ANTI-PD1 ANTIBODIES, ACTIVATABLE ANTI-PD1 ANTIBODIES, AND METHODS OF USE THEREOF

• US 20170044259A1
• Filed: 07/13/2016
• Published: 02/16/2017
• Est. Priority Date: 07/13/2015
• Status: Active Grant

• Current Assignee: CytomX Therapeutics, Inc
• Original Assignee: CytomX Therapeutics, Inc
• Inventors: Tipton, Kimberly Ann, West, James William, Chan, Chanty Mariategue
• Granted Patent: US 10,513,558 B2
• Time in Patent Office: Days
• 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 2039/545 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• 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: C07K 16/2818 against CD28 or CD152
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/3015 Breast
• CPC Class Codes: C07K 16/3023 Lung
• CPC Class Codes: C07K 16/303 Liver or Pancreas
• CPC Class Codes: C07K 16/3038 Kidney, bladder
• CPC Class Codes: C07K 16/3046 Stomach, Intestines
• CPC Class Codes: C07K 16/3053 Skin, nerves, brain
• CPC Class Codes: C07K 16/3069 Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/54 F(ab')2
• 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 2317/569 : Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• 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
• CPC Class Codes: C07K 2319/50 : containing protease site

Abstract

The invention relates generally to antibodies that specifically bind programmed cell death protein 1 (PD-1), activatable antibodies that specifically bind to PD-1 and methods of making and using these anti-PD-1 antibodies and anti-PD-1 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 PD-1, wherein the AB has one or more of the characteristics selected from the group consisting of:

(a) the AB inhibits binding of mammalian PD-1 to mammalian PDL1 with an EC₅₀ value less than 5 nM;

(b) the AB inhibits binding of mammalian PD-1 to mammalian PDL2 with an EC₅₀ value less than 5 nM; and

(c) the AB specifically binds to human PD-1 and cynomolgus monkey PD-1.

100. The antibody of claim 1, wherein the AB comprises:

(i) a combination of a variable heavy chain complementarity determining region 1 (VH CDR1), a variable heavy chain complementarity determining region 2 (VH CDR2), a variable heavy chain complementarity determining region 3 (VH CDR3), a variable light chain complementarity determining region 1 (VL CDR1), a variable light chain complementarity determining region 2 (VL CDR2), and a variable light chain complementarity determining region 3 (VL CDR3) selected from the group consisting of;

(a) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

653-657;

(b) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

658-663;

(c) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

664-668;

(d) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

669-677;

(e) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

678-682; and

(f) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

683-687;

(ii) a variable heavy chain complementarity determining region 1 (VH CDR1), a variable heavy chain complementarity determining region 2 (VH CDR2), a variable heavy chain complementarity determining region 3 (VH CDR3), a variable light chain complementarity determining region 1 (VL CDR1), a variable light chain complementarity determining region 2 (VL CDR2), and a variable light chain complementarity determining region 3 (VL CDR3) selected from the group consisting of;

(a) a VH CDR1 sequence comprising GFTFSGYAMS (SEQ ID NO;

     653);

a VH CDR2 sequence comprising YISNSGGNAH (SEQ ID NO;

     658);

a VH CDR3 sequence comprising EDYGTSPFVY (SEQ ID NO;

     664);

a VL CDR1 sequence comprising RASESVDAYGISFMN (SEQ ID NO;

     676);

a VL CDR2 sequence comprising AASNQGS (SEQ ID NO;

     678); and

a VL CDR3 sequence comprising QQSKDVPWT (SEQ ID NO;

     683); and

(b) a VH CDR1 sequence comprising GFTFSGYAMS (SEQ ID NO;

     653);

a VH CDR2 sequence comprising YISNSGGNAH (SEQ ID NO;

     658);

a VH CDR3 sequence comprising EDYGTSPFVY (SEQ ID NO;

     664);

a VL CDR1 sequence comprising RASESVDSYGISFMN (SEQ ID NO;

