ANTI-PDL1 ANTIBODIES, ACTIVATABLE ANTI-PDL1 ANTIBODIES, AND METHODS OF USE THEREOF

• US 20160311903A1
• Filed: 03/14/2016
• Published: 10/27/2016
• Est. Priority Date: 03/13/2015
• Status: Active Grant

• Current Assignee: CytomX Therapeutics, Inc
• Original Assignee: CytomX Therapeutics, Inc
• Inventors: Mei, Li, Nguyen, Margaret T.L., West, James William, Moore, Stephen James, Hostetter, Daniel Robert, Vasiljeva, Olga, Sagert, Jason Gary, Terrett, Jonathan Alexander
• Granted Patent: US 10,336,824 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0058 Antibodies
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 16/2827 against B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• 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 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/70 characterized by effect upon binding to a cell or to an antigen
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention relates generally to antibodies that bind programmed death ligand 1 (PDL1), activatable antibodies that specifically bind to PDL1 and methods of making and using these anti-PDL1 antibodies and anti-PDL1 activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

Claims

1. An isolated antibody or antigen binding fragment thereof (AB) that specifically binds to mammalian PDL1, wherein the AB has one or more of the following characteristics:

(a) the AB specifically binds to human PDL1 and murine PDL1;

(b) the AB specifically binds to human PDL1 and cynomolgus monkey PDL1;

(c) the AB specifically binds to human PDL1, murine PDL1, and cynomolgus monkey PDL1;

(d) the AB inhibits binding of human B7-1 and human PD1 to human PDL1 with an EC₅₀ value less than 10 nM;

(e) the AB inhibits binding of murine B7-1 and murine PD1 to murine PDL1 with an EC₅₀ value less than 10 nM; and

(f) the AB inhibits binding of cynomolgus monkey B7-1 and cynomolgus monkey PD1 to cynomolgus monkey PDL1 with an EC₅₀ value less than 10 nM.

85. The antibody of claim 1, wherein the antibody has one or more of the following characteristics selected from the group consisting of:

(i) the antibody comprises;

(a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO;

212;

(b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

213, 217, 221, 225, 229, 231, and 238-247;

(c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

214, 218, 222, 226, 230, 232, and 233-237;

(d) a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO;

209;

(e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

210, 215, 219, 223, and 227; and

(f) a variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

211, 216, 220, 224, and 228.

(ii) the antibody comprises a VL CDR1 sequence comprising RASQSISSYLN (SEQ ID NO;

     209);

a VL CDR2 sequence comprising AASSLQS (SEQ ID NO;

     215);

a VL CDR3 sequence comprising DNGYPST (SEQ ID NO;

     228);

a VH CDR1 sequence comprising SYAMS (SEQ ID NO;

     212);

a VH CDR2 sequence comprising SSIWRNGIVTVYADS (SEQ ID NO;

     246); and

a VH CDR3 sequence comprising WSAAFDY (SEQ ID NO;

     235);

(iii) the antibody comprises a variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

46, 48, 50, 52, 54, and 56, and a variable light chain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

12 and SEQ ID NO;

58; and

(iv) the antibody comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO;

46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO;

58, or a VH comprising the amino acid sequence of SEQ ID NO;

46 and a VL comprising the amino acid sequence of SEQ ID NO;

12.

86. An isolated nucleic acid molecule encoding the antibody of claim 1.

87. A vector comprising the isolated nucleic acid molecule of claim 86.

88. The isolated antibody of claim 1, wherein the AB specifically binds to mammalian PDL1 with a dissociation constant of

(a) 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.1 nM to 1 nM, 0.2 nM to 1 nM, 0.3 nM to 1 nM, 0.4 nM to 1 nM, 0.5 nM to 1 nM, 0.75 nM to 1 nM,

(b) 0.01 nM to 0.75 nM, 0.05 nM to 0.75 nM, 0.1 nM to 0.75 nM, 0.2 nM to 0.75 nM, 0.3 nM to 0.75 nM, 0.4 nM to 0.75 nM, 0.5 nM to 0.75 nM,

(c) 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.1 nM to 0.5 nM, 0.2 nM to 0.5 nM, 0.3 nM to 0.5 nM, 0.4 nM to 0.5 nM,

(d) 0.01 nM to 0.4 nM, 0.05 nM to 0.4 nM, 0.1 nM to 0.4 nM, 0.2 nM to 0.4 nM, 0.3 nM to 0.4 nM,

(e) 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM,

(f) 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM,

(g) 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or

(h) 0.01 nM to 0.05 nM.

