Activatable binding polypeptides and methods of identification and use thereof

Abstract

The present disclosure provides activatable binding polypeptides (ABPs), which contain a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM). The present disclosure provides activatable antibody compositions, which contain a TBM containing an antigen binding domain (ABD), a MM and a CM. Furthermore the present disclosure also provides ABPs which contain a first TBM, a second TBM and a CM. The ABPs exhibit an “activatable” conformation such that at least one of the TBMs is less accessible to target when uncleaved than after cleavage of the CM in the presence of a cleaving agent capable of cleaving the CM. The disclosure further provides libraries of candidate ABPs, methods of screening to identify such ABPs, and methods of use. The disclosure further provides ABPs having TBMs that bind VEGF, CTLA-4, or VCAM, ABPs having a first TBM that binds VEGF and a second TBM that binds FGF, as well as compositions and methods of use.

1 . An activatable binding polypeptide (ABP) comprising:
 a target binding moiety (TBM);   a masking moiety (MM) capable of inhibiting binding of the TBM to a target, wherein said MM does not have an amino acid sequence of a naturally occurring binding partner of said TBM; and   a cleavable moiety (CM), wherein said CM is positioned in the activatable binding polypeptide such that in a cleaved state in the presence of a target, the TBM binds the target, and in an uncleaved state in the presence of the target, binding of the TBM to the target is inhibited by the MM.   
     
     
         2 . The ABP of  claim 1 , wherein said MM is selected from a plurality of candidate polypeptides based on its ability to inhibit binding of the TBM to the target in an uncleaved state and allow binding of the TBM to the target in a cleaved state. 
     
     
         3 . The ABP of  claim 1 , wherein the MM inhibits binding of the TBM to the target via steric hindrance when the ABP is in an uncleaved state. 
     
     
         4 . The ABP of  claim 3 , wherein the MM comprises a cysteine residue and steric hindrance is achieved via disulfide bond linkage between said cysteine residue and an additional cysteine residue adjacent to or within the TBM. 
     
     
         5 . The ABP of  claim 1 , wherein the target of the TBM is an extracellular polypeptide. 
     
     
         6 . The ABP of  claim 1 , wherein the CM is located between the TBM and the MM in the ABP. 
     
     
         7 . The ABP of  claim 1 , wherein the CM is located within the MM. 
     
     
         8 . The ABP of  claim 1 , wherein the CM comprises a protease substrate. 
     
     
         9 . The ABP of  claim 8 , wherein the protease substrate comprises a plasmin substrate, a caspase substrate or a matrix metalloprotease (MMP) substrate. 
     
     
         10 . The ABP of  claim 9 , wherein the protease substrate comprises an MMP substrate. 
     
     
         11 . The ABP of  claim 10 , wherein said matrix metalloprotease substrate is a matrix metalloprotease 1 (MMP-1) substrate, a matrix metalloprotease 2 (MMP-2) substrate, a matrix metalloprotease 9 (MMP-9) substrate, or a matrix metalloprotease 14 (MMP-14) substrate. 
     
     
         12 . The ABP of  claim 8 , wherein said protease substrate is a substrate for an intracellular protease. 
     
     
         13 . The ABP of  claim 1 , wherein the CM comprises a cysteine-cysteine disulfide bond. 
     
     
         14 . A method of selecting for an activatable binding polypeptide (ABP), said method comprising:
 contacting a plurality of candidate activatable binding polypeptides (candidate ABPs) with a target capable of binding a target binding moiety of the candidate ABPs and a cleaving agent capable of cleaving a cleavable moiety (CM) of the ABPs;   screening a first population of members of said plurality which bind to said target in the presence of the cleaving agent;   contacting said first population with the target in the absence of the cleaving agent; and   screening a second population of members from said first population by depleting said first population for members that bind the target in the absence of the cleaving agent;   wherein said method provides for selection of candidate ABPs which exhibit decreased binding to the target in the absence of the cleaving agent as compared to target binding in the presence of the cleaving agent.   
     