     675);

a VL CDR2 sequence comprising AASNQGS (SEQ ID NO;

     678); and

a VL CDR3 sequence comprising QQSKDVPWT (SEQ ID NO;

     683);

or

(iii) a combination of a variable heavy chain (VH) and a variable light chain (VL) selected from the group consisting of;

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

21, 23, 25, 27, 29, 31, 33, 35, 37, 2052, and 2053, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59;

(b) a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

21, 2052, and 2053, and a VL comprising the amino acid sequence of SEQ ID NO;

47;

(c) a VH comprising the amino acid sequence of SEQ ID NO;

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

45; and

(d) a VH comprising the amino acid sequence of SEQ ID NO;

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

47.

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

102. A vector comprising the isolated nucleic acid molecule of claim 101.

103. 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 construct that encodes the antibody of claim 1 under conditions that lead to expression of the antibody, and recovering the antibody.

104. A method of reducing immune suppression comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of claim 1.

105. A method of reducing binding of a ligand selected from the group consisting of PDL1 or PDL2 to PD-1 on T cells comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody of claim 1.

106. The method of claim 105, wherein the ligand is located on a tumor or other immune cell.

107. A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of claim 1.

108. The method of claim 107, wherein the PDL1-mediated disorder or disease is cancer.

109. The method of claim 107, wherein the method comprises administering an additional agent.

110. The method of claim 109, wherein the additional agent is a therapeutic agent.

2. An activatable antibody that, in an activated state, specifically binds to mammalian PD-1, wherein said activatable antibody comprises:

an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1;

a masking moiety (MM) that inhibits the binding of the AB to mammalian PD-1 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 AB specifically blocks a natural ligand of PD-1 from binding to the mammalian PD-1.

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

(i) the activatable antibody in an uncleaved state specifically binds to the mammalian PD-1 with a dissociation constant of;

0.5 nM to 1 nM, 0.5 nM to 2 nM, 0.5 nM to 5 nM, 0.5 nM to 10 nM, 0.5 nM to 15 nM, 0.5 nM to 20 nM, 0.5 nM to 25 nM, 0.5 nM to 50 nM, 0.5 nM to 75 nM, 0.5 nM to 100 nM, 0.5 nM to 150 nM, 0.5 nM to 200 nM, 0.5 nM to 300 nM, 0.5 nM to 400 nM,

1 nM to 2 nM, 1 nM to 5 nM, 1 nM to 10 nM, 1 nM to 15 nM, 1 nM to 20 nM, 1 nM to 25 nM, 1 nM to 50 nM, 1 nM to 75 nM, 1 nM to 100 nM, 1 nM to 150 nM, 1 nM to 200 nM, 1 nM to 300 nM, 1 nM to 400 nM,

2 nM to 5 nM, 2 nM to 10 nM, 2 nM to 15 nM, 2 nM to 20 nM, 2 nM to 25 nM, 2 nM to 50 nM, 2 nM to 75 nM, 2 nM to 100 nM, 2 nM to 150 nM, 2 nM to 200 nM, 2 nM to 300 nM, 2 nM to 400 nM,

5 nM to 10 nM, 5 nM to 15 nM, 5 nM to 20 nM, 5 nM to 25 nM, 5 nM to 50 nM, 5 nM to 75 nM, 5 nM to 100 nM, 5 nM to 150 nM, 5 nM to 200 nM, 5 nM to 300 nM, 5 nM to 400 nM,

10 nM to 15 nM, 10 nM to 20 nM, 10 nM to 25 nM, 10 nM to 50 nM, 10 nM to 75 nM, 10 nM to 100 nM, 10 nM to 150 nM, 10 nM to 200 nM, 10 nM to 300 nM, 10 nM to 400 nM,

15 nM to 20 nM, 15 nM to 25 nM, 15 nM to 50 nM, 15 nM to 75 nM, 15 nM to 100 nM, 15 nM to 150 nM, 15 nM to 200 nM, 15 nM to 300 nM, 15 nM to 400 nM,

