or vectors described herein (e.g., polynucleotides and/or vectors encoding activatable antibodies). In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell. In other aspects, the disclosure relates to a method of making an activatable antibody, the method comprising culturing any of the host cells described herein under conditions suitable for production of the antibody or activatable antibody. In some embodiments, the method further comprises recovering the antibody produced by the cell or the activatable antibody.

Other aspects of the present disclosure relate to a method of treating or delaying progression of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a polypeptide encoded by one or more polynucleotides from any of the libraries described herein and/or any activatable antibody described herein (e.g., an activatable antibody to human CTLA4 or an activatable antibody to human CD 137). In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, and chemotherapy. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of: pomalist (pomalyst), rilamed (revlimd), lenalidomide (lenalidomide), pomalidomide (pomalidomide), thalidomide (thalidomide), DNA alkylated platinum-containing derivative cisplatin (cispain), 5-fluorouracil (5-fluorouracil), cyclophosphamide (cyclophosphamide), anti-CD 137 antibody, anti-CTLA 4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD 20 antibody, anti-CD 40 antibody, anti-DR 5 antibody, anti-CD 1d antibody, anti-CD 3 antibody, anti-SLAMF 7 antibody, anti-KIR receptor antibody, anti-OX 40 antibody, anti-HER 2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab (cetuximab), rituximab (rituximab), stpertuzumab (CSF), rituximab (trastuzumab), IFN therapy (revolum), chimeric antigen receptor-mediated radiotherapy, IFN-alpha, radioisotopic-receptor (T), single-radiation therapy, radioisotopic-T receptor (T), chimeric antibody, radioisotopic-T-receptor (T), chimeric antibody, radioisotopic-12, radioisotopic (T-receptor, antibody, anti-cancer vaccines and oncolytic viruses.

It will be appreciated that one, some or all of the properties of the various embodiments described above and herein may be combined to form further embodiments of the present disclosure. These and other aspects of the disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description.

Drawings

Figure 1 shows a schematic of an exemplary selection process for self-blocking peptides using Fab fragments of the target antibody displayed on the yeast surface.

Figure 2 shows a schematic of an exemplary selection process for self-blocking peptides using scFv fragments of the target antibody displayed on the yeast surface.

Figures 3A-3B show functional display of CTLA 4-targeted fabs and scfvs on yeast as determined by flow cytometry. Figure 3A shows functional display of CTLA 4-targeted fabs on the surface of yeast. Figure 3B shows functional display of CTLA 4-targeted scFv on the surface of yeast.

Figure 4 shows an exemplary selection process for activatable antibodies targeting human CTLA 4. The yeast library displaying the fusion protein was subjected to several rounds of FACS-based screening.

Figures 5A-5B show that exemplary CTLA4 can activate CTLA4 binding affinity of antibody clones as determined by flow cytometry. Figure 5A shows that CTLA4 in scFv format can activate the binding affinity of the antibody clone, including the case where the CTLA4 can activate antibody clone B13287 with the masking peptide remaining intact, or the masking peptide is cleaved by TEV protease, compared to the scFv fragment of the target antibody without the masking peptide. Figure 5B shows that CTLA4 in Fab form can activate CTLA4 binding affinity of antibody clone, including the case where CTLA4 can activate the masking peptide of antibody clone B13189 remains intact, or the masking peptide is cleaved by TEV protease, compared to Fab fragments of the target antibody without the masking peptide.

Figure 6A-6B show that exemplary CTLA4 can activate the masking efficiency of antibodies TY22401, TY22403, TY22402 and TY22404 compared to the parent antibody TY 21580. Figure 6A shows the association and dissociation curves for the indicated activatable antibodies as compared to the parent antibody TY21580, as determined by the ForteBio system. Figure 6B shows a graph of the relative ratio of bound activatable antibody compared to the parent antibody TY 21580.

Figures 7A-7C show the masking efficiency of exemplary CTLA4 can activate antibodies against recombinant human CTLA4-Fc, as determined by ELISA. Figure 7A shows first ELISA data indicating that CTLA4 can activate binding of antibodies TY22401, TY22402, TY22403, TY22404 to recombinant human CTLA4-Fc compared to the parent antibody TY 21580. Figure 7B shows second batch ELISA data indicating that CTLA4 can activate binding of antibodies TY22401, TY22402, TY22403, TY22404 to recombinant human CTLA4-Fc compared to the parent antibody TY 21580. Figure 7C shows that CTLA4 can activate binding of antibodies TY22563, TY22564, TY22565, TY22566 to recombinant human CTLA4-Fc compared to the parent antibody TY 21580.

Figure 8A-figure 8B show that CTLA4 can activate the activity of antibody TY22404 after removal of masking peptide. FIG. 8A shows the SDS-PAGE results of activatable antibody TY22404 without treatment, treated with protease uPA or treated with 5 or 10 units of protease MMP-9. Figure 8B shows the binding of activatable antibody TY22404 without treatment, treated with protease uPA, or treated with protease MMP-9, as determined by ELISA, compared to the parent antibody TY 21580.

Figures 9A-9C show Size Exclusion Chromatography (SEC) profiles of exemplary activatable antibodies under accelerated stress conditions. Figure 9A shows the SEC profile of activatable antibody TY22402 after six cycles of freeze and thaw compared to control conditions. Figure 9B shows the SEC profile of activatable antibody TY22402 after seven days at 50 ℃ compared to control conditions. Fig. 9C shows the percentage of the SEC major peak area of an exemplary activatable antibody after seven days at 50 ℃, after up to 28 days of storage at 40 ℃, or after six freeze and thaw cycles, as compared to control conditions.

FIG. 10 shows the percentage of SEC main peak area of activatable antibodies TY22401 and TY22402 after storage at about 8mg/mL or >150 mg/mL.

Figure 11 shows the masking efficiency of untreated activatable antibodies TY21580, TY22401, TY22402 and TY22566 incubated at pH 3.7 for 30 minutes or at pH 3.7 for 1 hour, as determined by the ForteBio system.

Figure 12A-figure 12B show human Peripheral Blood Mononuclear Cell (PBMC) activation by isotype control antibody, parent antibody TY21580 or exemplary CTLA4 activatable antibodies TY22401, TY22402 or TY22404 as measured by ELISA. Figure 12A shows the effect on IL-2 secretion from CD3 sensitized human PBMCs stimulated with isotype control, parent antibody TY21580 and exemplary CTLA4 activatable antibodies TY22401, TY22402 or TY 22404. Figure 12B shows the effect of IFN γ secretion from CD3 sensitized human PBMCs stimulated with isotype control, parent antibody TY21580 and exemplary CTLA4 activatable antibodies TY22401, TY22402 or TY 22404.

Figure 13 shows antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isotype control antibody, parent antibody TY21580 or exemplary activatable antibodies TY22401, TY21580 or TY22404 on HEK293F cells transiently overexpressing human CTLA4 as determined by ADCC reporter gene assay.

Figure 14A-figure 14B show the in vivo anti-tumor efficacy of the parent antibody TY21580, isotype control antibody or exemplary CTLA4 activatable antibodies TY22401, TY22402 or TY22566 in the MC38 isogenic mouse colorectal tumor model. FIG. 14A shows tumor growth curves for different treatment groups of female C57BL/6 mice bearing tumors established via MC 38. Data points represent group means; error bars represent SEM. Figure 14B shows individual tumor growth curves for groups treated with TY21580, TY22401, TY22402 and TY 22566.

Figure 15 shows the in vivo anti-tumor efficacy of an isotype control antibody, the parent antibody TY21580, or one of the three activatable antibodies in a CT26 syngeneic mouse colorectal tumor model. Tumor growth curves are shown for different treatment groups of female C57BL/6 mice bearing tumors established by CT 26. Data points represent group means; error bars represent SEM.

Figure 16 shows the in vivo anti-tumor efficacy of an isotype control antibody, the parent antibody TY21580, or one of the three activatable antibodies in a H22 syngeneic mouse liver tumor model. Tumor growth curves are shown for different treatment groups of female C57BL/6 mice bearing H22 established tumors. Data points represent group means; error bars represent SEM.

Figure 17A-figure 17B show the in vivo anti-tumor efficacy of the parent antibody TY21580, isotype control antibody, and exemplary activatable antibodies TY22401, TY22402 or TY22566 in a 3LL syngeneic mouse lung tumor model. FIG. 17A shows tumor growth curves for different treatment groups of female C57BL/6 mice bearing 3LL established tumors. Data points represent group means; error bars represent SEM. Figure 17B shows individual tumor growth curves for groups treated with TY21580, TY22401, TY22402 and TY 22566.

Fig. 18A-18C show the time course of blood concentration of Test Article (TA) administered intravenously to female BALB/C mice at a concentration of 10mg/kg, as determined by ELISA. FIG. 18A shows the time course of the blood concentration of activatable antibody TY22401 administered intravenously to female BALB/c mice at a concentration of 10mg/kg compared to the parent antibody TY 21580. FIG. 18B shows the time course of the blood concentration of activatable antibody TY22402 administered intravenously to female BALB/c mice at a concentration of 10mg/kg compared to the parent antibody TY 21580. Figure 18C shows the time course of the blood concentration of activatable antibody TY22404 administered intravenously to female BALB/C mice at a concentration of 10mg/kg compared to the parent antibody TY 21580.

Figure 19 shows the repeated dose toxicity of isotype control antibody, parent antibody TY21580 and exemplary activatable antibodies TY22566, TY22401 and TY22402 using the NOD mouse model. The percent survival over 20 days for each treatment group is shown.

Fig. 20A-20B show functional display of Fab and scFv targeting human CD137 on yeast, as determined by flow cytometry. Figure 20A shows functional display of CD 137-targeted scFv on the surface of yeast. Figure 20B shows functional display of CD 137-targeted fabs on the surface of yeast.

Figure 21 shows an exemplary selection process for activatable antibodies targeting human CD 137. The yeast library displaying the fusion protein was subjected to several rounds of FACS-based screening.

Fig. 22A-22B show that exemplary CD137 can activate CD137 binding affinity of antibody clones, as determined by flow cytometry. Figure 22A shows that CD137 in scFv format can activate the binding affinity of the antibody clone, including the case where CD137 can activate the masking peptide of antibody clone B13428 remains intact, or the masking peptide is cleaved by TEV protease, compared to the scFv fragment of the target antibody without the masking peptide. Figure 22B shows that CD137 in scFv format can activate the CD137 binding affinity of the antibody clone, including the case where the CD137 can activate the masking peptide of antibody clone B13439 remains intact, or the masking peptide is cleaved by TEV protease, compared to the scFv fragment of the target antibody without the masking peptide.

Figure 23 shows the masking efficiency of an exemplary activatable antibody against human CD137 as determined by flow cytometry compared to the parent antibody TY 21242.

Fig. 24A and 24B depict the masking efficiency of exemplary activatable antibodies containing variable length masking peptides compared to the parent antibody TY 21580. Masking efficiency was determined using an ELISA-based method. Figures 24A and 24B represent two experimental setups using the same experimental method to test various activatable anti-CTLA 4 antibodies.

Figure 25 depicts the masking efficiency of exemplary activatable antibodies containing different lengths of cleaved peptide compared to the parent antibody TY 21580. Masking efficiency was determined using an ELISA-based method.

Detailed Description

I. General techniques

The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are generally employed by those skilled in the art using conventional methods, such as the widely utilized methods described in, for example: sambrook et al, Molecular Cloning, A Laboratory Manual 3 rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; current Protocols in Molecular Biology (ed. F.M. Ausubel et al, (2003)); the book Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)); harlow and Lane eds (1988) Antibodies, A Laboratory Manual and Animal Cell Culture (R.I. Freshney eds (1987)); oligonucleotide Synthesis (m.j. gait eds., 1984); methods in Molecular Biology, human Press; cell Biology A Laboratory Notebook (J.E.Cellis eds., 1998) Academic Press; animal Cell Culture (r.i. freshney eds, 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts,1998) Plenum Press; cell and Tissue Culture Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell eds., 1993-8) J.Wiley and Sons; handbook of Experimental Immunology (eds. d.m.weir and c.c.blackwell); gene Transfer Vectors for Mammalian Cells (eds. J.M.Miller and M.P.Calos, 1987); PCR The Polymerase Chain Reaction, (Mullis et al eds., 1994); current Protocols in Immunology (J.E. Coligan et al, 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies A Practical Approach (D.Catty. eds., IRL Press, 1988-; monoclonal Antibodies A Practical Approach (P.Shepherd and C.dean ed., Oxford University Press, 2000); use Antibodies A Laboratory Manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press,1999), The Antibodies (eds. M.Zantetti and J.D.Capra, Harwood Academic Publishers,1995), and Cancer: Principles and Practice of Oncology (V.T.Devita et al, J.B.Lippinco Company, 1993).

Definition of

Before the present disclosure is described in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule(s)" optionally includes a combination of two or more of the molecule(s), and so forth.

The term "about" as used herein refers to the usual range of error for the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments relating to that value or parameter per se.

It will be appreciated that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of … …," and "consisting essentially of … …" aspects and embodiments.

The term "and/or" as used herein in phrases such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used herein in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The term "amino acid" refers to naturally occurring and synthetic amino acids as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, but has a C-terminal carboxyl group, an N-terminal amino group, or a side chain functional group that has been chemically modified to another functional group. The term "amino acid mimetic" refers to a compound that differs in structure from the general chemical structure of an amino acid, but functions in a similar manner as a naturally occurring amino acid.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See, e.g., Immunology-A Synthesis (2 nd edition, ed.s.Golub and D.R.Gren eds., Sinauer Associates, Sunderland, Mass. (1991)).

The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

"polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interspersed with non-nucleotide components. The polynucleotide may comprise one or more modifications performed after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps"; replacing one or more of the naturally occurring nucleotides with an analog; internucleotide modifications such as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) as well as with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen (psoralen), etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.); and unmodified forms of one or more polynucleotides. Furthermore, any hydroxyl groups typically present in sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5 'and 3' terminal OH groups may be phosphorylated or substituted with an amine or organic end-capping group moiety having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to obtain standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose that are generally known in the art, including, for example, 2' -O-methyl-ribose; 2' -O-allyl-ribose; 2 '-fluoro-ribose or 2' -azido-ribose; carbocyclic sugar analogs; an alpha-anomeric sugar; epimeric sugars such as arabinose, xylose or lyxose; a pyranose; a furanose; sedoheptulose; acyclic analogs and abasic nucleoside analogs such as methyl nucleoside. One or more phosphodiester linkages may be replaced by an alternative linking group. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is replaced by p (O) S ("thioester"), p (S) S ("dithioate"), (O) NR2 ("amidate"), p (O) R, P (O) OR ', CO, OR CH2 ("methylal"), where each R OR R' is independently H OR a substituted OR unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, OR aralkyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, which is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes the nucleic acid molecule being separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences naturally flanking the nucleic acid, i.e., sequences located at the 5 'and 3' ends of the nucleic acid of interest.

As used herein, a "library" refers to a collection of two or more entities having a common class. For example, a library containing polynucleotides may refer to a collection of two or more polynucleotides. The term "library" is used herein in the broadest sense and specifically covers sub-libraries which may or may not be combined.

As used herein, "unique" means that a member of a collection is different from other members of the collection. For example, a unique activatable antibody in a library may refer to an activatable antibody having a particular sequence not shared by other activatable antibodies in the library. Indeed, it will be appreciated that "unique" members of a physically-implemented form of a library may be present in more than one copy. For example, a library may contain a plurality of "unique" activatable antibodies, where one or more of the "unique" activatable antibody molecules are present in more than one copy.

As used herein, "diversity" refers to diversification and/or heterogeneity. For example, the diversity of antibodies in a library can refer to the presence of multiple antibodies with unique sequences in the library.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., Fab '-SH, F (ab')₂Fv and/or single chain variable fragments or scFv) as long as they exhibit the desired biological activity.

In some embodiments, the term "antibody" refers to an antigen binding protein (i.e., an immunoglobulin) having a substantially four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each heavy chain has a variable region at the N-terminus (abbreviated herein as V)_H) Followed by a constant region. The heavy chain constant region comprises three domains, i.e.C_H1、C_H2And C_H3. Each light chain has a variable region at the N-terminus (abbreviated herein as V) _I) Followed by a constant region at its other end. The light chain constant region comprises a domain, i.e., C_L。V_LAnd V_HAlignment, and C_LAligned with the first constant domain of the heavy chain (CH 1). V_HAnd V_LPaired together to form a single antigen binding site. IgM antibodies consist of 5 elementary heterotetramer units and an additional polypeptide called J chain, thus containing 10 antigen binding sites, while secretory IgA antibodies can aggregate to form multivalent aggregates comprising 2-5 elementary 4 chain units and J chains.

V_HRegion and V_LRegions can be based on structure and sequenceThe analysis was further subdivided into regions of hypervariability, termed hypervariable regions (HVRs). HVRs alternate with more conserved regions known as framework regions (FW) (see, e.g., Chen et al (1999) J. mol. biol. (1999)293, 865-881). Each V_HAnd V_LConsisting of three HVRs and four FWs arranged in the following order from amino-terminus to carboxy-terminus: FW-1_ HVR-1_ FW-2_ HVR-2_ FW-3_ HVR-3_ FW 4. Throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Similarly, the three HVRs of the light chain are designated HVR-L1, HVR-L2, and HVR-L3.

The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids (see, e.g., Fundamental Immunology chapter 7 (Paul, w. eds., 2 nd edition Raven Press, n.y. (1989)).

L chains from any vertebrate species can be assigned to one of two distinct types, termed κ and λ, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of the heavy chain of an antibody, antibodies can be assigned to different classes or isotypes. There are five antibody classes: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α (alpha), δ (delta), epsilon (epsilon), γ (gamma) and μ (mu), respectively. The IgG antibody class can be further classified into four subclasses according to gamma heavy chain Y1-Y4, namely IgG1, IgG2, IgG3 and IgG4, respectively. .

The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to a portion or portions of an antibody that retain the ability to bind to the antigen to which the antibody binds. Examples of "antigen-binding fragments" of antibodies include (i) Fab fragments, i.e., composed of V_L、V_H、C_LAnd C_H1Monovalent fragments consisting of domains; (ii) f (ab')₂Fragments, i.e. comprising two Fab fragments linked by a disulfide bridge at the hinge regionA bivalent fragment of a segment; (iii) from V_HAnd C_H1Domain-forming Fd fragments; (iv) v from one arm of an antibody_LAnd V_H(iv) Fv fragments consisting of domains, (V) dAb fragments (Ward et al, Nature 341:544-546(1989)), consisting of V _HDomain composition; and (vi) isolating the Complementarity Determining Regions (CDRs).

The term "CTLA 4" is used herein and includes human CTLA4 (e.g., UniProt accession number P16410) as well as variants, subtypes and species homologs thereof (e.g., mouse CTLA4(UniProt accession number P09793), rat CTLA4(UniProt accession number Q9Z1a7), dog CTLA4(UniProt accession number Q9XSI1), cynomolgus CTLA4(UniProt accession number G7PL88), and the like). Thus, a binding molecule (e.g., an activatable antibody) may also bind CTLA4 from a species other than human. In other cases, the binding molecule may be completely specific for human CTLA4, and may not exhibit species cross-reactivity or other types of cross-reactivity.

The term "CD 137" is used herein and includes human CD137 (e.g., GenBank accession No. NM _ 001561; NP _001552) as well as variants, subtypes, and species homologs thereof (e.g., mouse CD137(GenBank gene identifier 21942), rat CD137(GenBank gene identifier 500590), dog CD137(GenBank gene identifier 608274), cynomolgus monkey CTLA4(GenBank gene identifier 102127961), and the like). Thus, a binding molecule (e.g., an activatable antibody) may also bind CD137 from a species other than human. In other cases, the binding molecule may be completely specific for human CD137, and may not exhibit species cross-reactivity or other types of cross-reactivity.

The term "chimeric antibody" refers to an antibody comprising amino acid sequences derived from different animal species, such as those having a variable region derived from a human antibody and a murine immunoglobulin constant region.

The term "competitive binding" refers to the interaction of two antibodies when they bind to a binding target. A first antibody competes for binding with a second antibody if binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to binding of the first antibody in the absence of the second antibody. An alternative, wherein the binding of the second antibody to its epitope in the presence of the first antibody is also detectably reduced, may be, but need not be, the fact. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits the binding of another antibody to its cognate epitope, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their corresponding epitope or epitopes.

The term "epitope" refers to the portion of an antigen that is bound by an antibody (or antigen-binding fragment thereof). Epitopes can be formed from both contiguous amino acids or non-contiguous amino acids that are contiguous by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include various numbers of amino acids in unique spatial conformations. Methods for determining the spatial conformation of an Epitope include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all Methods used in combination with computational modeling of the complex structure of an antigen and its binding antibodies and its variants (see, e.g., epipope Mapping Protocols in Methods in Molecular Biology, vol 66, eds. g.e.morris (1996)). Once the desired epitope of an antigen is determined, antibodies can be raised to that epitope, e.g., using the techniques described herein. The generation and characterization of antibodies may also clarify information about desirable epitopes. Based on this information, it is then possible to competitively screen for antibodies that bind to the same epitope. One way to accomplish this is to conduct cross-competition studies to find antibodies that compete for binding to each other, i.e., antibodies compete for binding to antigen. A high throughput method for "partitioning" antibodies based on their cross-competition is described in PCT publication No. WO 03/48731.

The term "germline" refers to the nucleotide sequence of antibody genes and gene segments as they pass from parent to offspring through germ cells. Germline sequences differ from the nucleotide sequences encoding antibodies in mature B cells that have been altered during the process of B cell maturation by recombination and hypermutation events.

The term "glycosylation site" refers to an amino acid residue that is recognized by eukaryotic cells as a linking position for a sugar residue. The amino acids at which carbohydrates such as oligosaccharides are linked are typically asparagine (N-linked), serine (O-linked) and threonine (O-linked) residues. Specific attachment sites are generally indicated by amino acid sequences referred to herein as "glycosylation site sequences". The glycosylation site sequences for N-linked glycosylation are: -Asn-X-Ser-or-Asn-X-Thr-, wherein X can be any conventional amino acid except proline. The terms "N-linked" and "O-linked" refer to chemical groups that serve as attachment sites between sugar molecules and amino acid residues. The N-linked sugars are linked through an amino group; the O-linked sugars are linked through hydroxyl groups. The term "glycan occupancy" refers to the presence of a carbohydrate moiety attached to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on the polypeptide, none (occupied by 0 glycan sites), one (occupied by 1 glycan sites), or two (occupied by 2 glycan sites) sites may be occupied by a carbohydrate moiety.

The term "host cell" refers to a cellular system that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, for example, mammalian cultured cells derived from rodents (rat, mouse, guinea pig or hamster) such as CHO, BHK, NSO, SP2/0, YB 2/0; human cells, such as HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., e.coli cells), and cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

The term "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDRs or HVRs from a non-human animal or synthetic antibody, while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

The term "illustrative antibody" refers to any of the antibodies described herein. These antibodies can be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each antibody identified above encompasses all five classes with the same V_LRegion and V_HAn antibody of the amino acid sequence of the region. Furthermore, antibodies in the IgG class can be in any subclass (e.g., IgG1, IgG2, IgG3, and IgG 4). Thus, each antibody identified above in the IgG subclass encompasses all four subclasses with the same V_LRegion and V_HAn antibody of the amino acid sequence of the region. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes and in the four IgG subclasses are known in the art. The amino acid sequences of the full-length heavy and light chains of the IgG4 subclasses of each of the illustrative antibodies shown in table 1b are provided in this disclosure.

An "isolated" antibody or binding molecule is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, J.Chromatogr.B 848:79-87 (2007).

The term "k_a"refers to the association rate constant for a particular antibody-antigen interaction, and the term" k_d"refers to the off-rate constant for a particular antibody-antigen interaction.

The term "K_DBy "meansEquilibrium dissociation constant for a particular antibody-antigen interaction. It is composed of_dAnd k is_aRatio of (i.e. k)_d/k_a) Obtained and expressed as molar concentration (M). K_DUsed as a measure of the affinity of the binding of an antibody to its binding partner. K_DThe smaller, the more tightly bound the antibody, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar concentration in nanomolar (nM) dissociation constant binds a particular antigen more tightly than an antibody with a micromolar concentration in micromolar (μ M) dissociation constant. K of antibody_DThe values may be determined using well established methods in the art. K for determining antibody_DBy using ELISA. For example, an assay procedure using ELISA is described in at least example 3 of the present disclosure.

The term "mammal" refers to any animal species of the class mammalia. Examples of mammals include: a human; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; domestic animals such as cats, dogs, cattle, sheep, goats, horses and pigs; and captive wild animals such as lions, tigers, elephants, and the like.

The term "preventing" with respect to a disease condition in a mammal refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms of the disease.

As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. Amino acid sequence identity of polypeptides can be routinely determined using known computer programs such as Bestfit, FASTA or BLAST (see, e.g., Pearson, Methods enzymol.183:63-98 (1990); Pearson, Methods mol.biol.132:185-219 (2000); Altschul et al, J.mol.biol.215:403-410 (1990); Altschul et al, Nucleic Acids Res.25:3389-3402 (1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage identity is calculated over the full length of the reference amino acid sequence and gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed. This method, mentioned above in determining percent identity between polypeptides, is applicable to all proteins, fragments or variants thereof disclosed herein.