     
         15 . The method of  claim 14 , wherein said cleaving agent is a protease. 
     
     
         16 . The method of  claim 14 , wherein said cleaving agent is a disulfide bond reducing agent. 
     
     
         17 . The method of  claim 14 , wherein the target comprises a detectable label. 
     
     
         18 . The method of  claim 14 , wherein the first population is selected by detection of the detectable label. 
     
     
         19 . The method of  claim 14 , wherein the second population is produced by separating from the first population members that are detectably labeled. 
     
     
         20 . The method of  claim 14 , wherein each of said plurality of candidate activatable binding polypeptides is presented on a surface of a replicable biological entity in a display scaffold 
     
     
         21 . A library of candidate activatable binding polypeptides (ABPs), said library comprising a plurality of candidate ABPs displayed on the surface of a replicable biological entity. 
     
     
         22 . The library according to  claim 21 , wherein the replicable biological entity is a bacterial, yeast or mammalian cell. 
     
     
         23 . A composition comprising a nucleic acid construct comprising a nucleic acid coding for the ABP of  claim 1 . 
     
     
         24 . The composition of  claim 23 , wherein said nucleic acid construct further comprises a nucleic acid coding for a display scaffold wherein the nucleic acid coding for the ABP is operably inserted into the construct to provide for expression of a fusion protein for presentation of the ABP in the display scaffold on the surface of a host cell. 
     
     
         25 . The composition of  claim 24 , wherein the display scaffold is a circularly permuted outer membrane protein X (CPX). 
     
     
         26 . A composition, wherein said composition comprises a nucleic acid construct comprising a nucleic acid encoding a candidate activatable binding polypeptide, and further wherein said candidate activatable binding polypeptide comprises:
 (a) a target binding moiety (TBM);   (b) a cleavable moiety (CM); and   (c) a candidate masking moiety (MM), wherein the TBM, CM and candidate MM are positioned such that the ability of the candidate MM to inhibit binding of the TBM to a target in an uncleaved state and allow binding of the TBM to the target in a cleaved state can be determined.   
     
     
         27 . The composition of  claim 26 , wherein said nucleic acid construct further comprises a nucleic acid coding for a circularly permuted outer membrane protein X (CPX). 
     
     
         28 . A method of making a library of candidate activatable binding polypeptides, said method comprising:
 introducing into genomes of replicable biological entities a collection of recombinant DNA constructs that encode a plurality of candidate activatable binding polypeptides (ABPs), wherein each member of said plurality comprises a target binding moiety (TBM), a cleavable moiety (CM) and a candidate masking moiety (MM), said introducing producing recombinant replicable biological entities; and   culturing said recombinant replicable biological entities under conditions suitable for expression and display of the candidate ABPs.   
     
     
         29 . A pharmaceutical composition, wherein said pharmaceutical composition comprises a therapeutically effective amount of an activatable binding polypeptide (ABP) according to  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         30 . The pharmaceutical composition of  claim 29 , wherein the TBM of the ABP is capable of binding VEGF to effect inhibition of VEGF activity. 
     
     
         31 . A method of inhibiting angiogenesis in a mammalian subject, said method comprising
 administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of  claim 29 .   
     
     
         32 . The ABP of  claim 1 , wherein said target is any one of VCAM-1, VEGF-A, CTLA-4 or CD40L. 
     
     
         33 . The ABP of  claim 1  further comprising a detectable label. 
     
     
         34 . A method of labeling a target for detection comprising
 contacting an activatable binding polypeptide (ABP) with a sample suspected of containing both
 a target; and 
 a cleaving agent, wherein the ABP comprises
 a target binding moiety (TBM); 
 a detectable label; 
 a masking moiety (MM) capable of inhibiting binding of the TBM to a target, wherein said MM does not have an amino acid sequence of a naturally occurring binding partner of said TBM; and 
 a cleavable moiety (CM), wherein said CM is positioned in the activatable binding polypeptide such that in a cleaved state in the presence of a target, the TBM binds the target, and in an uncleaved state in the presence of the target, binding of the TBM to the target is inhibited by the MM, 
 
   wherein when said target and said cleaving agent are present in said sample, the cleaving agent cleaves the CM to produce a cleaved state in which the TBM of the ABP binds the target thereby labeling the target with the detectable label.   
     
