Modified antibody compositions, methods of making and using thereof

• US 8,513,390 B2
• Filed: 04/25/2012
• Issued: 08/20/2013
• Est. Priority Date: 01/12/2009
• Status: Active Grant

• Current Assignee: CytomX Therapeutics, Inc
• Original Assignee: CytomX Therapeutics, Inc
• Inventors: Stagliano, Nancy E., West, James W., Kamath, Kathryn, Bessette, Paul H., Sagert, Jason, Daugherty, Patrick, Gluck, Fred
• Primary Examiner(s): HUYNH, PHUONG N
• Application Number:
  US13/455,924
• Publication Number:
  US 20120207756A1
• Time in Patent Office: 482 Days
• Field of Search: None
• US Class Current: 530/387.3
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/507 Comprising a combination of two or more separate antibodies
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 47/6845 the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/22 : against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/241 : Tumor Necrosis Factors
• CPC Class Codes: C07K 16/28 : against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2818 : against CD28 or CD152
• CPC Class Codes: C07K 16/2845 : against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• CPC Class Codes: C07K 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2866 : against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/2875 : against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 against NGF C07K16/22, against TNF C07K16/241
• CPC Class Codes: C07K 16/2896 : against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 2317/20 : characterized by taxonomic origin
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• CPC Class Codes: C07K 2317/34 : Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/40 : characterized by post-translational modification
• CPC Class Codes: C07K 2317/51 : Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 : Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/31 : fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/50 : containing protease site
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 : having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6854 : Immunoglobulins

Abstract

The present disclosure provides modified antibodies which contain an antibody or antibody fragment (AB) modified with a masking moiety (MM). Such modified antibodies can be further coupled to a cleavable moiety (CM), resulting in activatable antibodies (AAs), wherein the CM is capable of being cleaved, reduced, photolysed, or otherwise modified. AAs can exhibit an activatable conformation such that the AB is more accessible to a target after, for example, removal of the MM by cleavage, reduction, or photolysis of the CM in the presence of an agent capable of cleaving, reducing, or photolysing the CM. The disclosure further provides methods of making and using such modified antibodies and activatable antibodies.

Claims

1. An activatable antibody that in an activated state binds Epidermal Growth Factor Receptor (EGFR),the activatable antibody comprising:

an antibody or an antigen binding fragment thereof (AB) that specifically binds to EGFR;

a masking moiety (MM) that inhibits the binding of the AB in an uncleaved state to EGFR and wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO;

1, 218-220, 236-265 and 266; and

at least one cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), legumain, and matriptase (MT-SP1) and the substrate for uPA comprises the amino acid sequence LSGRSDNH (SEQ ID NO;

     271).

2. The activatable antibody of claim 1, wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB to EGFR.

3. The activatable antibody of claim 1, wherein the MM does not interfere or compete with the AB for binding to EGFR in a cleaved state.

4. The activatable antibody of claim 1, wherein the protease is co-localized with EGFR in a tissue, and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

5. The activatable antibody of claim 1, wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows:

MM-CM-AB or AB-CM-MM.

6. The activatable antibody of claim 1, wherein the activatable antibody comprises a linking peptide between the MM and the CM.

7. The activatable antibody of claim 1, wherein the activatable antibody comprises a linking peptide between the CM and the AB.

8. The activatable antibody of claim 1, wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody has the structural arrangement in the uncleaved state from N-terminus to C-terminus as follows:

MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.

9. The activatable antibody of claim 1, wherein the AB has an equilibrium dissociation constant of at most 100 nM for binding to EGFR.

10. The activatable antibody of claim 1, wherein the MM is a polypeptide of about 2 to 40 amino acids in length.

11. The activatable antibody of claim 1, wherein the CM is positioned in the activatable antibody such that in the uncleaved state, binding of the activatable antibody to EGFR is reduced to occur with an equilibrium dissociation constant that is at least 100-fold greater than the equilibrium dissociation constant of an unmodified AB binding to EGFR, and whereas in the cleaved state, the AB binds EGFR.

12. The activatable antibody of claim 8, wherein the two linking peptides need not be identical to each other.

13. The activatable antibody of claim 1, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 100 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

14. The activatable antibody of claim 1, wherein the CM is a polypeptide of up to 15 amino acids in length.

15. The activatable antibody of claim 8, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

16. The activatable antibody of claim 1, wherein in the presence of EGFR, the MM reduces the ability of the AB to bind EGFR by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay.

