Activatable antibody, and method for fabricating and using the same

Abstract

To provide a library including polynucleotide.SOLUTION: A library including polynucleotides in which at least one of polynucleotides encodes a polypeptide including a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM) from N-terminal to C-terminal, and the FP includes an amino acid sequence according to formula (XIII): XmCXnCXo. In the formula, m denotes 2 to 10, n denotes 3 to 10, o denotes 1 to 10, each X independently denotes 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, the CM includes at least first cleavage site, and the TBM includes an antibody chain variable region and / or an antibody heavy chain variable region.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to International Patent Application No. PCT / CN2018 / 075065, filed February 2, 2018, which is incorporated by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file is incorporated by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 695402000641SEQLIST.TXT, Recorded: January 31, 2019, Size: 54KB).

[0003] The present disclosure relates to polynucleotides and polynucleotide libraries useful for screening and / or identifying one or more precision / context-dependent activatable binding polypeptides (e.g., activatable antibodies), as well as polypeptides and polypeptide libraries useful for screening and / or identifying precision / context-dependent activatable binding polypeptides (e.g., activatable antibodies that can bind to CTLA4 or CD137 when in an activated form), cells, methods, and kits related thereto. [Background technology]

[0004] Activatable binding polypeptides bind less to their targets when the antigen-binding moiety contained therein is uncleaved than after cleavage in the presence of one or more specific proteases. Thus, these activatable binding polypeptides provide antigen-specific binding proteins that are only capable of binding to their targets in certain circumstances (e.g., in protease-rich tumor microenvironments). Although many interesting activatable binding polypeptides have been developed, the process of developing such proteins is slow, labor-intensive, and expensive. Thus, there is a need for improved methods and products useful for identifying self-blocking peptides for activatable binding polypeptides.

[0005] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot / GenBank accession numbers, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0006] To meet these and other needs, disclosed herein are libraries of polynucleotides that are useful, for example, for screening and / or identifying activatable binding polypeptides (i.e., activatable antibodies). The disclosure is based, at least in part, on the discovery that the polypeptides described herein exhibit significantly improved masking efficiency prior to activation, allowing for better design, screening and / or identification of activatable binding polypeptides (i.e., activatable antibodies) with superior therapeutic index and safety profile. The disclosure is further based, at least in part, on the surprising discovery that the polynucleotide libraries described herein can be successfully constructed and screened to identify activatable binding polypeptides (see Examples 1 and 2 below). Disclosed herein are precision / context-dependent activatable binding polypeptides that, when active, bind to human CTLA4 (see Example 3) or to human CD137 (see Example 5), but do not bind when inactive, i.e., they bind to their target (when active) and remove the first peptide (FP) (i.e., masking moiety (MM) or self-blocking peptide) only after cleavage of the cleavable moiety (CM). The discovered first peptides (FP) (e.g., masking moiety) disclosed herein can efficiently mask antibody activity and / or reduce or completely inhibit antigen binding, while in some embodiments lacking the chemically labile residues methionine and / or tryptophan. Furthermore, the activatable antibodies identified using the polynucleotide libraries described herein are as efficient in treating multiple cancer types as their parent antibodies, and have significantly reduced cytotoxicity in susceptible animals (NOD mice, see Example 4).

[0007] Thus, in one aspect, provided herein is a library comprising polynucleotides, at least one of the polynucleotides encoding a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), wherein FP has the formula (XIII): m CX n CX o (SEQ ID NO:86), where m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, and o is 1 to 10; 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; CM comprises at least a first cleavage site; and TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the polynucleotides in the library encode at least 2, at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000 unique polypeptides, each unique polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), and FP is represented by the formula (XIII):XIII: m CX n CX o (SEQ ID NO:86), where m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, and o is 1 to 10; 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; CM comprises at least a first cleavage site; and TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, each of the polynucleotides in the library encodes a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), and FP is represented by Formula (XIII):XIII: m CX n CX o(SEQ ID NO:86), where m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, o is 1 to 10, 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, CM comprises at least a first cleavage site, and TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, FP is represented by Formula (XIV):(NNK) m TGY(NNK) n TGY(NNK) o (SEQ ID NO:87), wherein each N is independently A, G, T, or C, each K is independently T or G, and each Y is independently T or C.

[0008] In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the preceding embodiments, X in formula (XIII) is m Each X in is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may be combined with any of the preceding embodiments, X in formula (XIII) is n Each X in is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may be combined with any of the preceding embodiments, X in formula (XIII) is owherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may be combined with any of the preceding embodiments, m is 6. In some embodiments that may be combined with any of the preceding embodiments, m is 2 to 5, e.g., 2, 3, 4, or 5. In some embodiments that may be combined with any of the preceding embodiments, n is 6 to 8. In some embodiments that may be combined with any of the preceding embodiments, n is 6. In some embodiments that may be combined with any of the preceding embodiments, o is 1 to 2. In some embodiments that may be combined with any of the preceding embodiments, o is 2. In some embodiments that may be combined with any of the preceding embodiments, the FP further comprises an additional amino acid sequence at its N-terminus. In some embodiments, the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO: 16.

[0009] In some embodiments that may be combined with any of the preceding embodiments, 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. In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a first linker (L1) C-terminal to the first cleavage site. In some embodiments, L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24. In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a second cleavage site. In some embodiments, the second cleavage site is C-terminal to L1. In some embodiments, 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. In some embodiments, the first and second cleavage sites are different.In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a second linker (L2) C-terminal to the second cleavage site. In some embodiments, L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24. In some embodiments that may be combined with any of the preceding embodiments, the CM further comprises a linker N-terminal to the first cleavage site.

[0010] In some embodiments that may be combined with any of the preceding embodiments, the polypeptides encoded by one or more polynucleotides in the library include a first peptide (FP) and a polypeptide having a structure represented by the formula (III): 10 X 11 X 12 CX 13 X 14 and a cleavable moiety (CM) comprising an amino acid sequence according to 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, and X 10 is L, P, or Y, and X 11 is F, P, or Y, and X 12 is A, P, S, or Y, and X 13 is A, D, N, S, T, or Y, and X 14 is A, S, or Y. In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence according to formula (III). In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-46.

[0011] In some embodiments that may be combined with any of the preceding embodiments, the TBM comprises an antibody light chain variable region. In some embodiments, the TBM further comprises a heavy chain variable region C-terminal to the light chain variable region. In some embodiments, the library further comprises polynucleotides encoding one or more antibody heavy chain variable regions. In some embodiments, the heavy chain variable region and the light chain variable region form an antigen binding site that is capable of binding to a target in the absence of a masking moiety (MM).

[0012] In some embodiments that may be combined with any of the preceding embodiments, the TBM comprises an antibody heavy chain variable region. In some embodiments, the TBM further comprises a light chain variable region C-terminal to the heavy chain variable region. In some embodiments, the library further comprises polynucleotides encoding one or more antibody light chain variable regions. In some embodiments, the heavy chain variable region and the light chain variable region form an antigen binding site that is capable of binding to a target in the absence of a masking moiety (MM).

[0013] In some embodiments that can be combined with any of the preceding embodiments, at least one of the polynucleotides that encode the polypeptide is in a vector.In some embodiments, the vector is an expression vector or a display vector.In some embodiments that can be combined with any of the preceding embodiments, at least one of the polynucleotides that encode the polypeptide is in a cell.In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0014] Other aspects of the disclosure relate to methods of producing an activatable antibody, comprising culturing any of the cells described herein under conditions suitable for the production of an activatable antibody, hi some embodiments, the method further comprises recovering the activatable antibody produced by the cell, hi some embodiments, the method further comprises testing the activatable antibody for its ability to remain soluble while maintaining an activatable phenotype.

