Precision/context-dependent activatable antibodies, and methods of making and using the same

Abstract

Provided herein are libraries containing synthetic polynucleotides that encode activatable binding polypeptides. Further provided herein are activatable binding polypeptides and polypeptide libraries containing such activatable binding polypeptides. Also provided herein are vectors, vector libraries, cells, kits, and methods of making and using activatable polypeptide libraries.

1 . A library comprising polynucleotides, wherein at least one of the polynucleotides encodes a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM),
 wherein the FP comprises an amino acid sequence according to Formula (XIII): XmCXnCXo (SEQ ID NO: 86), wherein m is from 2-10, n is from 3-10, and o is from 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;   wherein the CM comprises at least a first cleavage site; and   wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.   
     
     
         2 . The library of  claim 1 , wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten unique polypeptides and each unique polypeptide comprise, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM),
 wherein the FP comprises an amino acid sequence according to Formula (XIII): XmCXnCXo (SEQ ID NO: 86), wherein m is from 2-10, n is from 3-10, and o is from 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;   wherein the CM comprises at least a first cleavage site; and   wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.   
     
     
         3 . (canceled) 
     
     
         4 . The library of  claim 1 , wherein the FP is encoded by a polynucleotide sequence comprising a nucleic acid sequence according to Formula (XIV): (NNK)mTGY(NNK)nTGY(NNK)o (SEQ ID NO: 87), wherein each N is independently A, G, T, or C, wherein each K is independently T or G, and wherein each Y is independently T or C. 
     
     
         5 . (canceled) 
     
     
         6 . The library of  claim 1 , wherein each X in Xm, Xn, and/or Xo of Formula (XIII) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The library of  claim 1 , wherein m is 2 or 3-6, wherein n is from 6-8, and/or wherein o is from 1-2. 
     
     
         10 - 13 . (canceled) 
     
     
         14 . The library of  claim 1  wherein the FP further comprises, at its N-terminus, the amino acid sequence of SEQ ID NO: 16. 
     
     
         15 . (canceled) 
     
     
         16 . The library of  claim 1 , wherein the first cleavage site is a protease cleavage site for a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 
     
     
         17 . The library of  claim 1 , wherein the CM further comprises a first linker (L1) C-terminal to the first cleavage site. 
     
     
         18 . The library of  claim 17 , wherein the L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 17-24. 
     
     
         19 . The library of  claim 1 , wherein the CM further comprises a second cleavage site. 
     
     
         20 . The library of  claim 19 , wherein the second cleavage site is C-terminal to the L1. 
     
     
         21 . The library of  claim 19 , wherein the second cleavage site is a protease cleavage site for a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 
     
     
         22 . The library of  claim 19 , wherein the first and second cleavage sites are different. 
     
     
         23 . The library of  claim 19 , wherein the CM further comprises a second linker (L2) C-terminal to the second cleavage site. 
     
     
         24 . The library of  claim 23 , wherein the L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 17-24. 
     
     
         25 . The library of  claim 1 , wherein the polypeptide comprises an amino acid sequence according to Formula (III):
 EVGSYX1X2X3 X4X5 X6CX7X8X9X10X11X12CX13 X14 SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 3), wherein X1 is A, D, I, N, P, or Y, X2 is A, F, N, S, or V, X3 is A, H, L, P, S, V, or Y, X4 is A, H, S, or Y, X5 is A, D, P, S, V, or Y, X6 is A, D, L, S, or Y, X7 is D, P, or V, X8 is A, D, H, P, S, or T, X9 is A, D, F, H, P, or Y, X10 is L, P, or Y, X11 is F, P, or Y, X12 is A, P, S, or Y, X13 is A, D, N, S, T, or Y, and X14 is A, S, or Y.   
     
     
         26 . The library of  claim 25 , wherein each of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence according to Formula (III). 
     
     
         27 . The library of  claim 1 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 25-46. 
     
     
         28 . The library of  claim 1 , wherein the TBM comprises:
 (a) an antibody light chain variable region, and a heavy chain variable region C-terminal to the light chain variable region; or   (b) an antibody heavy chain variable region, and a light chain variable region C-terminal to the heavy chain variable region.   
     