As used herein, the terms "bind," "specific binding," or "specific for … …" refer to a measurable and reproducible interaction, such as binding, between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds a target (which may be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or for a longer duration than it binds other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved between proteins from different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

The term "treating" with respect to a disease condition in a mammal refers to causing a desirable or beneficial effect in said mammal having said disease condition. Desirable or beneficial effects may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and/or metastasis, or other effects mediated by the number and/or activity of immune cells, etc.), or the prevention or inhibition of further development of the disease, disorder or condition. In the case of treating cancer in a mammal, a desirable or beneficial effect may include inhibiting further growth or spread of cancer cells, killing cancer cells, inhibiting recurrence of cancer, reducing pain associated with cancer, or improving survival of the mammal. The effect may be subjective or objective. For example, if the mammal is a human, the human may notice an improvement in energy or vigor or a reduction in pain as a sign of subjective improvement or response to therapy. Alternatively, the clinician may perceive a reduction in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or radiographic studies. Some laboratory signs that may be observed by a clinician regarding response to treatment include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme level tests. In addition, the clinician may observe a decrease in detectable tumor markers. Alternatively, other tests may be used to assess objective improvement, such as sonograms, nuclear magnetic resonance tests, and positron emission tests.

The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows propagation, selection, further manipulation or expression of the foreign nucleic acid molecule in the host cell or organism. The vector may be a plasmid, phage, transposon, cosmid, chromosome, virus or virion. One type of vector can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated along with the host genome (e.g., a non-episomal mammalian vector). Another type of vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Another specific type of vector capable of directing the expression of the expressible foreign nucleic acid to which they are operatively linked is often referred to as an "expression vector". Expression vectors typically have control sequences that drive expression of an expressible foreign nucleic acid. The simpler vectors, called "transcription vectors", are only capable of being transcribed and not translated: they can replicate in the target cell rather than express. The term "vector" encompasses all types of vectors, regardless of their function. Vectors capable of directing the expression of an expressible nucleic acid to which they are operatively linked are generally referred to as "expression vectors". Other examples of "vectors" may include display vectors (e.g., vectors that direct expression and display of an encoded polypeptide on the surface of a virus or cell, such as a bacterial cell, a yeast cell, an insect cell, and/or a mammalian cell).

As used herein, "subject," "patient," or "individual" may refer to a human or non-human animal. "non-human animal" can refer to any animal not classified as a human, such as a domestic, farm or zoo animal, a sport animal, a pet animal (such as a dog, horse, cat, cow, etc.), and an animal used in research. A research animal may refer without limitation to a nematode, arthropod, vertebrate, mammal, frog, rodent (e.g., mouse or rat), fish (e.g., zebrafish or puffer fish), bird (e.g., chicken), dog, cat, and non-human primate (e.g., rhesus monkey, cynomolgus monkey, chimpanzee, etc.). In some embodiments, the subject, patient, or individual is a human.

By "effective amount" is meant at least the following amount: the amount is effective to achieve one or more desired or indicated effects, including therapeutic or prophylactic results, at the requisite dosage and for the requisite period of time. An effective amount may be provided in one or more administrations. For the purposes of this disclosure, an effective amount of an antibody, drug, compound or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in clinical situations, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition (e.g., an effective amount as administered in monotherapy or in combination therapy). Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if a desired result is achieved or achieved in combination with one or more other agents.

Libraries of activatable binding polypeptides and production of libraries

Certain aspects of the present disclosure relate to a library of polynucleotides (e.g., encoding any of the polypeptides described herein) and/or polynucleotides, e.g., encoding polypeptides, including activatable antibodies, activatable antigen-binding fragments thereof, or derivatives of activatable antibodies, that can be used to screen for and/or identify one or more activatable binding polypeptides (i.e., one or more activatable antibodies).

The term "activatable binding polypeptide", "ABP" or "activatable antibody" includes polypeptides comprising a Target Binding Moiety (TBM), a Cleavable Moiety (CM) and a Masking Moiety (MM). In some embodiments, the TBM comprises an amino acid sequence that binds to a target. In some embodiments, the TBM comprises an Antigen Binding Domain (ABD) of an antibody or antibody fragment thereof. In some embodiments, the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH and the VL form a binding domain that binds a target in the absence of MM. In some embodiments, the VH and VL are covalently linked, for example in an scFv. In some embodiments, the VH and VL form a Fab fragment. In some embodiments, the VH is linked to an antibody heavy chain constant region and the VL is linked to an antibody light chain constant region.

In some embodiments, the activatable antibody comprises a polypeptide comprising, from N-terminus to C-terminus: the Masking Moiety (MM) -Cleavable Moiety (CM) -VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N-terminus to C-terminus: masking Moiety (MM) -Cleavable Moiety (CM) -VL-VH (e.g., scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N-terminus to C-terminus: the Masking Moiety (MM) -Cleavable Moiety (CM) -VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N-terminus to C-terminus: masking Moiety (MM) -Cleavable Moiety (CM) -VH-VL (e.g., scFv).

In some embodiments, the activatable antibody comprises a polypeptide comprising, from N-terminus to C-terminus: masking Moiety (MM) -L₁-Cleavable Moiety (CM) -L₂A VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N-terminus to C-terminus: masking Moiety (MM) -L ₁-Cleavable Moiety (CM) -L₂-VL-L₃VH (e.g.scFv). In some embodiments, the activatable antibody comprises a heavy chain variable region from NA polypeptide comprising, end-to-C-terminus, the structure: masking Moiety (MM) -Cleavable Moiety (CM) -L₁A VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N-terminus to C-terminus: masking Moiety (MM) -L₁-Cleavable Moiety (CM) -L₂-VH-L₃VL (e.g.scFv). In some embodiments, L is₁、L₂And/or L₃Is a joint. In some embodiments, L is₁、L₂And L₃Each of which is a linker that can have an independently selected length of 0 amino acids or 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids.

CM typically comprises an amino acid sequence that is cleavable, e.g., serves as a substrate for an enzyme, and/or a cysteine-cysteine pair capable of forming a reducible disulfide bond. Thus, when the terms "cleave", "cleavable", "cleaved", and the like are used in connection with CM, the terms encompass enzymatic cleavage, e.g., by proteases, as well as disruption of disulfide bonds between cysteine-cysteine pairs by disulfide bond reduction that may result from exposure to a reducing agent.

MM refers to an amino acid sequence in which MM interferes with or inhibits binding of TBM to its target when the CM of an activatable antibody is intact (e.g., not cleaved by the corresponding enzyme, and/or contains an unreduced cysteine-cysteine disulfide bond). In some embodiments, MM interferes with or inhibits binding of TBM to its target so efficiently that binding of TBM to its target is extremely low and/or below the limit of detection (e.g., binding cannot be detected in an ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with, or be included within, the MM. It should be noted that for convenience, "ABP" or "activatable antibody" is used herein to refer to ABP or activatable antibody in their uncleaved (or "native") state as well as in their cleaved state. It will be apparent to one of ordinary skill that, in some embodiments, a cleaved ABP may lack a MM due to, for example, cleavage of CM by a protease, which results in release of at least the MM (e.g., when the MM is not bound to ABP via a covalent bond (e.g., a disulfide bond between cysteine residues)). Exemplary ABPs are described in more detail below.

The libraries of the present disclosure can contain one or more polynucleotides encoding any of the polypeptides described herein (e.g., one or more of the activatable binding polypeptides described herein). In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of the libraries described herein encode a polypeptide comprising one or more full length antibody light and/or heavy chains. In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of the libraries described herein encode a polypeptide comprising one or more light chain and/or heavy chain Fab fragments. In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of the libraries described herein encode a polypeptide comprising one or more single-chain variable fragments (scfvs).

Other aspects of the disclosure relate to polypeptides (e.g., any of the polypeptides described herein) and/or libraries of polypeptides that can be used to screen for and/or identify one or more activatable binding polypeptides (i.e., one or more activatable antibodies), including activatable antibodies, activatable antigen-binding fragments thereof, or activatable antibody derivatives. The libraries of the present disclosure may contain one or more of the polypeptides described herein (e.g., one or more activatable binding polypeptides). In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the libraries described herein comprise one or more full length antibody light and/or heavy chains. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the libraries described herein comprise one or more light chain and/or heavy chain Fab fragments. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the libraries described herein comprise one or more single chain variable fragments (scfvs). In some embodiments, the polypeptide is expressed on the surface of a cell (e.g., displayed by a yeast or mammalian cell).

In some embodiments, the polypeptides of the disclosure comprise: (a) a First Peptide (FP); (b) a Cleavable Moiety (CM); and (c) a Target Binding Moiety (TBM). In some embodiments, FP is any first peptide described herein (e.g., comprising X according to formula (XIII): X) _mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y). In some embodiments, X is not W, M and/or C. In some embodiments, X of formula (XIII)_mEach X in (A) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P, X of formula (XIII)_nEach X in (A) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P, and/or X of formula (XIII)_oEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, m is 3 to 10. In some embodiments, FP is any first peptide described herein (e.g., comprising X according to formula (I): X)_mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P). In some embodiments, m is 3 to 10. In some embodiments, X is not W, M and/or C. In some embodiments, X of formula (I) _mEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P, and/or X of formula (I)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, FP is any first peptide described herein (e.g., comprising a peptide according to formula (XII): Z)_mCZ_nCZ_o(SEQ ID NO:71) wherein m is 2-10, n is 3-10, and o is 1-10, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P). In some embodiments, m is 3 to 10. In some embodiments, the CM is any cleavable moiety described herein (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site). In some embodiments, the CM is any cleavable moiety described herein (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site). In some embodiments, the TBM is any target binding moiety described herein (e.g., a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region).

In some embodiments, the First Peptide (FP) interferes with, blocks the binding of the target binding moiety to its target, reduces the ability of the target binding moiety to bind its target, prevents, inhibits, or competes with the target binding moiety for binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the First Peptide (FP) interferes with, blocks, decreases, prevents, inhibits, or competes with the target binding moiety for binding to its target only when the polypeptide has not been activated (e.g., activated by a pH change (increase or decrease), activated by a temperature shift (increase or decrease), activated after contact with a second molecule such as a small molecule or protein ligand, etc.). In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces a conformational change in the polypeptide (e.g., a shift in the First Peptide (FP)), resulting in the first peptide no longer preventing the activatable antibody from binding its target. In some embodiments, the First Peptide (FP) interferes with, blocks the binding of the target-binding moiety to its target, decreases the ability of the target-binding moiety to bind its target, prevents, inhibits, or competes with the target-binding moiety for binding to its target only when the Cleavable Moiety (CM) has not been cleaved by one or more proteases that cleave within the Cleavable Moiety (CM). In some embodiments, prior to activation, the first peptide (F) P) has a masking efficiency of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.). In some embodiments, the masking efficiency is measured as the difference in affinity of an activatable antibody comprising the First Peptide (FP) to bind its target (prior to activation) relative to the affinity of a polypeptide lacking the first peptide to bind its target (e.g., the difference in affinity of an activatable antibody comprising the First Peptide (FP) (prior to activation) relative to a parent antibody lacking the First Peptide (FP), for a target antigen such as CTLA4, or the difference in affinity of an activatable antibody comprising the First Peptide (FP) (prior to activation) for a target antigen such as CTLA4 relative to the difference in affinity of the activatable antibody for the target antigen after activation). In some embodiments, the masking efficiency is by EC bound with an activatable antibody comprising a First Peptide (FP)₅₀(prior to activation) divided by the EC of the parent antibody₅₀To measure (e.g. by measuring EC with ELISA) ₅₀(ii) a See, e.g., the method of example 3). In some embodiments, the masking efficiency is measured as the difference in the affinity of an activatable antibody comprising the First Peptide (FP) to bind its target prior to activation relative to the affinity of an activatable antibody comprising the First Peptide (FP) to bind its target after activation (e.g., the difference in the affinity of an activatable antibody to a target antigen, such as CTLA4, after activation relative to the activatable antibody prior to activation). In some embodiments, the First Peptide (FP) binds to a Target Binding Moiety (TBM) and prevents the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the First Peptide (FP) has a dissociation constant for binding to the target-binding moiety (TBM) that is greater than the dissociation constant for the target-binding moiety (TBM) for its target. In some embodiments, the First Peptide (FP) is a Masking Moiety (MM). The dissociation constant may be measured, for example, by techniques such as ELISA, surface plasmon resonance or bio-layer interferometry (BLI), or flow cytometry.

In some embodiments, the First Peptide (FP) does not interfere with, hinder, reduce the ability of, prevent, inhibit, or compete with the Target Binding Moiety (TBM) from binding to its target after the polypeptide has been activated (e.g., activated by treatment with one or more proteases that cleave within a Cleavable Moiety (CM), activated by a change in pH (increase or decrease), activated by a change in temperature (increase or decrease), activated after contact with a second molecule such as an enzyme, etc.). In some embodiments, after the Cleavable Moiety (CM) has been cleaved by one or more proteases that cleave within the Cleavable Moiety (CM), the First Peptide (FP) does not interfere with, block the binding of the target-binding moiety (TBM) to its target, decrease the ability of the target-binding moiety (TBM) to bind its target, prevent, inhibit the binding of the target-binding moiety (TBM) to its target, or compete with the binding of the target-binding moiety (TBM) to its target. In some embodiments, after activation, the First Peptide (FP) has a masking efficiency (e.g., relative affinity of the activatable antibody after activation compared to the affinity of the parent antibody) of at most about 1.75 (e.g., at most about 1.75, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, at most about 1.0, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, or at most about 0.5, etc.).

In some embodiments, the polypeptides of the present disclosure comprise the following structure from N-terminus to C-terminus: first Peptide (FP) -Cleavable Moiety (CM) -Target Binding Moiety (TBM). The libraries of the present disclosure may be used to screen for one or more activatable binding polypeptides (i.e., activatable antibodies) that bind, when in active form, any target of interest including, for example, CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, and/or B7-H4.

In some embodiments, the libraries of the present disclosure contain a library encoding at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, a polypeptide encoding at least one, a polypeptide encoding at least 2, a polypeptide encoding at least 3, a polypeptide encoding at least,At least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, at least 10³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A plurality of polynucleotides of a unique polypeptide as described herein, the polypeptide comprising: (a) a First Peptide (FP); (b) a Cleavable Moiety (CM); and (c) a Target Binding Moiety (TBM).

In some embodiments, a library of the present disclosure comprises a library encoding at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, at least 10³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A plurality of polynucleotides of a polypeptide as described herein, the polypeptide comprising: (a) a unique First Peptide (FP); (b) a Cleavable Moiety (CM); and (c) a Target Binding Moiety (TBM).

In some embodiments, a library of the present disclosure: 1) encodes and/or contains a number of unique peptides that is less than the number found in a typical random peptide library (e.g., comprising peptides according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86) or formula (I): x_mCX_nCZ_o(FP of the amino acid sequence of SEQ ID NO: 1); 2) encoding and/or containing a peptide comprising a pair of cysteine residues at fixed positions to ensure that the displayed peptide has a constrained conformation (e.g. comprising a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86) or formula (I): x_mCX_nCZ_o(FP of the amino acid sequence of SEQ ID NO: 1); and/or 3) encodes and/or contains a peptide having few or no chemically labile residues (such as methionine or tryptophan) (e.g., comprising a peptide according to formula (XIII): x _mCX_nCX_o(SEQ ID NO:86) or formula (I): x_mCX_nCZ_o(FP of the amino acid sequence of SEQ ID NO: 1). Advantageously, the libraries of the present disclosure have a significantly reduced library size relative to a random peptide library, thereby enabling the construction of peptides having much better (e.g., comprising X according to formula (XIII): X)_mCX_nCX_o(SEQ ID NO:86) or formula (I): x_mCX_nCZ_o(FP of the amino acid sequence of SEQ ID NO:1) coverage. Furthermore, the inclusion of a pair of cysteine residues at fixed positions ensures that the displayed peptide has a constrained conformation, tending to exhibit increased binding affinity and/or specificity. In addition, libraries of the present disclosure have peptides that include few to no residues such as methionine or tryptophan that are detrimental to the manufacturing process (e.g., comprising X according to formula (XIII): X)_mCX_nCX_o(SEQ ID NO:86) or formula (I): x_mCX_nCZ_o(FP of the amino acid sequence of SEQ ID NO: 1).

In some embodiments, a library of the present disclosure contains a plurality of polynucleotides, wherein at least one of the polynucleotides in the library encodes a polypeptide comprising: (a) comprising a compound according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, the one or more polynucleotides of the library The acid is in a vector (e.g., an expression vector or a display vector). In some embodiments, a library of the present disclosure contains a plurality of polynucleotides, wherein at least one of the polynucleotides in the library encodes a polypeptide comprising: (a) comprising a compound according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

In some embodiments, at least one of the polynucleotides in the library encodes a polypeptide comprising EVGSYX according to formula (III)₁X₂X₃X₄X₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CX₁₃X₁₄SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:3), wherein X₁Is A, D, I, N, P or Y, X₂Is A, F, N, S or V, X₃Is A, H, L, P, S, V or Y, X₄Is A, H, S or Y, X₅Is A, D, P, S, V or Y, X₆Is A, D, L, S or Y, X₇Is D, P or V, X₈Is A, D, H, P, S or T, X₉Is A, D, F, H, P or Y, X₁₀Is L, P or Y, X₁₁Is F, P or Y, X₁₂Is A, P, S or Y, X₁₃Is A, D, N, S, T or Y, and X₁₄Is A, S or Y. In some embodiments, at least one of the polynucleotides in the library encodes an activatable junctionThe polypeptide is synthesized (i.e., the antibody is activated). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

In some embodiments, at least one of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOS 25-46. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 250, at least 500, at least 10, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹Encoding a polypeptide comprising: (a) comprising a compound according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or a combination thereof of the polynucleotides in the library, At least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 250, at least 500, at least 10, at least³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹Encoding a polypeptide comprising: (a) comprising a compound according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising: (a) comprising a compound according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising: (a) comprising a compound according to formula (I): x _mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

In some embodiments, among the polynucleotides in the libraryEach encoding of (a) includes EVGSYX according to formula (III)₁X₂X₃X₄X₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CX₁₃X₁₄SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:3), wherein X1 is A, D, I, N, P or Y, X2 is A, F, N, S or V, X3 is A, H, L, P, S, V or Y, X4 is A, H, S or Y, X5 is A, D, P, S, V or Y, X6 is A, D, L, S or Y, X7 is D, P or V, X8 is A, D, H, P, S or T, X9 is A, D, F, H, P or Y, X10 is L, P or Y, X11 is F, P or Y, X12 is A, P, S or Y, X13 is A, D, N, S, T or Y, and X14 is A, S or Y. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOS 25-46. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10)³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86) wherein m is 2 to 10, n is 3 to 10,and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region; and d) an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10) ³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region; and d) an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, the structure: first Peptide (FP) -Cleavable Moiety (CM) -VL-VH. In some embodiments, at least one of the polypeptides is an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, the linker sequence separates VL and VH (i.e., the structure VL-linker-VH). The linker sequence may be any linker sequence known in the art, such as any of the linker sequences described herein. In some embodiments, the linker sequence is any copy number of GGGGS (SEQ ID NO:17) (e.g., repeats) 2 times, repeat 3 times, etc.).

In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10)³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) comprises a target-binding moiety (TBM) of an antibody light chain variable region, and the library further comprises one or more polynucleotides encoding an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10) ³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q,r, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) comprises a target-binding moiety (TBM) of an antibody light chain variable region, and the library further comprises one or more polynucleotides encoding an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the polynucleotide encoding the polypeptide comprising a Target Binding Moiety (TBM) comprising an antibody light chain variable region and the polynucleotide encoding the antibody heavy chain variable region are on the same vector (e.g., expressed from their own promoter) or on different vectors. In some embodiments, at least one of the polypeptides forms an activatable binding polypeptide (i.e., an activatable antibody) when coupled to an antibody heavy chain variable region.

In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10)³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a Target Binding Moiety (TBM) comprising an antibody heavy chain variable region; and d) an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the activatable binding polypeptide comprises a polypeptide comprising, from N-terminus to C-terminus: a first peptide(FP) -Cleavable Moiety (CM) -VH-VL. In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10) ³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a Target Binding Moiety (TBM) comprising an antibody heavy chain variable region; and d) an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the activatable binding polypeptide comprises a polypeptide comprising, from N-terminus to C-terminus: first Peptide (FP) -Cleavable Moiety (CM) -VH-VL. In some embodiments, at least one of the polypeptides is an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, the linker sequence separates VH and VL (i.e., the structure VH-linker-VL). The linker sequence may be any linker sequence known in the art, such as any of the linker sequences described herein. In some embodiments, the linker sequence is any copy number of GGGGS (SEQ ID NO:17) (e.g., 2 repeats, 3 repeats, etc.).

In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10)³At least 10⁴At least 10⁵At least 10⁶At least 10⁷ToLess than 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) comprises a target-binding moiety (TBM) of an antibody heavy chain variable region, and the library further comprises one or more polynucleotides encoding an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, a library of the present disclosure contains at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10) ³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least 10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹A) polynucleotide encoding a polypeptide comprising: (a) comprising a compound according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P; (b) a Cleavable Moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) target Binding Moieties (TBMs) comprising antibody heavy chain variable regions, and the librariesAlso included are one or more polynucleotides encoding the variable region of the antibody light chain. In some embodiments, m is 3 to 10. In some embodiments, the polynucleotide encoding the polypeptide comprising a Target Binding Moiety (TBM) comprising an antibody heavy chain variable region and the polynucleotide encoding the antibody light chain variable region are on the same vector (e.g., expressed from their own promoter) or on different vectors. In some embodiments, at least one of the polypeptides forms an activatable binding polypeptide (i.e., an activatable antibody) when coupled to an antibody light chain variable region.

The polynucleotides and/or polynucleotide libraries described herein may incorporate any of the HVR sequences (e.g., one, two, or three of the heavy chain variable region HVR sequences, and/or one, two, or three of the light chain variable region HVR sequences), the heavy chain variable region sequences, and/or the light chain variable region sequences of any of the antibodies described herein (e.g., anti-CTLA 4 antibody, anti-CD 137 antibody). The polynucleotides and/or polynucleotide libraries described herein may also incorporate any of the HVR sequences (e.g., one, two, or three of the heavy chain variable region HVR sequences, and/or one, two, or three of the light chain variable region HVR sequences), heavy chain variable region sequences, light chain variable region sequences, heavy chains, and/or light chains described in PCT application No. PCT/CN2017/098333 (incorporated herein by reference in its entirety) and/or PCT application No. PCT/CN2017/098299 (incorporated herein by reference in its entirety).

In some embodiments, a library of the present disclosure comprises one or more vectors (e.g., expression vectors and/or display vectors) encoding one or more polynucleotides (e.g., synthetic polynucleotides) of the present disclosure.

Also provided herein is a method of making a library, e.g., by providing and assembling polynucleotide sequences (e.g., one or more synthetic polynucleotides) of a library of the present disclosure. Also provided herein is a method of making a library, for example, by: multiple (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10) are selected ³At least 10⁴At least 10⁵At least 10⁶At least 10⁷At least 10⁸At least 10⁹At least 10¹⁰At least10¹¹At least 10¹²At least 10¹³At least 10¹⁴At least 10¹⁵At least 10¹⁶At least 10¹⁷At least 10¹⁸Or at least 10¹⁹One) a First Peptide (FP) sequence, a Cleavable Moiety (CM) sequence, and/or a target-binding moiety (TBM) sequence (e.g., any one or more of the FP, CM, and TBM sequences described herein), and assembling polynucleotide sequences encoding these sequences to generate a library of polynucleotides (e.g., synthetic polynucleotides) encoding a plurality of polypeptides. In some embodiments, at least one of the polypeptides encoded by the assembled library is an activatable binding polypeptide (i.e., an activatable antibody).

Polynucleotides encoding a polypeptide as described herein can be cloned into any suitable vector to express a portion or the entire polypeptide sequence. In some embodiments, the polynucleotide is cloned into a vector, thereby allowing the production of all or a portion of (i.e., creating a fusion protein) fused to a protein (e.g., a viral coat protein, a bacterial surface protein, a yeast surface protein, an insect cell surface protein, a mammalian cell surface protein) and displayed on the surface of the particle or cell as a portion or as the entire polypeptide. Several types of vectors are available and can be used to practice the present disclosure, such as phagemid vectors. Phagemid vectors typically contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other essential components as known to those of ordinary skill in the art. In some embodiments, polynucleotides encoding regions of polypeptides may be cloned into vectors for expression in bacterial cells for bacterial display or in yeast cells for yeast display. Exemplary vectors are described in U.S. pre-grant publication No. US 20160145604. In some embodiments, the vector is a display vector comprising, from 5 'to 3', a polynucleotide encoding an amino acid sequence to be displayed on a surface (e.g., the surface of a phage, bacteria, yeast, insect, or mammalian cell), a restriction site, a second polynucleotide encoding a surface peptide capable of being displayed on the surface, and a second restriction site. In some embodiments, the second polynucleotide encodes a phage coat protein, a yeast exowall protein (such as Aga2), a bacterial outer membrane protein, a cell surface tethering domain, or an adapter, or truncated forms or derivatives thereof. In some embodiments, the surface peptide is used for phage display, yeast display, bacterial display, insect display, or mammalian display, or shuttle display therebetween. In some embodiments, the amino acid sequence and the surface peptide are displayed on the surface as a fusion protein when expressed. In some embodiments, the vector further comprises a fusion tag 5 'to the first restriction site or 3' to the second restriction site.