89. A conjugated antibody comprising the antibody of claim 1 conjugated to an agent.

90. A method of producing an antibody by culturing a cell under conditions that lead to expression of the antibody, wherein the cell comprises a nucleic acid molecule encoding the antibody of claim 1.

91. A method of reducing PDL1 activity comprising administering an effective amount of the antibody of claim 1 to a subject in need thereof.

92. A method of blocking binding of a natural ligand to PDL1 comprising administering an effective amount of the antibody of claim 1 to a subject in need thereof.

93. A method of treating, alleviating a symptom of, or delaying the progression of a PDL1-mediated disorder or disease comprising administering an effective amount of the antibody of claim 1 to a subject in need thereof.

2. An activatable antibody that, in an activated state, specifically binds to mammalian PDL1, wherein said activatable antibody comprises:

an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PDL1;

a masking moiety (MM) that inhibits the binding of the AB to mammalian PDL1 when the activatable antibody is in an uncleaved state; and

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

3. The activatable antibody of claim 2, wherein the activatable antibody, in an activated state,

specifically blocks a natural ligand of PDL1 from binding to the mammalian PDL1.

4. The activatable antibody of claim 2, wherein the activatable antibody has one or more of the following characteristics selected from the group consisting of:

(i) the activatable antibody in an uncleaved state specifically binds to the mammalian PDL1 with a dissociation constant of

(a) 1 to 2 nM, 1 to 5 nM, 1 to 10 nM, 1 to 15 nM, 1 to 20 nM, 1 to 25 nM,

(b) 2 nM to 5 nM, 2 nM to 10 nM, 2 nM to 15 nM, 2 to 20 nM, 2 to 25 nM,

(c) 5 nM to 10 nM, 5 nM to 15 nM, 5 to 20 nM, 5 to 25 nM,

(d) 10 nM to 15 nM, 10 to 20 nM, 10 to 25 nM,

(e) 15 to 20 nM, 15 to 25 nM, or

(f) 20 to 25 nM;

(ii) the activatable antibody in an activated state specifically binds to the mammalian PDL1 with a dissociation constant of

(a) 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.1 nM to 1 nM, 0.2 nM to 1 nM, 0.3 nM to 1 nM, 0.4 nM to 1 nM, 0.5 nM to 1 nM, 0.75 nM to 1 nM,

(b) 0.01 nM to 0.75 nM, 0.05 nM to 0.75 nM, 0.1 nM to 0.75 nM, 0.2 nM to 0.75 nM, 0.3 nM to 0.75 nM, 0.4 nM to 0.75 nM, 0.5 nM to 0.75 nM,

(c) 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.1 nM to 0.5 nM, 0.2 nM to 0.5 nM, 0.3 nM to 0.5 nM, 0.4 nM to 0.5 nM,

(d) 0.01 nM to 0.4 nM, 0.05 nM to 0.4 nM, 0.1 nM to 0.4 nM, 0.2 nM to 0.4 nM, 0.3 nM to 0.4 nM,

(e) 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM,

(f) 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM,

(g) 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or

(h) 0.01 nM to 0.05 nM; and

(iii) the activatable antibody comprises an AB that specifically binds to the mammalian PDL1 with a dissociation constant of

(a) 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.1 nM to 1 nM, 0.2 nM to 1 nM, 0.3 nM to 1 nM, 0.4 nM to 1 nM, 0.5 nM to 1 nM, 0.75 nM to 1 nM,

(b) 0.01 nM to 0.75 nM, 0.05 nM to 0.75 nM, 0.1 nM to 0.75 nM, 0.2 nM to 0.75 nM, 0.3 nM to 0.75 nM, 0.4 nM to 0.75 nM, 0.5 nM to 0.75 nM,

(c) 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.1 nM to 0.5 nM, 0.2 nM to 0.5 nM, 0.3 nM to 0.5 nM, 0.4 nM to 0.5 nM,

(d) 0.01 nM to 0.4 nM, 0.05 nM to 0.4 nM, 0.1 nM to 0.4 nM, 0.2 nM to 0.4 nM, 0.3 nM to 0.4 nM,

(e) 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM,

(f) 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM,

(g) 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or

(h) 0.01 nM to 0.05 nM.