20 nM to 25 nM, 20 nM to 50 nM, 20 nM to 75 nM, 20 nM to 100 nM, 20 nM to 150 nM, 20 nM to 200 nM, 20 nM to 300 nM, 20 nM to 400 nM,

25 nM to 50 nM, 25 nM to 75 nM, 25 nM to 100 nM, 25 nM to 150 nM, 25 nM to 200 nM, 25 nM to 300 nM, 25 nM to 400 nM,

50 nM to 75 nM, 50 nM to 100 nM, 50 nM to 150 nM, 50 nM to 200 nM, 50 nM to 300 nM, 50 nM to 400 nM,

75 nM to 100 nM, 75 nM to 150 nM, 75 nM to 200 nM, 75 nM to 300 nM, 75 nM to 400 nM,

150 nM to 200 nM, 150 nM to 300 nM, 150 nM to 400 nM,

200 nM to 300 nM, 200 nM to 400 nM, or

300 nM to 400 nM;

(ii) the activatable antibody in an activated state specifically binds to the mammalian PD-1 with a dissociation constant of;

0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM,

0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 2 nM, 0.4 nM to 2 nM, 0.5 nM to 2 nM, 0.75 nM to 1 nM, 1 nM to 2 nM,

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,

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,

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,

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,

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,

0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM,

0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or

0.01 nM to 0.05 nM;

(iii) the activatable antibody comprises an AB that specifically binds to the mammalian PD-1 with a dissociation constant of;

0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM,

0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 2 nM, 0.4 nM to 2 nM, 0.5 nM to 2 nM, 0.75 nM to 1 nM, 1 nM to 2 nM,

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,

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,

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,

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,

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,

0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM,

0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or

0.01 nM to 0.05 nM, and

(iv) the activatable antibody comprises an AB that blocks the ability of a natural ligand to bind to the mammalian PDL1 with an EC₅₀ of;

0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.1 nM to 0.5 nM, 0.1 nM to 0.25 nM,

0.25 nM to 10 nM, 0.25 nM to 5 nM, 0.25 nM to 3 nM, 0.25 nM to 2 nM, 0.25 nM to 1 nM, 0.25 nM to 0.5 nM,

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,

1 nM to 10 nM, 1 nM to 5 nM, 1 nM to 3 nM, 1 nM to 2 nM,

2 nM to 10 nM, 2 nM to 5 nM, 2 nM to 3 nM,

3 nM to 10 nM, 3 nM to 5 nM, or

5 nM to 10 nM.

7. The activatable antibody of claim 2, wherein the mammalian PD-1 is selected from the group consisting of a human PD-1-, a murine PD-1, and a cynomolgus monkey PD-1;

or wherein the mammalian PD-1 is a human PD-1.

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

(a) the AB specifically binds human PD-1 and cynomolgus monkey PD-1;

(b) the AB inhibits binding of human PDL1 and human PDL2 to human PD-1;

(c) the AB inhibits binding of cynomolgus monkey PDL1 and cynomolgus monkey PDL2 to cynomolgus monkey PD-1;

(d) the AB specifically binds murine PD-1; and

(e) the AB specifically binds to human PD-1 or cynomolgus monkey PD-1 with a dissociation constant of less than 1 nM.

12. The activatable antibody of claim 4, wherein the natural ligand is a mammalian PDL1 or a mammalian PDL2, or wherein the natural ligand is selected from the group consisting of:

a human PDL1, a human PDL2, a murine PDL1, a murine PDL2, a cynomolgus monkey PDL1, and a cynomolgus monkey PDL2.

14. 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.

15. The activatable antibody of claim 14, 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 3 mg/kg to 10 mg/kg, 3 mg/kg or 10 mg/kg, or wherein the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 1 mg/kg to 20 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg or 20 mg/kg.

17. 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%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of NOD mice.