17. The activatable antibody of claim 1, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′

)₂ fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

18. The activatable antibody of claim 1, wherein the AB is or is from cetuximab, panitumumab, zalutumumab, matuzumab, nimotuzumab, ICR62, mAb 528, CH806, or MDX-447.

19. The activatable antibody of claim 1, wherein the AB is or is from cetuximab.

20. The activatable antibody of claim 1 further comprising a second AB wherein the target for the second AB is selected from the group consisting of the targets in Table 1.

21. The activatable antibody of claim 1, wherein the CM is a substrate for a second protease, wherein the second protease is an enzyme selected from the group consisting of the enzymes in Table 3.

22. The activatable antibody of claim 1, wherein the CM is a substrate for uPA and a second protease selected from the group consisting of legumain and matriptase (MT-SP1).

23. The activatable antibody of claim 1 wherein the Ab is conjugated to an agent.

24. The activatable antibody of claim 23, wherein the agent is a therapeutic agent.

25. The activatable antibody of claim 24, wherein the agent is an antineoplastic agent.

26. The activatable antibody of claim 23, wherein the agent is a toxin or fragment a thereof.

27. The activatable antibody of claim 23, wherein the agent is an agent selected from the group consisting of the agents in Table 4.

28. The activatable antibody of claim 23, wherein the agent is conjugated to the AB via a linker.

29. The activatable antibody of claim 28, wherein the linker is a cleavable linker.

30. The activatable antibody of claim 1 comprising a detectable moiety.

31. The activatable antibody of claim 30, wherein the detectable moiety is a diagnostic agent.

32. The activatable antibody of claim 1, wherein the serum half-life of the activatable antibody is at least 5 days when administered to an organism.

33. The activatable antibody of claim 1, wherein the affinity of binding of the activatable antibody to EGFR is higher in a target tissue when compared to the binding of the activatable antibody to EGFR in a non-target tissue.

34. The activatable antibody of claim 33, wherein the target tissue is a diseased tissue.

35. The activatable antibody of claim 34, wherein the diseased tissue is breast tissue, head tissue, neck tissue, lung tissue, pancreatic tissue, nervous system tissue, liver tissue, prostate tissue, urogenital tissue, cervical tissue, colon tissue or colorectal tissue.

36. The activatable antibody of claim 1, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 1000 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

37. The activatable antibody of claim 1, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 10,000 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

38. The activatable antibody of claim 8, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)_n, (GGS)_n, (GSGGS)_n (SEQ ID NO:

12) and (GGGS)_n (SEQ ID NO;

     13), where n is an integer of at least one.

39. The activatable antibody of claim 8, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:

14), GGSGG (SEQ ID NO;

     15), GSGSG (SEQ ID NO;

     16), GSGGG (SEQ ID NO;

     17), GGGSG (SEQ ID NO;

     18), and GSSSG (SEQ ID NO;

     19).

40. The activatable antibody of claim 1, wherein the first CM is a polypeptide that that functions as a substrate for a protease selected from the group consisting of uPA, legumain, and MT-SP1 and a second CM and wherein the substrate for uPA comprises the amino acid sequence LSGRSDNH (SEQ ID NO:

271).

41. The activatable antibody of claim 40, wherein the first CM is cleaved by a first cleaving agent selected from the group consisting of uPA, legumain, and MT-SP1 in a target tissue and wherein the second CM is cleaved by a second cleaving agent in a target tissue.

42. The activatable antibody of claim 41, wherein the first cleaving agent and the second cleaving agent are the same enzyme selected from the group consisting of uPA, legumain, and MT-SP1, and where the first CM and the second CM are different substrates for the enzyme.

43. The activatable antibody of claim 41, wherein the first cleaving agent and the second cleaving agent are different enzymes.

44. The activatable antibody of claim 41, wherein the first cleaving agent and the second cleaving agent are co-localized in the target tissue.

45. The activatable antibody of claim 40, wherein the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

46. The activatable antibody of claim 1, wherein the MM comprises the amino acid sequence CISPRGC (SEQ ID NO:

1).

47. The activatable antibody of claim 1, wherein the MM comprises the amino acid sequence CISPRGCPDGPYVM (SEQ ID NO:

218) or CISPRGCPDGPYVMY (SEQ ID NO;

     238).