7. The activatable antibody of claim 2, wherein the mammalian PDL1 has one or more of the following characteristics selected from the group consisting of:

(i) the mammalian PDL1 is selected from the group consisting of a human PDL1, a murine PDL1, a rat PDL1, and a cynomolgus monkey PDL1; and

(ii) the mammalian PDL1 is a human PDL1.

8. The activatable antibody of claim 2, wherein the AB has one or more of the following characteristics selected from the group consisting of:

(i) the AB specifically binds to human PDL1, murine PDL1 or cynomolgus monkey PDL1 with a dissociation constant of less than 1 nM;

(ii) the AB specifically binds to human PDL1 and murine PDL1;

(iii) the AB specifically binds to human PDL1 and cynomolgus monkey PDL1;

(iv) the AB specifically binds to human PDL1, murine PDL1, and cynomolgus monkey PDL1;

(v) the AB inhibits binding of human B7-1 and human PD1 to human PDL1;

(vi) the AB inhibits binding of murine B7-1 and murine PD1 to murine PDL1; and

(vii) the AB inhibits binding of cynomolgus monkey B7-1 and cynomolgus monkey PD1 to cynomolgus monkey PDL1.

12. The activatable antibody of claim 2, wherein the AB blocks the ability of a natural ligand to bind to the mammalian PDL1 with an EC₅₀ of

(a) 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 3 nM, 0.5 nM to 2 nM, 0.5 nM to 1 nM,

(b) 1 nM to 10 nM, 1 nM to 5 nM, 1 nM to 3 nM, 1 nM to 2 nM,

(c) 2 nM to 10 nM, 2 nM to 5 nM, 2 nM to 3 nM,

(d) 3 nM to 10 nM, 3 nM to 5 nM, or

(e) 5 nM to 10 nM.

13. The activatable antibody of claim 12, wherein the natural ligand has one or more of the following characteristics selected from the group consisting of:

(i) the natural ligand is a mammalian PD1;

(ii) the natural ligand is selected from the group consisting of;

a human PD1, a murine PD1, and a cynomolgus monkey PD1;

(iii) the natural ligand is a mammalian B7-1; and

(iv) the natural ligand is selected from the group consisting of;

a human B7-1, a murine B7-1, and a cynomolgus monkey B7-1.

17. The activatable antibody of claim 2, wherein:

(a) the AB induces type 1 diabetes in a non-obese diabetic (NOD) mouse; and

(b) the activatable antibody in an uncleaved state inhibits the induction of type 1 diabetes in a NOD mouse.

18. The activatable antibody of claim 17, wherein the activatable antibody inhibits the induction of type 1 diabetes in the NOD mouse after administration of the activatable antibody at a single dose of

(a) 0.1 mg/kg to 3 mg/kg, 0.5 mg/kg to 3 mg/kg, 1 mg/kg to 3 mg/kg, 2 mg/kg to 3 mg/kg,

(b) 0.1 mg/kg to 2 mg/kg, 0.5 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg,

(c) 0.1 mg/kg to 1 mg/kg, 0.5 mg/kg to 1 mg/kg, or

(d) 0.1 mg/kg to 0.5 mg/kg.

19. The activatable antibody of claim 2, wherein:

(a) the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50% of a population of non-obese diabetic (NOD) mice, and

(b) the AB induces type 1 diabetes in greater than 50% of a population of NOD mice.

20. The activatable antibody of claim 19, wherein the activatable antibody does not induce type 1 diabetes in greater than 50% of the population of NOD mice after administration to each mouse in the population a single dose of the activatable antibody at a dosage of:

(a) 0.1 mg/kg to 3 mg/kg, 0.5 mg/kg to 3 mg/kg, 1 mg/kg to 3 mg/kg, 2 mg/kg to 3 mg/kg,

(b) 0.1 mg/kg to 2 mg/kg, 0.5 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg,

(c) 0.1 mg/kg to 1 mg/kg, 0.5 mg/kg to 1 mg/kg, or

(d) 0.1 mg/kg to 0.5 mg/kg.

21. The activatable antibody of claim 19, wherein:

(a) the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50% of a population of non-obese diabetic (NOD) mice when administered at a single dose of 1 mg/kg; and

(b) the AB induces type 1 diabetes in greater than 50% of a population of NOD mice, when administered at a single dose of 1 mg/kg.