Certain aspects of the present disclosure relate to a population of cells containing one or more of the vectors described herein. Polypeptides encoded by polynucleotides produced by any of the techniques described herein, or other suitable techniques, can be expressed and screened to identify activatable binding polypeptides having a desired structure and/or activity. Expression of the polypeptide can be performed, for example, using a cell-free extract (e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g., yeast display). In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell (such as a Chinese Hamster Ovary (CHO) cell). Methods for transfecting bacterial cells, yeast cells, or mammalian cells are known in the art and described in the references cited herein. Expression of polypeptides (e.g., one or more activatable binding polypeptides) in these cell types (e.g., from a library of the present disclosure) and screening for the activatable binding polypeptide of interest is described in more detail below.

Alternatively, the polynucleotide may be expressed in an E.coli expression system such as described by Pluckthun and Skerra. (meth. enzymol.,1989,178: 476; Biotechnology,1991,9: 273). The mutein may be expressed for secretion in culture medium and/or in the cytoplasm of the bacteria, as described by Better and Horwitz, meth.enzymol.,1989,178: 476. In some embodiments, the polypeptide is linked to the 3' end of a sequence encoding a signal sequence, such as an ompA, phoA or pelB signal sequence (Lei et al, J.Bacteriol.,1987,169: 4379). These gene fusions are assembled in a bicistronic construct, and thus, they can be expressed from a single vector and secreted into the periplasmic space of E.coli, where they will refold and can be recovered in active form. (Skerra et al, Biotechnology,1991,9: 273). For example, a gene encoding a polypeptide comprising a First Peptide (FP), a Cleavable Moiety (CM), and a Target Binding Moiety (TBM) comprising an antibody light chain can be expressed in parallel with an antibody heavy chain gene to produce a polypeptide of interest.

In other embodiments, a composition such as, for example, US 20040072740; US 20030100023; and secretion signal and lipidation moieties as described in US20030036092, the polypeptide sequences of the present disclosure are expressed on the membrane surface of prokaryotes such as e.

Alternatively, polypeptide sequences of the present disclosure can be expressed and screened by anchored periplasmic expression (APEx two-hybrid surface display) as described, for example, in Jeong et al, PNAS,2007,104:8247, or by other anchoring methods as described, for example, in Mazor et al, Nature Biotechnology,2007,25: 563.

Higher eukaryotic cells, such as mammalian cells, e.g., myeloma cells (e.g., NS/0 cells), hybridoma cells, Chinese Hamster Ovary (CHO) cells, and Human Embryonic Kidney (HEK) cells, can also be used to express polypeptides of the disclosure. Polypeptides expressed in mammalian cells (e.g., activatable binding polypeptides) can be designed to be secreted into the culture medium or expressed on the surface of the cell.

In other embodiments, polypeptides (e.g., activatable binding polypeptides) may be selected using mammalian cell display (Ho et al, PNAS,2006,103: 9637). In some embodiments, as described above and exemplified below, the polypeptide (e.g., activatable binding polypeptide) may be selected after being fused to all or a portion of the viral coat protein (i.e., producing a fusion protein), for example, using phage display, and displayed on the surface of the particle or cell, either partially or entirely, of the polypeptide sequence.

Certain aspects of the present disclosure relate to a non-human animal comprising a polynucleotide or polynucleotide library of the present disclosure. For example, a non-human animal of the present disclosure can be modified such that its genome comprisesPolynucleotides encoding the polypeptides of the disclosure (e.g., activatable binding polypeptides). In some embodiments, the transgenic animal (e.g., a mouse) expresses a polypeptide encoded by a polynucleotide. Techniques for modifying the genome of non-human animals are known in the art (e.g., for generating xenolouse^TMThe method of (1).

Screening for activatable binding polypeptides obtained from the libraries of the present disclosure can be performed by any suitable means (e.g., determining target binding before and after activation, such as treatment of the polypeptide with one or more proteases that cleave the sequence within a Cleavable Moiety (CM)). For example, binding activity can be assessed by standard immunoassays and/or affinity chromatography. Screening of polypeptides of the present disclosure for catalytic functions, such as proteolytic functions, can be accomplished using standard assays, such as hemoglobin patch assays. Determination of binding affinity of a polypeptide (e.g., an activatable binding polypeptide) for a target can be determined in vitro using a variety of well-known techniques, e.g., ELISA, BIACORE based on surface plasmon resonance to measure the rate of binding of a protein to a given target ^TMInstruments, or e.g. using ForteBioRED96 platform (Pall Life Sciences) biolayer interferometry exemplified below. (BLI), in vivo assays can be performed using any of a number of animal models, followed by subsequent testing in humans, as appropriate. Cell-based bioassays are also contemplated. The polypeptide (e.g., activatable binding polypeptide) may be further selected for functional activity, e.g., antagonistic or agonistic activity. For example, in some embodiments, BLI is used to measure binding affinity between a polypeptide comprising one or more fab fragments and one or more targets by tagging the antigen with a human IgG1-Fc tag and capture (e.g., before and after activation) by an anti-hIgG-Fc capture (AHC) biosensor. The polypeptide may be tagged with a His6 tag at the C-terminus of the CH1 domain, overexpressed in host cells such as e.coli, and purified, for example, using Ni-NTA resin. Affinity can then be measured using an AHC sensor (anti-human IgG-Fc capture dip and readBiosensor) that is dipped into a well containing purified Fab-containing polypeptide diluted with kinetic buffer, e.g., to 5-10 μ g/mL.

After identifying the binding agent (e.g., by determining that the polypeptide is capable of binding to the target or antigen when "active" (e.g., after treatment with a protease) but not when "inactive" (e.g., before treatment with a protease), the nucleic acid can be extracted. The extracted DNA can then be used directly to transform e.coli host cells, or the coding sequence can be amplified, e.g., using PCR with appropriate primers, and sequenced by any typical sequencing method. The DNA sequences of the binding agent may be subjected to restriction enzyme digestion, followed by insertion into a vector for protein expression.

First Peptide (FP)

In some embodiments, the disclosure relates to polynucleotides and/or polynucleotide libraries encoding one or more polypeptides comprising a First Peptide (FP). In some embodiments, the disclosure relates to polypeptides and/or polypeptide libraries comprising at least one polypeptide comprising a First Peptide (FP). In some embodiments, the First Peptide (FP) comprises a peptide according to formula (XIII): x_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. In some embodiments, m is 3 to 10. In some embodiments, X is not W, M and/or C. In some embodiments, X of formula (XIII) _mEach X in (A) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P, X of formula (XIII)_nEach X in (A) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P, and/or X of formula (XIII)_oEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, the FP comprises a compound according to formula (XIV): (NNK)_mTGY(NNK)_nTGY(NNK)_o(SEQ ID N87), wherein each N is independently A, G, T or C, wherein each K is independently T or G, and wherein each Y is independently T or C, and wherein each H is independently A, T or C.

In some embodiments, the First Peptide (FP) comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, m is 3 to 10. In some embodiments, X is not W, M and/or C. In some embodiments, X of formula (I) _mEach X in (A) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P, and X of formula (I)_nEach X in (a) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, the FP comprises a compound according to formula (II): (NNK)_mTGY(NNK)_nTGY(NHC)_o(SEQ ID NO:2), wherein each N is independently A, G, T or C, wherein each K is independently T or G, wherein each Y is independently T or C, and wherein each H is independently A, T or C.

In some embodiments, the First Peptide (FP) comprises a peptide according to formula (XII): z_mCZ_nCZ_o(SEQ ID NO:71), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, m is 3 to 10.

In some embodiments, m is 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, m is 6 to 8. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6.

In some embodiments, n is 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, n is 6 to 8. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 6. In some embodiments, n is 8.

In some embodiments, o is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, o is 1-2. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 2.

In some embodiments, the First Peptide (FP) comprises a peptide according to formula (IV): z₆CX₆CZ₂(SEQ ID NO:55), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the First Peptide (FP) comprises a peptide according to formula (V): z₆CX₈CZ₂(SEQ ID NO:56), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the First Peptide (FP) comprises a peptide according to formula (VI): (Z)₆)C(Z₆)C(Z₂)(SEQ ID NO57), wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the First Peptide (FP) comprises a peptide according to formula (VII): (Z)₆)C(Z₈)C(Z₂) (SEQ ID NO:58), wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the First Peptide (FP) comprises an amino acid sequence selected from the group consisting of: x_mCADAPNHCXX(SEQ ID NO:88)、X_mCHHSPANCXX(SEQ ID NO:89)、X_mCPILRHRCXX(SEQ ID NO:90)、X_mCKWRPSRCXX(SEQ ID NO:91)、X_mCRVLPRRCXX(SEQ ID NO:92)、X_mCLWRHRSCXX (SEQ ID NO:93) and X_mCPRLRRKCXX (SEQ ID NO:94), wherein m is 2-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. In some embodiments, each X is not M, W or C. In some embodiments, each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P. In some embodiments, m is 2. In some embodiments, the First Peptide (FP) comprises the amino acid sequence EVGSYPTDLDACADAPNHCHF (SEQ ID NO:95), EVGSYSSTHAHCHHSPANCIS (SEQ ID NO:96), EVGSYDTDYDFCPILRHRCDS (SEQ ID NO:97), EVGSYNDYNYHCKWRPSRCHN (SEQ ID NO:98), EVGSYYHDYDDCRVLPRRCFN (SEQ ID NO:99), EVGSYSNNFASCLWRHRSCAD (SEQ ID NO:100), or EVGSYTDNYDYCPRLRRKCYH (SEQ ID NO: 101). In some embodiments, the Target Binding Moiety (TBM) comprises the sequence of one or more of the anti-CD 137 antibodies described herein, including antibodies described for particular amino acid sequences of HVRs, variable regions (VL, VH), and/or light and heavy chains (e.g., IgG1, IgG2, IgG 4). In some embodiments, the Target Binding Moiety (TBM) comprises a full length antibody light chain of one or more of the anti-CD 137 antibodies described herein.

In some embodiments, the First Peptide (FP) comprises a peptide selected from the group consisting ofAmino acid sequence: x_mCPDHPYPCXX(SEQ ID NO:102)、X_nCDAFYPYCXX(SEQ ID NO:103)、X_mCDSHYPYCXX (SEQ ID NO:104) and X_mCVPYYYACXX (SEQ ID NO:105), wherein m is 2-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. In some embodiments, the First Peptide (FP) comprises the amino acid sequence EVGSYNFVADSCPDHPYPCSA (SEQ ID NO:110), EVGSYIVHHSDCDAFYPYCDS (SEQ ID NO:111), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO:112), EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:113), EVGSYYSAYPACDSHYPYCQS (SEQ ID NO:114), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO:115), EVGSYPQPSSDCVPYYYACAY (SEQ ID NO:116), or EVGSYPNPASDCVPYYYACAY (SEQ ID NO: 117). In some embodiments, the Target Binding Moiety (TBM) comprises the sequence of one or more of the anti-CTLA 4 antibodies described herein, including antibodies described with respect to particular amino acid sequences of HVRs, variable regions (VL, VH) and/or light and heavy chains (e.g., IgG1, IgG2, IgG 4). In some embodiments, the Target Binding Moiety (TBM) comprises a full length antibody light chain of one or more of the anti-CTLA 4 antibodies described herein.

In some embodiments, the First Peptide (FP) comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 72-85.

In some embodiments, any of the First Peptides (FP) described herein can further comprise one or more additional amino acid sequences (e.g., one or more polypeptide tags). Examples of suitable additional amino acid sequences can include, without limitation, purification tags (such as his tags, flag tags, maltose binding protein, and glutathione-S-transferase tags), detection tags (such as tags that can be detected photometrically (e.g., red or green fluorescent protein, etc.)), tags having detectable enzymatic activity (e.g., alkaline phosphatase, etc.), tags containing secretory sequences, leader sequences, and/or stabilizing sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), and the like. In some embodiments, the one or more additional amino acid sequences are N-terminal to the First Peptide (FP). In some embodiments, the additional amino acid sequence comprises or consists of the sequence EVGSY (SEQ ID NO: 16).

In some embodiments, the first peptide is a masking peptide that binds to a Target Binding Moiety (TBM) prior to activation (e.g., prior to treatment with one or more proteases that cleave within a Cleavable Moiety (CM), prior to undergoing a (local) pH change (increase or decrease), prior to temperature shift (increase or decrease), prior to contact with a second molecule such as a small molecule or protein ligand, etc.), and inhibits the polypeptide from binding its target, but does not bind TBM and/or inhibits the polypeptide from binding its target after activation (e.g., after treatment with one or more proteases that cleave within a Cleavable Moiety (CM), after being subjected to a (local) pH change (increase or decrease), after a temperature shift (increase or decrease), after contact with a second molecule such as a small molecule or protein ligand, etc.). In some embodiments, a First Peptide (FP) (e.g., a masking moiety) inhibits binding of a polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) to its target when the CM is not cleaved, but does not inhibit binding of a polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) to its target when the CM is cleaved. In some embodiments, a First Peptide (FP) (e.g., a masking moiety) has a dissociation constant for binding to TBM that is greater than (e.g., at least about 1.5-fold greater, at least about 2-fold greater, at least about 2.5-fold greater, at least about 3-fold greater, at least about 3.5-fold greater, at least about 4-fold greater, at least about 4.5-fold greater, at least about 5-fold greater, at least about 10-fold greater, at least about 100-fold greater, at least about 500-fold greater, etc.) the dissociation constant of the polypeptide (e.g., an activatable polypeptide (i.e., an activatable antibody)) for its target.

Cleavable Moiety (CM)

In some embodiments, the disclosure relates to polynucleotides and/or polynucleotide libraries encoding one or more polypeptides comprising a Cleavable Moiety (CM). In some embodiments, the present disclosure relates to polypeptides and/or polypeptide libraries comprising at least one polypeptide comprising a Cleavable Moiety (CM).

In some embodiments, the Cleavable Moiety (CM) comprises at least a first Cleavage Site (CS)₁) (e.g., a first protease cleavage site). In some embodiments, the first cleavage site is a first protease cleavage site. Any suitable protease cleavage site recognized and/or cleaved by any protease known in the art (e.g., a protease known to be co-localized with the target of the CM-containing polypeptide) can be used, including, for example, protease cleavage sites recognized and/or cleaved by: urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and/or MMP-27); tobacco Etch Virus (TEV) protease; a fibrinolytic enzyme; thrombin; PSA; PSMA; ADAMS/ADAMTS (e.g., ADAM 8, ADAM 9, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM DEC1, ADAMTS1, ADAMTS4, and/or ADAMTS 5); caspase (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and/or caspase-14); aspartic proteases (e.g., RACE and/or Renin (Renin)); aspartic cathepsins (e.g., cathepsin D and/or cathepsin E); cysteine cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V/L2 and/or cathepsin X/Z/P); cysteine proteases (e.g., Cruzipain, Legumain, and/or Otubain-2); KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and/or KLK 14); metalloproteases (e.g., transmembrane peptidase (Meprin), Neprilysin, PSMA, and/or BMP-1); serine proteases (e.g. activated protein C, cathepsin a, cathepsin G, Chymase (chynase) and/or coagulation factor proteases such as FVIIa, FIXa, FXa, FXIa, FXIIa); elastase (elastase); granzyme B; guanidinobenzyme; HtrA 1; human neutrophil elastase; lactoferrin (lactoferrin); marapsin; NS 3/4A; PACE 4; tPA; tryptase (tryptase); type II transmembrane serine Protease (TTSP) (e.g., DESC1, DPP-4, FAP, Hepsin (Hepsin), Matriptase-2 (Matriptase-2), MT-SP 1/Matriptase, TMPRSS2, TMPRSS3, and/or TMPRSS 4); and the like. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from the group consisting of: uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from the group consisting of: uPA, MMP-2, MMP-9, and/or TEV protease. In some embodiments, the protease cleavage site comprises an amino acid sequence selected from SGRSA (SEQ ID NO:13), PLGLAG (SEQ ID NO:14), and ENLYFQG (SEQ ID NO: 15).

In some embodiments, the polypeptide comprising the First Peptide (FP) and the Cleavable Moiety (CM) comprises a peptide according to formula (VIII): the amino acid sequence of EVGSY (Z6) C (Z6) C (Z2) SGRSA (SEQ ID NO:4), wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the polypeptide comprising the First Peptide (FP) and the Cleavable Moiety (CM) comprises a peptide according to formula (IX): the amino acid sequence of EVGSY (Z6) C (X6) C (Z2) SGRSA (SEQ ID NO:5), wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the polypeptide comprising the First Peptide (FP) and the Cleavable Moiety (CM) comprises a peptide according to formula (X): the amino acid sequence of EVGSY (Z6) C (Z8) C (Z2) SGRSA (SEQ ID NO:6), wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the polypeptide comprising the First Peptide (FP) and the Cleavable Moiety (CM) comprises a peptide according to formula (XI): the amino acid sequence of EVGSY (Z6) C (X8) C (Z2) SGRSA (SEQ ID NO:7), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

In some embodiments, the Cleavable Moiety (CM) further comprises a first linker (L)₁). In some embodiments, the first linker (L)₁) At the first Cleavage Site (CS)₁) (e.g., the first protease cleavage site). In some embodiments, the Cleavable Moiety (CM) comprises the following structure from N-terminus to C-terminus: (CS)₁)-L₁。

Any suitable joint (e.g., flexible joint) known in the art may be used, including, for example: a glycine polymer (G) n, wherein n is an integer of at least 1 (e.g., at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.); glycine-serine polymers (GS) n, wherein n is an integer of at least 1 (e.g., at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as GGGGS (SEQ ID NO:17), SGGS (SEQ ID NO:18), GGSG (SEQ ID NO:19), GGSGG (SEQ ID NO:20), gsgsgsg (SEQ ID NO:21), GSGGG (SEQ ID NO:22), GGGSG (SEQ ID NO:23) and/or GSSSG (SEQ ID NO: 24); glycine-alanine polymer; alanine-serine polymers; and the like. The linker sequence can be of any length, such as about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymer or glycine-serine polymer), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, and the like. In some embodiments, the linker is any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 17-24. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO 17 or 18.

In some embodiments, the Cleavable Moiety (CM) further comprises at least a second cleavage site (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the Cleavable Moiety (CM) further comprises a second Cleavage Site (CS)₂). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site may be any suitable protease cleavage site recognized and/or cleaved by any of the proteases described above. In some embodiments, the first (CS)₁) And a second (CS)₂) A cleavage site is a protease cleavage site that is recognized and/or cleaved by the same protease. In some embodiments, the first (CS)₁) And a second (CS)₂) A cleavage site is a protease cleavage site recognized and/or cleaved by a different protease (e.g., a first protease cleavage site is recognized and/or cleaved by uPA, while a second protease cleavage site is recognized and/or cleaved by MMP-2; a first protease cleavage site is recognized and/or cleaved by uPA and a second protease cleavage site is recognized and/or cleaved by MMP-9; a first protease cleavage site is recognized and/or cleaved by uPA and a second protease cleavage site is recognized and/or cleaved by TEV protease; etc.). In some embodiments, at least a second Cleavage Site (CS) ₂) At the first joint (L)₁) The C-terminal of (1). In some embodiments, the Cleavable Moiety (CM) comprises the following structure from N-terminus to C-terminus: (CS)₁)-L₁-(CS₂)。

In some embodiments, the Cleavable Moiety (CM) further comprises at least a second linker (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the Cleavable Moiety (CM) further comprises a second linker (L)₂). Second joint (L)₂) May be any suitable linker as described above. In some embodiments, the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 17-24. In some embodiments, the first (L)₁) And second (a)L₂) The linkers are identical (e.g., both linkers comprise the sequence of SEQ ID NO:17 or 18). In some embodiments, the first (L)₁) And second (L)₂) The joints being different (e.g. first joint (L)₁) Comprising the amino acid sequence of SEQ ID NO 17, and a second linker (L)₂) Comprising the amino acid sequence of SEQ ID NO 18, etc.). In some embodiments, at least a second linker (L)₂) At the second Cleavage Site (CS)₂) The C-terminal of (1). In some embodiments, the Cleavable Moiety (CM) comprises the following structure from N-terminus to C-terminus: (CS)₁)-L₁-(CS₂)-L₂。

Exemplary FP-CM sequences

In some embodiments, the polypeptides of the present disclosure comprise the following structure from N-terminus to C-terminus: (FP) - (PCS) ₁)-L₁-(PCS₂)-L₂. In some embodiments, the polypeptides of the present disclosure comprise the following amino acid sequences: EVGSYDALHYACPPDYYACYYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 25); EVGSYNSYHAYCPHPLYPCTASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 26); EVGSYASSAVLCVTAYFSCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 27); EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 28); EVGSYNFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 29); EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 30); EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 31); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 32); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 33); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 34); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 35); EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 36); EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 37); EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 38); EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 39); EVGSYPTDLDACADAPNHCHFSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 40); EVGSYSSTHAHCHHSPANCISSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 41); EVGSYDTDYDFCPILRHRCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 42); EVGSYNDYNYHCKWRPSRCHNSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 43); EVGSYYHDYDDCRVLPRRCFNSGRS AGGGGSPLGLAGSGGS (SEQ ID NO: 44); EVGSYSNNFASCLWRHRSCADSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 45); and/or EVGSYTDNYDYCPRLRRKCYHSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 46). In some embodiments, the polypeptides of the present disclosure comprise the following structure from N-terminus to C-terminus: (FP) - (PCS)₁)-L₁-(PCS₂)-L₂-(TBM)。

Target Binding Moieties (TBM)

In some embodiments, the disclosure relates to polynucleotides and/or polynucleotide libraries encoding one or more polypeptides comprising a Target Binding Moiety (TBM). In some embodiments, the present disclosure relates to polypeptides and/or polypeptide libraries comprising at least one polypeptide comprising a Target Binding Moiety (TBM). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region and/or an antibody heavy chain variable region. In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region. In some embodiments, the Target Binding Moiety (TBM) comprises an antibody heavy chain variable region. In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region and an antibody heavy chain variable region. In some embodiments, the antibody heavy chain variable region is C-terminal to the antibody light chain variable region. In some embodiments, the antibody light chain variable region is C-terminal to the antibody heavy chain variable region. In some embodiments, a Target Binding Moiety (TBM) of the present disclosure comprises an antibody light chain variable region and/or an antibody heavy chain variable region specific for any target of interest including, for example, CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, and/or B7-H4.

In some embodiments, the Target Binding Moiety (TBM) comprises a full length antibody light chain and/or a full length antibody heavy chain. The antibody light chain may be a kappa or lambda light chain. Antibody heavy chains may be in any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is in the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclasses. The antibody heavy chains described herein may be converted from one class or subclass to another using methods known in the art.

Any one or more of the Target Binding Moieties (TBMs) described herein may incorporate PCT application number PCT/CN2017/098333 (incorporated herein by reference in its entirety), PCT application number PCT/CN2017/098299 (incorporated herein by reference in its entirety), PCT application number PCT/CN2017/098332 (incorporated herein by reference in its entirety), and/or "composition Comprising Cross-reactive Anti-CTLA4 Antibodies filed concurrently with attorney docket number 69540 and 2000540, and Methods of Making and Using the Same "of PCT application (incorporated herein by reference in its entirety), any of the HVR sequences of any of the antibodies described in the PCT application (e.g., one, two or three of the heavy chain variable region HVR sequences, and/or one, two or three of the light chain variable region HVR sequences), a heavy chain variable region sequence, and/or a light chain variable region sequence.

Any one or more of the Target Binding Moieties (TBMs) described herein may incorporate any HVR sequence (e.g., one, two, or three of the heavy chain variable region HVR sequences, and/or one, two, or three of the light chain variable region HVR sequences), heavy chain variable region sequence, and/or light chain variable region sequence of any antibody described herein (e.g., anti-CTLA 4 antibody, anti-CD 137 antibody).

In some embodiments, the Target Binding Moiety (TBM) comprises the sequence of one or more of the anti-CTLA 4 antibodies described herein, including antibodies described with respect to particular amino acid sequences of HVRs, variable regions (VL, VH) and/or light and heavy chains (e.g., IgG1, IgG2, IgG 4). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region comprising HVR-L1 comprising amino acid sequence RASQSVRGRFLA (SEQ ID NO:62), HVR-L2 comprising amino acid sequence DASNRATGI (SEQ ID NO:63), and/or HVR-L3 comprising amino acid sequence YCQQSSSWPPT (SEQ ID NO: 64). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the Target Binding Moiety (TBM) comprises an antibody heavy chain variable region comprising HVR-H1 comprising amino acid sequence YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and/or HVR-H3 comprising amino acid sequence ARSYVYFDY (SEQ ID NO: 61). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the Target Binding Moiety (TBM) comprises: a) an antibody light chain variable region comprising HVR-L1 comprising amino acid sequence RASQSVRGRFLA (SEQ ID NO:62), HVR-L2 comprising amino acid sequence DASNRATGI (SEQ ID NO:63), and/or HVR-L3 comprising amino acid sequence YCQQSSSWPPT (SEQ ID NO: 64); and b) an antibody heavy chain variable region comprising HVR-H1 comprising amino acid sequence YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and/or HVR-H3 comprising amino acid sequence ARSYVYFDY (SEQ ID NO: 61). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:48 and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47.