     
         35 . The method of  claim 34 , further comprising detecting the presence or absence of said detectable label. 
     
     
         36 . The method of  claim 34 , wherein said contacting occurs in vivo. 
     
     
         37 . The method of  claim 34 , wherein said contacting occurs in vitro. 
     
     
         38 . An activatable binding polypeptide (ABP) comprising:
 a first target binding moiety (TBM);   a second TBM; and   a cleavable moiety (CM), wherein said CM is positioned in the activatable binding polypeptide such that in a cleaved state in the presence of a target, the first and second TBMs bind target, and in an uncleaved state the ABP is in a conformation such that the first TBM interferes with target binding by the second TBM.   
     
     
         39 . The ABP of  claim 38 , wherein in the uncleaved state, the ABP is in a conformation such that the first and second TBMs interfere with binding of target to the first and second TBMs. 
     
     
         40 . The ABP of  claim 38 , wherein the first and second TBMs are capable of binding different targets. 
     
     
         41 . The ABP of  claim 40 , wherein said different targets are FGF2 and VEGF. 
     
     
         42 . The ABP of  claim 38 , wherein said first TBM is selected from a plurality of candidate polypeptides based on the ability of said first TBM to inhibit binding of said second TBM to a target when the ABP is in an uncleaved state and allow binding of said second TBM to the target when the ABP is in a cleaved state. 
     
     
         43 . The ABP of  claim 38 , wherein said first TBM interferes with target binding by said second TBM via steric hindrance when the ABP is in an uncleaved state. 
     
     
         44 . The ABP of  claim 43 , wherein said ABP comprises a first cysteine residue within or adjacent to said first TBM and a second cysteine residue within or adjacent to said second TBM, and wherein steric hindrance is achieved via disulfide bond linkage between said first and second cysteine residue. 
     
     
         45 . The ABP of  claim 38 , wherein the target of said first TBM and said second TBM is an extracellular polypeptide. 
     
     
         46 . The ABP of  claim 38 , wherein the CM is located between said first TBM and said second TBM in the ABP. 
     
     
         47 . The ABP of  claim 38 , wherein the CM is located within either said first TBM or said second TBM. 
     
     
         48 . The ABP of  claim 38 , wherein the CM comprises a protease substrate. 
     
     
         49 . The ABP of  claim 48 , wherein the protease substrate comprises a plasmin substrate, a caspase substrate or a matrix metalloprotease (MMP) substrate. 
     
     
         50 . The ABP of  claim 48 , wherein the protease substrate comprises an MMP substrate. 
     
     
         51 . The ABP of  claim 50 , wherein said MMP substrate is a matrix metalloprotease 1 (MMP-1) substrate, a matrix metalloprotease 2 (MMP-2) substrate, a matrix metalloprotease 9 (MMP-9) substrate, or a matrix metalloprotease 14 (MMP-14) substrate. 
     
     
         52 . The ABP of  claim 48 , wherein said protease substrate is a substrate for an intracellular protease. 
     
     
         53 . The ABP of  claim 38 , wherein the CM comprises a cysteine-cysteine disulfide bond. 
     
     
         54 . A method of selecting for a dual target binding activatable binding polypeptide (ABP), said method comprising:
 contacting a plurality of candidate activatable binding polypeptides (ABPs), wherein each member of said plurality comprises a first target binding moiety (TBM), a second TBM and a cleavable moiety (CM), with a target capable of binding said first TBM and a cleaving agent capable of cleaving the CM;   selecting a first population of members of said plurality which bind to said target in the presence of the cleaving agent;   contacting said first population with said target in the absence of the cleaving agent; and   selecting a second population of members from said first population by depleting said first population for members that bind to said target in the absence of the cleaving agent,   wherein said method provides for selection of candidate ABPs which exhibit decreased binding to said target in the absence of the cleaving agent as compared to binding to said target in the presence of the cleaving agent.   
     