[0015] Other aspects of the disclosure include methods of screening for activatable antibodies that bind to a target using any of the libraries described herein, comprising: a) contacting an expression product of the library with the target before the CM is cleaved; b) contacting an expression product of the library with the target after the CM is cleaved; and c) isolating one or more expression products that bind to the target after the CM is cleaved but not before the CM is cleaved. Also provided herein are methods of screening for activatable antibodies that bind to a target using any of the libraries described herein, comprising: a) contacting an expression product of the library with the target before the CM is cleaved; b) contacting an expression product of the library with the target after the CM is cleaved; and c) isolating one or more expression products that bind to the target after the CM is cleaved but have reduced binding affinity to the target before the CM is cleaved, compared to the binding affinity after the CM is cleaved. In some embodiments, the expression products are less than the K of the expression products before the CM is cleaved. D However, the K of the expression product after CM is cleaved Dis isolated when the abundance of the antibody is at least twice as high (eg, at least 5 times, at least 10 times, at least 15 times, or more). In some embodiments, the CM comprises at least a first 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. In some embodiments, the target is CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, and / or B7-H4. In some embodiments, the target is CTLA4 or CD137.

[0016] Other aspects of the disclosure relate to a polypeptide encoded by one or more polynucleotides of any of the libraries described herein, or a library of polypeptides encoded by one or more polynucleotides of any of the libraries described herein.

[0017] Another aspect of the present disclosure relates to kits that include any of the libraries described herein.

[0018] Another aspect of the present disclosure relates to a library comprising antigen-binding domains, at least one of the antigen-binding domains 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 FP has the formula (XIII):m CX n CX o (SEQ ID NO: 86), where m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10; 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; the CM comprises at least a first cleavage site; and the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, at least two, at least three, at least four, at least five, at least ten, at least fifty, at least one hundred, at least one thousand 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), and the FP comprises the formula (XIII):XIII: m CX n CX o (SEQ ID NO:86), where m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, and o is 1 to 10; 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; CM comprises at least a first cleavage site; and TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that may be combined with any of the preceding embodiments, each of the antigen-binding domains encodes a unique polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), and FP is represented by Formula (XIII):XIII: m CX n CX o(SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, o is 1-10, 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, CM comprises at least a first cleavage site, and the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that can be combined with any of the preceding embodiments, the TBM comprises an antibody light chain variable region and the antigen binding domain further comprises an antibody heavy chain variable region. In some embodiments that can be combined with any of the preceding embodiments, the TBM comprises an antibody heavy chain variable region and the antigen binding domain further comprises an antibody light chain variable region. In some embodiments that can be combined with any of the preceding embodiments, each X is not M, W, or C. In some embodiments that can be combined with any of the preceding embodiments, the X in formula (XIII) m Each X in is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may be combined with any of the preceding embodiments, X in formula (XIII) is n Each X in is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may be combined with any of the preceding embodiments, X in formula (XIII) is o wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, each of the antigen binding domains in the library is displayed on a phage or a cell (e.g., a yeast cell).

[0019] Another aspect of the present disclosure relates to 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 FP is represented by Formula (I): m CX n CZ o(SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, o is 1-10, 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, 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, CM comprises at least a first cleavage site, and TBM comprises an antibody light chain variable region. Other aspects of the disclosure relate to antibodies comprising a heavy chain and a light chain, wherein the light chain is any of the antibody light chains described herein. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the preceding embodiments, X of formula (XIII) is not M, W, or C. m wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0020] Another aspect of the present disclosure relates to 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 FP is represented by Formula (I): m CX n CZ o (SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, o is 1-10, 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, 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, CM comprises at least a first cleavage site, and TBM comprises an antibody heavy chain variable region. Another aspect of the disclosure relates to an antibody comprising a heavy chain and a light chain, wherein the heavy chain is any of the antibody heavy chains described herein. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the preceding embodiments, X of formula (XIII) is not M, W, or C.m wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0021] Another aspect of the present disclosure relates to a cell comprising at least one polypeptide displayed on a surface, the at least 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 has the formula (I): X m CX n CZ o (SEQ ID NO: 1), wherein m is 2-10 (e.g., 3-10), n is 3-10, o is 1-10, 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, 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, CM comprises at least a first cleavage site, and TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the preceding embodiments, X of formula (I) 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, 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, CM comprises at least a first cleavage site, and TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. m In some embodiments that may be combined with any of the preceding embodiments, X in formula (I) is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. n wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0022] Another aspect of the present disclosure relates to an activatable antibody comprising a 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 is represented by the formula (I): m CX n CZ o(SEQ ID NO:1), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, 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, and 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 binding of the activatable antibody to human CTLA4 if the CM is not cleaved, the CM comprises at least a first cleavage site, the TBM comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), and the activatable antibody binds to human CTLA4 via the VH and VL if the CM is cleaved. In some embodiments, the TBM comprises a VL and the activatable antibody further comprises a VH. In some embodiments, the TBM comprises a VH and the activatable antibody further comprises a VL. In some embodiments, the TBM comprises, from N-terminus to C-terminus, a VH and a VL, or a VL and a VH. In some embodiments, the CM comprises at least a first protease cleavage site and is cleaved by one or more proteases 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. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the preceding embodiments, X in formula (I) is mIn some embodiments that may be combined with any of the preceding embodiments, X in formula (I) is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. n wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may be combined with any of the preceding embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 72-78. In some embodiments that may be combined with any of the preceding embodiments, VL comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 62, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments that may be combined with any of the preceding embodiments, VL comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments that may be combined with any of the preceding embodiments, VH comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 59, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments that may be combined with any of the preceding embodiments, VH comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, m is 3-10.

[0023] Another aspect of the present disclosure relates to an activatable antibody comprising a 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 is represented by the formula (I): m CX n CZ o(SEQ ID NO:1), wherein m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, and o is 1 to 10, 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, and 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 binding of the activatable antibody to human CD137 when the CM is not cleaved, the CM comprises at least a first cleavage site, the TBM comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), and the activatable antibody binds to human CD137 via the VH and VL when the CM is cleaved. In some embodiments, the TBM comprises a VL and the activatable antibody further comprises a VH. In some embodiments, the TBM comprises a VH and the activatable antibody further comprises a VL. In some embodiments, the TBM comprises, from N-terminus to C-terminus, a VH and a VL, or a VL and a VH. In some embodiments, the CM comprises at least a first protease cleavage site and is cleaved by one or more proteases 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. In some embodiments that may be combined with any of the preceding embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the preceding embodiments, X in formula (I) is mIn some embodiments that may be combined with any of the preceding embodiments, X in formula (I) is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. n wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may be combined with any of the preceding embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-85 and 88-94. In some embodiments that may be combined with any of the preceding embodiments, VL comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments that may be combined with any of the preceding embodiments, VL comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments that may be combined with any of the preceding embodiments, VH comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 65, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments that may be combined with any of the preceding embodiments, VH comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, m is 3-10.

[0024] Other aspects of the disclosure relate to polynucleotides encoding any of the activatable antibodies described herein. In other aspects, the disclosure relates to vectors comprising any of the polynucleotides described herein (e.g., polynucleotides encoding activatable antibodies). In some embodiments, the vector is an expression vector and / or a display vector. In other aspects, the disclosure relates to host cells comprising any of the polynucleotides and / or vectors described herein (e.g., polynucleotides and / or vectors encoding activatable antibodies). In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell. In other aspects, the disclosure relates to methods of making an activatable antibody, comprising culturing any of the host cells described herein under conditions suitable for the production of an antibody or activatable antibody. In some embodiments, the method further comprises recovering the antibody or activatable antibody by the cell.