     
         29 - 33 . (canceled) 
     
     
         34 . The library of  claim 1 , wherein at least one of the polynucleotides encoding the polypeptide is in a vector. 
     
     
         35 . (canceled) 
     
     
         36 . The library of  claim 1 , wherein at least one of the polynucleotides encoding the polypeptide is in a cell. 
     
     
         37 . (canceled) 
     
     
         38 . A method of producing an activatable antibody comprising culturing a cell comprising the library of  claim 36  under conditions suitable for producing the polypeptide comprising the FP, CM, and TBM, wherein the polypeptide is an activatable antibody. 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . A method of using the library of  claim 1  to screen for a activatable antibody that binds to a target, the method comprising:
 a) contacting the expression products of the library with the target before the CM is cleaved; 
 b) contacting the expression products of the library with the target after the CM is cleaved; and 
 c) isolating one or more of the expression products that binds to the target after the CM is cleaved, but has reduced binding to the target before the CM is cleaved. 
 
     
     
         42 - 44 . (canceled) 
     
     
         45 . A polypeptide encoded by one or more polynucleotides from the library of  claim 1 . 
     
     
         46 . A kit comprising the library of  claim 1 . 
     
     
         47 . A library comprising antigen binding domains, wherein at least one of the antigen binding domains comprises a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM),
 wherein the FP comprises an amino acid sequence according to Formula (XIII): XmCXnCXo (SEQ ID NO: 86), wherein m is from 2-10, n is from 3-10, and o is from 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;   wherein the CM comprises at least a first cleavage site; and   wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.   
     
     
         48 . The library of  claim 47 , wherein at least two, at least three, at least four, at least five, or at least ten of the antigen binding domains comprise a unique polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM),
 wherein the FP comprises an amino acid sequence according to Formula (XIII): XmCXnCXo (SEQ ID NO: 86), wherein m is from 2-10, n is from 3-10, and o is from 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;   wherein the CM comprises at least a first cleavage site; and   wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.   
     
     
         49 - 52 . (canceled) 
     
     
         53 . The library of  claim 47 , wherein each X in Xm, Xn, and/or Xo in formula (XIII) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . The library of  claim 47 , wherein each of the antigen binding domains is displayed on a cell surface or on a phage. 
     
     
         57 . An antibody light chain comprising a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM),
 wherein the FP comprises an amino acid sequence according to Formula (I): XmCXnCZo (SEQ ID NO: 1), wherein m is from 2-10, n is from 3-10, and o is from 1-10, wherein each Xis independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P;   wherein the CM comprises at least a first cleavage site; and   wherein the TBM comprises an antibody light chain variable region.   
     
     
         58 . An antibody comprising a heavy chain and a light chain, wherein the light chain is a light chain of  claim 57 . 
     
     
         59 . An antibody heavy chain comprising a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM),
 wherein the FP comprises an amino acid sequence according to Formula (I): XmCXnCZo (SEQ ID NO: 1), wherein m is from 2-10, n is from 3-10, and o is from 1-10, wherein each Xis independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P;   wherein the CM comprises at least a first cleavage site; and   wherein the TBM comprises an antibody heavy chain variable region.   
     
     
         60 . An antibody comprising a heavy chain and a light chain, wherein the heavy chain is a heavy chain of  claim 59 . 
     
     
         61 . A cell comprising at least one polypeptide displayed on its surface, wherein the at least polypeptide comprises, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM),
 wherein the FP comprises an amino acid sequence according to Formula (I): XmCXnCZo (SEQ ID NO: 1), wherein m is from 2-10, n is from 3-10, and o is from 1-10, wherein each Xis independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P;   wherein the CM comprises at least a first cleavage site; and   wherein the TBM comprises an antibody light chain variable region and/or an antibody heavy chain variable region.   
     
     
         62 - 65 . (canceled) 
     
     
         66 . An activatable antibody comprising:
 a first polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM),   wherein the MM comprises an amino acid sequence according to Formula (I): XmCXnCZo (SEQ ID NO: 1), wherein m is from 2-10, n is from 3-10, and o is from 1-10, wherein each Xis independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the MM inhibits the binding of the activatable antibody to human CD137 when the CM is not cleaved;   wherein the CM comprises at least a first cleavage site; and   wherein:   a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH);   b) the TBM comprises an antibody heavy chain variable region (VH), and the activatable antibody further comprises a second polypeptide comprising an antibody light chain variable region (VL);   c) the TBM comprises from the N-terminus to the C-terminus, an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); or   d) the TBM comprises from the N-terminus to the C-terminus, an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL); and   wherein the activatable antibody binds to human CD137 via the VH and VL when the CM is cleaved.   
     