48. The activatable antibody of claim 8, wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO:

111).

49. The activatable antibody of claim 8, wherein LP2 comprises the amino acid sequence GS or GSSG.

50. The activatable antibody of claim 8, wherein the CM is a substrate for an enzyme selected from the group consisting of uPA, legumain and MT-SP1 and wherein the substrate for uPA comprises the amino acid sequence LSGRSDNH (SEQ ID NO:

271), wherein the MM comprises the amino acid sequence CISPRGCPDGPYVM (SEQ ID NO;

     218), wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO;

     111), and wherein LP2 comprises the amino acid sequence GS.

51. An activatable antibody that in an activated state binds Epidermal Growth Factor Receptor (EGFR), the activatable antibody comprising:

an antibody or an antigen binding fragment thereof (AB) that specifically binds to EGFR;

a masking moiety (MM) that inhibits the binding of the AB in an uncleaved state to EGFR, wherein the MM comprises the amino acid sequence CISPRGCPDGPYVM (SEQ ID NO;

     218) or CISPRGCPDGPYVMY (SEQ ID NO;

     238); and

at least one cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

52. The activatable antibody of claim 51, wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB to EGFR.

53. The activatable antibody of claim 51, wherein the MM does not interfere or compete with the AB for binding to EGFR in a cleaved state.

54. The activatable antibody of claim 51, wherein the protease is co-localized with EGFR in a tissue, and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

55. The activatable antibody of claim 51, wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows:

MM-CM-AB or AB-CM-MM.

56. The activatable antibody of claim 51, wherein the activatable antibody comprises a linking peptide between the MM and the CM.

57. The activatable antibody of claim 51, wherein the activatable antibody comprises a linking peptide between the CM and the AB.

58. The activatable antibody of claim 51, wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows:

MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.

59. The activatable antibody of claim 51, wherein the AB has an equilibrium dissociation constant of at most 100 nM for binding to EGFR.

60. The activatable antibody of claim 51, wherein the MM is a polypeptide of no more than 40 amino acids in length.

61. The activatable antibody of claim 51, wherein the CM is positioned in the activatable antibody such that in the uncleaved state, binding of the activatable antibody to EGFR is reduced to occur with an equilibrium dissociation constant that is at least 100-fold greater than the equilibrium dissociation constant of an unmodified AB binding to EGFR, and whereas in the cleaved state, the AB binds EGFR.

62. The activatable antibody of claim 58, wherein the two linking peptides need not be identical to each other.

63. The activatable antibody of claim 51, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 100 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

64. The activatable antibody of claim 51, wherein the CM is a polypeptide of up to 15 amino acids in length.

65. The activatable antibody of claim 58, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

66. The activatable antibody of claim 51, wherein in the presence of EGFR, the MM reduces the ability of the AB to bind EGFR by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay.

67. The activatable antibody of claim 51, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′

)₂ fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

68. The activatable antibody of claim 51, wherein the AB is or is from cetuximab, panitumumab, zalutumumab, matuzumab, nimotuzumab, ICR62, mAb 528, CH806, or MDX-447.

69. The activatable antibody of claim 51, wherein the AB is or is from cetuximab.

70. The activatable antibody of claim 51 further comprising a second AB wherein the target for the second AB is selected from the group consisting of the targets in Table 1.

71. The activatable antibody of claim 51, wherein the CM is a substrate for an enzyme selected from the group consisting of the enzymes in Table 3.

72. The activatable antibody of claim 71, wherein the CM is a substrate for an enzyme selected from the group consisting of urokinase-type plasminogen activator (uPA), legumain and matriptase (MT-SP1).

73. The activatable antibody of claim 51 wherein the Ab is conjugated to an agent.

74. The activatable antibody of claim 73, wherein the agent is a therapeutic agent.

75. The activatable antibody of claim 74, wherein the agent is an antineoplastic agent.

76. The activatable antibody of claim 73, wherein the agent is a toxin or a fragment thereof.

77. The activatable antibody of claim 73, wherein the agent is an agent selected from the group consisting of the agents in Table 4.

78. The activatable antibody of claim 73, wherein the agent is conjugated to the AB via a linker.

79. The activatable antibody of claim 78, wherein the linker is a cleavable linker.

80. The activatable antibody of claim 51 further comprising a detectable moiety.

81. The activatable antibody of claim 80, wherein the detectable moiety is a diagnostic agent.

82. The activatable antibody of claim 51, wherein the serum half-life of the activatable antibody is at least 5 days when administered to an organism.