[0025] Other aspects of the present disclosure relate to methods of treating or delaying the progression of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a polypeptide encoded by one or more polynucleotides from any of the libraries described herein and / or any of the activatable antibodies described herein (e.g., an activatable antibody to human CTLA4 or an activatable antibody to human CD137). In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, and chemotherapy. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of pomalyst, Revlimid, lenalidomide, pomalidomide, thalidomide, DNA alkylating platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anti-cancer vaccines, and oncolytic viruses.

[0026] It should be understood that one, some, or all of the characteristics of the various embodiments described above and herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description. [Brief description of the drawings]

[0027] [Figure 1]FIG. 1 shows a schematic diagram of an exemplary selection process for self-blocking peptides using Fab fragments of target antibodies displayed on the yeast surface. [Diagram 2] FIG. 1 shows a schematic diagram of an exemplary selection process for self-blocking peptides using scFv fragments of target antibodies displayed on the yeast surface. [Diagram 3] Figure 1 shows functional display of Fab and scFv targeting CTLA4 on yeast as determined by flow cytometry. A shows functional display of Fab targeting CTLA4 on the surface of yeast. B shows functional display of scFv targeting CTLA4 on the surface of yeast. [Figure 4] 1 shows an exemplary selection process for activatable antibodies targeting human CTLA4. A yeast library displaying fusion proteins was subjected to several rounds of FACS-based screening. [Diagram 5] 1 shows the CTLA4 binding affinity of exemplary CTLA4 activatable antibody clones as determined by flow cytometry. A shows the binding affinity of CTLA4 activatable antibody clones in scFv format, including CTLA4 activatable antibody clone B13287 with intact masking peptide or with TEV protease cleaved masking peptide, compared to the scFv fragment of the targeting antibody without the masking peptide. B shows the CTLA4 binding affinity of CTLA4 activatable antibody clones in Fab format, including CTLA4 activatable antibody clone B13189 with intact masking peptide or with TEV protease cleaved masking peptide, compared to the Fab fragment of the targeting antibody without the masking peptide. [Figure 6] Figure 1 shows the masking efficiency of exemplary CTLA4 activatable antibodies TY22401, TY22403, TY22402, and TY22404 compared to parent antibody TY21580. A shows the association and dissociation curves of the indicated activatable antibodies compared to parent antibody TY21580 as determined by the ForteBio system. B shows a graph of the relative ratio of activatable antibody bound compared to parent antibody TY21580. [Figure 7] 1 shows the masking efficiency of exemplary CTLA4 activatable antibodies against recombinant human CTLA4-Fc as determined by ELISA. A shows a first batch of ELISA data showing the binding of CTLA4 activatable antibodies TY22401, TY22402, TY22403, TY22404 to recombinant human CTLA4-Fc compared to parent antibody TY21580. B shows a second batch of ELISA data showing the binding of CTLA4 activatable antibodies TY22401, TY22402, TY22403, TY22404 to recombinant human CTLA4-Fc compared to parent antibody TY21580. C shows the binding of CTLA4 activatable antibodies TY22563, TY22564, TY22565, TY22566 to recombinant human CTLA4-Fc compared to parent antibody TY21580. [Figure 8] Figure 1 shows the activity of CTLA4 activatable antibody TY22404 upon removal of the masking peptide. A shows SDS-PAGE results for activatable antibody TY22404 untreated, treated with protease uPA, or treated with 5 or 10 units of protease MMP-9. B shows binding of activatable antibody TY22404 untreated, treated with protease uPA, or treated with protease MMP-9 compared to parent antibody TY21580 as determined by ELISA. [Figure 9A] 1 shows size-exclusion chromatography (SEC) profiles of exemplary activatable antibodies under accelerated stress conditions. 1 shows the SEC profile of activatable antibody TY22402 after 6 cycles of freeze-thaw compared to control conditions. [Figure 9B] 1 shows size exclusion chromatography (SEC) profiles of exemplary activatable antibodies under accelerated stress conditions. 1 shows the SEC profile of activatable antibody TY22402 after 7 days at 50° C. compared to control conditions. [Figure 9C]1 shows size exclusion chromatography (SEC) profiles of an exemplary activatable antibody under accelerated stress conditions. The percentage of SEC main peak area of ​​an exemplary activatable antibody after 7 days at 50° C., after storage at 40° C. for up to 28 days, or after 6 cycles of freeze-thawing is shown compared to control conditions. [Figure 10] Shown is the percentage of the SEC main peak area for activatable antibodies TY22401 and TY22402 after storage at about 8 mg / mL or above 150 mg / mL. [Figure 11] 1 shows the masking efficiency of untreated activatable antibodies TY21580, TY22401, TY22402, and TY22566 incubated at pH 3.7 for 30 minutes or incubated at pH 3.7 for 1 hour as determined by the ForteBio system. [Figure 12A] 1 shows human peripheral blood mononuclear cell (PBMC) activation by isotype control antibody, parent antibody TY21580, or exemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22404 as measured by ELISA. 2 shows the effect on IL-2 secretion from CD3-primed human PBMC stimulated with isotype control antibody, parent antibody TY21580, and exemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22404. [Figure 12B] 1 shows human peripheral blood mononuclear cell (PBMC) activation by isotype control antibody, parent antibody TY21580, or exemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22404 as measured by ELISA. 2 shows the effect on IFNγ secretion from CD3-primed human PBMC stimulated with isotype control antibody, parent antibody TY21580, and exemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22404. [Figure 13]1 shows antibody-dependent cell-mediated cytotoxicity (ADCC) activity of an isotype control antibody, parent antibody TY21580, or exemplary activatable antibodies TY22401, TY21580, or TY22404 against HEK293F cells transiently overexpressing human CTLA4 as determined by an ADCC reporter gene assay. [Figure 14A] Figure 1 shows the in vivo antitumor effect of parental antibody TY21580, isotype control antibody, or exemplary CTLA4 activatable antibody TY22401, TY22402, or TY22566 in MC38 syngeneic mouse colorectal tumor model. Tumor growth curves of female MC38 tumor-bearing C57BL / 6 mice in different treatment groups. Data points represent group mean values, error bars represent SEM. [Figure 14B] 1 shows the in vivo anti-tumor efficacy of parental antibody TY21580, isotype control antibody, or exemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22566 in the MC38 syngeneic mouse colorectal tumor model. Individual tumor growth curves for groups treated with TY21580, TY22401, TY22402, and TY22566 are shown. [Figure 15] Figure 1 shows the in vivo antitumor effects of an isotype control antibody, parent antibody TY21580, or one of the three activatable antibodies in a CT26 syngeneic mouse colorectal tumor model. Tumor growth curves of female CT26 tumor-bearing C57BL / 6 mice in different treatment groups are shown. Data points represent group means, and error bars represent SEM. [Figure 16] Figure 1 shows the in vivo antitumor effects of isotype control antibody, parent antibody TY21580, or one of the three activatable antibodies in the H22 syngeneic mouse liver tumor model. Tumor growth curves of female H22 tumor-bearing C57BL / 6 mice in different treatment groups. Data points represent group means, error bars represent SEM. [Figure 17A]Figure 1 shows the in vivo antitumor efficacy of parental antibody TY21580, isotype control antibody, and exemplary activatable antibodies TY22401, TY22402, or TY22566 in a 3LL syngeneic mouse lung tumor model. Tumor growth curves of female 3LL tumor-bearing C57BL / 6 mice in different treatment groups. Data points represent group means and error bars represent SEM. [Figure 17B] 1 shows the in vivo anti-tumor efficacy of parental antibody TY21580, an isotype control antibody, and exemplary activatable antibodies TY22401, TY22402, or TY22566 in a 3LL syngeneic mouse lung tumor model. Individual tumor growth curves for groups treated with TY21580, TY22401, TY22402, and TY22566 are shown. [Figure 18] 1 shows the time course of blood concentrations as determined by ELISA of test article (TA) administered intravenously at 10 mg / kg to female BALB / c mice. A shows the time course of blood concentrations of activatable antibody TY22401 administered intravenously at 10 mg / kg to female BALB / c mice compared to the parent antibody TY21580. B shows the time course of blood concentrations of activatable antibody TY22402 administered intravenously at 10 mg / kg to female BALB / c mice compared to the parent antibody TY21580. C shows the time course of blood concentrations of activatable antibody TY22404 administered intravenously at 10 mg / kg to female BALB / c mice compared to the parent antibody TY21580. [Figure 19]