18. The activatable antibody of claim 17, wherein the activatable antibody does not induce type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% 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:

3 mg/kg to 10 mg/kg, 3 mg/kg or 10 mg/kg.

19. The activatable antibody of claim 18, wherein the activatable antibody and the AB have one or more of the characteristics selected from the group consisting of:

(a) the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of non-obese diabetic (NOD) mice when administered at a single dose of 3 mg/kg; and

the AB induces type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of NOD mice, when administered at a single dose of 3 mg/kg; and

(b) the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of non-obese diabetic (NOD) mice when administered at a single dose of 10 mg/kg; and

the AB induces type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of NOD mice, when administered at a single dose of 10 mg/kg; and

(c) the AB of the activatable antibody induces type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of the population of the NOD mice after administration to each mouse in the population a single dose of the AB at a dosage of;

3 mg/kg to 20 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg or 20 mg/kg.

22. The activatable antibody of claim 2, wherein 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, wherein the population of NOD mice are concurrently dosed with an anti-CTLA4 antibody.

23. The activatable antibody of claim 22, wherein the population of NOD mice are each administered:

(a) a single dose of the activatable antibody at a dosage of 10 mg/kg;

(b) a single dose of the anti-CTLA4 antibody at a dosage of 10 mg/kg;

or

(c) a single dose of the activatable antibody at a dosage of 10 mg/kg and a single dose of the anti-CTLA4 antibody at a dosage of 10 mg/kg.

26. The activatable antibody of claim 14, wherein the NOD mouse is a female NOD/ShiLtJ mouse sub strain.

27. The activatable antibody of claim 26, wherein the population of NOD mice are each 5 weeks old when the activatable antibody is first administered, or wherein the population of NOD mice are each 10 weeks old when the activatable antibody is first administered.

29. The activatable antibody of claim 2, wherein the AB comprises:

(a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

653-657;

(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;

658-663;

(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;

664-668;

(d) a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs;

669-677;

(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;

678-682; 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;

683-687.

30. The activatable antibody of claim 2, wherein the AB comprises a VH CDR1 sequence comprising GFTFSGYAMS (SEQ ID NO:

653);

a VH CDR2 sequence comprising YISNSGGNAH (SEQ ID NO;

     658);

a VH CDR3 sequence comprising EDYGTSPFVY (SEQ ID NO;

     664);

a VL CDR1 sequence comprising RASESVDAYGISFMN (SEQ ID NO;

     676);

a VL CDR2 sequence comprising AASNQGS (SEQ ID NO;

     678); and

a VL CDR3 sequence comprising QQSKDVPWT (SEQ ID NO;

     683), or wherein the AB comprises a VH CDR1 sequence comprising GFTFSGYAMS (SEQ ID NO;

     653);

a VH CDR2 sequence comprising YISNSGGNAH (SEQ ID NO;

     658);

a VH CDR3 sequence comprising EDYGTSPFVY (SEQ ID NO;

     664);

a VL CDR1 sequence comprising RASESVDSYGISFMN (SEQ ID NO;

     675);

a VL CDR2 sequence comprising AASNQGS (SEQ ID NO;

     678); and

a VL CDR3 sequence comprising QQSKDVPWT (SEQ ID NO;

     683).

32. The activatable antibody of claim 2, wherein the AB comprises a combination of a variable heavy chain (VH) and a variable light chain (VL) selected from the group consisting of:

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

21, 23, 25, 27, 29, 31, 33, 35, 37, 2052, and 2053, and a variable light chain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59;

(b) a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

21, 2052, and 2053, and a VL comprising the amino acid sequence of SEQ ID NO;

45 and 47;

(c) a VH comprising the amino acid sequence of SEQ ID NO;

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

45; and

(d) a VH comprising the amino acid sequence of SEQ ID NO;

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

47.