     
         55 . The method of  claim 54 , wherein said cleaving agent is a protease. 
     
     
         56 . The method of  claim 54 , wherein said cleaving agent is a disulfide bond reducing agent. 
     
     
         57 . The method of  claim 54 , wherein said target comprises a detectable label. 
     
     
         58 . The method of  claim 57 , wherein the first population is selected by detection of the detectable label. 
     
     
         59 . The method of  claim 57 , wherein the second population is produced by separating from the first population members that are detectably labeled. 
     
     
         60 . The method of  claim 54 , wherein each of said plurality of candidate activatable binding polypeptides is presented on a surface of a replicable biological entity in a display scaffold. 
     
     
         61 . The method of  claim 54 , wherein binding of the second TBM to target is assessed by providing the amino acid sequence of the second TBM in a display scaffold and detecting binding of target to the second TBM. 
     
     
         62 . A library of candidate dual target binding activatable binding polypeptides (ABPs), said library comprising a plurality of candidate dual target binding ABPs displayed on the surface of a replicable biological entity. 
     
     
         63 . The library according to  claim 62 , wherein the replicable biological entity is a bacterial, yeast or mammalian cell. 
     
     
         64 . A composition comprising a nucleic acid construct comprising a nucleic acid coding for the ABP of  claim 38 . 
     
     
         65 . The composition of  claim 64 , wherein said nucleic acid construct further comprises a nucleic acid coding for a display scaffold wherein the nucleic acid coding for the ABP is operably inserted into the construct to provide for expression of a fusion protein for presentation of the ABP in the display scaffold on the surface of a host cell. 
     
     
         66 . The composition of  claim 65 , wherein the display scaffold is a circularly permuted outer membrane protein X (CPX). 
     
     
         67 . A composition, wherein said composition comprises a nucleic acid construct comprising a nucleic acid encoding a candidate activatable binding polypeptide, and further wherein said candidate activatable binding polypeptide comprises:
 (a) a first target binding moiety (TBM);   (b) a cleavable moiety (CM); and   (c) a second TBM, wherein the first TBM, CM and second TBM are positioned such that the ability of said first TBM to inhibit binding of said second TBM to a target in an uncleaved state and allow binding of said second TBM to the target in a cleaved state can be determined.   
     
     
         68 . The composition of  claim 67 , wherein said nucleic acid construct further comprises a nucleic acid coding for a circularly permuted outer membrane protein X (CPX). 
     
     
         69 . A method of making a library of candidate activatable binding polypeptides, said method comprising:
 introducing into genomes of replicable biological entities a collection of recombinant DNA constructs that encode a plurality of dual target binding candidate activatable binding polypeptides (ABPs), wherein each member of said plurality comprises a first target binding moiety (TBM), a cleavable moiety (CM) and a second TBM, said introducing producing recombinant replicable biological entities; and   culturing said recombinant replicable biological entities under conditions suitable for expression and display of the candidate dual target binding ABPs.   
     
     
         70 . A pharmaceutical composition, wherein said pharmaceutical composition comprises a therapeutically effective amount of a dual target binding activatable binding polypeptide (ABP) according to  claim 38  and a pharmaceutically acceptable excipient. 
     
     
         71 . The pharmaceutical composition of  claim 70 , wherein the first TBM of the ABP binds VEGF to effect VEGF inhibition and the second TBM binds fibroblast growth factor-2 (FGF2) to effect FGF2 inhibition. 
     
     
         72 . A method of inhibiting angiogenesis in a mammalian subject, said method comprising
 administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of  claim 70 .   
     
     
         73 . An activatable binding polypeptide (ABP) comprising:
 a. at least one antigen binding domain (ABD) capable of binding a target;   b. at least one masking moiety (MM) coupled to said ABD, capable of interfering with specific binding of the ABD to the target; and   c. at least one cleavable moiety (CM) coupled to said ABD, wherein said CM is positioned in the ABP such that in an uncleaved state the MM interferes with specific binding of the ABD to the target and in a cleaved state the MM does not interfere with specific binding of the ABD to the target.   
     