     
         67 . The activatable antibody of  claim 66 , wherein the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH). 
     
     
         68 . The activatable antibody of  claim 66 , wherein m is 2 or 3-6, wherein n is from 6-8, and/or wherein o is 1 or 2. 
     
     
         69 - 72 . (canceled) 
     
     
         73 . The activatable antibody of  claim 66 , wherein each X in Xm and/or Xn of formula (I) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. 
     
     
         74 . (canceled) 
     
     
         75 . The activatable antibody of  claim 66 , wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO 79-85 and XmCADAPNHCXX (SEQ ID NO:88), XmCHHSPANCXX (SEQ ID NO:89), XmCPILRHRCXX (SEQ ID NO:90), XmCKWRPSRCXX (SEQ ID NO:91), XmCRVLPRRCXX (SEQ ID NO:92), XmCLWRHRSCXX (SEQ ID NO:93), and XmCPRLRRKCXX (SEQ ID NO:94), where m is from 2-10, and where each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 
     
     
         76 . The activatable antibody of  claim 66 , wherein the MM further comprises, at its N-terminus, the amino acid sequence of SEQ ID NO: 16. 
     
     
         77 . (canceled) 
     
     
         78 . The activatable antibody of  claim 66 , wherein the first cleavage site is a protease cleavage site for a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 
     
     
         79 . The activatable antibody of  claim 66 , wherein the CM further comprises a first linker (L1) C-terminal to the first cleavage site. 
     
     
         80 . The activatable antibody of  claim 79 , wherein the L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 17-24. 
     
     
         81 . The activatable antibody of  claim 79 , wherein the CM further comprises a second cleavage site C-terminal to the L1. 
     
     
         82 . (canceled) 
     
     
         83 . The activatable antibody of  claim 81 , wherein the second cleavage site is a protease cleavage site for a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 
     
     
         84 . The activatable antibody of  claim 81 , wherein the first and second cleavage sites are different. 
     
     
         85 . The activatable antibody of  claim 81 , wherein the CM further comprises a second linker (L2) C-terminal to the second cleavage site. 
     
     
         86 . The activatable antibody of  claim 85 , wherein the L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 17-24. 
     
     
         87 . (canceled) 
     
     
         88 . The activatable antibody of  claim 66 , wherein the activatable antibody comprises an amino acid sequence according to Formula (III):
 EVGSYX1X2X3 X4X5 X6CX7X8X9X10X11X12CX13 X14 SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 3), wherein X1 is A, D, I, N, P, or Y, X2 is A, F, N, S, or V, X3 is A, H, L, P, S, V, or Y, X4 is A, H, S, or Y, X5 is A, D, P, S, V, or Y, X6 is A, D, L, S, or Y, X7 is D, P, or V, X8 is A, D, H, P, S, or T, X9 is A, D, F, H, P, or Y, X10 is L, P, or Y, X11 is F, P, or Y, X12 is A, P, S, or Y, X13 is A, D, N, S, T, or Y, and X14 is A, S, or Y.   
     
     
         89 . The activatable antibody of  claim 66 , wherein the activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 40-46. 
     
     
         90 . The activatable antibody of  claim 66 , wherein:
 (a) the VL comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70;   (b) the VL comprises the amino acid sequence of SEQ ID NO: 50;   (c) the VH comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 65, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 66, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 67; and/or   (d) the VH comprises the amino acid sequence of SEQ ID NO: 49.   
     
     
         91 - 93 . (canceled) 
     
     
         94 . A method of treating or delaying progression of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the activatable antibody of  claim 66 . 
     
     
         95 . The method of  claim 94 , further comprising administering to the subject an effective amount of at least one additional therapeutic agent selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, target therapies, radiation therapies, and chemotherapies. 
     
     
         96 . (canceled) 
     
     
         97 . (canceled)