In some embodiments, the Target Binding Moiety (TBM) comprises the sequence of one or more of the anti-CD 137 antibodies described herein, including antibodies described for particular amino acid sequences of HVRs, variable regions (VL, VH), and/or light and heavy chains (e.g., IgG1, IgG2, IgG 4). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region comprising HVR-L1 comprising amino acid sequence RASQSIGSYLA (SEQ ID NO:68), HVR-L2 comprising amino acid sequence DASNLETGV (SEQ ID NO:69), and/or HVR-L3 comprising amino acid sequence YCQQGYYLWT (SEQ ID NO: 70). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the Target Binding Moiety (TBM) comprises an antibody heavy chain variable region comprising HVR-H1 comprising amino acid sequence FSLSTGGVGVGWI (SEQ ID NO:65), HVR-H2 comprising amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and/or HVR-H3 comprising amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 67). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49. In some embodiments, the Target Binding Moiety (TBM) comprises: a) an antibody light chain variable region comprising HVR-L1 comprising amino acid sequence RASQSIGSYLA (SEQ ID NO:68), HVR-L2 comprising amino acid sequence DASNLETGV (SEQ ID NO:69), and/or HVR-L3 comprising amino acid sequence YCQQGYYLWT (SEQ ID NO: 70); and b) an antibody heavy chain variable region comprising HVR-H1 comprising amino acid sequence FSLSTGGVGVGWI (SEQ ID NO:65), HVR-H2 comprising amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and/or HVR-H3 comprising amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 67). In some embodiments, the Target Binding Moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:50 and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49.

Polypeptides and polypeptide libraries

Other aspects of the disclosure relate to polypeptides (e.g., any of the polypeptides described herein) and/or libraries of polypeptides that can be used to screen, identify and/or select one or more activatable binding polypeptides (i.e., one or more activatable antibodies), including activatable antibodies, activatable antigen-binding fragments thereof, or derivatives of activatable antibodies. The libraries of the present disclosure may contain one or more of the polypeptides described herein (e.g., one or more activatable binding polypeptides). In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the libraries described herein comprise one or more antigen binding domains. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the libraries described herein comprise one or more full length antibody light and/or heavy chains. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the libraries described herein comprise one or more light chain and/or heavy chain Fab fragments. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the libraries described herein comprise one or more single chain variable fragments (scfvs). In some embodiments, the polypeptide is expressed on the surface of a cell (e.g., displayed by a yeast or mammalian cell).

In some embodiments, a polypeptide of the disclosure (e.g., in a library) comprises: (a) a First Peptide (FP); (b) a Cleavable Moiety (CM); and (c) a Target Binding Moiety (TBM). In some embodiments, FP is any first peptide described herein (e.g., comprising X according to formula (XIII): X)_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y). In some embodiments, FP is any first peptide described herein (e.g., comprising X according to formula (I): X)_mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P). In some embodiments, m is 3 to 10. In some embodiments, the CM is any cleavable moiety described herein (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site). In some embodiments, the TBM is any target binding moiety described herein (e.g., a Target Binding Moiety (TBM) comprising an antibody light chain variable region and/or an antibody heavy chain variable region).

In some embodiments, provided herein is an antigen binding domain and/or a library comprising antigen binding domains, wherein at least one (e.g., one, some, or all) of the antigen binding domains comprise a polypeptide of the present disclosure. In some embodiments, at least one (e.g., one, some, or all) of the antigen binding domains comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (XIII): X_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, SAmino acids of T, V, W and Y); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety comprising an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one (e.g., one, some, or all) of the antigen binding domains comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (I): X _mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety comprising an antibody light chain variable region. In some embodiments, the antigen binding domain further comprises an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one (e.g., one, some, or all) of the antigen binding domains comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (XIII): X_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety comprising an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one (e.g., one, some, or all) of the antigen binding domains comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (I): X _mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, each X is independently selected from AAmino acids of C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety comprising an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the antigen binding domain further comprises an antibody light chain variable region.

In some embodiments, also provided herein is an antibody fragment or scFv comprising any of the polypeptides described herein. In some embodiments, the antibody fragment or scFv comprises a polypeptide comprising a Target Binding Moiety (TBM) comprising an antibody light chain variable region. In some embodiments, the antibody fragment or scFv comprises a polypeptide comprising a Target Binding Moiety (TBM) comprising an antibody heavy chain variable region. In some embodiments, provided herein is a library having antibody fragments or scfvs, wherein at least one of the antibody fragments or scfvs comprises any of the polypeptides described herein. In some embodiments, at least one (e.g., one, some, or all) of the antibody fragments or scfvs in the library comprise a polypeptide comprising a Target Binding Moiety (TBM) comprising an antibody light chain variable region. In some embodiments, at least one (e.g., one, some, or all) of the antibody fragments or scfvs in the library comprise a polypeptide comprising a Target Binding Moiety (TBM) comprising an antibody heavy chain variable region. Also provided herein are cells and/or libraries of cells that express on their surface one or more of the antibody fragments and/or scfvs described herein.

In some embodiments, the disclosure relates to an antibody light chain comprising a polypeptide of the disclosure. In some embodiments, the antibody light chain comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (XIII): X_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, WAnd amino acids of Y); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the antibody light chain comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (I): X_mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a Target Binding Moiety (TBM) comprising an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the disclosure relates to a library comprising antibody light chains, wherein at least one (e.g., one, some, or all) of the antibody light chains in the library are antibody light chains as described above. In some embodiments, the present disclosure relates to an antibody comprising an antibody light chain and an antibody heavy chain, wherein the antibody light chain is an antibody light chain as described above. In some embodiments, the antibody heavy chain is any antibody heavy chain known in the art (including any antibody heavy chain described herein). In some embodiments, the disclosure relates to a library comprising antibodies, wherein at least one (e.g., one, some, or all) of the antibodies are antibodies as described above.

In some embodiments, the disclosure relates to an antibody heavy chain comprising a polypeptide of the disclosure. In some embodiments, an antibody heavy chain comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (XIII): X_mCX_nCX_o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, TAmino acids of V, W and Y); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a Target Binding Moiety (TBM) comprising an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, an antibody heavy chain comprises a polypeptide comprising from N-terminus to C-terminus: (a) a First Peptide (FP) (e.g., comprising X according to formula (I): X_mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P); (b) a cleavable moiety (e.g., a Cleavable Moiety (CM) comprising at least a first protease cleavage site); and (c) a Target Binding Moiety (TBM) comprising an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the disclosure relates to a library comprising antibody heavy chains, wherein at least one (e.g., one, some, or all) of the antibody heavy chains in the library are antibody heavy chains as described above. In some embodiments, the present disclosure relates to an antibody comprising an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain is an antibody heavy chain as described above. In some embodiments, the antibody light chain is any antibody light chain known in the art (including any antibody light chain described herein). In some embodiments, the disclosure relates to a library comprising antibodies, wherein at least one (e.g., one, some, or all) of the antibodies are antibodies as described above.

Polypeptides of the disclosure (e.g., any of the antibodies described above) can be produced using, for example, recombinant methods and compositions as described in U.S. patent No. 4,816,567. In some embodiments, isolated nucleic acids encoding any polypeptide (e.g., any of the antibodies described above) are provided. The nucleic acid may encode V comprising an antibody_LAnd/or a V comprising an antibody_HE.g., the light chain and/or heavy chain of an antibody. In some embodiments, provided herein is one or moreA plurality of vectors (e.g., expression vectors) comprising the nucleic acids. In some embodiments, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) vector comprising a nucleic acid encoding a polypeptide comprising V_LThe amino acid sequence of a polypeptide of the present disclosure and comprising V_H(ii) an amino acid sequence (e.g., an activatable binding polypeptide (i.e., an activatable antibody)), (2) a vector comprising a nucleic acid encoding a polypeptide comprising a V_HThe amino acid sequence of a polypeptide of the present disclosure and comprising V_L(3) comprises a sequence encoding a polypeptide comprising a V, and (3) a polypeptide comprising a polypeptide having an amino acid sequence of (e.g., an activatable binding polypeptide (i.e., an activatable antibody)), (ii) a polypeptide comprising an amino acid sequence of (e.g., an activatable binding polypeptide), and (iii) a polypeptide comprising _LA first vector comprising a nucleic acid encoding an amino acid sequence comprising a polypeptide of the present disclosure, and a second vector comprising a nucleic acid encoding a polypeptide comprising V_HA second vector comprising a nucleic acid comprising the amino acid sequence of (a) (e.g., an activatable binding polypeptide (i.e., an activatable antibody)), or (4) a vector comprising a nucleic acid comprising an amino acid sequence of (a) a_HA first vector comprising a nucleic acid encoding an amino acid sequence comprising a polypeptide of the present disclosure, and a second vector comprising a nucleic acid encoding a polypeptide comprising V_LA second carrier for the nucleic acid of (e.g., an activatable binding polypeptide (i.e., an activatable antibody)). In some embodiments, the host cell is eukaryotic, such as a yeast cell, an insect cell, a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a method of making a polypeptide, e.g., an activatable binding polypeptide (i.e., an activatable antibody), is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the polypeptide, e.g., an activatable binding polypeptide (i.e., an activatable antibody), provided above under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide, e.g., an activatable binding polypeptide (i.e., an activatable antibody), from the host cell (or host cell culture medium).

For recombinant production of a polypeptide of the disclosure (e.g., an activatable binding polypeptide (i.e., an activatable antibody)), a nucleic acid encoding, for example, a polypeptide as described above (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. The nucleic acids can be readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to genes encoding one or more polypeptides.

Suitable host cells for cloning or expressing a vector encoding a polypeptide, e.g., encoding an activatable binding polypeptide (i.e., an activatable antibody), include prokaryotic or eukaryotic cells. For example, polypeptides such as activatable binding polypeptides (i.e., activatable antibodies) can be produced in bacteria, particularly when glycosylation and Fc effector function are not required (see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; see also Charlton, Methods in Molecular Biology, Vol.248 (coded by B.K.C.Los., Humana Press, Totowa, NJ,2003), pp.245-. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable for polypeptide encoding (e.g., can activate binding polypeptides (i.e., can activate antibody) encoding vectors cloning or expression hosts, including fungi and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of polypeptides with a partially or fully human glycosylation pattern. See Gerngross, nat. Biotech.22: 1409-.

Host cells suitable for expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIIES for antibody production in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney transformed by SV40CV1 strain (COS-7); human embryonic kidney lines (293 or 293 cells, as described, e.g., in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR ^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (edited by B.K.C.Lo, Humana Press, Totowa, NJ), pp.255-268 (2003).

Activatable binding polypeptides and their production

In some embodiments, provided herein are activatable binding polypeptides (e.g., activatable antibodies) screened, identified and/or selected from any of the polynucleotide and/or polypeptide libraries described herein.

In some embodiments, the activatable antibodies of the present disclosure are situation-dependent (e.g., are activated (are only able to bind their target) in certain situations, such as in a protease-rich tumor microenvironment). In some embodiments, the activatable antibodies of the present disclosure provide improved safety (e.g., show reduced toxicity, do not induce significant changes in the weight of many organs, do not alter liver histopathology, hematology, and/or blood biochemistry, etc.) over more traditional non-activatable antibodies. In some embodiments, the activatable antibodies of the present disclosure have improved pharmacokinetic properties (e.g., have a longer in vivo half-life) compared to more traditional non-activatable antibodies.

In some embodiments, an activatable binding polypeptide of the disclosure comprises: (a)a First Peptide (FP) (e.g., a masking moiety), (b) a cleavable moiety, and (c) a target-binding moiety. In some embodiments, the First Peptide (FP) binds a Target Binding Moiety (TBM) that can activate the binding domain and reduces or inhibits binding of the activatable binding moiety to its target (e.g., human CTLA4 or human CD137) as compared to binding of a corresponding binding polypeptide to the target lacking the masking moiety and/or as compared to binding of the parent antibody to the target. In some embodiments, prior to activation, the masking portion (MM) has a masking efficiency of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.). In some embodiments, the masking efficiency is measured as the difference in the affinity of an activatable antibody comprising a Masking Moiety (MM) to bind its target (prior to activation) relative to the affinity of a polypeptide lacking a masking moiety to bind its target (e.g., the difference in the affinity of an activatable antibody comprising a Masking Moiety (MM) (prior to activation) relative to a parent antibody lacking a Masking Moiety (MM), to a target antigen such as CTLA4 or CD137, or the difference in the affinity of an activatable antibody comprising a Masking Moiety (MM) (prior to activation) to a target antigen such as CTLA4 or CD137 relative to the affinity of the activatable antibody to the target antigen after activation). In some embodiments, masking efficiency is through the binding of EC with an activatable antibody comprising a Masking Moiety (MM) ₅₀(prior to activation) divided by the EC of the parent antibody₅₀To measure (e.g. by measuring EC with ELISA)₅₀(ii) a See, e.g., the method of example 3). In some embodiments, the masking efficiency is measured as the difference in affinity of an activatable antibody comprising a Masking Moiety (MM) to bind its target prior to activation relative to the affinity of an activatable antibody comprising a Masking Moiety (MM) to bind its target after activation (e.g., the difference in affinity for a target antigen of an activatable antibody prior to activation relative to the activatable antibody after activation). In some embodiments, the Masking Moiety (MM) binds to the target binding moietyAnd (TBM) and prevents the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody).

In some embodiments, an "activatable" binding polypeptide refers to a binding polypeptide that: exhibit a first level of binding to a target when in an inhibited, masked, and/or unlysed state, and exhibit a second level of binding to the target when in an uninhibited, unmasked, and/or lysed state, wherein the second level of target binding is greater than the first level of target binding. In some embodiments, the chance that the activatable binding polypeptide will be in proximity to the target is increased after cleavage (e.g., by one or more proteases) within the cleavable moiety.

In some embodiments, the binding affinity of a polypeptide to its target (e.g., human CTLA4 or CD137) is increased at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, after activation by a change in pH (increase or decrease), after activation by contact with a second molecule, such as a small molecule or protein ligand, etc.) when compared to before activation of the polypeptide, At least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold or more), the polypeptides of the disclosure are generally considered to be "activatable" binding polypeptides. In some embodiments, the EC of a polypeptide if following "activation" is used ₅₀Reduced to at least about 1/2 (e.g., at least about 1/2, at least about 1/2.5, at least about 1/3, at least about 1/3.5, at least about 1/4, at least about 1/4.5, at least about 1/5, at least about 1/5.5, at least about 1/6, at least about 1/6.5, at least about 1/7, at least about 1/7.5, at least about 1/8, at least about 1/8.5, at least about 1/9, at least about 1/9.5, at least about 1/10, at least about 1/2, at least about 1/2.5, at least about 3978, etcAbout 1/25, at least about 1/50, at least about 1/75, at least about 1/100, at least about 1/250, at least about 1/500, at least about 1/750 or at least about 1/1000, or reduced to less) (e.g., as measured by ELISA or FACS assays; see examples below), then the polypeptides of the disclosure are generally considered "activatable. In some embodiments, the EC of the polypeptide if after treatment with a protease that cleaves within the cleavable moiety₅₀To at least about 1/2 (e.g., as measured by ELISA or FACS assays; see examples below), then the polypeptides of the disclosure are generally considered "activatable".

In some embodiments, when the masking moiety binds to the target binding moiety of the activatable binding polypeptide, the K of the activatable binding polypeptide with respect to its target_DIs when the masking moiety is not bound to the target-binding moiety (e.g., after "activation" of the activatable binding polypeptide (such as after protease treatment to effect cleavage within the cleavable moiety)) and/or is the K of the parent antibody with respect to the target _DAbout 2 (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) times greater. Methods of measuring affinity are known in the art, including, for example, by the method described in example 3 below.

In some embodiments, K for the target relative to the parent antibody, relative to when the masking moiety is not bound to the target-binding moiety (e.g., after "activation" of the activatable binding polypeptide, such as after protease treatment to effect cleavage within the cleavable moiety), and/or relative to the parent antibody_D(ii) when the masking moiety binds to the target binding moiety of the activatable binding polypeptide, K of the activatable binding polypeptide with respect to its target_DBy at least about 25% (e.g., at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%). Methods of measuring affinity are known in the art, including, for example, by the method described in example 3 below.

In some embodiments, the masking moiety sterically hinders binding of the activatable binding polypeptide to its target, and/or allosterically hinders binding of the activatable binding polypeptide to its target. In some embodiments, the masking moiety does not comprise an amino acid sequence that can activate a natural binding partner of the binding polypeptide.

In some embodiments, the dissociation constant of the masking moiety for the target-binding moiety is greater than the dissociation constant of the activatable binding polypeptide for the target (when in an active, activated form). In some embodiments, the dissociation constant of the masking moiety with respect to the target-binding moiety is greater than about 2 (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) times the dissociation constant of the activatable binding polypeptide with respect to the target (when in activated form). In some embodiments, the dissociation constant of the masking moiety with respect to the target-binding moiety is about equal to the dissociation constant of the activatable binding polypeptide with respect to the target (when in the activated form). In some embodiments, the First Peptide (FP) binds to the Target Binding Moiety (TBM) and prevents the polypeptide from binding to its target only when the polypeptide has not been activated (e.g., activated by treatment with one or more proteases that cleave within a Cleavable Moiety (CM), activated by a change in pH (increase or decrease), activated by a change in temperature (increase or decrease), activated after contact with a second molecule such as a small molecule or protein ligand, etc.). In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces a conformational change in the polypeptide (e.g., a shift in the First Peptide (FP)) such that the first peptide no longer prevents the polypeptide from binding its target.

The activatable binding polypeptides described herein (i.e., activatable antibodies) may be further modified. In some embodiments, the activatable binding polypeptide is attached to an additional molecular entity. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or labels, and antibodies.

In some casesIn embodiments, the activatable binding polypeptides of the disclosure are linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic or other cancer therapeutic agents and radioisotopes. Specific examples of cytotoxic agents include paclitaxel (taxol), cytochalasin b (cytochalasin b), gramicidin d (gramicidin d), ethidium bromide (ethidium bromide), emetine (emetine), mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine (vincristine), vinblastine (vinblastatin), colchicine (colchicin), doxorubicin (doxobicin), daunorubicin (daunorubicin), dihydroxy anthrax dione (dihydroanthracin dione), mitoxantrone (mitoxantrone), milaxomycin (hramycin), actinomycin d (practicin d), 1-dehydrotestosterone (1-desogestristone), glucocorticoid (glucodecorin), procaine (procaine), and analogs of procaine (procaine) and procaine. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate (methotrexate), 6-mercaptopurine (6-mercapture), 6-thioguanine (6-thioguanine), cytarabine (cytarabine), 5-fluorouracil (5-fluorouracil), dacarbazine (decarbazine), alkylating agents (e.g., dichloromethyldiethylamine (meclorethamine), thiotepa (thiopea), chlorambucil (chlorambucil), melphalan (melphalan), carmustine (carmustine) (BSNU) and lomustine (CCNU), cyclophosphamide (cytochalamide), busulfan (busufan), dibromomannitol (dibromonitol), streptozocin (streptozocin), mitomycin (mitomycin C) and doxorubicin (doxorubicin) (e.g., doxorubicin (DDP), doxorubicin (DDP), and doxorubicin (DDP) (e.g., doxorubicin (gentamicin), and doxorubicin (DDP) (e), and doxorubicin (DDP) (e) (e.g., gentamycin (gentamycin) (CCNU (gentamycin)),) and doxorubicin (gentamicin (doxorubicin) (e.g., doxorubicin (gentamycin) (gentamycin (doxorubicin) (DDP) (gentamycin) (gentamycin (doxorubicin) (gentamycin) (MCP) (gentamycin) (e) and doxorubicin, Bleomycin (bleomycin), mithramycin (mithramycin) and Antromycin (AMC), and antimitotic agents such as vincristine and vinblastine. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ¹³¹Indium, indium¹¹¹Yttrium, yttrium⁹⁰And lutetium¹⁷⁷. Methods for attaching polypeptides to pharmaceutical agents are known in the art, such as using various linker techniques. Examples of linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. For a further discussion of linkers and methods for linking therapeutic agents to antibodies, see, e.g., Saito et al, adv. drug Deliv. Rev.55:199-215 (2003); trail, et al, Cancer Immunol.Immunother.52:328-337 (2003); payne, Cancer Cell 3: 207-; allen, nat. Rev. cancer 2: 750-; pastan and Kreitman, curr. Opin. Investig. drugs 3: 1089-; senter and Springer (2001) adv. drug Deliv. Rev.53: 247-264.

Activatable binding polypeptides targeting CTLA4

In some embodiments, the disclosure relates to activatable binding polypeptides (i.e., activatable antibodies) that bind to human CTLA4, including activatable anti-CTLA 4 antibodies, activatable antigen-binding fragments of anti-CTLA 4 antibodies, and/or activatable derivatives of anti-CTLA 4 antibodies. In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a Masking Moiety (MM), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the MM comprises a peptide according to formula (I): x _mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH). In some embodiments, m is 3 to 10. In some embodiments, MM inhibits binding of the activatable antibody to human CTLA4 when the CM is not cleaved. In some embodiments, the activatable antibody is capable of binding to human CTLA4 when CM is cleaved. In some embodiments, the MM comprises an amino acid sequence selected from SEQ ID NOS 72-78, as listed in Table A.

Table a masking peptide sequences of activatable antibodies

In some embodiments, the activatable binding polypeptide comprises any of the anti-CTLA 4 antibodies described herein, including antibodies described with respect to particular amino acid sequences of HVRs, variable regions (VL, VH), and/or light and heavy chains (e.g., IgG1, IgG2, IgG 4). In some embodiments, the anti-CTLA 4 antibody is a human antibody. In some embodiments, the anti-CTLA 4 antibody is a humanized and/or chimeric antibody.

In some embodiments, the activatable binding polypeptide comprises: a) HVR-H1 comprising amino acid sequence YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and HVR-H3 comprising amino acid sequence ARSYVYFDY (SEQ ID NO: 61); and/or b) HVR-L1 comprising amino acid sequence RASQSVRGRFLA (SEQ ID NO:62), HVR-L2 comprising amino acid sequence DASNRATGI (SEQ ID NO:63), and HVR-L3 comprising amino acid sequence YCQQSSSWPPT (SEQ ID NO: 64). In some embodiments, the activatable binding polypeptide comprises: a) 47 or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO; and/or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO 48.

In some embodiments, the disclosure relates to activatable binding polypeptides that bind human CTLA4 when in active form (e.g., the activatable binding polypeptide is active after cleavage in a cleavable moiety (e.g., with one or more proteases), but inactive before cleavage in a cleavable moiety (e.g., with one or more proteases)) and have at least one of the following functional properties (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine): (a) at a K of 500nM or less _DCombined human and cynomolgus monkeyMouse, rat and/or dog CTLA 4; (b) has antagonistic activity against human CTLA 4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS and/or B7-H4 at concentrations up to 100 nM; (d) CTLA4 cross-reactive with monkeys, mice, rats, and/or dogs; (e) induction of ADCC effects (e.g. on tregs); (f) activating human PBMCs (e.g., stimulating secretion of IL-2 and/or IFN γ); (g) can inhibit tumor cell growth; (h) has therapeutic effect on cancer; and (i) inhibits binding of human CTLA4 to human CD80 and/or human CD 86. Also provided herein are one or more activatable binding polypeptides that cross-compete with one or more of the CTLA 4-targeting activatable binding polypeptides and/or anti-CTLA 4 antibodies described herein for binding to human CTLA 4.

In some embodiments, the activatable binding polypeptide, when in an inactive form, has a K of about 500nM or greater_DBind to human, cynomolgus monkey, mouse, rat and/or dog CTLA 4. In some embodiments, when in an active form, a binding polypeptide can be activated with a K of about 500nM or less (e.g., about 500nM or less, about 450nM or less, about 400nM or less, about 350nM or less, about 300nM or less, about 250nM or less, about 200nM or less, about 150nM or less, about 100nM or less, about 90nM or less, about 80nM or less, about 70nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 25nM or less, about 20nM or less, about 10nM or less, about 1nM or less, about 0.1nM or less, etc.) _DBind to human, cynomolgus monkey, mouse, rat and/or dog CTLA 4. In some embodiments, the activatable binding polypeptide, when in an active form, has a K of about 350nM or less_DBind to human, cynomolgus monkey, mouse, rat and/or dog CTLA 4. In some embodiments, the activatable binding polypeptide, when in an active form, has a K of about 100nM or less_DBinds to human CTLA 4. In some embodiments, the activatable binding polypeptide, when in an active form, has a K of about 50nM or less_DBinds to human CTLA 4. In some embodiments, the activatable binding polypeptide, when in an active form, has a K of about 10nM or less_DBinds to human CTLA 4. Measurement of K for activatable binding polypeptides_DThe method of (a) can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like. In some embodiments, K_DMeasured by ELISA (see, e.g., example 3 below).

In some embodiments, the activatable binding polypeptide, when in an inactive form, has no antagonistic activity against human CTLA 4. In some embodiments, the activatable binding polypeptide, when in an active form, has antagonistic activity against human CTLA4 (e.g., induces ADCC effects (such as on tregs), activates PBMCs (such as by activating, inducing, and/or stimulating IL-2 and/or IFN γ secretion), blocks binding of human CTLA4 to human CD80 and/or human CD86, etc.). In some embodiments, the activatable binding polypeptide, when in active form, blocks one or more activities of human CTLA4 (e.g., blocks one or more activities of human CTLA4 when a cell, such as a human cell, expressing human CTLA4 is contacted by the activated activatable binding polypeptide).