     
         74 . The ABP of  claim 73  wherein the ABD is from a Fab fragment, ScFv, or SCAB. 
     
     
         75 . The ABP of  claim 73  wherein the CM is coupled to the C-terminus of the ABD. 
     
     
         76 . The ABP of  claim 73  wherein the CM is coupled to the N-terminus of the ABD. 
     
     
         77 . The ABP of  claim 76  wherein the CM is coupled to the N-terminus of a VL chain of the ABD. 
     
     
         78 . The ABP of  claim 73  wherein the target is selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, EGFR, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1, TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV, Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. 
     
     
         79 . The ABP of  claim 73  wherein the CM is a substrate for an enzyme selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 
     
     
         80 . The ABP of  claim 73  wherein the CM is a substrate for MMP-9. 
     
     
         81 . The ABP of  claim 73  further comprising a linker peptide, wherein said linker peptide is positioned between the MM and the CM. 
     
     
         82 . The ABP of  claim 73  further comprising a linker peptide, wherein said linker peptide is positioned between the ABD and the CM. 
     
     
         83 . The ABP of  claim 73  further comprising a detectable moiety. 
     
     
         84 . The ABP of  claim 83  wherein the detectable moiety is a diagnostic agent. 
     
     
         85 . A composition comprising an antigen binding domain (ABD) capable of binding a target coupled to at least one masking moiety (MM) wherein said MM interferes with specific binding of the ABD to the target. 
     
     
         86 . The composition of  claim 85  further comprising a cleavable moiety (CM) wherein said composition comprises two configurations, a first configuration wherein the CM is in an uncleaved state and the MM interferes with specific binding of the ABD to the target and a second configuration wherein the CM is in a cleaved state and the MM does not interfere with specific binding of the ABD to the target. 
     
     
         87 . A method of modifying a composition containing an antigen binding domain (ABD) capable of binding a target, the method comprising coupling a masking moiety (MM) and a cleavable moiety (CM) to said ABD such that in a uncleaved state the MM interferes with the ABD to specifically bind the target and in a cleaved state the MM does not interfere with the ABD to specifically bind the target. 
     
     
         88 . The method of  claim 87  wherein the MM is coupled to the C-terminus of the ABD. 
     
     
         89 . The method of  claim 87  wherein the MM is coupled to the N-terminus of the ABD. 
     
     
         90 . The composition of  claim 87  wherein the ABD is from a Fab fragment, ScFv, or SCAB. 
     
     
         91 . The method of  claim 87  wherein the target is selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, EGFR, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1, TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV, Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. 
     
     
         92 . The method of  claim 87  wherein the ABD is from an antibody selected from the group consisting of bevacizumab, ranibizumab, trastuzumab, infliximab, adalimumab, efalizumab, gemtuzumab ozogamicin, tositumomab, ibritumomab tiuxetan, eculizumab, alemtuzumab, rituximab, abiciximab, cetuximab, daclizumab, basiliximab, gemtuzumab, panitumumab, eculizumab, natalizumab, omalizumab, ipilimumab, tremelimumab and palivizumab. 
     
     
         93 . The method of  claim 87  wherein the ABD is from an antibody or an antibody fragment to a target selected from the group consisting of VEGF, EGFR, CTLA-4, TNFα, Integrinα4, IL2R, Complement C5, CD 11a, CD20, CD25, CD33, CD52, Glycoprotein receptor IIb/IIIa, IgE, Her2, and F protein of RSV. 
     
     
         94 . The method of  claim 87  wherein the ABD is from an antibody or an antibody fragment thereof to VEGF. 
     
     
         95 . The method of  claim 87  wherein the ABD is from bevacizumab or ranibizumab. 
     