83. The activatable antibody of claim 51, wherein the affinity of binding of the activatable antibody to EGFR is higher in a target tissue when compared to the binding of the activatable antibody to EGFR in a non-target tissue.

84. The activatable antibody of claim 83, wherein the target tissue is a diseased tissue.

85. The activatable antibody of claim 84, wherein the diseased tissue is breast tissue, head tissue, neck tissue, lung tissue, pancreatic tissue, nervous system tissue, liver tissue, prostate tissue, urogenital tissue, cervical tissue, colon tissue or colorectal tissue.

86. The activatable antibody of claim 51, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 1000 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

87. The activatable antibody of claim 51, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 10,000 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

88. The activatable antibody of claim 58, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)_n, (GGS)_n, (GSGGS)_n (SEQ ID NO:

12) and (GGGS)_n (SEQ ID NO;

     13), where n is an integer of at least one.

89. The activatable antibody of claim 58, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:

14), GGSGG (SEQ ID NO;

     15), GSGSG (SEQ ID NO;

     16), GSGGG (SEQ ID NO;

     17), GGGSG (SEQ ID NO;

     18), and GSSSG (SEQ ID NO;

     19).

90. The activatable antibody of claim 51, wherein the activatable antibody comprises at least a first CM and a second CM.

91. The activatable antibody of claim 90, wherein the first CM is cleaved by a first cleaving agent in a target tissue and wherein the second CM is cleaved by a second cleaving agent in a target tissue.

92. The activatable antibody of claim 91, wherein the first cleaving agent and the second cleaving agent are the same enzyme, and where the first CM and the second CM are different substrates for the enzyme.

93. The activatable antibody of claim 91, wherein the first cleaving agent and the second cleaving agent are different enzymes.

94. The activatable antibody of claim 91, wherein the first cleaving agent and the second cleaving agent are co-localized in the target tissue.

95. The activatable antibody of claim 90, wherein the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

96. The activatable antibody of claim 51, wherein the CM is a substrate for uPA and comprises the amino acid sequence LSGRSDNH (SEQ ID NO:

271).

97. The activatable antibody of claim 58, wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO:

111).

98. The activatable antibody of claim 58, wherein LP2 comprises the amino acid sequence GS or GSSG.

99. The activatable antibody of claim 58, wherein the CM is a substrate for an enzyme selected from the group consisting of urokinase-type plasminogen activator (uPA) and comprises the amino acid sequence LSGRSDNH (SEQ ID NO:

271), legumain and matriptase (MT-SP1), wherein the MM comprises the amino acid sequence CISPRGCPDGPYVM (SEQ ID NO;

     218), wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO;

     111), and wherein LP2 comprises the amino acid sequence GS.

100. An activatable antibody that in an activated state binds Epidermal Growth Factor Receptor (EGFR),the activatable antibody comprising:

an antibody or an antigen binding fragment thereof (AB) that specifically binds to EGFR;

a masking moiety (MM) that inhibits the binding of the AB in an uncleaved state to EGFR, wherein the MM comprises the amino acid sequence CISPRGCPDGPYVM (SEQ ID NO;

     218);

at least one cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), legumain and matriptase (MT-SP

     1) and wherein the substrate for uPA comprises the amino acid sequence LSGRSDNH (SEQ ID NO;

     271);

a first linking peptide (LP1) comprising the amino acid sequence GGSGGS (SEQ ID NO;

     111); and

a second linking peptide (LP2) comprising the amino acid sequence GS,

wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows;

MM-LP1-CM-LP2-AB or AB-LP2-CM-LP 1-MM.

101. The activatable antibody of claim 100, wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB to EGFR.

102. The activatable antibody of claim 100, wherein the MM does not interfere or compete with the AB for binding to EGFR in a cleaved state.

103. The activatable antibody of claim 100, wherein the protease is co-localized with EGFR in a tissue, and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

104. The activatable antibody of claim 100, wherein the AB has an equilibrium dissociation constant of at most 100 nM for binding to EGFR.