In some embodiments, the activatable binding polypeptide, when in an inactive form, is not cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and/or dog CTLA 4. In some embodiments, the activatable binding polypeptide, when in active form, is cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and/or dog CTLA 4. In some embodiments, the activatable binding polypeptide, when in active form, is cross-reactive with monkey CTLA 4. In some embodiments, the activatable binding polypeptide, when in active form, is cross-reactive with mouse CTLA 4. In some embodiments, the activatable binding polypeptide, when in active form, is cross-reactive with rat CTLA 4. In some embodiments, the activatable binding polypeptide, when in active form, is cross-reactive with canine CTLA 4. In some embodiments, when in active form, the binding polypeptide can be activated to bind to mouse CTLA 4; monkey and rat CTLA 4; monkey and dog CTLA 4; mouse and rat CTLA 4; mouse and dog CTLA 4; rat and dog CTLA 4; monkey, mouse and rat CTLA 4; monkey, mouse and dog CTLA 4; monkey, rat and dog CTLA 4; mouse, rat, and dog CTLA 4; or monkey, mouse, rat and dog CTLA 4. In some embodiments, the activatable binding polypeptide, when in an active form, is cross-reactive at about 350nM (e.g., at about 1nM, at about 10nM, at about 25nM, at about 50nM, at about 75nM, at about 100nM, at about 150nM, at about 200nM, at about 250nM, at about 300nM, at about 350 nM). Methods of measuring cross-reactivity are known in the art and include, without limitation, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like.

In some embodiments, the activatable binding polypeptide, when in an inactive form, does not induce ADCC effects (e.g., on human cells such as tregs). In some embodiments, the activatable binding polypeptide has reduced ADCC effect (e.g., on human cells such as tregs) when in an inactive form as compared to a control binding polypeptide (e.g., a parent antibody lacking the First Peptide (FP) and the Cleavable Moiety (CM)). In some embodiments, the activatable antibody, when in an active form, induces ADCC effects (e.g., on human cells such as tregs). Methods of measuring ADCC effects (e.g., in vitro methods) are known in the art, including, but not limited to, by the methods described in example 4 below. In some embodiments, the activatable binding polypeptide, when in an inactive form, induces an ADCC effect less than about 10% (e.g., induces ADCC less than about 10%, less than about 5%, less than about 1%, etc.) relative to a control (e.g., a parent antibody lacking the First Peptide (FP) and the Cleavable Moiety (CM)). In some embodiments, the activatable binding polypeptide, when in active form, induces an ADCC effect by more than about 10% (e.g., induces ADCC by more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, etc.) relative to a control (e.g., isotype control).

In some embodiments, the activatable binding polypeptide is capable of inhibiting tumor cell growth and/or proliferation. In some embodiments, tumor cell growth and/or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with the activatable binding polypeptide relative to a corresponding tumor cell not contacted with the activatable binding polypeptide (or relative to a corresponding tumor cell contacted with an isotype control antibody). In some embodiments, the activatable binding polypeptide is capable of causing a reduction in tumor volume in a subject when the activatable binding polypeptide is administered to the subject. In some embodiments, the activatable binding polypeptide is capable of reducing the tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the initial tumor volume in the subject (e.g., prior to administration of the activatable binding polypeptide; compared to a corresponding tumor in a subject administered an isotype control antibody). Methods of monitoring tumor cell growth/proliferation, tumor volume, and/or tumor inhibition are known in the art, including, for example, by the methods described in example 4 below.

In some embodiments, the activatable binding polypeptide has a therapeutic effect on cancer. In some embodiments, the binding polypeptide can be activated to reduce one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer is partially or completely alleviated when administered an activatable binding polypeptide.

In some embodiments, the present disclosure provides isolated activatable binding polypeptides that, when in active form, compete or cross-compete for binding to human CTLA4 with an antibody comprising: a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 59; HVR-H2 comprising the amino acid sequence of SEQ ID NO 60; and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 61; and/or b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 62; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the present disclosure provides isolated activatable binding polypeptides that, when in active form, compete or cross-compete for binding to human CTLA4 with an antibody comprising: a) 47 or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO; and/or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO 48. The ability of an activatable binding polypeptide to compete or cross-compete for binding with an antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, an antibody (e.g., as described above) can be allowed to bind to human CTLA4 under saturating conditions, followed by measuring the ability of the test to activate the binding polypeptide (when in active form) to bind to CTLA 4. If the test activatable binding polypeptide is capable of binding CTLA4 simultaneously with an antibody, the test activatable binding polypeptide binds to a different epitope than the antibody. However, if the test activatable binding polypeptide is not capable of simultaneously binding CTLA4, then the test activatable binding polypeptide binds to the same epitope, an overlapping epitope, or an epitope in close proximity to the epitope bound by the antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS or surface plasmon resonance.

In some embodiments, the activatable binding polypeptide (when in an inactive form) does not inhibit binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD 86). In some embodiments, the activatable binding polypeptide (when in active form) inhibits binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD 86). In some embodiments, the activatable binding polypeptide inhibits binding between CTLA4 and its ligand in vitro. In some embodiments, the activatable binding polypeptide has a half maximal Inhibitory Concentration (IC) of about 500nM or less (e.g., about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 100nM or less, about 50nM or less, about 25nM or less, about 10nM or less, about 1nM or less, etc.) for inhibiting binding of CTLA4 to CD80 and/or CD86₅₀). In some embodiments, the activatable binding polypeptide has a half maximal Inhibitory Concentration (IC) of about 100nM or less for inhibiting binding of CTLA4 to CD80 and/or CD86₅₀). In some embodiments, the activatable binding polypeptide is completely inhibited when provided at a concentration of about 100nM or greater (e.g., about 100nM or greater, about 500nM or greater, about 1 μ M or greater, about 10 μ M or greater, etc.) Binding of human CTLA4 to CD80 and/or CD 86. As used herein, the term "complete inhibition" refers to an activatable binding polypeptide that is capable of reducing binding between a first protein and a second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods of measuring the ability of a polypeptide to inhibit binding of a first protein (e.g., human CTLA4) and a second protein (e.g., human CD80 or human CD86) are known in the art, including without limitation by BIAcore analysis, ELISA assays, and flow cytometry.

Activatable binding polypeptides targeting CD137

In some embodiments, the disclosure relates to activatable binding polypeptides that bind to human CD137 (i.e., activatable antibodies), including activatable anti-CD 137 antibodies, antigen-binding fragments of activatable anti-CD 137 antibodies, and/or derivatives of activatable anti-CD 137 antibodies. In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a Masking Moiety (MM), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the MM comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH). In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a Masking Moiety (MM), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the MM comprises a peptide according to formula (I): x _mCX_nCZ_o(SEQ ID NO:1) wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y(ii) the amino acids of group, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein the TBM comprises an antibody light chain variable region (VH); and (b) an antibody heavy chain variable region (VL). In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a Masking Moiety (MM), a Cleavable Moiety (CM), and a target-binding moiety (TBM), wherein the MM comprises a peptide according to formula (I): x_mCX_nCZ_o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, MM inhibition may activate binding of the antibody to human CD137 when the CM is not cleaved. In some embodiments, the activatable antibody is capable of binding to human CD137 when the CM is cleaved. In some embodiments, the MM comprises an amino acid sequence selected from SEQ ID NOS 79-85 and 88-94, as set forth in Table B.

Masking peptide sequences of activatable antibodies

m is 2 to 10; and each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

In some embodiments, the activatable binding polypeptide comprises any of the anti-CD 137 antibodies described herein, including antibodies described for particular amino acid sequences of HVRs, variable regions (VL, VH), and/or light and heavy chains (e.g., IgG1, IgG2, IgG 4). In some embodiments, the anti-CD 137 antibody is a human antibody. In some embodiments, the anti-CD 137 antibody is a humanized and/or chimeric antibody.

In some embodiments, the activatable binding polypeptide comprises: a) HVR-H1 comprising amino acid sequence FSLSTGGVGVGWI (SEQ ID NO:65), HVR-H2 comprising amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and HVR-H3 comprising amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 67); and/or b) HVR-L1 comprising amino acid sequence RASQSIGSYLA (SEQ ID NO:68), HVR-L2 comprising amino acid sequence DASNLETGV (SEQ ID NO:69), and HVR-L3 comprising amino acid sequence YCQQGYYLWT (SEQ ID NO: 70). In some embodiments, the activatable binding polypeptide comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:49 or a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 49; and/or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:50 or a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 50.

In some embodiments, the disclosure relates to activatable binding polypeptides that, when in an active form (e.g., the activatable binding polypeptide is active after cleavage in a cleavable moiety (e.g., with one or more proteases), but is inactive prior to cleavage in a cleavable moiety (e.g., with one or more proteases)), bind human CD137 and have at least one of the following functional properties (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least sevenOr all eight): (a) at a K of 500nM or less_DBinds to human CD 137; (b) has agonistic activity on human CD 137; (c) does not bind to human OX40, CD40, GITR and/or CD27 receptors at concentrations up to 1000 nM; (d) (ii) is cross-reactive with monkey, mouse, rat and/or dog CD 137; (e) does not induce ADCC effect; (f) can inhibit tumor cell growth; (g) has therapeutic effect on cancer; and (h) inhibiting the binding between CD137 and CD 137L. In some embodiments, the activatable binding polypeptides disclosed herein may also inhibit, e.g., completely inhibit, the binding between CD137 and its ligand CD 137L. Also provided herein are one or more activatable-binding polypeptide anti-CD 137 antibodies or antigen-binding fragments that cross-compete with one or more of the CD 137-targeted activatable binding polypeptides and/or anti-CD 137 antibodies described herein for binding to human CD 137.

In some embodiments, the activatable binding polypeptide (when in an inactive form) has a K of about 500nM or greater_DBinds to human CD 137. In some embodiments, a binding polypeptide (when in an active form) can be activated with a K of about 500nM or less (e.g., about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 150nM or less, about 100nM or less, about 90nM or less, about 80nM or less, about 75nM or less, about 70nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 25nM or less, about 20nM or less, about 10nM or less, about 1nM or less, about 0.1nM or less, etc.)_DBinds to human CD 137. In some embodiments, the activatable binding polypeptide has a K of about 100nM or less_DBinds to human CD 137. In some embodiments, the activatable binding polypeptide has a K of about 50nM or less_DBinds to human CD 137. Measurement of K for activatable binding polypeptides_DThe method of (a) can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like. In some embodiments, K_DMeasured by ELISA (see, e.g., example 5 below).

In some embodiments, an activatable binding polypeptide described herein (when in active form) has agonist activity to human CD 137. In some embodiments, the activatable binding polypeptide induces one or more (e.g., one or more, two or more, three or more, etc.) activities of human CD137 when a cell expressing human CD137 (e.g., a human cell) is contacted by the (activity) activatable binding polypeptide. Various CD137 activities are known in the art and may include, without limitation, induction of NF- κ B-dependent transcription, induction of T cell proliferation, prolongation of T cell survival, co-stimulation of activated T cells, induction of cytokine secretion (such as IL-2), and induction of monocyte activation. In some embodiments, the one or more CD137 activities are not binding of CD137 to its ligand. Methods for measuring CD137 activity (e.g., induction of NF- κ B-dependent transcription and/or T cell proliferation, etc.) are known in the art. In some embodiments, the binding polypeptide can be activated to increase NF-. kappa.B-dependent transcription in cells expressing human CD137 (e.g., human cells). In some embodiments, NF- κ B-dependent transcription is increased by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 99% or more in CD 137-expressing cells (e.g., human cells) contacted with an (active) activatable binding polypeptide relative to corresponding cells not contacted with an activatable binding polypeptide (e.g., corresponding cells contacted with an isotype control antibody) or corresponding cells contacted with an activatable binding polypeptide when in an inactive form. In some embodiments, NF- κ B-dependent transcription is increased by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, or more in cells expressing CD137 (e.g., human cells) contacted with the activatable binding polypeptide (when in an active form) relative to corresponding cells not contacted with the activatable binding polypeptide (e.g., corresponding cells contacted with an isotype control antibody) or corresponding cells contacted with the activatable binding polypeptide when in an inactive form.

In some embodiments, the activatable binding polypeptide (when in an inactive form) is not cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and/or dog CD 137. In some embodiments, the activatable binding polypeptide (when in active form) is cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and/or dog CD 137. In some embodiments, the activatable binding polypeptide is cross-reactive with monkey CD 137. In some embodiments, the activatable binding polypeptide is cross-reactive with mouse CD 137. In some embodiments, the activatable binding polypeptide is cross-reactive with rat CD 137. In some embodiments, the activatable binding polypeptide is cross-reactive with dog CD 137. In some embodiments, the activatable binding polypeptide binds to monkey and mouse CD 137; monkey and rat CD 137; monkey and dog CD 137; mouse and rat CD 137; mouse and dog CD 137; rat and dog CD 137; monkey, mouse and rat CD 137; monkey, mouse and dog CD 137; monkey, rat and dog CD 137; mouse, rat, and dog CD 137; or monkey, mouse, rat and dog CD137 were cross-reactive. In some embodiments, the activatable binding polypeptide is cross-reactive at about 100nM (e.g., at about 1nM, at about 10nM, at about 25nM, at about 50nM, at about 75nM, at about 100 nM). Methods of measuring cross-reactivity are known in the art and include, without limitation, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like.

In some embodiments, the activatable binding polypeptide does not induce ADCC effects. Methods of measuring ADCC effects are known in the art. In some embodiments, the activatable binding polypeptide (when in active or inactive form) does not induce ADCC effects by more than about 10% (does not induce ADCC by more than about 10%, more than about 5%, more than about 1%, more than about 0.1%, more than about 0.01%) relative to a control.

In some embodiments, the present disclosure provides an isolated activatable binding polypeptide that, when in an active form, competes or cross-competes for binding to human CD137 with an antibody comprising: a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 65; HVR-H2 comprising the amino acid sequence of SEQ ID NO 66; and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 67; and/or b) HVR-L1 comprising the amino acid sequence of SEQ ID NO 68; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the present disclosure provides an isolated activatable binding polypeptide that, when in an active form, competes or cross-competes for binding to human CD137 with an antibody comprising: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 49; and/or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO 50. The ability of an activatable binding polypeptide to compete or cross-compete for binding with an antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, an antibody (e.g., as described above) can be allowed to bind to human CD137 under saturating conditions, followed by measurement of the ability of the test to activate the binding polypeptide (when in active form) to bind to CD 137. If the test activatable binding polypeptide is capable of binding CD137 simultaneously with the antibody, then the test activatable binding polypeptide binds to a different epitope than the antibody. However, if the test activatable binding polypeptide is not capable of simultaneously binding CD137, then the test activatable binding polypeptide binds to the same epitope, an overlapping epitope, or an epitope in close proximity to the epitope bound by the antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS or surface plasmon resonance.

In some embodiments, the activatable binding polypeptide (when in an inactive form) does not inhibit binding between CD137 and its ligand (e.g., human CD137 and human CD 137L). In some embodiments, the activatable binding polypeptide (when in active form) inhibits binding between CD137 and its ligand (e.g., human CD137 and human CD 137L). In some embodiments, the activatable binding polypeptide inhibits binding between CD137 and its ligand in vitro. In some embodiments, the activatable binding polypeptide (when in an active form) has a half maximal Inhibitory Concentration (IC) for inhibiting the binding of CD137 to its ligand of about 500nM or less (e.g., about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 100nM or less, about 50nM or less, about 25nM or less, about 10nM or less, about 1nM or less, etc.)₅₀). In some embodiments, the activatable binding polypeptide has a half maximal Inhibitory Concentration (IC) of about 100nM or less for inhibiting the binding of CD137 to its ligand₅₀). In some embodiments, the activatable binding polypeptide completely inhibits binding of human CD137 to its ligand when provided at a concentration of about 100nM or greater (e.g., about 100nM or greater, about 500nM or greater, about 1 μ Μ or greater, about 10 μ Μ or greater, etc.). Methods of measuring the ability of a polypeptide to inhibit the binding of a first protein (e.g., CD137) and a second protein (e.g., CD137L) are known in the art, including without limitation by BIAcore analysis, ELISA assays, and flow cytometry.

Antibodies

In some embodiments, the disclosure relates to an activatable binding polypeptide comprising an antibody described herein (e.g., a CTLA4 or CD137 antibody described above). The antibodies described herein (e.g., CTLA4 or CD137 antibodies) can be in any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibodies described herein (e.g., CTLA4 or CD137 antibodies) are in the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclasses. Antibodies described herein (e.g., CTLA4 or CD137 antibodies) can be converted from one class or subclass to another using methods known in the art. An exemplary method for producing antibodies in a desired class or subclass comprises the steps of: isolating nucleic acids encoding the heavy chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody) and nucleic acids encoding the light chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody), isolating nucleic acids encoding V_HSequence of region such that V_HThe sequences are linked to sequences encoding the heavy chain constant region of the desired class or subclass, the light chain gene and heavy chain constructs are expressed in the cell, and the antibody is collected.

Antigen binding fragments

In some embodiments, the disclosure relates to an activatable binding polypeptide comprising an antigen-binding fragment, such as an antigen-binding fragment of an antibody described herein (e.g., a CTLA4 or CD137 antibody).

The antigen-binding fragment may comprise any sequence of any of the antibodies described herein. In some embodiments, the antigen-binding fragment comprises (1) a light chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody); (2) the heavy chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody); (3) the variable region of a light chain from an antibody described herein (e.g., a CTLA4 or CD137 antibody); (4) the variable region of a heavy chain from an antibody described herein (e.g., a CTLA4 or CD137 antibody); (5) one or more HVRs (e.g., one, two, three, four, five, or six HVRs) of an antibody described herein (e.g., a CTLA4 or CD137 antibody); or (6) the amino acid sequences of three HVRs from the light chain of an antibody described herein (e.g., CTLA4 or CD137 antibody) and three HVRs from the heavy chain of an antibody described herein (e.g., CTLA4 or CD137 antibody).

In some embodiments, the present disclosure provides an antigen-binding fragment of an antibody (which binds to human CTLA4) comprising a heavy chain variable region comprising: HVR-H1 comprising amino acid sequence YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and HVR-H3 comprising amino acid sequence ARSYVYFDY (SEQ ID NO: 61); and/or a light chain variable region comprising: HVR-L1 comprising amino acid sequence RASQSVRGRFLA (SEQ ID NO:62), HVR-L2 comprising amino acid sequence DASNRATGI (SEQ ID NO:63), and HVR-L3 comprising amino acid sequence YCQQSSSWPPT (SEQ ID NO: 64). In some embodiments, the present disclosure provides an antigen-binding fragment of an antibody, the antigen-binding fragment comprising: a) 47 or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO; and/or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO 48.

In some embodiments, the present disclosure provides an antigen-binding fragment of an antibody (that binds human CD137) comprising a heavy chain variable region comprising: HVR-H1 comprising amino acid sequence FSLSTGGVGVGWI (SEQ ID NO:65), HVR-H2 comprising amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and HVR-H3 comprising amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 67); and/or a light chain variable region comprising: HVR-L1 comprising amino acid sequence RASQSIGSYLA (SEQ ID NO:68), HVR-L2 comprising amino acid sequence DASNLETGV (SEQ ID NO:69), and HVR-L3 comprising amino acid sequence YCQQGYYLWT (SEQ ID NO: 70). In some embodiments, the present disclosure provides an antigen-binding fragment of an antibody, the antigen-binding fragment comprising: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 49; and/or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO 50.

In some embodiments, antigen-binding fragments of antibodies described herein (e.g., CTLA4 or CD137 antibodies) include: (i) fab fragment consisting of V_L、V_H、C_LAnd C_H1 domain; (ii) f (ab')₂A fragment which is A bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) from V_HAnd C_H1 domain; (iv) v from one arm of an antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (v) dAb fragments (Ward et al (1989) Nature 341:544-546) consisting of V_HDomain composition; (vi) (vii) isolated CDRs, and (vii) a single chain antibody (scFv), which is a V comprising an antibody_LV with regions attached to antibodies_HPolypeptides of regions (see, e.g., Bird et al (1988) Science 242: 423-.

Antibody derivatives

In some embodiments, the present disclosure provides an activatable binding polypeptide comprising a derivative of an antibody described herein (e.g., a CTLA4 or CD137 antibody).

In some embodiments, the antibody derivative is obtained by modifying the amino acid sequence of a parent antibody while maintaining the overall molecular structure of the amino acid sequence of the parent antibody. The amino acid sequence of any region of the parent antibody chain may be modified, such as the framework, HVR, or constant regions. The type of modification includes a substitution, insertion, deletion, or combination thereof of one or more amino acids of the parent antibody.

In some embodiments, an antibody derivative comprises a V that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOs 47-50 _LOr V_HAnd (4) a zone. In some embodiments, an antibody derivative comprises a region of HVR-H1 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs 59 or 65. In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least 66% of an amino acid sequence as set forth in any of SEQ ID NOs 60 or 66A region of HVR-H2 amino acid sequence that is 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, an antibody derivative comprises a region of HVR-H3 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOs 61 or 67. In some embodiments, an antibody derivative comprises a region of HVR-L1 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 62 or 68. In some embodiments, an antibody derivative comprises a region of HVR-L2 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOs 63 or 69. In some embodiments, an antibody derivative comprises a region of HVR-L3 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID nos. 64 or 70.

In some particular embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions to the amino acid sequence of an antibody described herein (e.g., a CTLA4 or CD137 antibody).

Amino acid substitutions encompass both conservative and non-conservative substitutions. The term "conservative amino acid substitution" means the replacement of one amino acid by another, wherein the two amino acids have similarities in certain physicochemical properties, such as the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, substitution may typically be made within each of the following groups: (a) nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids such as arginine, lysine and histidine; and (d) negatively charged (acidic) amino acids such as aspartic acid and glutamic acid.

Modifications may be made in any position of the amino acid sequence of the antibody, including HVRs, framework or constant regions. In one embodiment, the disclosure provides a V comprising an illustrative antibody described herein (e.g., a CTLA4 or CD137 antibody)_HAnd V_LAntibody derivatives of HVR sequences, but containing different framework sequences than those of the illustrative antibody. The framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA Sequences for Human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" Human germline sequence database (Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, U.S. Deparatment of Health and Human Services, NIH publication Nos. 91-3242 (1991); Tomlinson et al, J.mol.biol.24: 776-. Framework sequences useful in the construction of antibody derivatives include those that are similar in structure to the framework sequences used by the illustrative antibodies of the present disclosure. For example, the HVR-H1, HVR-H2, and HVR-H3 sequences and HVR-L1, HVR-L2, and HVR-L3 sequences of the illustrative antibodies can be grafted onto a framework region having the same sequence as the framework region found in the germline immunoglobulin gene from which the framework sequence is derived, or the HVR sequences can be grafted onto a framework region containing one or more mutations as compared to the germline sequence.

In some embodiments, the antibody derivative is a chimeric antibody comprising the amino acid sequence of an illustrative antibody described herein (e.g., a CTLA4 or CD137 antibody). In one example, one or more HVRs from one or more illustrative antibodies are combined with HVRs from an antibody from a non-human animal, such as a mouse or rat. In another example, the HVRs of a chimeric antibody are all derived from one or more of the illustrative antibodies. In some particular embodiments, the chimeric antibody comprises one, two, or three HVRs from the heavy chain variable region of the illustrative antibody and/or one, two, or three HVRs from the light chain variable region of the illustrative antibody. Chimeric antibodies can be produced using conventional methods known in the art.

Another type of modification is to make V_HAnd/or V_LAmino acid residues within the HVR region of the chain are mutated. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effect on antibody binding or other functional property of interest can be assessed in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutation may be an amino acid addition and/or deletion. In addition, at most one, two, three, four, or five residues within the HVR region are typically altered. In some embodiments, the antibody derivative comprises 1, 2, 3, or 4 amino acid substitutions in a heavy chain HVR and/or in a light chain HVR. In another embodiment, the amino acid substitution is a change of one or more cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. The cysteine may be a typical or atypical cysteine. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of the illustrative antibody.

Can also be paired with V_HAnd/or V_LFramework residues within the regions are modified. Typically, the framework variants are prepared to reduce the immunogenicity of the antibody. One approach is to "back mutate" one or more framework residues into the corresponding germline sequence. Antibodies that have undergone cellular mutation of the recipient may contain framework residues that differ from the germline sequence from which the antibody is derived. The residues can be identified by comparing the antibody framework sequences to the germline sequence from which the antibody is derived. To return the sequence of framework regions toTheir germline configuration allows somatic mutations to be "back-mutated" into germline sequences by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

In addition, modifications may be made within the Fc region of the illustrative antibodies, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This process is further described in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another instance, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody.

In addition, an antibody of the present disclosure can be modified to alter its potential glycosylation sites or pattern according to routine experimentation known in the art. In another aspect, the disclosure provides derivatives of the antibodies described herein (e.g., CTLA4 or CD137 antibodies) that contain at least one mutation in the variable region of the light or heavy chain that alters the glycosylation pattern in the variable region. Such antibody derivatives may have increased affinity and/or altered specificity with respect to binding to the antigen. Mutations may add new glycosylation sites in the V-region, alter the position of one or more V-region glycosylation sites, or remove pre-existing V-region glycosylation sites. In one embodiment, the present disclosure provides derivatives of the antibodies described herein (e.g., CTLA4 or CD137 antibodies) having potential N-linked glycosylation sites at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in one heavy chain variable region are removed. In another embodiment, the present disclosure provides derivatives of the antibodies described herein (e.g., CTLA4 or CD137 antibodies) having potential N-linked glycosylation sites at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in both heavy chain variable regions are removed. Methods of altering the glycosylation pattern of an antibody are known in the art, such as those described in U.S. patent No. 6,933,368, the disclosure of which is incorporated herein by reference.