     
         96 . The method of  claim 87  wherein the ABD is from an antibody or an antibody fragment thereof to TNFα. 
     
     
         97 . The method of  claim 87  wherein the ABD is from infliximab or adalimumab. 
     
     
         98 . The method of  claim 87  wherein the ABD is from an antibody or an antibody fragment thereof to CD20. 
     
     
         99 . The method of  claim 87  wherein the ABD is from tositumomab, ibritumomab tiuxetan, or rituximab. 
     
     
         100 . The method of  claim 87  wherein the ABD is from an antibody or an antibody fragment thereof to EGFR. 
     
     
         101 . The method of  claim 87  wherein the ABD is from cetuximab or panitumumab. 
     
     
         102 . The method of  claim 87  wherein the CM is a substrate for an enzyme selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 
     
     
         103 . A method of screening candidate peptides to identify a masking moiety (MM) peptide with specific binding affinity for an antibody or fragment thereof comprising an antigen binding domain (ABD), said method comprising:
 a. providing a library of peptide scaffolds, wherein each peptide scaffolds comprises:
 i. a transmembrane protein (TM); 
 ii. a candidate peptide; 
   b. contacting the antibody or fragment thereof comprising an ABD with said library; and   c. identifying a MM peptide having specific binding affinity for the antibody or fragment thereof comprising an ABD.   
     
     
         104 . The method of  claim 103  wherein the antibody or fragment thereof comprises an ABD capable of binding a target wherein the target is selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, EGFR, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1, TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV, Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. 
     
     
         105 . The method of  claim 103  wherein the ABD is from an antibody selected from the group consisting of bevacizumab, ranibizumab, trastuzumab, infliximab, adalimumab, efalizumab, gemtuzumab ozogamicin, tositumomab, ibritumomab tiuxetan, eculizumab, alemtuzumab, rituximab, abiciximab, cetuximab, daclizumab, basiliximab, gemtuzumab, panitumumab, eculizumab, natalizumab, omalizumab, ipilimumab, tremelimumab, and palivizumab. 
     
     
         106 . The method of  claim 103  wherein the ABD is from an antibody or an antibody fragment to a target selected from the group consisting of VEGF, EGFR, CTLA-4, TNFα, Integrinα4, IL2R, Complement C5, CD 11a, CD20, CD25, CD33, CD52, Glycoprotein receptor IIb/IIIa, IgE, Her2, and F protein of RSV. 
     
     
         107 . The method of  claim 103  wherein the ABD is from an antibody or an antibody fragment thereof to VEGF. 
     
     
         108 . The method of  claim 103  wherein the ABD is from bevacizumab or ranibizumab. 
     
     
         109 . The method of  claim 103  wherein the ABD is from an antibody or an antibody fragment thereof to TNFα. 
     
     
         110 . The method of  claim 103  wherein the ABD is from infliximab or adalimumab. 
     
     
         111 . The method of  claim 103  wherein the ABD is from an antibody or an antibody fragment thereof to CD20. 
     
     
         112 . The method of  claim 103  wherein the ABD is from tositumomab, ibritumomab tiuxetan, or rituximab. 
     
     
         113 . The method of  claim 103  wherein the ABD is from an antibody or an antibody fragment thereof to EGFR. 
     
     
         114 . The method of  claim 103  wherein the ABD is from cetuximab or panitumumab. 
     
     
         115 . The method of  claim 103  wherein the library comprises viruses, cells or spores. 
     
     
         116 . The method of  claim 103  wherein the library comprises  E. coli.    
     
     
         117 . The method of  claim 103  wherein the peptide scaffold further comprises a detectable moiety. 
     
     
         118 . A method of treating a condition in a subject including administering to the subject a composition comprising an antibody or fragment thereof containing an antigen binding domain (ABD) capable of binding a target coupled to a masking moiety (MM) and a cleavable moiety (CM), such that in an uncleaved state the MM interferes with the ABD to specifically bind the target and in a cleaved state the MM does not interfere with the ABD to specifically bind the target. 
     