[0023] Figure 1 shows repeat dose toxicity of an isotype control antibody, parent antibody TY21580, and exemplary activatable antibodies TY22566, TY22401, and TY22402 using the NOD mouse model. Percent survival over 20 days is shown for each treatment group. [Figure 20] Figure 1 shows functional display of Fab and scFv targeting human CD137 on yeast as determined by flow cytometry. A shows scFv targeting CD137 on the surface of yeast. B shows Fab targeting CD137 on the surface of yeast. [Figure 21]1 shows an exemplary selection process for activatable antibodies targeting human CD137. A yeast library displaying fusion proteins was subjected to several rounds of FACS-based screening. [Figure 22A] 1 shows the CD137 binding affinity of exemplary CD137-activatable antibody clones as determined by flow cytometry. Binding affinity of CD137-activatable antibody clones in scFv format, including CD137-activatable antibody clone B13428, with intact masking peptide or with masking peptide cleaved by TEV protease, compared to scFv fragment of target antibody without masking peptide. [Figure 22B] 1 shows the CD137 binding affinity of exemplary CD137-activatable antibody clones as determined by flow cytometry. The CD137 binding affinity of CD137-activatable antibody clones in scFv format, including CD137-activatable antibody clone B13439, with either an intact masking peptide or a masking peptide cleaved by TEV protease, is shown, compared to a scFv fragment of the target antibody without the masking peptide. [Figure 23] 1 shows the masking efficiency of exemplary activatable antibodies against human CD137 compared to the parent antibody TY21242, as determined by flow cytometry. [Figure 24] Figure 1 shows the masking efficiency of exemplary activatable antibodies containing masking peptides of varying lengths compared to the parent antibody TY21580. Masking efficiency was determined using an ELISA-based method. A and B represent two experiments set up using the same experimental method to test various activatable anti-CTLA4 antibodies. [Diagram 25] 1 shows the masking efficiency of exemplary activatable antibodies containing truncated peptides of different lengths compared to the parent antibody TY21580. Masking efficiency was determined using an ELISA-based method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] I. General Techniques The techniques and procedures described or referenced herein are generally well understood by those of skill in the art and may be implemented using conventional methodology, e.g., as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMA Usubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames and G.R. Taylor eds. (1995)); Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.,1987);PCR: The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(JEColigan et al.,eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory) Press, 1999), The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., J. B. Lippincott Company, 1993).

[0029] II. Definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0030] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and so forth.

[0031] As used herein, the term "about" refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0032] It is to be understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0033] As used herein, the term "and / or" is intended to mean that phrases such as "A and / or B" include both A and B, A or B, A (single), and B (single). Similarly, as used herein, the term "and / or" is intended to mean that phrases such as "A, B, and / or C" include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0034] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a natural amino acid, but the C-terminal carboxy group, the N-terminal amino group, or a functional group on the side chain has been chemically modified to another functional group. The term "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions similarly to a natural amino acid.

[0035] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage, see, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0036] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

[0037] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can contain modification(s) made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps" which replace one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH may be phosphorylated or replaced with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyls may be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0038] The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes a nucleic acid molecule that is separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid of interest).

[0039] As used herein, "library" refers to a set of two or more entities that share a class. For example, a library containing polynucleotides may refer to a set of two or more polynucleotides. The term "library" is used in the broadest sense herein, specifically encompassing sub-libraries that may or may not be combined.

[0040] As used herein, "unique" refers to a member of a set that is distinct from other members of the set. For example, a unique activatable antibody in a library may refer to an activatable antibody that has a particular sequence that is not shared by other activatable antibodies in the library. In practice, it should be understood that a "unique" member of a physical realization of a library may exist in more than one copy. For example, a library may contain multiple "unique" activatable antibodies, with one or more of the "unique" activatable antibody molecules occurring in more than one copy.

[0041] As used herein, "diversity" refers to variety and / or heterogeneity. For example, the diversity of antibodies in a library refers to the large variety of antibodies with unique sequences present in the library.

[0042] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., Fab, Fab', Fab'-SH, F(ab')2, Fv and / or single chain variable fragments or scFv), so long as they exhibit the desired biological activity.

[0043] In some embodiments, the term "antibody" refers to an antigen-binding protein (i.e., an immunoglobulin) with a basic four polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain contains a variable region (referred to herein as V) at the N-terminus. H The heavy chain constant region is divided into three domains, C H1 , C H2 and C H3 Each light chain consists of a variable region (referred to herein as V I The light chain constant region consists of one domain, C L It consists of. V L is V H Aligned with C L is aligned with the first constant domain (CH1) of the heavy chain. H and V L The pairings of the tetramers together form a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain and thus contain 10 antigen-binding sites, whereas secreted IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units plus the J chain.

[0044] V H and V L Based on structural and sequence analysis, the regions can be further subdivided into regions of hypervariability called hypervariable regions (HVRs). HVRs are interspersed with more conserved regions called framework regions (FWs) (see, e.g., Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). H and V Lis composed of three HVRs and four FWs, arranged from amino to carboxy terminus in the following order: FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Similarly, the three HVRs of the light chain are referred to as HVR-L1, HVR-L2, and HVR-L3.

[0045] The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 or more amino acids (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY) (1989)).

[0046] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Antibodies can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain of their heavy chains (CH). There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG class of antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, by the gamma heavy chains Y1-Y4, respectively.

[0047] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retain the ability to bind to the antigen to which the antibody binds. Examples of "antigen-binding fragments" of antibodies include: (i) V L , V H , CL , and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and C H1 (iv) an Fd fragment consisting of the V domain of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a V H domains (Ward et al., Nature 341:544-546 (1989)), and (vi) isolated complementarity determining regions (CDRs).

[0048] The term "CTLA4" is used in this application to include human CTLA4 (e.g., UniProt Accession No. P16410), as well as variants, isoforms, and species homologs thereof (e.g., mouse CTLA4 (UniProt Accession No. P09793), rat CTLA4 (UniProt Accession No. Q9Z1A7), canine CTLA4 (UniProt Accession No. Q9XSI1), cynomolgus monkey CTLA4 (UniProt Accession No. G7PL88), etc.). Thus, a binding molecule (e.g., an activatable antibody) may also bind to CTLA4 from species other than human. In other cases, a binding molecule may be completely specific for human CTLA4 and may not exhibit species or other type cross-reactivity.