22. The activatable antibody of claim 17, wherein the NOD mouse is a female NOD/ShiLtJ mouse substrain.

23. The activatable antibody of claim 17, wherein the activatable antibody has one or more of the following characteristics selected from the group consisting of:

(i) the activatable antibody inhibits the induction of type 1 diabetes in a NOD mouse by at least 3-fold compared to the AB; and

(ii) the activatable antibody exhibits a safety margin that is at least a three-fold safety margin relative to the AB.

25. The activatable antibody of claim 2, wherein the activatable antibody in an uncleaved state binds to a smaller percentage of a population of peripheral blood CD4+ CD8+ T lymphocytes than does the AB.

26. The activatable antibody of claim 25, wherein the peripheral blood CD4+ CD8+ T lymphocytes are murine.

27. The activatable antibody of claim 26, wherein the murine peripheral blood CD4+ CD8+ T lymphocytes are derived from a tumor-bearing mouse.

28. The activatable antibody of claim 2, wherein the activatable antibody has one or more of the following characteristics selected from the group consisting of:

(i) the AB comprises;

(a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO;

212;

(b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

213, 217, 221, 225, 229, 231, and 238-247;

(c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

214, 218, 222, 226, 230, 232, and 233-237;

(d) a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO;

209;

(e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

210, 215, 219, 223, and 227; and

(f) a variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

211, 216, 220, 224, and 228;

(ii) the AB comprises a VL CDR1 sequence comprising RASQSISSYLN (SEQ ID NO;

     209);

a VL CDR2 sequence comprising AASSLQS (SEQ ID NO;

     215);

a VL CDR3 sequence comprising DNGYPST (SEQ ID NO;

     228);

a VH CDR1 sequence comprising SYAMS (SEQ ID NO;

     212);

a VH CDR2 sequence comprising SSIWRNGIVTVYADS (SEQ ID NO;

     246); and

a VH CDR3 sequence comprising WSAAFDY (SEQ ID NO;

     235);

(iii) the AB comprises a variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

46, 48, 50, 52, 54, and 56, and a variable light chain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

12 and SEQ ID NO;

58; and

(iv) the AB comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO;

46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO;

58, or a VH comprising the amino acid sequence of SEQ ID NO;

46 and a VL comprising the amino acid sequence of SEQ ID NO;

12.

32. The activatable antibody of claim 2, wherein the MM has one or more of the following characteristics selected from the group consisting of:

(i) the MM has a dissociation constant for binding to the AB that is greater than the dissociation constant of the AB to PDL1;

(ii) the MM does not interfere or compete with the AB for binding to PDL1 when the activatable antibody is in a cleaved state;

(iii) the MM is a polypeptide of no more than 40 amino acids in length;

(iv) the MM polypeptide sequence is different from that of human PDL1;

(v) the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB;

(vi) the MM polypeptide sequence is no more than 25% identical to any natural binding partner of the AB; and

(vii) the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

59-81, 208, and 426.

39. The activatable antibody of claim 2, wherein the CM has one or more of the following characteristics selected from the group consisting of:

(i) the CM is a substrate for a protease that is active in diseased tissue;

(ii) the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

338-394, and 435-445, 883-921, 1009, and 1010; and

(iii) the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

377-394, 883-921, 1009, and 1010.

42. The activatable antibody of claim 2, 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.

43. The activatable antibody of claim 2, wherein the AB is linked to the CM.

44. The activatable antibody of claim 43, wherein the AB is linked directly to the CM.

45. The activatable antibody of claim 43, wherein the AB is linked to the CM via a linking peptide.

46. The activatable antibody of claim 2, wherein the MM is linked to the CM such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows:

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

47. The activatable antibody of claim 46, wherein the activatable antibody comprises a linking peptide between the MM and the CM, a linking peptide between the CM and the AB, or both a linking peptide between the MM and the CM and a linking peptide between the CM and the AB.

49. The activatable antibody of claim 46, 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.

50. The activatable antibody of claim 49, wherein the two linking peptides need not be identical to each other.

51. The activatable antibody of claim 49, wherein each of LP and LP2 is a peptide of about 1 to 20 amino acids in length.

52. The activatable antibody of claim 2, comprising an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence selected from the group consisting of SEQ ID NOs;

83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, and 157, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, and 1144-1191, 1200, and 1201;

(ii) an amino acid sequence selected from the group consisting of SEQ ID NOs;

931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, and 1201;

(iii) the amino acid sequence of SEQ ID NO;

428;

(iv) the amino acid sequence of SEQ ID NO;

1008; and

(v) an amino acid sequence selected from the group consisting of SEQ ID NO;

430, 432, 434, and 1202.