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

(a) the MM has a dissociation constant for binding to the AB that is greater than the dissociation constant of the AB to PD-1;

(b) the MM does not interfere or compete with the AB for binding to PD-1 when the activatable antibody is in a cleaved state;

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

(d) the MM polypeptide sequence is different from that of human PD-1;

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

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

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

66-213, 384-514, and 548-571;

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

384-514 and 548-571; and

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

66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, and 99.

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

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

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

214, 294-361, 1092-1112, 1157, 1158, 1161, 1162, 1165, 1166, 1169, 1520, and 1695-1704; and

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

214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157.

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

(a) the AB is an antigen binding fragment 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;

(b) the AB is a monoclonal antibody;

(c) the AB is linked to the CM;

(d) the AB is linked directly to the CM; and

(e) the AB is linked to the CM via a linking peptide.

51. 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.

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

54. 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.

57. 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;

215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 919, 921, 923, 925, 927, 929, 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, 1028, 1029, 1041-1076, 1138, 1139, 1143, 1144, 1147, 1148, 1151, 1152, 1155, 1156, 1159, 1160, 1163, 1164, 1167, 1168, 1170, 1171, 1174, 1175, 1178, 1179, 1182, 1183, 1186, 1187, 1190, 1191, 1194, 1195, 1198, 1199, 1202, 1203, 2055, 2054, 2057, 2056, 2059, and 2058;

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

919, 921, 923, 925, 927, 929, 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, 1028, 1041-1076, 1138, 1144, 1148, 1152, 1156, 1160, 1164, 1168, 1170, 1174, 1178, 1182, 1186, 1190, 1194, 1198, 1202, 2055, 2057, and 2059;

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

215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 1029, 1139, 1143, 1147, 1151, 1155, 1159, 1163, 1167, 1171, 1175, 1179, 1183, 1187, 1191, 1195, 1199, 1203, 2054, 2056, and 2058;

(iv) an amino acid sequence selected from the group consisting of SEQ ID NOS;

515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1030, 1032, 1034, 1036, 1039, 1077-1090, 1113-1120, 1123, 1124, 1127, 1128, 1131, 1132, 1134, and 1135;

(v) an amino acid sequence selected from the group consisting of SEQ ID NOS;

999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1030, 1032, 1034, 1036, 1039, 1070-1090, 1119, 1123, 1127, 1131, and 1134; and

(vi) an amino acid sequence selected from the group consisting of SEQ ID NOS;

515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 1120, 1124, 1128, 1132, and 1135.

63. The activatable antibody of claim 2, wherein the AB comprises:

(i) a combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO;

1346 and the CDR sequences of the light chain amino acid sequence of SEQ ID NO;

626;

or (ii) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO;

     1705);

the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO;

     1706);

the VH CDR3 sequence comprises TNDDY (SEQ ID NO;

     1707);

the VL CDR1 sequence comprises QSVSSY (SEQ ID NO;

     1708);

the VL CDR2 sequence comprises DAS (SEQ ID NO;

     1709); and

the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO;

     1710);

or (iii) the AB comprises a heavy chain comprising the amino acid sequence of SEQ ID NO;

1346 and a light chain comprising the amino acid sequence of SEQ ID NO;

626.

64. The activatable antibody of claim 63, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

1206-1295.

66. The activatable antibody of claim 63 comprising an amino acid sequence selected from the group consisting of:

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

1296, 1297, 1300, 1301, 1304, 1305, 1308, 1309, 1312, 1313, 1316, 1317, 1320, 1321, 1324, 1325, 1328, 1329, 1332, 1333, 1336, 1337, 1340, and 1341;

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

1297, 1301, 1305, 1309, 1313, 1317, 1321, 1325, 1329, 1333, 1337, and 1341; and

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

1296, 1300, 1304, 1308, 1312, 1316, 1320, 1324, 1328, 1332, 1336, and 1340.