[0049] The term "CD137" is used in this application and includes human CD137 (e.g., GenBank Accession Nos. NM001561, NP001552), as well as variants, isoforms, and species homologs thereof (e.g., mouse CD137 (GenBank Gene ID 21942), rat CD137 (GenBank Gene ID 500590), canine CD137 (GenBank Gene ID 608274), cynomolgus monkey CTLA4 (GenBank Gene ID 102127961), etc.). Thus, a binding molecule (e.g., an activatable antibody) may also bind to CD137 from species other than human. In other cases, a binding molecule may be completely specific for human CD137 and may not exhibit species or other type cross-reactivity.

[0050] The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from different animal species, such as those having a variable region derived from a human antibody and a murine immunoglobulin constant region.

[0051] The term "compete for binding" refers to the interaction of two antibodies in binding to a binding target. A first antibody competes with a second antibody for binding if the binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody can be, but need not be, the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope, whether to the same extent, more or less, the antibodies are said to "cross-compete" with each other for binding of their respective epitope(s).

[0052] The term "epitope" refers to a portion of an antigen to which an antibody (or an antigen-binding fragment thereof) binds. Epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include a variable number of amino acids in unique spatial conformations. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, 2D nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or any method used in combination with a computational model of the antigen and its complex structure with its bound antibody and its variants (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). Once the desired epitope of an antigen is determined, antibodies against that epitope can be generated, for example, using the techniques described herein. Antibody generation and characterization can also elucidate information about the desired epitope. From this information, it is possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to perform cross-competition studies to find antibodies that competitively bind to each other, i.e., the antibodies compete for binding to the antigen. A high-throughput process for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO03 / 48731.

[0053] The term "germline" refers to the nucleotide sequences of antibody genes and gene segments as inherited from parents to offspring via germ cells. Germline sequences are distinguished from the nucleotide sequences encoding antibodies of mature B cells that have been altered by recombination and hypermutation events during the B cell maturation process.

[0054] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a location for the attachment of a sugar residue. The amino acids to which carbohydrates, such as oligosaccharides, are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. The particular attachment site is typically signaled by a sequence of amino acids, referred to herein as a "glycosylation site sequence." The glycosylation site sequence for N-linked glycosylation is -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid except proline. The terms "N-linked" and "O-linked" refer to chemical groups that serve as attachment sites between a sugar molecule and an amino acid residue. N-linked sugars are attached through an amino group. O-linked sugars are attached through a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety linked to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on a polypeptide, either zero (0-glycan site occupation), one (1-glycan site occupation), or both (2-glycan site occupation) sites can be occupied by carbohydrate moieties.

[0055] The term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; human cells (e.g., HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. coli cells), as well as cells contained within transgenic animals or cultured tissues. This term encompasses not only the particular subject cell, but also the progeny of such cells. Since certain modifications may occur in subsequent generations due to either mutations or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

[0056] A "human antibody" is one that possesses the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire, or an amino acid sequence that corresponds to other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0057] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. A humanized antibody may contain some or all of the CDRs or HVRs from a non-human animal or synthetic antibody, but the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0058] The term "exemplary antibody" refers to any one of the antibodies described herein. These antibodies may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above may be of any class, including, but not limited to, V. L and V H It encompasses all five classes of antibodies that have the same amino acid sequence for the V region. Furthermore, antibodies of the IgG class can be of any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each of the above-identified antibodies of the IgG subclasses includes the V L and V H It encompasses antibodies of all four subclasses that have the same amino acid sequence for that region. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes as well as the four IgG subclasses are known in the art. The amino acid sequences of the full-length heavy and light chains for each IgG4 subclass of the exemplary antibodies shown in Table 1b are provided in the present disclosure.

[0059] An "isolated" antibody or binding molecule is one that is isolated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0060] "k a The term "k" refers to the association rate constant of a particular antibody-antigen interaction, while "k d The term "antibody-antigen interaction" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0061] "K D The term "equilibrium dissociation constant" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. This is k d vs. k a The ratio of (i.e., k d / k a ) and expressed as molar concentration (M). D is used as a measure of the affinity of binding of an antibody to a binding partner. D The smaller the K, the stronger the antibody binds, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant will bind to a particular antigen more strongly than an antibody with a micromolar (μM) dissociation constant. D Values ​​can be determined using methods well established in the art. D One method for determining is by using ELISA. For example, an assay procedure using ELISA is described in at least Example 3 of the present disclosure.

[0062] The term "mammal" refers to any animal species of the mammalian class. Examples of mammals include humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; farm animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0063] The terms "prevent" or "prophylaxis" in reference to a particular disease state in a mammal refer to preventing or delaying the onset of the disease or preventing the manifestation of its clinical or subclinical symptoms.

[0064] As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of amino acids that are identical between sequences.The amino acid sequence identity of polypeptides can be conventionally determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, Methods Enzymol.183:63-98(1990); Pearson, Methods Mol.Biol.132:185-219(2000); Altschul et al., J.Mol.Biol.215:403-410(1990); Altschul et al., Nucleic Acids Res.25:3389-3402(1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, parameters are set such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, allowing a difference in homology of up to 5% of the total number of amino acid residues in the reference sequence. This aforementioned method of determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.

[0065] As used herein, the terms "bind", "bind to", "specifically bind to", or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for longer than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0066] The terms "treat", "treating" or "treatment" refer to a particular disease state in a mammal, and to causing a desired or beneficial effect in a mammal having a disease state. A desired or beneficial effect may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by immune cell numbers and / or activity, etc.), or preventing or inhibiting further progression of the disease, condition, or disorder. In the context of treating cancer in a mammal, a desired or beneficial effect may include inhibiting further growth or metastasis of cancer cells, killing cancer cells, inhibiting recurrence of cancer, reducing pain associated with cancer, or improving the survival of the mammal. The effect may be either subjective or objective. For example, if the animal is a human, the human may perceive improved vitality or survival, or reduced pain, as subjective symptoms of improvement or response to treatment. Alternatively, the clinician may perceive a reduction in tumor size or burden based on physical examination, clinical laboratory values, tumor markers, or x-ray findings. Some clinical signs that clinicians may observe regarding treatment response include normalization of laboratory values ​​such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, clinicians may observe a decrease in detectable tumor markers. Alternatively, other tests, such as ultrasound imaging, nuclear magnetic resonance imaging, and positron emission tomography, may be used to assess objective improvement.

[0067] The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows the foreign nucleic acid molecule to be propagated, selected, further manipulated, or expressed in a host cell or organism. The vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion. Some types of vectors can be integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome (e.g., non-episomal mammalian vectors). Other types of vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Another specific type of vector capable of directing the expression of an expressible foreign nucleic acid to which they are operably linked is usually referred to as an "expression vector". Expression vectors generally have control sequences that drive the expression of an expressible foreign nucleic acid. Simpler vectors, known as "transcription vectors", are capable of only transcription but not translation, and they can replicate but not express in target cells. The term "vector" encompasses all types of vectors, regardless of their function. Vectors capable of directing the expression of an expressible nucleic acid to which they are operatively linked are commonly referred to as "expression vectors." Other examples of "vectors" can include display vectors (e.g., vectors that direct the expression and display of an encoded polypeptide on the surface of a virus or a cell, such as a bacterial cell, yeast cell, insect cell, and / or mammalian cell).

[0068] As used herein, a "subject," "patient," or "individual" may refer to a human or non-human animal. A "non-human animal" may refer to any animal not classified as a human, such as farm animals, livestock, or zoo animals, sport animals, pet animals (e.g., dogs, horses, cats, cows, etc.), and animals used in research. A research animal may refer to, but is not limited to, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is a human.