Examples of other antibody derivatives provided by the present disclosure include single chain antibodies, minibifunctional antibodies, domain antibodies, nanobodies, and single antibodies. "Single chain antibody" (scFv) consisting of V_LThe domain is linked to V_HSingle polypeptide chain composition of a domain wherein V_LDomains and V_HThe domains pair to form monovalent molecules. Single chain antibodies can be prepared according to methods known in the art (see, e.g., Bird et al, (1988) Science 242: 423-. A "minibifunctional antibody" consists of two chains, each comprising a heavy chain variable region linked to a light chain variable region on the same polypeptide chain, connected by a short peptide linker, wherein the two regions on the same chain do not pair with each other, but rather with a complementary domain on the other chain to form a bispecific molecule. Methods for preparing minibifunctional antibodies are known in the art (see, e.g., Holliger P. et al, (1993) Proc. Natl. Acad. Sci. USA 90: 6444-. Domain antibodies (dabs) are small functional binding units of antibodies, corresponding to the variable region of the heavy or light chain of an antibody. Domain antibodies are well expressed in bacterial, yeast and mammalian cell systems. Additional details of domain antibodies and methods for their production are known in the art (see, e.g., U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European patents 0368684 and 0616640; WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019, and WO 03/002609). Nanobodies are derived from the heavy chain of an antibody. Nanobodies typically comprise a single variable domain and two constant domains (CH2 and CH3) and retain the antigen binding ability of the original antibody. Nanobodies may be prepared by methods known in the art (see, e.g., U.S. patent No. 6,765,087, U.S. patent No. 6,838,254, WO 06/079372). A single antibody consists of one light and one heavy chain of an IgG4 antibody. The single antibody may be prepared by removing the hinge region of the IgG4 antibody. Additional details of monoclonal antibodies and methods for their preparation can be found in WO2007/059782 In (1).

VII composition

In other aspects, the disclosure provides a composition comprising one or more of the polypeptides (e.g., activatable binding polypeptides) described herein. In some embodiments, the composition is a pharmaceutical composition comprising a polypeptide (e.g., an activatable binding polypeptide) and a pharmaceutically acceptable carrier. The compositions may be prepared by conventional methods known in the art.

The term "pharmaceutically acceptable carrier" refers to any non-active substance suitable for use in a formulation for delivery of a polypeptide (e.g., an activatable binding polypeptide). The carrier may be an anti-adherent, binder, coating agent, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

The composition may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. One particular form of composition suitable for delivery of the polypeptide (e.g., activatable binding polypeptide) is a sterile liquid, such as a solution, suspension or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating the polypeptide (e.g., activatable binding polypeptide) in the desired amount in an appropriate carrier, followed by sterile microfiltration. Dispersions can be prepared by incorporating the polypeptide (e.g., the activatable binding polypeptide) into a sterile vehicle that contains a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquids, the methods of preparation include vacuum drying and freeze-drying (lyophilization) to produce a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The various dosage forms of the compositions can be prepared by conventional techniques known in the art.

The relative amounts of the polypeptides (e.g., activatable binding polypeptides) included in the composition will vary depending on a number of factors, such as the particular polypeptide and carrier used, the dosage form, and the desired release and pharmacokinetic profiles. The amount of polypeptide (e.g., activatable binding polypeptide) in a single dosage form will generally be that amount which produces a therapeutic effect, but may also be a smaller amount. Typically, this amount will range from about 0.01% to about 99%, from about 0.1% to about 70%, or from about 1% to about 30%, relative to the total weight of the dosage form.

In addition to the polypeptide (e.g., the activatable binding polypeptide), one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described herein below. The appropriate amount of additional therapeutic agent to be included in the composition can be readily selected by one of skill in the art and will vary depending on a number of factors, such as the particular agent and carrier used, the dosage form, and the desired release and pharmacokinetic profile. The amount of additional therapeutic agent included in a single dosage form will generally be that amount of the agent which produces a therapeutic effect, but may be a smaller amount.

Any of the polypeptides (e.g., activatable binding polypeptides) and/or compositions (e.g., pharmaceutical compositions) described herein can be used in the preparation of a medicament (e.g., an agent for treating or delaying the progression of a cancer in a subject in need thereof).

Use of activatable binding polypeptides and pharmaceutical compositions

The polypeptides (e.g., activatable binding polypeptides) and pharmaceutical compositions thereof provided by the present disclosure may be used for therapeutic, diagnostic, or other purposes, such as modulating an immune response, treating cancer, enhancing the efficacy of other cancer therapies, enhancing the efficacy of vaccines, or treating autoimmune diseases. Thus, in other aspects, the disclosure provides methods of using a polypeptide (e.g., an activatable binding polypeptide) or a pharmaceutical composition thereof. In one aspect, the present disclosure provides a method of treating a disorder in a mammal, the method comprising administering to the mammal in need of treatment an effective amount of a polypeptide (e.g., an activatable binding polypeptide) provided by the present disclosure or a composition thereof. In some embodiments, the polypeptide is an activatable binding polypeptide that binds CTLA4 (e.g., human CTLA4) or CD137 (e.g., human CD137) when in active form. In some embodiments, the mammal is a human.

In some embodiments, the disorder is cancer. A variety of cancers can be treated or prevented with the methods, uses, or agents provided by the present disclosure. Examples of such cancers include lung cancers such as bronchial carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (noncancerous), and sarcoma (cancerous); heart cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondroma, chondroma, chondroblastoma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulocytoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, oligodendrogliomas, medulloblastomas, chordomas, schwannoma, ependymoma, meningioma, pituitary adenomas, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratomas, dermatocysts, and hemangiomas; cancers in the digestive system such as leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, large intestinal polyps, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, suprarenal adenoid tumor, and renal pelvis transitional cell carcinoma; bladder cancer; hematological cancers such as acute lymphocytic (lymphoblastic) leukemia, acute myelogenous (myelocytic, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myelogenous (myelogenous, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myelogenous leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancer; eye-related cancers such as retinoblastoma and intraocular melanoma; cancers of the male reproductive system such as benign prostatic hypertrophy, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system such as uterine cancer (endometrial cancer), cervical cancer (cervical cancer), ovarian cancer (ovarian carcinoma), vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of parathyroid gland; pancreatic cancer; and hematological cancers such as leukemia, myeloma, non-hodgkin's lymphoma, and hodgkin's lymphoma.

In another aspect, the present disclosure provides a method of enhancing an immune response in a mammal, the method comprising administering to the mammal an effective amount of a polypeptide (e.g., an activatable binding polypeptide) provided by the present disclosure or a composition thereof. In some embodiments, the polypeptide is an activatable binding polypeptide that binds CTLA4 (e.g., human CTLA4) or CD137 (e.g., human CD137), and the mammal is a human. The term "enhance an immune response" or grammatical variations thereof means to stimulate, provoke, augment, improve or enhance any response of the immune system of a mammal. The immune response may be a cellular response (i.e. mediated by cells, such as cytotoxic T lymphocytes) or a humoral response (i.e. mediated by antibodies), and may be a primary or secondary immune response. Examples of enhancing the immune response include activating PBMCs and/or T cells (including increasing secretion of one or more cytokines such as IL-2 and/or IFN γ). Enhancement of immune responses can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release, tumor regression, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Generally, the methods of the present disclosure result in an enhancement of the immune response achieved by the mammal when compared to the immune response achieved by an untreated mammal or a mammal not treated with the recited methods.

In practicing the methods of treatment, the polypeptide (e.g., activatable binding polypeptide) can be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Thus, in another aspect, the disclosure provides a combination therapy comprising a polypeptide (e.g., an activatable binding polypeptide) in combination with one or more additional therapies or therapeutic agents for separate, sequential or simultaneous administration. The term "additional therapeutic agent" can refer to any therapeutic agent other than the polypeptide provided by the present disclosure (e.g., an activatable binding polypeptide). In a particular aspect, the present disclosure provides a combination therapy for treating cancer in a mammal, comprising administering to the mammal an effective amount of a polypeptide provided herein (e.g., an activatable binding polypeptide) in combination with one or more additional therapeutic agents. In another embodiment, the mammal is a human.

A wide variety of cancer therapeutics can be used in combination with the polypeptides (e.g., activatable binding polypeptides) provided by the present disclosure. One of ordinary skill in the art will recognize the existence and development of other cancer therapies that may be used in combination with the methods and polypeptides of the present disclosure, and will not be limited to those therapy forms set forth herein. Examples of classes of additional therapeutic agents that may be used in combination therapy to treat cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents. In some embodiments, the additional therapeutic agent is a viral gene therapy, an immune checkpoint inhibitor, a targeted therapy, a radiation therapy, and/or a chemotherapeutic agent.

The term "chemotherapeutic agent" refers to a chemical or biological substance that can cause cancer cells to die, or interfere with the growth, division, repair, and/or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO 2006/129163 and US 20060153808, the disclosures of which are incorporated herein by reference. Examples of specific chemotherapeutic agents include: (1) alkylating agents such as chlorambucil (chlorambucil) (LEUKERAN), Cyclophosphamide (CYTOXAN), ifosfamide (ifosfamide) (IFEX), dichloromethyl diethylamine (mechlorethamine hydrochloride) (mustagen), thiotepa (thioplac) (thioplac (THIOPLEX)), streptozotocin (streptozocin (ZANOSAR)), carmustine (carmustine) (BICNU), griffonia (GLIADEL WAFER)), lomustine (lomustine) (cenu), and dacarbazine (dacarbazine) (carmustine (DTIC-DOME)); (2) alkaloids or phytovinca alkaloids, including cytotoxic antibiotics such as doxorubicin (doxorubicin) (ADRIAMYCIN), epirubicin (epirubicin) (elence), Pleocicin (PHARMORUBICIN), daunorubicin (daunorubicin) (seco), daunorubicin (daunorubicin), Daunorubicin (DAUNOXOME), nemulin (nemorubicin), idarubicin (idarubicin) (idarubicin preservative-free solution (IDAMYCIN PFS), lanudorine (zados), mitoxantrone (mitoxantrone) (DHAD, novalutalone (NOVANTRONE)), dactinomycin (dactinomycin) (actinomycin d (actimycin d), cosemegen (cosmengen)), mithramycin (plicamycin) (mitomycin (MITHRACIN)), mitomycin (mitomycin) (bramycin (vinorelbine)), and vinorelbine (vinblastine (vinorelbine (bramycin)), vinblastine (bramycin)), and vinorelbine (vinblastine (bramycin (brane (bramycin)) Vincristine (vincristine) (ONCOVIN) and vindesine (vindesine) (etheine) powder for cancer); (3) antimetabolites such as capecitabine (capecitabine) (hiloda (XELODA)), cytarabine (cytarabine) (setxate-U (cytasa-U)), fludarabine (fludarabine) (FLUDARA)), gemcitabine (gemcitabine) (jianzar), hydroxyurea (hydraurea) (HYDRA), methotrexate (methotrexate) (fowlx, methoxate (trexate), troxiel (xall)), nelarabine (nelabane) (arrhen (aron)), trimetrexate (neexin), and pemetrexed (pemetrexed) (eindad (alimatta)); (4) pyrimidine antagonists, such as 5-fluorouracil (5-FU); capecitabine (hiloda), raltitrexed (ramytrexed) (tomdex), tegafur-Uracil (UFTORAL), and gemcitabine (jiaojian); (5) taxanes such as docetaxel (docetaxel) (TAXOTERE), paclitaxel (paclitaxel) (TAXOL); (6) platinum drugs such as cisplatin (cispain) (platanol) and carboplatin (PARAPLATIN) and oxaliplatin (oxaliplatin) (ELOXATIN)); (7) topoisomerase inhibitors such as irinotecan (irinotecan) (cape Tomato), topotecan (topotecan) (carcinoid (HYCAMTIN)), etoposide (etoposide) (pyriprox (ETOPHOS), metopidil (VEPESSID), Topusa (TOPOSAR) and teniposide (Vernonide (VUMON)); (8) epipodophyllotoxin (podophyllotoxin) derivatives such as etoposide (bupirimate, terbutryn, topiramate); (9) folic acid derivatives such as leucovorin (WELLCOVORIN); (10) nitrosoureas (nitrosourea) such as carmustine (binitrous), lomustine (strobion); (11) inhibitors of receptor tyrosine kinases including Epidermal Growth Factor Receptor (EGFR), Vascular Endothelial Growth Factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), Hepatocyte Growth Factor Receptor (HGFR), and platelet-derived growth factor receptor (PDGFR), such as gefitinib (gefitinib) (IRESSA), erlotinib (erlotinib) (tacevec), bortezomib (bortezomib) (VELCADE), imatinib mesylate (imatinib mesylate) (GLEEVEC), gefitinib (gefitinib), lapatinib (lapatinib), sorafenib (sorafenib), thalidomide (thalidomide), Sunitinib (SUTENT) (sunitinib), acituzumab (hexitumab), herceptib (rituximab) (HERCEPTIN), rituximab (rituximab) (ritrin (rituximab)), and platelet-derived growth factor receptor (PDGFR) Cetuximab (cetuximab) (ERBITUX), bevacizumab (bevacizumab) (AVASTIN)) and ranibizumab (ranibizumab) (LUCENTIS), lym-1 (oncolin (ONCOLYM)), antibodies against insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO 2002/053596; (12) angiogenesis inhibitors such as bevacizumab (avastin), suramin (suramin) (germann), angiostatin (angiostatin), SU5416, thalidomide and matrix metalloproteinase inhibitors such as batimastat (batimastat) and marimastat (marimastat) and those disclosed in WO 2002055106; and (13) proteasome inhibitors such as bortezomib (verkod).

The term "immunotherapeutic" refers to a chemical or biological substance that enhances the immune response in a mammal. Examples of immunotherapeutic agents include: bacille calmette-guerin (BCG); cytokines, such as interferon; vaccines such as MyVax personalized immunotherapy, Onyvax-P, oncoffeine (Oncophage), GRNVAC1, favade (Favld), pronitrogen (Provenge), Gevax (GVAX), lovaxin c (lovaxin c), bivax (BiovaxID), Gimx (GMXX) and newvax (NeuVax); and antibodies such as alemtuzumab (camptos (camp)), bevacizumab (avastin), cetuximab (erbitux), ozomicin gemtuzumab (gemtub ozogamicin) (milotarg), ibritumomab tiuxetan (zevallin), panitumumab (panitumumab) (vectib (VECTIBIX)), rituximab (rituximab ), trastuzumab (herceptin), tositumomab (tositumomab) (BEXXAR), ipilimumab (ipilimumab) (leafo (YERVOY)), tremelimumab (tremelimumab), CAT-3888, agonistic antibodies to the OX40 receptor (such as those disclosed in WO 2009/079335), agonistic antibodies to the CD40 receptor (such as those disclosed in WO 2003/040170), and TLR-9 agonists (such as those disclosed in WO2003/015711, WO2004/016805, and WO 2009/022215).

The term "hormonal therapeutic agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones, or inhibits or counteracts the effects of hormones on the growth and/or survival of cancerous cells. Examples of such agents suitable for the methods herein include those disclosed in US 20070117809. Examples of specific hormonal therapy agents include tamoxifen (tamoxifen) (NOLVADEX), toremifene (toremifene) (fareton), fulvestrant (fulvestrant) (FASLODEX), anastrozole (anamedex), exemestane (exemestane) (arnosine), letrozole (letrozole) (fleron (FEMARA)), megestrol acetate (megestro), goserelin (zolerelin) (ZOLADEX), and leuprolide (leuprolide) (lipranolol (pron)). The binding molecules of the present disclosure can also be used in combination with non-pharmaceutical hormone therapies, such as (1) surgical methods that remove all or a portion of an organ or gland involved in hormone production, such as the ovary, testis, adrenal gland, and pituitary gland, and (2) radiation therapy, wherein the organ or gland of the patient is subjected to radiation in an amount sufficient to inhibit or eliminate the production of the targeted hormone.

In some embodiments, the additional therapeutic agent is one or more of: pomalyst, rilimid, lenalidomide, pomalidomide, thalidomide, DNA-alkylated platinum-containing derivative cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD 137 antibody, anti-CTLA 4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD 20 antibody, anti-CD 40 antibody, anti-DR 5 antibody, anti-CD 1d antibody, anti-TIM 3 antibody, anti-SLAMF 7 antibody, anti-KIR receptor antibody, anti-OX 40 antibody, anti-HER 2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pertuzumab, radiotherapy, single dose radiation, fractionated radiation CSF, focal radiation, total organ radiation, IL-12, IFN α, GM-CSF, chimeric antigen receptor, inherited transferred T cells, anti-cancer vaccine, and oncolytic virus.

Combination therapy for the treatment of cancer also encompasses the combination of binding molecules with surgery to remove the tumor. The binding molecule may be administered to the mammal before, during or after surgery.

Combination therapy for the treatment of cancer also encompasses the combination of polypeptides (e.g., activatable binding polypeptides) with radiation therapy such as ionizing (electromagnetic) radiation therapy (e.g., X-ray or gamma ray) and particle beam radiation therapy (e.g., high linear energy radiation). The radiation source may be external or internal to the mammal. The polypeptide can be administered to the mammal before, during, or after radiation therapy.

The polypeptides (e.g., activatable binding polypeptides) and compositions thereof provided by the present disclosure may be administered by any suitable enteral or parenteral route of administration. The term "enteral route of administration" refers to administration through any part of the gastrointestinal tract. Examples of enteral routes include oral, transmucosal, buccal and rectal routes, or the intragastric route. By "parenteral route of administration" is meant a route of administration other than the enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or topical administration. The polypeptides (e.g., activatable binding polypeptides) and compositions of the present disclosure may be administered using any suitable method, such as by oral ingestion, nasogastric tubing, gastrostomy tubing, injection, infusion, implantable infusion pump, and osmotic pump. Suitable routes and methods of administration may vary depending on many factors, such as the particular polypeptide used, the rate of absorption desired, the particular formulation or dosage form used, the type or severity of the condition being treated, the particular site of action, and the condition of the patient, and may be readily selected by one of skill in the art.

The term "effective amount" of a binding molecule can refer to an amount effective to achieve the intended therapeutic goal. For example, in the context of enhancing an immune response, an "effective amount" can be any amount effective to stimulate, provoke, augment, improve or boost any response of the immune system of a mammal. In the context of treating a disease, an "effective amount" can be any amount sufficient to result in any desirable or beneficial effect in the treated mammal. In particular, in the case of treating cancer, examples of desirable or beneficial effects include inhibiting further growth or spread of cancer cells, killing cancer cells, inhibiting recurrence of cancer, reducing pain associated with cancer, or improving survival in a mammal. An effective amount of a polypeptide described herein (e.g., an activatable binding polypeptide) can be in the range of about 0.001 to about 500mg, or about 0.01 to about 100mg/kg, per kg body weight of the mammal. For example, the amount can be about 0.3mg, 1mg, 3mg, 5mg, 10mg, 50mg, or 100mg per kg body weight of the mammal. In some embodiments, an effective amount of a polypeptide of the disclosure (e.g., an activatable binding polypeptide) is in the range of about 0.01-30mg per kg body weight of the mammal. In some other embodiments, an effective amount of a polypeptide of the disclosure (e.g., an activatable binding polypeptide) is in the range of about 0.05-15mg per kg body weight of the mammal. The precise dosage level to be administered can be readily determined by one of skill in the art and will depend upon a number of factors, such as the type and severity of the condition to be treated, the particular polypeptide employed, the route of administration, the time of administration, the duration of treatment, the particular additional therapy employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The polypeptide (e.g., activatable binding polypeptide) or composition thereof may be administered at multiple times. The interval between single doses may be, for example, daily, weekly, monthly, every three months, or yearly. An exemplary treatment regimen entails administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Dosage regimens for the polypeptides of the disclosure (e.g., activatable binding polypeptides) may include 1mg per kg body weight or 3mg per kg body weight by intravenous administration using one of the following dosing schedules: (i) every four weeks for six doses, followed by every three months; (ii) every three weeks; (iii) 3mg per kg body weight, 1 time, followed by every three weeks, 1mg per kg body weight.

IX. kit

In another aspect, provided herein is a kit comprising a library of polynucleotides of the present disclosure. In some embodiments, the kit further comprises a package insert comprising instructions for expressing, modifying, screening, or otherwise using the library, e.g., to identify the activatable binding polypeptide of interest. In some embodiments, the kit further comprises one or more buffers, e.g., for storing, transferring, transfecting, or otherwise using one or more of the polynucleotides (e.g., synthetic polynucleotides). In some embodiments, the kit further comprises one or more containers for storing one or more of the polynucleotides. In some embodiments, the kit further comprises one or more vectors, e.g., for transfecting a host cell with one or more of the polynucleotides.

In another aspect, provided herein is a kit comprising an activatable binding polypeptide and/or composition described herein. In some embodiments, the kit further comprises a package insert comprising instructions for using the activatable binding polypeptide and/or composition. In some embodiments, the kit further comprises one or more buffers that can activate the binding polypeptide and/or composition, e.g., for storage, transfer, administration, or otherwise use. In some embodiments, the kit further comprises one or more containers (e.g., syringes, etc.) for storing or administering the activatable binding polypeptide and/or composition.

The previous written description is considered to be sufficient to enable one skilled in the art to practice the disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Examples

Example 1: method for identifying self-blocking peptides for activatable binding polypeptides

As noted above, there is a need for improved methods and products that can be used to identify self-blocking peptides for activatable binding polypeptides. Thus, described herein is a new system that has been designed and implemented to efficiently discover masking portions with good exploitability. In this system, the target antibody fragment Fab (fig. 1) or scFv (fig. 2) is first displayed on the yeast surface and confirmed to be functional in binding its antigen. Next, the improved peptide library was fused directly to the N-terminus of the light chain, and a yeast library displaying the fusion protein on the yeast surface was constructed. The yeast library was then subjected to several rounds of FACS-based screening: first, yeast clones with low binding to antigen were enriched, followed by treatment of the enriched yeast clones with protease to remove the N-terminal peptide, and clones with high binding to antigen were selected (fig. 1 and 2). After 4-5 rounds of sorting, plasmids were extracted from these clones and the masking peptide sequence was confirmed by DNA sequencing.

There are several unique features built into this new system that make it powerful in identifying masking peptides for target antibodies with good developability:

1) The peptide library was fused directly to the N-terminus of the target antibody fragment, not to any foreign backbone proteins, and the masking peptide was found in the same situation as the final product. This eliminates contamination of false positive peptide sequences and significantly reduces the workload for their downstream characterization.

2) The protease cleavage mediated activation mechanism is integrated into the screening process. This will ensure that the peptides found not only mask antigen binding prior to activation, but also no longer block antigen binding after protease cleavage. These are prerequisites to be considered qualified as good masking peptides for any activatable antibody.

3) An improved design of peptide libraries was used. Unlike the random peptide libraries commonly used, a pair of cysteine residues are introduced into fixed positions in the peptide library to ensure that the displayed peptide has a constrained conformation. It was observed that constrained peptides tend to exhibit increased binding affinity and specificity (Uchiyama et al (2005)99(5): 448-56). Unlike the widely used NNK (or NNS) codons that encode all 20 residues including chemically labile residues such as M and W, NHC codons were employed in some or all of the peptide libraries. The NHC codon encodes 12 amino acid residues (D, A, Y, S, T, N, I, L, F, V, H and P), and excludes residues that are detrimental to the manufacturing process, such as methionine, tryptophan, or cysteine. Furthermore, the use of NHC codons also significantly reduces the theoretical peptide library size relative to NNK (or NNS) codons, thus enabling the construction of libraries with much better coverage. These libraries perform well when tested against different target antibodies.

Example 2: design of Constrained Peptide Libraries (CPLs)

Four exemplary Constrained Peptide Libraries (CPLs) were designed (table 1).

Table 1: designed CPL

Each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P

At their core is the sequence Z6CX6CZ2(SEQ ID NO:55) or Z6CX8CZ2(SEQ ID NO:56) and the two fixed cysteine residues form a disulfide bond to constrain the peptide conformation. In the synthesized oligonucleotide, degenerate codon NHC was used at all positions except the inside of the loop, with NNK codon also used in CPL011 and CPL 013. Compared to NNK or NNS codons, NHC codons encode 12 residues (table 2), thus covering significant diversity, but lacking the chemically labile residues methionine, tryptophan and cysteine. Furthermore, the reduced theoretical diversity compared to NNK or NNS codons enables the construction of libraries with better coverage.

Table 2: NHC codon

These masking peptide sequences are followed by an invariant cleavage peptide sequence (SGRSAGGGGSPLGLAGSGGS, SEQ ID NO:12) containing two protease recognition sites: SGRSA (SEQ ID NO:13) for the protease urokinase-type plasminogen activator (uPA), and PLGLAG (SEQ ID NO:14) for the protease matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). These recognition sites have been used by many groups in the in vivo tumor cell-specific activation of targeting agents (see, e.g., Ke et al (1997) J Biol Chem 272(33): 20456-62; Gerspach et al (2006) Cancer Immunol 55(12): 1590-. During yeast-based screening, the MMP-9 recognition sequence was replaced with the Tobacco Etch Virus (TEV) protease recognition sequence (ENLYFQG, SEQ ID NO:15) due to the availability and specificity of the TEV protease.