     
         119 . A method of diagnosing a condition in a subject including administering to the subject a composition comprising an antibody or fragment thereof containing an antigen binding domain (ABD) capable of binding a target coupled to a masking moiety (MM) and a cleavable moiety (CM), such that in a cleaved state the MM interferes with the ABD to specifically bind the target and in an uncleaved state the MM does not interfere with the ABD to specifically bind the target. 
     
     
         120 . The method of  claim 118  or  119  wherein the ABD is from a Fab fragment, ScFv, or SCAB. 
     
     
         121 . The method of  claim 118  or  119  wherein the ABD is from an antibody selected from the group consisting of bevacizumab, ranibizumab, trastuzumab, infliximab, adalimumab, efalizumab, gemtuzumab ozogamicin, tositumomab, ibritumomab tiuxetan, eculizumab, alemtuzumab, rituximab, abiciximab, cetuximab, daclizumab, basiliximab, gemtuzumab, panitumumab, eculizumab, natalizumab, omalizumab, ipilimumab, tremelimumab, and palivizumab. 
     
     
         122 . The method of  claim 118  or  119  wherein the target is selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, EGFR, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1, TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV, Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. 
     
     
         123 . The method of  claim 118  or  119  wherein the ABD is from an antibody or an antibody fragment to a target selected from the group consisting of VEGF, EGFR, CTLA-4, TNFα, Integrinα4, IL2R, Complement C5, CD 11a, CD20, CD25, CD33, CD52, Glycoprotein receptor IIb/IIIa, IgE, Her2, and F protein of RSV. 
     
     
         124 . The method of  claim 118  or  119  wherein the ABD is from an antibody or an antibody fragment thereof to VEGF. 
     
     
         125 . The method of  claim 118  or  119  wherein the ABD is from bevacizumab or ranibizumab. 
     
     
         126 . The method of  claim 118  or  119  wherein the ABD is from an antibody or an antibody fragment thereof to TNFα. 
     
     
         127 . The method of  claim 118  or  119  wherein the ABD is from infliximab or adalimumab. 
     
     
         128 . The method of  claim 118  or  119  wherein the ABD is from an antibody or an antibody fragment thereof to CD20. 
     
     
         129 . The method of  claim 118  or  119  wherein the ABD is from tositumomab, ibritumomab tiuxetan, or rituximab. 
     
     
         130 . The method of  claim 118  or  119  wherein the ABD is from an antibody or an antibody fragment thereof to EGFR. 
     
     
         131 . The method of  claim 118  or  119  wherein the ABD is from cetuximab or panitumumab. 
     
     
         132 . An enzyme-activatable anti-VEGF-A antibody or fragment thereof. 
     
     
         133 . The antibody of  claim 132  wherein the antibody is ranibizumab. 
     
     
         134 . The antibody of  claim 132  wherein the antibody is activated by MMP-9. 
     
     
         135 . An enzyme-activatable CTLA-4 antibody or fragment thereof. 
     
     
         136 . The antibody of  claim 135  wherein the antibody is activated by MMP-9. 
     
     
         137 . An enzyme-activatable VCAM-1 antibody of fragment thereof. 
     
     
         138 . The antibody of  claim 137  wherein the antibody is activated by MMP-9. 
     
     
         139 . The enzyme of any one of  claims 132 ,  135  and  137 , wherein the enzyme selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 
     
     
         140 . The antibody of any one of  claims 132 ,  135  and  137 , wherein the antibody fragment is a scFv, Fab, or SCAB. 
     
     
         141 . The antibody of  claim 135  wherein the antibody is ipilimumab or tremelimumab. 
     
     
         142 . The method of  claim 87 ,  103 ,  118 , or  119  wherein the ABD is from an antibody or an antibody fragment thereof to CTLA-4. 
     
     
         143 . The method of  claim 87 ,  103 ,  118 , or  119  wherein the ABD is from ipilimumab or tremelimumab. 
     
     
         144 . A reaction mixture comprising an ABP, a protease capable of cleaving said ABP, and a target of said ABP.