[0069] "Effective amount" refers to at least an amount effective at the dosage and for the period of time necessary to achieve one or more desired or indicated effects, including therapeutic or prophylactic results. An effective amount may be provided in one or more administrations. For purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition (e.g., an effective amount when administered as a monotherapy or combination therapy). Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved when combined with one or more other agents.

[0070] III. Generation of Activatable Binding Polypeptides and Libraries Certain aspects of the present disclosure relate to libraries of polynucleotides (e.g., encoding any of the polypeptides described herein) and / or polynucleotides encoding polypeptides that are useful for screening and / or identifying one or more activatable binding polypeptides (i.e., one or more activatable antibodies), including, for example, an activatable antibody, an activatable antigen-binding fragment thereof, or a derivative of an activatable antibody.

[0071] The term "activatable binding polypeptide", "ABP", or "activatable antibody" includes a polypeptide comprising a target binding portion (TBM), a cleavable portion (CM), and a masking portion (MM). In some embodiments, the TBM comprises an amino acid sequence that binds to a target. In some embodiments, the TBM comprises an antigen binding domain (ABD) of an antibody or an antibody fragment thereof. In some embodiments, the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), where the VH and VL form a binding domain that binds to the target in the absence of MM. In some embodiments, the VH and VL are covalently linked, for example in an scFv. In some embodiments, the VH and VL form a Fab fragment. In some embodiments, the VH is linked to an antibody heavy chain constant region and the VL is linked to an antibody light chain constant region.

[0072] In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., an scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VH-VL (e.g., an scFv).

[0073] In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL-L3-VH (e.g., an scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-L1-VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure masking moiety (MM)-L1-cleavable moiety (CM)-L2-VH-L3-VL (e.g., an scFv). In some embodiments, L1, L2, and / or L3 are linkers that can have an independently selected length of either 0 amino acids or 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids.

[0074] A CM generally comprises an amino acid sequence that is cleavable, e.g., that serves as a substrate for an enzyme, and / or a cysteine-cysteine ​​pair that can form a reducible disulfide bond. Thus, when the terms "cleavage," "cleavable," "cleaved," and the like are used in connection with a CM, the terms encompass disruption of the disulfide bond between the cysteine-cysteine ​​pair by, e.g., enzymatic cleavage by a protease, as well as reduction of the disulfide bond, which may result from exposure to a reducing agent.

[0075] MM refers to the amino acid sequence when the CM of an activatable antibody is intact (e.g., not cleaved by the corresponding enzyme and / or contains an unreduced cysteine-cysteine ​​disulfide bond), and the MM interferes with or inhibits the binding of the TBM to its target. In some embodiments, the MM interferes with or inhibits the binding of the TBM to its target so efficiently that binding of the TBM to its target is extremely low and / or below the limit of detection (e.g., binding cannot be detected by ELISA or flow cytometry assays). The amino acid sequence of the CM may overlap with or be contained within the MM. For convenience, it should be noted that "ABP" or "activatable antibody" is used herein to refer to the ABP or activatable antibody in both their uncleaved (or "native") state, as well as their cleaved state. It will be apparent to one of skill in the art that in some embodiments, the cleaved ABP may lack the MM, e.g., due to cleavage of the CM by a protease, resulting in release of at least the MM (e.g., if the MM is not bound to the ABP by a covalent bond (e.g., a disulfide bond between cysteine ​​residues). Exemplary ABPs are described in further detail below.

[0076] A library of the disclosure may contain one or more polynucleotides encoding any of the polypeptides described herein (e.g., one or more of the activatable binding polypeptides described herein). In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of a library described herein encode a polypeptide comprising a full-length antibody light chain and / or a full-length antibody heavy chain(s). In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of a library described herein encode a polypeptide comprising a light chain and / or a heavy chain Fab fragment(s). In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of a library described herein encode a polypeptide comprising a single chain variable fragment(s) (scFv).

[0077] Other aspects of the disclosure relate to libraries of polypeptides useful for screening and / or identifying one or more activatable binding polypeptides (i.e., one or more activatable antibodies), including polynucleotides (e.g., any of the polypeptides described herein) and / or activatable antibodies, activatable antigen-binding fragments thereof, or derivatives of activatable antibodies. The libraries of the disclosure may contain one or more of the polypeptides described herein (e.g., one or more activatable binding polypeptides). In some embodiments, one or more of the polypeptides of the libraries described herein (i.e., one, some, or all) comprise full-length antibody light chain and / or heavy chain(s). In some embodiments, one or more of the polypeptides of the libraries described herein (i.e., one, some, or all) comprise light chain and / or heavy chain Fab fragment(s). In some embodiments, one or more of the polypeptides of the libraries described herein (i.e., one, some, or all) comprise single chain variable fragment(s) (scFv). In some embodiments, the polypeptides are expressed on a cell surface (e.g., yeast or mammalian cell display).

[0078] In some embodiments, a polypeptide of the present disclosure comprises (a) a first peptide (FP), (b) a cleavable moiety (CM), and (c) a target binding moiety (TBM). In some embodiments, the FP is any of the first peptides described herein (e.g., those represented by formula (XIII): m CX n CX o (SEQ ID NO: 86), where m is 2-10, n is 3-10, o is 1-10, and 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. In some embodiments, each X is not W, M, and / or C. In some embodiments, the X in formula (XIII) is mEach X 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. n each X in is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; and / or X in formula (XIII) o wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, m is 3 to 10. In some embodiments, FP is any of the first peptides described herein (e.g., those of Formula (I): X m CX n CZ o (SEQ ID NO:1), wherein m is 2-10, n is 3-10, o is 1-10, each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and 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. In some embodiments, m is 3-10. In some embodiments, X is not W, M, and / or C. In some embodiments, X of Formula (I) m Each X in 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. n wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, FP is any of the first peptides described herein (e.g., those of formula (XII):Z m CZ n CZ o(SEQ ID NO: 71), wherein m is 2-10, n is 3-10, o is 1-10, and 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. In some embodiments, m is 3-10. In some embodiments, the CM is any of the cleavable moieties described herein (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site). In some embodiments, the CM is any of the cleavable moieties described herein (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site). In some embodiments, the TBM is any of the target binding moieties described herein (e.g., a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region).

[0079] In some embodiments, the first peptide (FP) interferes with, occludes, reduces the ability of, prevents, inhibits, or competes with the target binding moiety for binding to its target (e.g., an inactive activatable antibody). In some embodiments, the first peptide (FP) interferes with, occludes, reduces, prevents, inhibits, or competes with the target binding moiety for binding to its target only if the polypeptide has not been activated (e.g., activated by a change in pH (increasing or decreasing), activated by a change in temperature (increasing or decreasing), activated after contact with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces a conformational change in the polypeptide (e.g., dislocation of the first peptide (FP)) such that the first peptide no longer interferes with binding of the activatable antibody to its target. In some embodiments, the first peptide (FP) interferes with, occludes, reduces the ability of, prevents, inhibits, or competes with the target binding moiety for binding to its target only if the cleavable moiety (CM) is not cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, the first peptide (FP) has a masking efficiency of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.) prior to activation.In some embodiments, masking efficiency is measured by the difference in affinity of an activatable antibody comprising a masking moiety (before activation) for binding to its target compared to the affinity of a polypeptide lacking the first peptide (FP) for binding to its target (e.g., the difference in affinity of an activatable antibody comprising the first peptide (FP) (before activation) for a target antigen (such as CTLA4) compared to a parent antibody lacking the first peptide (FP), or the difference in affinity of an activatable antibody comprising the first peptide (FP) (before activation) for a target antigen (such as CTLA4) compared to the affinity of the activatable antibody after activation). In some embodiments, masking efficiency is the EC of binding of an activatable antibody comprising the first peptide (FP) (before activation). 50 , EC of the parent antibody 50 (e.g., EC by ELISA) 50 (see, e.g., methods in Example 3). In some embodiments, the masking efficiency is measured by the difference in the affinity of an activatable antibody comprising the first peptide (FP) before activation to bind to its target (e.g., the difference in the affinity of an activatable antibody comprising the first peptide (FP) for binding to its target antigen (such as CTLA4) before activation compared to the affinity of an activatable antibody comprising the first peptide (FP) for binding to its target after activation). In some embodiments, the first peptide (FP) binds to a target binding moiety (TBM) and prevents the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the first peptide (FP) has a dissociation constant for binding to the target binding moiety (TBM) that is greater than the dissociation constant of the target binding moiety (TBM) for its target. In some embodiments, the first peptide (FP) is a masking moiety (MM). The dissociation constant can be measured, for example, by techniques such as ELISA, surface plasmon resonance or biolayer interferometry (BLI), or flow cytometry.