CPL and invariant lytic peptide were fused to the N-terminus of the light chain of a target antibody in scFv or Fab format, which was linked to the Aga2 protein displayed on the yeast surface. The inclusion of an alternative TEV protease recognition site is important in identifying the correct type of masking peptide sequence, i.e. antigen binding is blocked prior to protease cleavage and antigen binding is able to be achieved after protease cleavage. The examples described below demonstrate that the lytic activation mechanism of activatable antibodies, initially shown in the case of yeast, is reproduced in the case of fully IgG molecules expressed in mammalian cells.

Example 3: construction and validation of activatable antibodies targeting CTLA4

Displaying functional target antibodies on yeast surface

Low copy number CEN/ARS based vectors are used to express target antibodies (antibody TY21580 targeting human CTLA 4) in yeast saccharomyces cerevisiae (s.cerevisiae) under the control of the inducible GAL1-10 promoter. surface display of scFv was achieved by fusion of the Aga2 protein at its C-terminus under the control of the GAL1 promoter, similar to the previously disclosed arrangement (Boder and Wittrup (1997) Nat Biotechnol 15(6): 553-7). For Fab, their surface display was achieved by Aga2 protein fused to the N-terminus of the heavy chain (fusion of VH and CH 1) under the control of GAL1 promoter, while the light chain (fusion of VL and CL) was under the control of GAL10 promoter. The Fab is displayed on the yeast surface by association of the light chain with the membrane-anchored heavy chain.

Surface display of Fab or scFv was verified by staining with an antibody recognizing the fusion affinity tag, and the functionality of Fab or scFv displayed on yeast was investigated using biotinylated human CTLA 4. Briefly, yeast cells (1X10^6) were collected 48 hours after induction in galactose medium, washed once with PBSA buffer, and then incubated with 10nM biotinylated antigen for 1 hour at room temperature. Yeast cells were then washed twice with PBSA buffer and incubated with PE-conjugated streptavidin (1:500 dilution) (eBioscience #2-4317-87) for 30 min at 4 ℃. The yeast cells were then analyzed by flow cytometry. As shown in figures 3A-3B, both fabs (figure 3A) and scfvs (figure 3B) targeting CTLA4 were successfully displayed on the yeast surface and were able to strongly bind their antigens.

Construction of a CPL-containing Yeast library

The synthesized oligonucleotide encoding CPL was fused to the oligonucleotide encoding the cleavage peptide by 5 PCR cycles. The primers used (F primer and R primer) are listed in Table 3. The composition of the PCR reaction was: 1 XPrimeSTAR buffer, 2.5mM dNTP, 100. mu.M each of F and R primers, and 100. mu.M each of template 1(CPL oligonucleotide) and template 2 (oligonucleotide encoding cleavage peptide), and 2.5U PrimeSTAR HS DNA polymerase. The PCR procedure used was: a)1 cycle of 96 ℃ for 5 minutes; 2) 96 ℃ (15 seconds), 60 ℃ (15 seconds), 72 ℃ (6 seconds) for 5 cycles; and 3)1 cycle of 72 ℃ for 3 minutes. Exonuclease I is used to digest single stranded DNA, followed by gel electrophoresis to purify the PCR product. The purified PCR product was then digested with BamHI and KpnI and cloned into a bacterial filter vector digested with the same two restriction enzymes. In the filter vector, CPL and lytic peptide are placed downstream of the bacterial secretion signal peptide and upstream of the beta-lactamase lacking the signal sequence. Functional beta-lactamases selected on ampicillin plates indicated in-frame fusions of CPL and cleavage peptides, thereby eliminating any out-of-frame errors (N-1 or N-2) introduced into the synthesized degenerate oligonucleotides. In addition, some of the poorly folded sequences were also reduced from the pool. Ligation products were transformed into electrocompetent bacterial cells, and the diversity of CPL libraries was typically between 5x10^9 and 1x10^ 10. Sequencing of individual clones indicated that very high in-frame rates (in many cases, almost 100%) were achieved by this method.

Table 3: PCR primer

To prepare a CPL-containing yeast library, plasmids were extracted from the bacterial library and used as templates for PCR amplification of DNA fragments encoding CPL and lytic peptides. The primers used (PL0009_ F and BL1024_ R) are listed in Table 3. The amplified PCR fragments were purified by gel electrophoresis and transformed into electrocompetent yeast cells along with linearized plasmids expressing the target antibody fused to Aga 2. The homologous sequences on both ends of the PCR fragment and the plasmid ensure efficient homologous recombination inside the yeast cell. The diversity of the constructed yeast libraries is typically between 1x10^9 to 2x10^ 9.

FACS-based screening of masking peptides against CTLA4 antibodies

A total of 1x10^8 yeast cells from the CPL yeast library were used to screen for masking peptides against target antibodies. For each round of sorting by MoFlo XDP, yeast cells induced in galactose medium were collected, washed once with PBSA buffer, and then incubated with 10nM (reduced to 1nM in each later round) biotinylated antigen for 1 hour at room temperature. Yeast cells were then washed twice with PBSA buffer and incubated with PE-conjugated streptavidin (1:500 dilution) (eBioscience #2-4317-87) for 30 min at 4 ℃. After two more washes with PBSA buffer, the yeast cells were adjusted to 2-3OD/mL and subjected to sorting. As shown in figure 4, 10nM biotinylated CTLA4-Fc was used and weak binding agents were enriched in round 1. After yeast cells from round 1 were grown in glucose medium, induced and treated with AcTEV protease (6U/OD cells) (Thermo Fisher Scientific #12575015) at 30 ℃ for 2 hours in galactose medium, and the strong binding agent was purified. Starting from round 3 sorting, the concentration of biotinylated CTLA4-Fc was reduced to 1nM and weak binding agents were collected. In round 4, each yeast cell fraction was also treated with AcTEV in parallel to verify protease cleavage mediated activation of the target antibody. As shown in figure 4, it is evident that AcTEV lysis results in a significant increase in the population of cells that bind strongly to antigen, indicating that the screening strategy is effective. Single clones from round 5 sorting were plated on selective media and grown individually to further confirm lysis-mediated antigen-binding activation.

As shown in fig. 5A-5B, selected CTLA4 in scFv (fig. 5A) or Fab (fig. 5B) form can activate antibody clones to exhibit little binding to antigen in the presence of masking peptides. However, when yeast cells were treated with TEV protease to remove the masking peptide, binding to the antigen was significantly increased. The combination of incorporating TEV recognition sites in the cleavage peptide and using TEV protease to verify the selected clones significantly increased the success rate of selecting masking peptides.

To identify the masking peptide sequence, a shuttle plasmid (general # GK2002-200) was extracted from the selected yeast clone and transformed into competent E.coli cells. Plasmids were prepared and the regions encoding the masking peptides were sequenced and aligned. As expected, these sequences can be divided into several groups, indicating the clear enrichment achieved by each round of sorting. Four sets of masking peptide sequences are listed in table 4 along with the invariant cleavage peptide sequences.

Table 4: masking peptide sequences

IgG transformation and expression

Four sets of masking peptides listed in table 4, and additional four masking peptide sequences derived from two of them (B13192 and B13197) to eliminate potential glycosylation sites (table 5) were transformed into IgG 1.

Table 5: additional masking peptide sequences

The heavy and light chains were cloned separately into the mammalian expression vector pcdna3.3(Thermo Fisher Scientific, catalog No. K830001) and the masking peptide and invariant cleavage peptide were fused to the N-terminus of the light chain in the same manner as displayed on the yeast surface. The VH and VL sequences of the parent CTLA4 antibody (TY21580) are listed below (see also PCT International application entitled "Compositions Comprising Cross-reactive Anti-CTLA4 Antibodies, and Methods of Making and Using the Same," filed concurrently herewith under attorney docket number 69540-:

anti-CTLA 4 heavy chain variable region (SEQ ID NO: 47):

EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS

anti-CTLA 4 light chain variable region (SEQ ID NO: 48):

DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR。

each pair of plasmids was transiently transfected into HEK293F cells. After six days, the supernatant was collected, clarified by centrifugation and filtration, and IgG purified by standard protein a affinity chromatography (MabSelect SuRe, GE Healthcare). IgG was eluted and neutralized and buffer exchanged into PB buffer (20mM sodium phosphate, 150mM NaCl, pH 7.0). Protein concentration was determined by ultraviolet spectrophotometry and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing and non-reducing conditions. Importantly, the expression levels of activatable antibodies in HEK293 cells were similar to their parent antibodies and their purification yields after protein a resin were also similar, indicating that the presence of masking and lytic peptides did not have a negative effect on antibody expression in mammalian cells.

Measurement of masking efficiency

The ForteBio Octet RED96 system (Pall, USA) was used to rapidly assess the efficiency of masking peptides. Briefly, activatable antibodies (and their parent antibody TY21580) were diluted to 30 μ g/mL in KB buffer (PBS buffer supplemented with 0.02% tween 20 and 0.1% BSA) in a parallel fashion and captured by an anti-human IgG capture (AHC) biosensor (Pall, USA). The sensor was then associated with His-tagged CTLA4 protein (25nM) for 300 seconds, followed by dissociation in KB buffer for another 300 seconds. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio data analysis 7.1(Pall, USA) according to the manufacturer's guidelines. As shown in fig. 6A-6B, the response achieved with the activatable antibody was significantly lower than the response achieved with the parent antibody, indicating that the masking peptide effectively blocks the binding of the antibody to its antigen. However, of the four activatable antibodies, TY22401 was less effective, which is consistent with the results from the ELISA assay discussed below.

Recombinant human CTLA4-Fc was diluted to 1 μ g/mL in PBS and coated on Maxisorp plates overnight at 4 ℃. The plates were blocked with PBS supplemented with 3% skim milk for 1 hour at 37 ℃. After washing, 100 μ Ι _ of 3-fold serial dilutions of antibody were added to each well. After incubation for 1 hour at 37 ℃, the plates were washed four times and 100 μ Ι _ of HRP-conjugated anti-human IgG (Fab-specific) antibody (1:6000 dilution) was added to each well. Plates were incubated at 37 ℃ for 1 hour, washed four times, then 50 μ Ι _ of TMB substrate solution was added to each well, and plates were incubated at room temperature. Using 50 mu L H per well₂SO₄After the reaction was terminated, the absorbance at 450nm was measured. Evaluation of EC by fitting ELISA data using an asymmetric sigmoid (five-parameter logistic equation) model of GraphPad Prism 6 software₅₀. Two experiments were performed on activatable antibodies TY22401, TY22402 and TY22404, resulting in two calculated masking efficiencies for each of these activatable antibodies. Masking efficiency of each activatable antibody by using EC for binding of activatable antibody₅₀EC divided by parent antibody (TY21580)₅₀To calculate. As shown in fig. 7A-7C and table 6, all activatable antibodies showed significantly reduced binding to its antigen compared to the parent antibody, and the calculated masking efficiency was in the range of 48 to 2213. The difference in masking efficiency may be due to the difference in EC ₅₀The measurement of values and the variation in data fit, and the masking efficiency of each activatable antibody may fall within the calculated range (e.g., the masking efficiency of activatable antibody TY22402 is between 377 and 2213). These results indicate when lactation is occurringVarious masking peptides identified from CPL maintain their masking efficiency in animal cells and when expressed as part of a complete IgG molecule.

Table 6: activatable antibody ELISA before protease cleavage

Removal of masking peptides restores antibody activity

The activatable antibodies were purified by treatment with a protease that recognizes the cleavage sequence and then tested to determine whether removal of the masking peptide restored their activity. As an example, 20. mu.g of TY22404(0.5mg/mL) was treated with 1. mu.g of recombinant human uPA (Acrobiosystems, # PLU-H5229) in reaction buffer (50mM Tris-HCl, 0.01% Tween 20, pH 8.5); or 5 or 10 units of recombinant human MMP-9(BioVision, #7867-500) in reaction buffer (50mM Tris, 150mM NaCl, 5mM CaCl)₂、20μM ZnCl₂pH 7.5) TY 22404. The reaction was carried out at 37 ℃ for 21 hours. Removal of the masking peptide from the light chain was confirmed by SDS-PAGE analysis, fig. 8A. Masking efficiency was then measured by ELISA as described above. As shown in figure 8B and table 7, after removal of the masking peptide, the activatable antibody becomes indistinguishable from the parent antibody in its binding to the antigen.

Table 7: activatable antibody ELISA after protease cleavage

Developability Profile of activatable antibodies

For manufacturing purposes, it is critical that the activatable antibodies found have a good developability profile. Several different tests were performed with purified activatable antibodies expressed in mammalian cells. Activatable antibody was adjusted to 1mg/mL in 20mM histidine (pH 5.5) and antibody quality analysis was performed using analytical size exclusion chromatography using Waters 2695 and Waters 2996UV detector and TSKgel g3000SWXL column (300mM × 7.8mM) (Tosoh Bioscience). For each assay, 10 μ g of antibody was injected and fractionated with buffer (200mM sodium phosphate, pH 7.0) at a flow rate of 0.5 mL/min.

Three accelerated stress tests were performed: the activatable antibody was incubated at 50 ℃ for 7 days, at 40 ℃ for 28 days, and six freeze-thaw cycles. The freeze-thaw test was performed by: mu.L of the sample (1mg/mL in 20mM histidine (pH 5.5)) was frozen at-80 ℃ for 30 minutes and then thawed at room temperature for 60 minutes. As shown in fig. 9A-9C, all activatable antibodies remained stable and exhibited little aggregation after 7 days of storage at 50 ℃ or 28 days of storage at 40 ℃. After six freeze-thaw cycles, they showed a slight deterioration; however, the major monomer peak remained around 95%, indicating that these activatable antibodies were extremely stable under these accelerated stress tests. Without wishing to be bound by theory, it is noteworthy that the activatable antibody has not been subjected to extensive buffer optimization procedures, and therefore, with optimized buffers and excipients, the stability of the activatable antibody may be further improved.

Next, the activatable antibody was concentrated in 20mM histidine (pH 5.5) to more than 150mg/mL (Table 8). No precipitation of activatable antibody was observed and the viscosity of the samples was very manageable. The concentrated activatable antibody is then diluted to 20mg/mL or 1mg/mL to analyze High Molecular Weight (HMW) species. As shown in FIG. 10 and Table 8, no significant increase in HMW species was observed, indicating that these activatable antibodies were extremely soluble and stable in the test buffer up to high concentrations.

Table 8: concentration of activatable antibody >150mg/mL

Sample identifier:	initial concentration (mg/mL):	high concentration (mg/mL):
			TY22401	10.9	187.2
TY22402	8.4	160.0

to investigate the stability of the activatable antibody at low pH, the purified activatable antibody (in 20mM histidine (pH 5.5), at 10 mg/mL) was titrated to 1mg/mL with citric acid and the pH was adjusted to 3.7 and kept at room temperature for 30 and 60 minutes. Thereafter, the sample was neutralized to pH 7.0 with 1M Tris base. Masking efficiency of activatable antibodies was measured with ForteBio as described above. As shown in figure 11, the masking efficiency remained unchanged after 30 or 60 minutes of low pH incubation, indicating that the masking peptides retained their blocking efficacy after low pH incubation.

In summary, the data indicate that the discovered activatable antibodies remain stable under various stress conditions and, therefore, they have a good exploitability profile.

Example 4: in vitro and in vivo characterization of activatable antibodies targeting CTLA4

It was previously shown that the parent antibody TY21580 alone did not stimulate human T cell activation or human PBMC cell activation (see PCT International application entitled "Compositions Comprising Cross-reactive Anti-CTLA4 Antibodies, and Methods of Making and Using the Same", filed concurrently herewith under attorney docket No. 69540-2000540, which is incorporated herein by reference in its entirety). It is known that CTLA-4 activity on T cells is associated with a first (TCR/CD3) signal and a second signal involving B7-CD 28/CTLA-4. Consistently, it was shown that the parent antibody TY21580 significantly enhanced human PBMC cell activation at low concentrations of the anti-CD 3 antibody.

In vitro functional characterization

Here, the activity of activatable antibodies targeting CTLA4 with respect to human PMBC activation was evaluated in the presence of low concentrations of anti-CD 3 antibody. Human PBMC were freshly isolated from blood of healthy donors (#44) by density gradient centrifugation using Histopaque-1077 (Sigma). anti-CD 3(OKT-3) antibody was coated overnight on 96-well plates at 4 ℃. After washing, 1x10^5 freshly isolated human PBMCs were added to each well followed by the addition of test article at different concentrations. Induction of IL-2 was measured 48 hours after stimulation using the human IL-2ELISA Ready-SET-go (Invitrogen) kit. IFN-. gamma.in the supernatant was measured using the human IFN-. gamma.ELISA Ready-SET-go (Invitrogen) kit. As shown in fig. 12A-fig. 12B, at high concentrations TY22404 induced IL-2 production and TY22401 induced IFN- γ production. However, the activity of the activatable antibody is significantly lower than that of the parent TY21580 antibody.

Next, the activatable antibodies were tested for antibody-dependent cytotoxic activity and compared to the parent antibody TY21580 for antibody-dependent cytotoxic activity. ADCC reporter assay is used to assess ADCC activity of an activatable antibody. HEK293F cells overexpressing human CTLA4 (HEK 293F/hTLA-4 cells) were used as target cells; jurkat cell lines overexpressing CD16a and NFAT-Luc (Jurkat/CD16a cells) were used as effector cells. 1x10^5 Jurkat/CD16a cells and 1x10^4 HEK 293F/hTLA-4 cells (E: T ratio 10:1) were mixed with different concentrations of antibody. After 6 hours of incubation, 100 μ L of One-Glo reagent was added to the cells and the cells were lysed for 10 minutes. The supernatant was removed for luminescence measurements using a SpectraMax i3x plate reader. As shown in figure 13, the activatable antibody exhibited several logs lower ADCC activity compared to the parent antibody TY 21580. The ADCC activity of TY22401 is higher than that of TY22402 and TY 22404. In summary, in vitro data indicate that the activatable antibody that is better masked has less ADCC activity.

The anti-tumor activity of the activatable antibody was then evaluated in a number of syngeneic mouse tumor models, including the MC38 colorectal tumor model, the CT26 colorectal tumor model, the H22 liver tumor model, and the 3LL lung tumor model, and compared to the anti-tumor activity of the parent antibody TY 21580.

Antitumor efficacy in the MC38 colorectal tumor model

C57BL/6 mice (n-8/group, female, 6-8 weeks old) were inoculated subcutaneously with MC38(NTCC-MC38) murine colon cancer cells. When a tumor (70mm3) was established, treatment was initiated with either an isotype control antibody, the parent antibody TY21580, or one of the three activatable antibodies by intraperitoneal injection twice a week. Tumor growth was monitored twice a week and the mean tumor volume was assessed over time ± s.e.m. (figure 14A) and individual tumor growth curves (figure 14B). As shown in fig. 14A-fig. 14B, all three activatable antibodies showed potent anti-tumor activity in the MC38 isogenic mouse tumor model, similar to the parent antibody TY 21580.

Antitumor efficacy in CT26 colorectal tumor model

BALB/c mice (n 8/group, female, 7-8 weeks old) were inoculated subcutaneously with CT26(Shanghai Institutes for Biological Sciences) murine colon cancer cells. When a tumor (100mm3) was established, treatment was initiated at 5mg/kg with either the isotype control antibody, the parent antibody TY21580, or one of the three activatable antibodies by intraperitoneal injection twice a week. Tumor growth was monitored twice a week and reported as mean tumor volume over time ± s.e.m. As shown in figure 15, all three activatable antibodies showed potent anti-tumor activity in the CT26 isogenic mouse tumor model, similar to the parent antibody TY 21580.

Antitumor efficacy in H22 liver tumor model

BALB/c mice (n 8/group, female, 7-8 weeks old) were inoculated subcutaneously with H22(China Center for Type Culture Collection) murine hepatoma cells. When a tumor (100mm3) was established, treatment was initiated at 5mg/kg with either the isotype control antibody, the parent antibody TY21580, or one of the three activatable antibodies by intraperitoneal injection twice a week. Tumor growth was monitored twice a week and reported as mean tumor volume over time ± s.e.m. As shown in figure 16, all three activatable antibodies showed potent anti-tumor activity in the H22 isogenic mouse tumor model, similar to the parent antibody TY 21580.

Antitumor efficacy in 3LL lung cancer model

C57BL/6 mice (n-10/group, female, 6-8 weeks old) were inoculated subcutaneously with 3ll (jcrb) murine lung cancer cells. When a tumor (75mm3) was established, treatment was initiated with either an isotype control antibody, the parent antibody TY21580, or one of the three activatable antibodies by intraperitoneal injection twice a week. Tumor growth was monitored twice a week and the mean tumor volume was assessed over time ± s.e.m. (figure 17A) and individual tumor growth curves (figure 17B). As shown in fig. 17A-17B, all three activatable antibodies showed potent anti-tumor activity in a 3LL isogenic mouse tumor model, similar to the parent antibody TY 21580.

Pharmacokinetic analysis

Pharmacokinetic studies were performed in BALB/c female mice at about 8 weeks of age. Three mice per group were injected intraperitoneally with test article at 10 mg/kg. Blood samples (approximately 50ul each) were collected at 3, 6, 24, 48, 96 and 168 hours post-dose. Blank control blood was collected from three naive female mice that had not been administered antibody. Serum concentrations of each test antibody were determined by ELISA, with anti-human IgG Fc antibody for capture and HRP-labeled anti-human IgG (Fab-specific) antibody (Sigma) for detection (fig. 18A-18C). Compared to previous data collected for the parent antibody TY21580, the activatable antibodies TY22401 (fig. 18A), TY22402 (fig. 18B) and TY22404 (fig. 18C) had much slower clearance times and much longer half-lives. TY22401 has a half-life of 196 hours and the drug concentration at 168 hours is about 55 μ g/mL. TY22402 has a half-life of 134 hours and the drug concentration at 168 hours is about 40 μ g/mL. TY22404 has a half-life of 254 hours and the drug concentration at 168 hours is about 45 μ g/mL. In contrast, the parent antibody TY21580 had a half-life of 107 hours and the drug concentration at 168 hours was about 17. mu.g/mL.

Repeated dose toxicity study

When assessing the effect of TY21580 on the age of onset of diabetes in NOD mice, it was found that high doses of TY21580 could lead to death in animals of NOD mice but not normal BALB/c mice. Here, NOD mouse model was used to evaluate the safety of activatable antibodies compared to TY 21580. NOD mice were treated with either isotype control antibody, parent antibody TY21580, or one of the three activatable antibodies by intraperitoneal injection at 50mg/kg on days 0, 3, 7 and 12 (n ═ 5/group, female, 6 weeks old). In the TY21580 treated group, 1 animal died after the third dose and 3 animals died after the fourth dose. As shown in figure 19, at study termination, all animals treated with either the isotype control or any of the three activatable antibodies survived and were in good health. These data indicate that the activatable antibody has an acceptable safety/toxicity profile in mice, and that the activatable antibody is much safer than the parent antibody TY21580 in NOD mice.

Example 5: construction and validation of activatable antibodies targeting CD137

Activatable antibodies targeting human CD137 were developed in a manner similar to the protocol described above in example 3 for developing anti-CTLA 4 activatable antibodies. Fab fragments (fig. 20A) or scFv (fig. 20B) of the parental CD137 antibody were displayed on the surface of yeast by fusion to Aga2 protein and their ability to bind CD137 was confirmed by flow cytometry. The VH and VL sequences of the parent CD137 antibody (TY21242) are listed below (see also PCT international application number PCT/CN2017/098332, herein incorporated by reference in its entirety):

anti-CD 137 heavy chain variable region (SEQ ID NO: 49):

EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSS

anti-CD 137 light chain variable region (SEQ ID NO: 50):

DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIK。

yeast libraries were constructed with CPL fused to the N-terminus of the light chain and subjected to FACS-based screening procedures. Single clones from round 4 or round 5 sorting (fig. 21) were plated on selective media and grown individually to confirm lysis-mediated antigen binding activation. As shown in fig. 22A-22B, selected CD137 activatable antibody clones exhibited little binding to antigen in the presence of masking peptide; however, when TEV protease is used to treat yeast cells to remove masking peptides, binding to the antigen is significantly increased.

As observed with CTLA4 activatable antibodies, the identified masking sequences can be divided into several groups, indicating the clear enrichment achieved by each round of sorting. Seven sets of masking peptide sequences are listed in table 9 along with the invariant cleavage peptide sequences. Several of these sequence groups (TY22594, TY22595, TY22596, TY22598, TY22599) are derived from a CPL011 library containing NNK codons in the loop between the two fixed Cys residues. Interestingly, for all of these sequence groups, two or more Arg residues were present in the loop, suggesting that charge-charge interactions may be involved between the masking peptide and the CDRs of the parent antibody. In fact, negatively charged Asp residues are present in VH CDR2 and VH CDR 3.

Table 9: masking peptide sequences

The masking sequence and invariant cleavage sequence were then tested in the context of a complete IgG4 molecule expressed in mammalian cells. Their expression levels were similar to their parent antibodies, and their purification yields after protein a resin were also similar, indicating that the presence of masking and cleavage peptides does not have any negative effect on the expression of the antibodies in mammalian cells.