68. The activatable antibody of claim 2, wherein the AB comprises:

(i) a combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO;

1514 and the CDR sequences of the light chain amino acid sequence of SEQ ID NO;

638 or (ii) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO;

     1711);

the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO;

     1712);

the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO;

     1713);

the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO;

     1714);

the VL CDR2 sequence comprises LAS (SEQ ID NO;

     1715); and

the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO;

     1716);

or

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

1514 and a light chain comprising the amino acid sequence of SEQ ID NO;

638.

69. The activatable antibody of claim 68, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

1351-1465.

71. The activatable antibody of claim 69 comprising an amino acid sequence selected from the group consisting of:

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

1466, 1467, 1470, 1471, 1474, 1475, 1478, 1479, 1482, 1483, 1486, 1487, 1490, 1491, 1494, 1495, 1498, 1499, 1502, 1503, 1506, 1507, 1510, and 1511;

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

1467, 1471, 1475, 1479, 1483, 1487, 1491, 1495, 1499, 1503, 1507, and 1511; and

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

1466, 1470, 1474, 1478, 1482, 1486, 1490, 1494, 1498, 1502, 1506, and 1510.

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

74. The conjugated activatable antibody of claim 73, wherein the agent has one or more of the following characteristics selected from the group consisting of:

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

(b) the agent is conjugated to the activatable antibody via a cleavable linker,

(c) the linker is conjugated to the activatable antibody via a non-cleavable linker;

(d) the agent is a detectable moiety; and

(e) the agent is a diagnostic agent.

79. A pharmaceutical composition comprising the conjugated activatable antibody of claim 73 and a carrier.

80. A pharmaceutical composition comprising the activatable antibody of claim 2.

81. The pharmaceutical composition of claim 79 comprising an additional agent.

82. The pharmaceutical composition of claim 80 comprising an additional agent.

83. The pharmaceutical composition of claim 81, wherein the additional agent is a therapeutic agent.

84. The pharmaceutical composition of claim 82, wherein the additional agent is a therapeutic agent.

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

86. A vector comprising the isolated nucleic acid molecule of claim 85.

87. 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 85.

88. A method of manufacturing an activatable antibody that, in an activated state, binds to PD-1, the method comprising:

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

(b) recovering the activatable antibody.

89. A method of reducing binding of a ligand selected from the group consisting of PDL1 or PDL2 to PD-1 on T cells comprising administering an effective amount of the activatable antibody of claim 2 to a subject in need thereof.

92. A method of reducing immune suppression comprising administering to a subject in need thereof a therapeutically effective amount of the activatable antibody of claim 2.

93. The method of claim 92, wherein the immune suppression is mediated by engagement of PD-1 on T cells to PDL1 or PDL2 on tumor or other immune cells.

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

95. The method of claim 84, wherein the PDL1-mediated disorder or disease is cancer.

96. The method of claim 95, wherein the cancer is selected from the group consisting of:

(a) a bladder cancer, a bone cancer, a breast cancer, a carcinoid, a cervical cancer, a cholangiocarcinoma, 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 thymus cancer, a thyroid cancer, a urogenital cancer, and a urothelial cancer; and

(b) 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, thymoma, 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.

98. The method of claim 89, wherein the method comprises administering an additional agent.

99. The method of claim 98, wherein the additional agent is a therapeutic agent.

5-6. -6. (canceled)

8-9. -9. (canceled)

11. (canceled)

13. (canceled)

16. (canceled)

20-21. -21. (canceled)

24-25. -25. (canceled)

28. (canceled)

31. (canceled)

33-34. -34. (canceled)

36-43. -43. (canceled)

45-46. -46. (canceled)

48-50. -50. (canceled)

53. (canceled)

55. The activatable antibody of claim 55, wherein LP1 and LP2 have one or more of the characteristics selected from the group consisting of:

(a) the two linking peptides need not be identical to each other; and

(b) each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

56. (canceled)

58-62. -62. (canceled)

65. (canceled)

67. (canceled)

70. (canceled)

72. (canceled)

75-78. -78. (canceled)

90-91. -91. (canceled)

97. (canceled)