[0080] In some embodiments, the first peptide (FP) does not interfere with, occlude, reduce the ability of, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target after the polypeptide is activated (e.g., activated by treatment with one or more proteases that cleave within the cleavable moiety (CM), activated by a change in pH (increased or decreased), activated by a change in temperature (increased or decreased), activated after contact with a second molecule (such as an enzyme), etc.). In some embodiments, the first peptide (FP) does not interfere with, occlude, reduce the ability of, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target after the cleavable moiety (CM) is cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, the first peptide (FP) has a masking efficiency after activation (e.g., the relative affinity of the activatable antibody after activation compared to the affinity of the parent antibody) of at most about 1.75 (e.g., at most about 1.75, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, at most about 1.0, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, or at most about 0.5, etc.).

[0081] In some embodiments, the polypeptides of the disclosure comprise, from N-terminus to C-terminus, the structure: first peptide (FP)-cleavable moiety (CM)-target binding moiety (TBM). The libraries of the disclosure can be used to screen for one or more activatable binding polypeptides (i.e., activatable antibodies) that, when in an activated form, bind to any target of interest, including, for example, CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, and / or B7-H4.

[0082] In some embodiments, the libraries of the disclosure include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, at least 10, at least 25 ... 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 The polypeptide comprises a plurality of polynucleotides encoding individual unique polypeptides.

[0083] In some embodiments, the libraries of the disclosure include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, at least 10, at least 25 ... 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 106 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 The polypeptide comprises a plurality of polynucleotides encoding the respective polypeptides.

[0084] In some embodiments, the libraries of the present disclosure contain: 1) a smaller number of unique peptides (e.g., peptides of formula (XIII):X) than are found in a typical random peptide library. m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o (FP comprising an amino acid sequence according to SEQ ID NO:1); 2) a peptide comprising a pair of cysteine ​​residues at fixed positions (e.g., a peptide represented by formula (XIII): X m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o (FP comprising an amino acid sequence according to SEQ ID NO:1); and / or 3) a peptide having few or no chemically labile residues (methionine or tryptophan) (e.g., a peptide of formula (XIII): X m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ oAdvantageously, the libraries of the present disclosure dramatically reduce library size compared to random peptide libraries and encode and / or contain peptides (e.g., FPs comprising an amino acid sequence according to formula (XIII): X m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o (FPs comprising an amino acid sequence according to SEQ ID NO:1). Moreover, the inclusion of a pair of cysteine ​​residues at fixed positions ensures that the presented peptides have a constrained conformation, which tends to show increased binding affinity and / or specificity. Furthermore, the libraries of the present disclosure contain peptides (e.g., peptides of formula (XIII):XIII) that contain little or no residues that are unfavorable to the manufacturing process, such as methionine or tryptophan. m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o (FP comprising an amino acid sequence according to SEQ ID NO:1).

[0085] In some embodiments, a library of the present disclosure contains a plurality of polynucleotides, and at least one of the polynucleotides in the library has a structure represented by (a) Formula (XIII):X m CX n CX o(b) a first peptide (FP) comprising an amino acid sequence according to (SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and (c) a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector). In some embodiments, a library of the disclosure contains a plurality of polynucleotides, and at least one of the polynucleotides in the library has a structure represented by (a) Formula (I):X m CX n CZ o (b) a cleavable moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (eg, an expression vector or a display vector).

[0086] In some embodiments, at least one of the polynucleotides in the library has formula (III): 10 X 11 X 12 CX 13 X 14 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; and X 10 is L, P, or Y, and X 11 is F, P, or Y, and X 12 is A, P, S, or Y, and X 13 is A, D, N, S, T, or Y, and X 14 is A, S, or Y. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

[0087] In some embodiments, at least one of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 25-46. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

[0088] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 250, at least 500, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 The compound has the formula (XIII): m CX n CX o(b) a cleavable moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 250, at least 500, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 The compound has the formula (I): X m CX n CZ o(b) a cleavable moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (eg, an expression vector or a display vector).

[0089] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 250, at least 500, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 The formula (III) is EVGSYX1X2X3X4X5X6CX7X8X9X 10 X 11 X 12 CX 13 X 14 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; and X 10 is L, P, or Y, and X 11 is F, P, or Y, and X 12 is A, P, S, or Y, and X 13 is A, D, N, S, T, or Y, and X 14 is A, S, or Y. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

[0090] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 250, at least 500, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 Each polynucleotide encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 25-46. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

[0091] In some embodiments, each of the polynucleotides in the library has the formula (a) (XIII): m CX n CX o(SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; (b) a cleavable moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising: (a) a first peptide (FP) comprising an amino acid sequence according to Formula (I): X m CX n CZ o (b) a cleavable moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (eg, an expression vector or a display vector).

[0092] In some embodiments, each of the polynucleotides in the library has the formula (III): 10 X 11 X 12 CX 13 X 14X1 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; and X 10 is L, P, or Y, and X 11 is F, P, or Y, and X 12 is A, P, S, or Y, and X 13 is A, D, N, S, T, or Y, and X 14 is A, S, or Y. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

[0093] In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 25-46. In some embodiments, at least one of the polynucleotides in the library encodes an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

[0094] In some embodiments, the library of the present disclosure comprises: (a) a compound of formula (XIII):X m CX n CX o(e.g., at least 1, at least 2, at least 5, at least 10, at least 100, at least 100, at least 100, or at least 100, more preferably ... 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 In some embodiments, m is 3 to 10. In some embodiments, the library of the present disclosure contains (a) a polynucleotide of formula (I): X m CX n CZ oand each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H, and P; (b) a cleavable portion (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a target binding portion (TBM) comprising an antibody light chain variable region; and d) an antibody heavy chain variable region. 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19In some embodiments, m is 3 to 10. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, the structure: first peptide (FP)-cleavable moiety (CM)-VL-VH. In some embodiments, at least one of the polypeptides is an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, a linker sequence separates the VL and VH (i.e., the structure VL-linker-VH). The linker can be any linker sequence known in the art, for example, any of the linker sequences described herein. In some embodiments, the linker sequence is any number of copies of GGGGS (SEQ ID NO: 17) (e.g., repeated twice, repeated three times, etc.).