The masking efficiency was then measured by flow cytometry. Briefly, yeast cells displaying human CD137 on their surface were washed twice with PBSA buffer and 50. mu.L (1X10^6) of cells were dispensed into each well of a 96-well plate. Cells were then incubated with 3-fold serial dilutions of the antibody on ice for 1 hour, washed once with PBSA buffer, and then incubated with 100. mu.L of PE-conjugated mouse anti-human Fc antibody (1. mu.g/ml) onceIncubate on ice for 30 minutes. The cells are then washed once, followed by flow cytometry (b) ((b))CytoFlex). As shown in figure 23 and table 10, all activatable antibodies showed significantly reduced binding to human CD137 on the cell surface compared to the parent antibody TY21242, and the calculated masking efficiency ranged from 20-fold (for TY22596) to over 300-fold (for TY22586, TY22595, and TY 22599). These results indicate that the masking peptides identified from the CPL library displayed on yeast maintain their masking efficiency when expressed in mammalian cells.

Table 10: masking efficiency of CD137 activatable antibodies

Too low-binding is so weak that it is not detectable in this assay.

In summary, the data indicate that a variety of potent masking peptides were successfully found for each target antibody using the methods described herein.

Example 6: effect of masking peptide length on masking efficiency of activatable antibodies targeting CTLA4

Two activatable antibodies TY22402 and TY22404 were chosen to test the dependence of the masking efficiency on the length of the masking peptide to suit their specific application. The masking peptides of TY22402 and TY22404 were shortened from 21 residues to 16 or 12 residues by removing the residue from the N-terminus, thus leaving only 5 or 2 residues before the first cysteine residue in the masking peptide (table 11). These activatable antibodies were expressed and purified from mammalian cells and their masking efficiency was measured and compared to the parent antibody TY21580 as described in example 3. The results from both experiments indicate that these activatable antibodies can be prepared using different masking peptides ranging from 2 to 11 residues in length before the first cysteine residue to modulate antibody masking efficiency (fig. 24A and 24B; tables 12 and 13). This appears to suggest that the core masking motif contains a cysteine loop and its immediate residues and is sufficient to maintain masking efficiency.

Table 11: masking peptides of varying peptide length

Table 12 shows the masking efficiency of the antibody in fig. 24A. Table 13 shows the masking efficiency of the antibodies in fig. 24B.

Table 12: masking efficiency of antibodies with different masking peptide lengths

Sample identifier	EC50(nM)	Efficiency of masking
			TY21580	0.2223
TY22402	53.99	243
			TY22775	37.31	168
TY22404	68.40	308
			TY22776	65.90	296

Table 13: masking efficiency of antibodies with different masking peptide lengths

Example 7: effect of cleavage peptide length on masking efficiency of activatable antibodies targeting CTLA4

TY22404 was chosen to test the dependence of the masking efficiency on the length of the cleaved peptides to suit their specific application. The cleavage peptides of TY22404 were shortened to various lengths (table 14). Activatable antibodies were expressed and purified from mammalian cells and their masking efficiency was measured and compared to the parent antibody TY21580 as described in example 3. As shown in fig. 25 and table 15, the results indicate that these activatable antibodies can be prepared using different lytic peptides whose length ranges from 5 to 20 residues to modulate antibody masking efficiency. The strong association between masking and cleavage motifs is striking; when the peptide length is truncated from 41 amino acids to 17 amino acids, the masking efficiency of TY23291 is enhanced to at least 30-fold compared to TY 22404. These results indicate that several novel masking peptides can be designed and engineered. In addition, the coupling between the masking motif and the cleavage motif can be further explored.

Table 14: masking peptides with different cleavage peptide lengths

Table 15 shows the masking efficiency of the antibodies in fig. 25.

Table 15: masking efficiency of antibodies with different cleavage peptide lengths

Sample identifier	EC50(nM)	Efficiency of masking
			TY21580	0.2505
TY22404	117.4	469
			TY23286	1496	5972
TY23289	133.2	532
			TY23280	2952	11784
TY23291	3656	14595

Sequence listing

<110> Tian performance pharmaceutical Co., Ltd. (Adagene Inc.)

<120> activatable antibodies and methods of making and using the same

<130> 69540-20006.00

<140> not yet allocated

<141> 2019-02-02

<150> PCT/CN2019/074581

<151> 2019-02-02

<150> PCT/CN2018/075065

<151> 2018-02-02

<160> 133

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 3

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 5

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<220>

<221> variants

<222> 3

<223> to repeat at least three times and up to ten times

<220>

<221> variants

<222> 5

<223> to repeat at least once and up to ten times

<400> 1

Xaa Cys Xaa Cys Xaa

1 5

<210> 2

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> misc_feature

<222> 1, 2, 7, 8, 13

<223> n = A, T, C or G

<220>

<221> misc_feature

<222> 3, 9

<223> n = T or G

<220>

<221> misc_feature

<222> 6, 12

<223> n = T or C

<220>

<221> misc_feature

<222> 14

<223> n = A, T or C

<220>

<221> misc_feature

<222> 1, 2, 3

<223> to repeat at least twice and up to ten times

<220>

<221> misc_feature

<222> 7, 8, 9

<223> to repeat at least three times and up to ten times

<220>

<221> misc_feature

<222> 13, 14, 15

<223> to repeat at least once and up to ten times

<400> 2

nnntgnnnnt gnnnc 15

<210> 3

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 6

<223> Xaa = Ala, Asp, Ile, Asn, Pro or Tyr

<220>

<221> variants

<222> 7

<223> Xaa = Ala, Phe, Asn, Ser or Val

<220>

<221> variants

<222> 8

<223> Xaa = Ala, His, Leu, Pro, Ser, Val or Tyr

<220>

<221> variants

<222> 9

<223> Xaa = Ala, His, Ser or Tyr

<220>

<221> variants

<222> 10

<223> Xaa = Ala, Asp, Pro, Ser, Val or Tyr

<220>

<221> variants

<222> 11

<223> Xaa = Ala, Asp, Leu, Ser or Tyr

<220>

<221> variants

<222> 13

<223> Xaa = Asp, Pro or Val

<220>

<221> variants

<222> 14

<223> Xaa = Ala, Asp, His, Pro, Ser or Thr

<220>

<221> variants

<222> 15

<223> Xaa = Ala, Asp, Phe, His, Pro or Tyr

<220>

<221> variants

<222> 16

<223> Xaa = Leu, Pro or Tyr

<220>

<221> variants

<222> 17

<223> Xaa = Phe, Pro or Tyr

<220>

<221> variants

<222> 18

<223> Xaa = Ala, Pro, Ser or Tyr

<220>

<221> variants

<222> 20

<223> Xaa = Ala, Asp, Asn, Ser, Thr or Tyr

<220>

<221> variants

<222> 21

<223> Xaa = Ala, Ser or Tyr

<400> 3

Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 4

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 20, 21

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<400> 4

Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala

20 25

<210> 5

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 6, 7, 8, 9, 10, 11, 20, 21

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<220>

<221> variants

<222> 13, 14, 15, 16, 17, 18

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<400> 5

Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala

20 25

<210> 6

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<400> 6

Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala

20 25

<210> 7

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 6, 7, 8, 9, 10, 11, 22, 23

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<220>

<221> variants

<222> 13, 14, 15, 16, 17, 18, 19, 20

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<400> 7

Glu Val Gly Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Cys Xaa Xaa Ser Gly Arg Ser Ala

20 25

<210> 8

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> misc_feature

<222> 16, 22, 28

<223> n = A, T, C or G

<220>

<221> misc_feature

<222> 17, 23, 29

<223> n = A, T or C

<220>

<221> misc_feature

<222> 16, 17, 18

<223> present in six repetitions

<220>

<221> misc_feature

<222> 22, 23, 24

<223> present in six repetitions

<220>

<221> misc_feature

<222> 28, 29, 30

<223> to repeat twice

<400> 8

gaggttggat cctacnnctg tnnctgcnnc tcaggtcgtt ccgct 45

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> misc_feature

<222> 16, 22, 23, 28

<223> n = A, T, C or G

<220>

<221> misc_feature

<222> 17, 29

<223> n = A, T or C

<220>

<221> misc_feature

<222> 16, 17, 18

<223> present in six repetitions

<220>

<221> misc_feature

<222> 22, 23, 24

<223> present in six repetitions

<220>

<221> misc_feature

<222> 28, 29, 30

<223> to repeat twice

<400> 9

gaggttggat cctacnnctg tnnntgcnnc tcaggtcgtt ccgct 45

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> misc_feature

<222> 16, 22, 28

<223> n = A, T, C or G

<220>

<221> misc_feature

<222> 17, 23, 29

<223> n = A, T or C

<220>

<221> misc_feature

<222> 16, 17, 18

<223> present in six repetitions

<220>

<221> misc_feature

<222> 22, 23, 24

<223> present in eight repetitions

<220>

<221> misc_feature

<222> 28, 29, 30

<223> to repeat twice

<400> 10

gaggttggat cctacnnctg tnnctgcnnc tcaggtcgtt ccgct 45

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> misc_feature

<222> 16, 22, 23, 28

<223> n = A, T, C or G

<220>

<221> misc_feature

<222> 17, 29

<223> n = A, T or C

<220>

<221> misc_feature

<222> 16, 17, 18

<223> present in six repetitions

<220>

<221> misc_feature

<222> 22, 23, 24

<223> present in eight repetitions

<220>

<221> misc_feature

<222> 28, 29, 30

<223> to repeat twice

<400> 11

gaggttggat cctacnnctg tnnntgcnnc tcaggtcgtt ccgct 45

<210> 12

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 12

Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro Leu Gly Leu Ala Gly

1 5 10 15

Ser Gly Gly Ser

<210> 13

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 13

Ser Gly Arg Ser Ala

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 14

Pro Leu Gly Leu Ala Gly

1 5

<210> 15

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 15

Glu Asn Leu Tyr Phe Gln Gly

1 5

<210> 16

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 16

Glu Val Gly Ser Tyr

1 5

<210> 17

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 17

Gly Gly Gly Gly Ser

1 5

<210> 18

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 18

Ser Gly Gly Ser

<210> 19

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 19

Gly Gly Ser Gly

<210> 20

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 20

Gly Gly Ser Gly Gly

1 5

<210> 21

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 21

Gly Ser Gly Ser Gly

1 5

<210> 22

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 22

Gly Ser Gly Gly Gly

1 5

<210> 23

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 23

Gly Gly Gly Ser Gly

1 5

<210> 24

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 24

Gly Ser Ser Ser Gly

1 5

<210> 25

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 25

Glu Val Gly Ser Tyr Asp Ala Leu His Tyr Ala Cys Pro Pro Asp Tyr

1 5 10 15

Tyr Ala Cys Tyr Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 26

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 26

Glu Val Gly Ser Tyr Asn Ser Tyr His Ala Tyr Cys Pro His Pro Leu

1 5 10 15

Tyr Pro Cys Thr Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 27

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 27

Glu Val Gly Ser Tyr Ala Ser Ser Ala Val Leu Cys Val Thr Ala Tyr

1 5 10 15

Phe Ser Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 28

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 28

Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro

1 5 10 15

Tyr Pro Cys Ser Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 29

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 29

Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro

1 5 10 15

Tyr Pro Cys Ser Ala Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 30

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 30

Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr

1 5 10 15

Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 31

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 31

Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr

1 5 10 15

Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 32

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 32

Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr

1 5 10 15

Pro Tyr Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 33

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 33

Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr

1 5 10 15

Pro Tyr Cys Asn Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 34

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 34

Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 35

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 35

Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Glu

20 25 30

Asn Leu Tyr Phe Gln Gly Ser Gly Gly Ser

35 40

<210> 36

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 36

Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr

1 5 10 15

Pro Tyr Cys Gln Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 37

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 37

Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr

1 5 10 15

Pro Tyr Cys Asn Ser Ala Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 38

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 38

Glu Val Gly Ser Tyr Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 39

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 39

Glu Val Gly Ser Tyr Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 40

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 40

Glu Val Gly Ser Tyr Pro Thr Asp Leu Asp Ala Cys Ala Asp Ala Pro

1 5 10 15

Asn His Cys His Phe Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 41

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 41

Glu Val Gly Ser Tyr Ser Ser Thr His Ala His Cys His His Ser Pro

1 5 10 15

Ala Asn Cys Ile Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 42

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 42

Glu Val Gly Ser Tyr Asp Thr Asp Tyr Asp Phe Cys Pro Ile Leu Arg

1 5 10 15

His Arg Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 43

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 43

Glu Val Gly Ser Tyr Asn Asp Tyr Asn Tyr His Cys Lys Trp Arg Pro

1 5 10 15

Ser Arg Cys His Asn Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 44

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 44

Glu Val Gly Ser Tyr Tyr His Asp Tyr Asp Asp Cys Arg Val Leu Pro

1 5 10 15

Arg Arg Cys Phe Asn Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 45

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 45

Glu Val Gly Ser Tyr Ser Asn Asn Phe Ala Ser Cys Leu Trp Arg His

1 5 10 15

Arg Ser Cys Ala Asp Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 46

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 46

Glu Val Gly Ser Tyr Thr Asp Asn Tyr Asp Tyr Cys Pro Arg Leu Arg

1 5 10 15

Arg Lys Cys Tyr His Ser Gly Arg Ser Ala Gly Gly Gly Gly Ser Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 47

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 47

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 Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr His Trp Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Tyr Val Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 48

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 48

Asp Ile Gln Leu 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 Ser Val Arg Gly Arg

20 25 30

Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Ser Trp Pro

85 90 95

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

100 105

<210> 49

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 49

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 Ser Leu Ser Thr Gly

20 25 30

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

35 40 45

Trp Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

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

85 90 95

Cys Ala Arg Gly Gly Ser Asp Thr Val Ile Gly Asp Trp Phe Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 50

Asp Ile Gln Leu 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 Ser Ile Gly Ser Tyr

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr 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 Gly Tyr Tyr Leu Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 51

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 51

tcgggtgagg ttggatccta c 21

<210> 52

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 52

gtacaggttc tcggtaccac c 21

<210> 53

<211> 65

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 53

tggagacaca gacaggatca ctggagactg ggtcagcagg atatcggatc ctgaaccgcc 60

tgaac 65

<210> 54

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 54

cttcgctgtt tttcaatatt ttctgttatt gcttcagttt tagcaggatc cgaggttgga 60

tcctac 66

<210> 55

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 2, 3, 4, 5, 6, 15, 16

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<220>

<221> variants

<222> 8, 9, 10, 11, 12, 13

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<400> 55

Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa

1 5 10 15

<210> 56

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 2, 3, 4, 5, 6, 17, 18

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<220>

<221> variants

<222> 8, 9, 10, 11, 12, 13, 14, 15

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<400> 56

Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys

1 5 10 15

Xaa Xaa

<210> 57

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<400> 57

Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa

1 5 10 15

<210> 58

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<400> 58

Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys

1 5 10 15

Xaa Xaa

<210> 59

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 59

Tyr Ser Ile Ser Ser Gly Tyr His Trp Ser Trp Ile

1 5 10

<210> 60

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 60

Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu

1 5 10 15

Lys Ser Arg Leu

<210> 61

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 61

Ala Arg Ser Tyr Val Tyr Phe Asp Tyr

1 5

<210> 62

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 62

Arg Ala Ser Gln Ser Val Arg Gly Arg Phe Leu Ala

1 5 10

<210> 63

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 63

Asp Ala Ser Asn Arg Ala Thr Gly Ile

1 5

<210> 64

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 64

Tyr Cys Gln Gln Ser Ser Ser Trp Pro Pro Thr

1 5 10

<210> 65

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 65

Phe Ser Leu Ser Thr Gly Gly Val Gly Val Gly Trp Ile

1 5 10

<210> 66

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 66

Leu Ala Leu Ile Asp Trp Ala Asp Asp Lys Tyr Tyr Ser Pro Ser Leu

1 5 10 15

Lys Ser Arg Leu

<210> 67

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 67

Ala Arg Gly Gly Ser Asp Thr Val Ile Gly Asp Trp Phe Ala Tyr

1 5 10 15

<210> 68

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 68

Arg Ala Ser Gln Ser Ile Gly Ser Tyr Leu Ala

1 5 10

<210> 69

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 69

Asp Ala Ser Asn Leu Glu Thr Gly Val

1 5

<210> 70

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 70

Tyr Cys Gln Gln Gly Tyr Tyr Leu Trp Thr

1 5 10

<210> 71

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<220>

<221> variants

<222> 3

<223> to repeat at least three times and up to ten times

<220>

<221> variants

<222> 5

<223> to repeat at least once and up to ten times

<220>

<221> variants

<222> 1, 3, 5

<223> Xaa = Asp, Ala, Tyr, Ser, Thr, Asn, Ile, Leu, Phe, Val, His or Pro

<400> 71

Xaa Cys Xaa Cys Xaa

1 5

<210> 72

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 72

Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro Tyr Pro Cys Ser Ala

1 5 10 15

<210> 73

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 73

Ile Val His His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser

1 5 10 15

<210> 74

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 74

Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr Pro Tyr Cys Asn Ser

1 5 10 15

<210> 75

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 75

Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr

1 5 10 15

<210> 76

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 76

Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr Pro Tyr Cys Gln Ser

1 5 10 15

<210> 77

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 77

Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr

1 5 10 15

<210> 78

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 78

Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr

1 5 10 15

<210> 79

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 79

Pro Thr Asp Leu Asp Ala Cys Ala Asp Ala Pro Asn His Cys His Phe

1 5 10 15

<210> 80

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 80

Ser Ser Thr His Ala His Cys His His Ser Pro Ala Asn Cys Ile Ser

1 5 10 15

<210> 81

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 81

Asp Thr Asp Tyr Asp Phe Cys Pro Ile Leu Arg His Arg Cys Asp Ser

1 5 10 15

<210> 82

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 82

Asn Asp Tyr Asn Tyr His Cys Lys Trp Arg Pro Ser Arg Cys His Asn

1 5 10 15

<210> 83

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 83

Tyr His Asp Tyr Asp Asp Cys Arg Val Leu Pro Arg Arg Cys Phe Asn

1 5 10 15

<210> 84

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 84

Asn Asn Phe Ala Ser Cys Leu Trp Arg His Arg Ser Cys Ala Asp

1 5 10 15

<210> 85

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 85

Thr Asp Asn Tyr Asp Tyr Cys Pro Arg Leu Arg Arg Lys Cys Tyr His

1 5 10 15

<210> 86

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<220>

<221> variants

<222> 3

<223> to repeat at least three times and up to ten times

<220>

<221> variants

<222> 5

<223> to repeat at least once and up to ten times

<220>

<221> variants

<222> 1, 3, 5

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<400> 86

Xaa Cys Xaa Cys Xaa

1 5

<210> 87

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> misc_feature

<222> 1, 2, 7, 8, 13, 14

<223> n = A, T, C or G

<220>

<221> misc_feature

<222> 3, 9, 15

<223> n = T or G

<220>

<221> misc_feature

<222> 6, 12

<223> n = T or C

<220>

<221> misc_feature

<222> 1, 2, 3

<223> to repeat at least twice and up to ten times

<220>

<221> misc_feature

<222> 7, 8, 9

<223> to repeat at least three times and up to ten times

<220>

<221> misc_feature

<222> 13, 14, 15

<223> to repeat at least once and up to ten times

<400> 87

nnntgnnnnt gnnnn 15

<210> 88

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 88

Xaa Cys Ala Asp Ala Pro Asn His Cys Xaa Xaa

1 5 10

<210> 89

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 89

Xaa Cys His His Ser Pro Ala Asn Cys Xaa Xaa

1 5 10

<210> 90

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 90

Xaa Cys Pro Ile Leu Arg His Arg Cys Xaa Xaa

1 5 10

<210> 91

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 91

Xaa Cys Lys Trp Arg Pro Ser Arg Cys Xaa Xaa

1 5 10

<210> 92

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 92

Xaa Cys Arg Val Leu Pro Arg Arg Cys Xaa Xaa

1 5 10

<210> 93

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 93

Xaa Cys Leu Trp Arg His Arg Ser Cys Xaa Xaa

1 5 10

<210> 94

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 94

Xaa Cys Pro Arg Leu Arg Arg Lys Cys Xaa Xaa

1 5 10

<210> 95

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 95

Glu Val Gly Ser Tyr Pro Thr Asp Leu Asp Ala Cys Ala Asp Ala Pro

1 5 10 15

Asn His Cys His Phe

<210> 96

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 96

Glu Val Gly Ser Tyr Ser Ser Thr His Ala His Cys His His Ser Pro

1 5 10 15

Ala Asn Cys Ile Ser

<210> 97

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 97

Glu Val Gly Ser Tyr Asp Thr Asp Tyr Asp Phe Cys Pro Ile Leu Arg

1 5 10 15

His Arg Cys Asp Ser

<210> 98

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 98

Glu Val Gly Ser Tyr Asn Asp Tyr Asn Tyr His Cys Lys Trp Arg Pro

1 5 10 15

Ser Arg Cys His Asn

<210> 99

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 99

Glu Val Gly Ser Tyr Tyr His Asp Tyr Asp Asp Cys Arg Val Leu Pro

1 5 10 15

Arg Arg Cys Phe Asn

<210> 100

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 100

Glu Val Gly Ser Tyr Ser Asn Asn Phe Ala Ser Cys Leu Trp Arg His

1 5 10 15

Arg Ser Cys Ala Asp

<210> 101

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 101

Glu Val Gly Ser Tyr Thr Asp Asn Tyr Asp Tyr Cys Pro Arg Leu Arg

1 5 10 15

Arg Lys Cys Tyr His

<210> 102

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 102

Xaa Cys Pro Asp His Pro Tyr Pro Cys Xaa Xaa

1 5 10

<210> 103

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 103

Xaa Cys Asp Ala Phe Tyr Pro Tyr Cys Xaa Xaa

1 5 10

<210> 104

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 104

Xaa Cys Asp Ser His Tyr Pro Tyr Cys Xaa Xaa

1 5 10

<210> 105

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<220>

<221> variants

<222> 1, 10, 11

<223> Xaa = Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr

<220>

<221> variants

<222> 1

<223> to repeat at least twice and up to ten times

<400> 105

Xaa Cys Val Pro Tyr Tyr Tyr Ala Cys Xaa Xaa

1 5 10

<210> 106

<400> 106

000

<210> 107

<400> 107

000

<210> 108

<400> 108

000

<210> 109

<400> 109

000

<210> 110

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 110

Glu Val Gly Ser Tyr Asn Phe Val Ala Asp Ser Cys Pro Asp His Pro

1 5 10 15

Tyr Pro Cys Ser Ala

<210> 111

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 111

Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr

1 5 10 15

Pro Tyr Cys Asp Ser

<210> 112

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 112

Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr

1 5 10 15

Pro Tyr Cys Asn Ser

<210> 113

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 113

Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr

<210> 114

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 114

Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr

1 5 10 15

Pro Tyr Cys Gln Ser

<210> 115

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 115

Glu Val Gly Ser Tyr Tyr Ser Ala Tyr Pro Ala Cys Asp Ser His Tyr

1 5 10 15

Pro Tyr Cys Asn Ser

<210> 116

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 116

Glu Val Gly Ser Tyr Pro Gln Pro Ser Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr

<210> 117

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 117

Glu Val Gly Ser Tyr Pro Asn Pro Ala Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr

<210> 118

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 118

Glu Val Gly Ser Tyr Ile Val His His Ser Asp Cys Asp Ala Phe Tyr

1 5 10 15

Pro Tyr Cys Asp Ser Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 119

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 119

Glu Val Gly His Ser Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser

1 5 10 15

Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly

20 25 30

Ser Gly Gly Ser

<210> 120

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 120

Glu Asp Cys Asp Ala Phe Tyr Pro Tyr Cys Asp Ser Ser Gly Arg Ser

1 5 10 15

Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly Ser Gly Gly Ser

20 25 30

<210> 121

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 121

Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro

20 25 30

Leu Gly Leu Ala Gly Ser Gly Gly Ser

35 40

<210> 122

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 122

Glu Val Gly Ser Ser Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr

1 5 10 15

Ser Gly Arg Ser Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly

20 25 30

Ser Gly Gly Ser

<210> 123

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 123

Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser

1 5 10 15

Ala Gly Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly Ser Gly Gly Ser

20 25 30

<210> 124

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 124

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Ala Gln Ile

100 105 110

Tyr Val Ile Asp Pro Glu

115

<210> 125

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 125

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile

100 105 110

Tyr Val Ile Asp Pro Glu

115

<210> 126

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 126

Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

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

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Ile Cys

50 55 60

Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu

65 70 75 80

Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr

85 90 95

Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val

100 105 110

Ile Asp Pro

115

<210> 127

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 127

Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly

1 5 10 15

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

20 25 30

Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val

35 40 45

Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Asp Ser Ile

50 55 60

Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly

65 70 75 80

Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met

85 90 95

Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr

100 105 110

Val Ile

<210> 128

<211> 120

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 128

Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

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

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu

115 120

<210> 129

<211> 120

<212> PRT

<213> little mouse (Mus musculus)

<400> 129

Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His

1 5 10 15

Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu

35 40 45

Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp

50 55 60

Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr

65 70 75 80

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

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu

115 120

<210> 130

<211> 31

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 130

Glu Val Gly Ser Tyr Pro Asn Pro Ser Ser Asp Cys Val Pro Tyr Tyr

1 5 10 15

Tyr Ala Cys Ala Tyr Ser Gly Arg Ser Ala Pro Leu Gly Leu Ala

20 25 30

<210> 131

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 131

Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser

1 5 10 15

Ala Pro Leu Gly Leu Ala

<210> 132

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 132

Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Ser Gly Arg Ser

1 5 10 15

Ala

<210> 133

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 133

Glu Asp Cys Val Pro Tyr Tyr Tyr Ala Cys Ala Tyr Pro Leu Gly Leu

1 5 10 15

Ala

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