56. A conjugated activatable antibody comprising the activatable antibody of claim 2 conjugated to an agent.

57. The conjugated activatable antibody of claim 56, wherein the agent has one or more of the following characteristics selected from the group consisting of:

(i) the agent is a toxin or fragment thereof;

(ii) the agent is a microtubule inhibitor;

(iii) the agent is a nucleic acid damaging agent;

(iv) the agent is a dolastatin or a derivative thereof;

(v) the agent is an auristatin or a derivative thereof;

(vi) the agent is a maytansinoid or a derivative thereof;

(vii) the agent is a duocarmycin or a derivative thereof;

(viii) the agent is a calicheamicin or a derivative thereof;

(ix) the agent is auristatin E or a derivative thereof;

(x) the agent is monomethyl auristatin E (MMAE);

(xi) the agent is monomethyl auristatin D (MMAD);

(xii) the agent is DM1;

(xiii) the agent is DM4;

(xiv) the agent is a detectable moiety;

(xv) the agent is a diagnostic agent;

(xvi) the agent is conjugated to the antibody via a linker;

(xvii) the agent is conjugated to the antibody via a cleavable linker; and

(xviii) the agent is conjugated to the antibody via a non-cleavable linker.

70. A pharmaceutical composition comprising the activatable antibody of claim 2 and a carrier.

71. The pharmaceutical composition of claim 70 comprising an additional agent.

72. The pharmaceutical composition of claim 71, wherein the additional agent is a therapeutic agent.

73. An isolated nucleic acid molecule encoding the activatable antibody of claim 2.

74. A vector comprising the isolated nucleic acid molecule of claim 73.

75. A method of producing an activatable antibody by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises the nucleic acid molecule of claim 73.

76. A method of manufacturing an activatable antibody that, in an activated state, binds to PDL1, the method comprising:

(a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody of claim 2; and

(b) recovering the activatable antibody.

77. A method of reducing PDL1 activity comprising administering an effective amount of the activatable antibody of claim 2 to a subject in need thereof.

78. A method of blocking binding of a natural ligand to PDL1 comprising administering an effective amount of the activatable antibody of claim 2 to a subject in need thereof.

79. A method of treating, alleviating a symptom of, or delaying the progression of a PDL1-mediated disorder or disease comprising administering a therapeutically effective amount of the activatable antibody of claim 2 to a subject in need thereof.

80. The method of claim 79, wherein the PDL1-mediated disorder or disease is cancer.

81. The method of claim 80, wherein the cancer is a bladder cancer, a bone cancer, a breast cancer, a carcinoid, a cervical cancer, a colon cancer, an endometrial cancer, a glioma, a head and neck cancer, a liver cancer, a lung cancer, a lymphoma, a melanoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a sarcoma, a skin cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer.

82. The method of claim 80, wherein the cancer is selected from the group consisting of melanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC), castration-resistant prostate cancer (CRPC), hepatocellular carcinoma (HCC), squamous cell carcinoma of the head and neck, carcinomas of the esophagus, ovary, gastrointestinal tract and breast, or a hematologic malignancy such as multiple myeloma, B-cell lymphoma, T-cell lymphoma, Hodgkin'"'"'s lymphoma/primary mediastinal B-cell lymphoma, and chronic myelogenous leukemia.

83. The method of claim 79, wherein the method comprises administering an additional agent.

84. The method of claim 83, wherein the additional agent is a therapeutic agent.

94. The activatable antibody of claim 19, wherein the NOD mouse is a female NOD/ShiLtJ mouse substrain.

95. The activatable antibody of claim 19, wherein the activatable antibody has one or more of the following characteristics selected from the group consisting of:

(i) the activatable antibody inhibits the induction of type 1 diabetes in a NOD mouse by at least 3-fold compared to the AB; and

(ii) the activatable antibody exhibits a safety margin that is at least a three-fold safety margin relative to the AB.

5-6. -6. (canceled)

9-11. -11. (canceled)

14-16. -16. (canceled)

24. (canceled)

29-31. -31. (canceled)

33-38. -38. (canceled)

40-41. -41. (canceled)

48. (canceled)

53-55. -55. (canceled)

58-69. -69. (canceled)