105. The activatable antibody of claim 100, wherein the MM is a polypeptide of no more than 40 amino acids in length.

106. The activatable antibody of claim 100, wherein the CM is positioned in the activatable antibody such that in the uncleaved state, binding of the activatable antibody to EGFR is reduced to occur with an equilibrium dissociation constant that is at least 100-fold greater than the equilibrium dissociation constant of an unmodified AB binding to EGFR, and whereas in the cleaved state, the AB binds EGFR.

107. The activatable antibody of claim 100, wherein the LP1 and LP2 need not be identical to each other.

108. The activatable antibody of claim 100, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 100 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

109. The activatable antibody of claim 100, wherein the CM is a polypeptide of up to 15 amino acids in length.

110. The activatable antibody of claim 100, wherein the LP1 is a peptide of about 6 to 20 amino acids in length and the LP2 is a peptide of about 2 to 20 amino acids in length.

111. The activatable antibody of claim 100, wherein in the presence of EGFR, the MM reduces the ability of the AB to bind EGFR by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay.

112. The activatable antibody of claim 100, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′

)₂ fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

113. The activatable antibody of claim 100, wherein the AB is or is from cetuximab, panitumumab, zalutumumab, matuzumab, nimotuzumab, ICR62, mAb 528, CH806, or MDX-447.

114. The activatable antibody of claim 100, wherein the AB is or is from cetuximab.

115. The activatable antibody of claim 100 further comprising a second AB wherein the target for the second AB is selected from the group consisting of the targets in Table 1.

116. The activatable antibody of claim 100, wherein the CM is a substrate for uPA, legumain or MT-SP1 and a second protease, wherein the second protease is an enzyme selected from the group consisting of the enzymes in Table 3.

117. The activatable antibody of claim 100 wherein the Ab is conjugated to an agent.

118. The activatable antibody of claim 117, wherein the agent is a therapeutic agent.

119. The activatable antibody of claim 118, wherein the agent is an antineoplastic agent.

120. The activatable antibody of claim 117, wherein the agent is a toxin or fragment thereof.

121. The activatable antibody of claim 117, wherein the agent is an agent selected from the group consisting of the agents in Table 4.

122. The activatable antibody of claim 117, wherein the agent is conjugated to the AB via a linker.

123. The activatable antibody of claim 122, wherein the linker is a cleavable linker.

124. The activatable antibody of claim 100 comprising a detectable moiety.

125. The activatable antibody of claim 124, wherein the detectable moiety is a diagnostic agent.

126. The activatable antibody of claim 100, wherein the serum half-life of the activatable antibody is at least 5 days when administered to an organism.

127. The activatable antibody of claim 100, wherein the affinity of binding of the activatable antibody to EGFR is higher in a target tissue when compared to the binding of the activatable antibody to EGFR in a non-target tissue.

128. The activatable antibody of claim 127, wherein the target tissue is a diseased tissue.

129. The activatable antibody of claim 128, wherein the diseased tissue is breast tissue, head tissue, neck tissue, lung tissue, pancreatic tissue, nervous system tissue, liver tissue, prostate tissue, urogenital tissue, cervical tissue, colon tissue or colorectal tissue.

130. The activatable antibody of claim 100, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 1000 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

131. The activatable antibody of claim 100, wherein the coupling of the MM reduces the ability of the AB to bind EGFR such that the dissociation constant (K_d) of the AB when coupled to the MM towards EGFR is at least 10,000 times greater than the K_d of the AB when not coupled to the MM towards EGFR.

132. The activatable antibody of claim 100, wherein the first CM is a polypeptide that that functions as a substrate for a protease selected from the group consisting of uPA, legumain, and MT-SP1 and a second CM and wherein the substrate for uPA comprises the amino acid sequence LSGRSDNH (SEQ ID NO:

271).

133. The activatable antibody of claim 132, wherein the first CM is cleaved by a first cleaving agent selected from the group consisting of uPA, legumain, and MT-SP1 in a target tissue and wherein the second CM is cleaved by a second cleaving agent in a target tissue.

134. The activatable antibody of claim 133, wherein the first cleaving agent and the second cleaving agent are the same enzyme selected from the group consisting of uPA, legumain, and MT-SP1, and where the first CM and the second CM are different substrates for the enzyme.

135. The activatable antibody of claim 133, wherein the first cleaving agent and the second cleaving agent are different enzymes.

136. The activatable antibody of claim 133, wherein the first cleaving agent and the second cleaving agent are co-localized in the target tissue.

137. The activatable antibody of claim 132, wherein the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.