[0095] In some embodiments, the library of the present disclosure comprises: (a) a compound of formula (XIII):X m CX n CX o and (b) a cleavable portion (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding portion (TBM) comprising an antibody light chain variable region. 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 1014 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 In some embodiments, the library of the present disclosure contains (a) polynucleotides of formula (I): X m CX n CZ o and (b) a cleavable portion (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding portion (TBM) comprising an antibody light chain variable region. 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19In some embodiments, the library contains at least one polynucleotide (m) encoding an antibody heavy chain variable region, and the library further comprises one or more polynucleotides encoding an antibody heavy chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the polynucleotide encoding a polypeptide comprising a target binding moiety (TBM) comprising an antibody light chain variable region and the polynucleotide encoding the antibody heavy chain variable region are on the same vector (e.g., expressed from their own promoters) or on different vectors. In some embodiments, at least one of the polypeptides forms an activatable binding polypeptide (i.e., an activatable antibody) when bound to an antibody heavy chain variable region.

[0096] In some embodiments, the library of the present disclosure comprises: (a) a compound of formula (XIII):X m CX n CX o (e.g., at least 1, at least 2, at least 5, at least 10, at least 100, at least 100, at least 100, or at least 100, more preferably ... 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 1016 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 In some embodiments, m is 3 to 10. In some embodiments, the activatable binding polypeptide comprises a polypeptide comprising, from the N-terminus to the C-terminus, the structure: first peptide (FP)-cleavable moiety (CM)-VH-VL. In some embodiments, the library of the disclosure comprises (a) a polynucleotide of formula (I): X m CX n CZ o and each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H, and P; (b) a cleavable portion (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a target binding portion (TBM) comprising an antibody heavy chain variable region; and d) an antibody light chain variable region. 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19In some embodiments, m is 3 to 10. In some embodiments, the activatable binding polypeptide comprises a polypeptide comprising, from N-terminus to C-terminus, the structure: first peptide (FP)-cleavable moiety (CM)-VH-VL. In some embodiments, at least one of the polypeptides is an activatable binding polypeptide (i.e., an activatable antibody). In some embodiments, a linker sequence separates the VH and VL (i.e., the structure VH-linker-VL). The linker can be any linker sequence known in the art, for example, any of the linker sequences described herein. In some embodiments, the linker sequence is any number of copies of GGGGS (SEQ ID NO: 17) (e.g., repeated twice, repeated three times, etc.).

[0097] In some embodiments, the library of the present disclosure comprises: (a) a compound of formula (XIII):X m CX n CX o and (b) a cleavable portion (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding portion (TBM) comprising an antibody heavy chain variable region. 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 In some embodiments, the library of the present disclosure comprises (a) a polynucleotide sequence of formula (I): X m CX n CZ o and (b) a cleavable portion (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); and (c) a target binding portion (TBM) comprising an antibody heavy chain variable region. 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19In some embodiments, the library contains at least one polynucleotide (m) encoding an antibody light chain variable region, and the library further comprises one or more polynucleotides encoding an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the polynucleotide encoding a polypeptide comprising a target binding moiety (TBM) comprising an antibody heavy chain variable region and the polynucleotide encoding the antibody light chain variable region are on the same vector (e.g., expressed from their own promoters) or on different vectors. In some embodiments, at least one of the polypeptides forms an activatable binding polypeptide (i.e., an activatable antibody) when bound to an antibody light chain variable region.

[0098] The polynucleotides and / or polynucleotide libraries described herein may incorporate any of the HVR sequences (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the light chain variable region HVR sequences), heavy chain variable region sequences, and / or light chain variable region sequences of any of the antibodies described herein (e.g., anti-CTLA4 antibodies, anti-CD137 antibodies). The polynucleotides and / or polynucleotide libraries described herein may also incorporate HVR sequences (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the light chain variable region HVR sequences), heavy chain variable region sequences, light chain variable region sequences, and / or heavy chains and / or light chains described in PCT Application No. PCT / CN2017 / 098333, which is incorporated by reference herein in its entirety, and / or PCT Application No. PCT / CN2017 / 098299, which is incorporated by reference herein in its entirety.

[0099] In some embodiments, a library of the disclosure comprises one or more vectors (e.g., expression vectors and / or display vectors) that encode one or more polynucleotides (e.g., synthetic polynucleotides) of the disclosure.

[0100] Further provided herein are methods of preparing libraries, e.g., by providing and assembling polynucleotide sequences (e.g., synthetic polynucleotide(s)) of the libraries of the present disclosure. For example, a plurality (e.g., at least 1, at least 2, at least 5, at least 10, at least 100, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 Pieces, at least 10 16 Pieces, at least 10 17 Pieces, at least 10 18 pcs, or at least 10 19 Also provided herein are methods of making a library by selecting a first peptide (FP) sequence, a cleavable moiety (CM) sequence, and / or a target binding moiety (TBM) sequence (e.g., any one or more of the FP, CM, and TBM sequences described herein) and assembling polynucleotide sequences encoding these sequences to create a library of polynucleotides (e.g., synthetic polynucleotides) encoding a plurality of polypeptides. In some embodiments, at least one of the polypeptides encoded by the assembled library is an activatable binding polypeptide (i.e., an activatable antibody).

[0101] A polynucleotide encoding a polypeptide described herein can be cloned into any vector suitable for expression of a portion or the entire polypeptide sequence. In some embodiments, the polynucleotide is cloned into a vector that allows for the production of a portion or the entire polypeptide fused to all or a portion of a protein (e.g., a viral coat protein, a bacterial surface protein, a yeast surface protein, an insect cell surface protein, a mammalian cell surface protein) (i.e., creating a fusion protein) and displayed on the surface of a particle or cell. Several types of vectors are available, for example, phagemid vectors may be used to implement the present disclosure. Phagemid vectors generally contain various components, including promoters, signal sequences, phenotypic selection genes, origins of replication, and other necessary components, as known to those skilled in the art. In some embodiments, a polynucleotide encoding a polypeptide region can be cloned into a vector for expression in bacterial cells for bacterial display or in yeast cells for yeast display. Exemplary vectors are described in U.S. Proc. Publication No. US20160145604. In some embodiments, the vector is a display vector that includes, from 5' to 3', a polynucleotide encoding an amino acid sequence to be displayed on a surface (e.g., the surface of a phage, bacteria, yeast, insect, or mammalian cell), a restriction site, a second polynucleotide encoding a surface peptide that can be displayed on the surface, and a second restriction site. In some embodiments, the second polynucleotide encodes a phage coat protein, a yeast outer wall protein (such as Aga2), a bacterial outer membrane protein, a cell surface tether domain, or an adaptor, or a truncated or derivative thereof. In some embodiments, the surface peptide is for phage display, yeast display, bacterial display, insect display, or mammalian display, or shuttle display therebetween. In some embodiments, when expressed, the amino acid sequence and the surface peptide are displayed as a fusion protein on the surface. In some embodiments, the vector further includes a fusion tag 5' to the first restriction site or 3' to the second restriction site.

[0102] Certain aspects of the present disclosure relate to populations of cells containing the vector(s) described herein. Polypeptides encoded by polynucleotides generated by any of the techniques described herein, or other suitable techniques, can be expressed and screened to identify activatable binding polypeptides having the desired structure and / or activity. Expression of the polypeptides can be carried out, for example, using cell-free extracts (e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g., yeast display). In some embodiments, the cells are bacterial cells, yeast cells, insect cells, or mammalian cells (such as Chinese Hamster Ovary (CHO) cells). Methods for transfecting bacterial, yeast, or mammalian cells are known in the art and described in the references cited herein. Expression of polypeptides (e.g., one or more activatable binding polypeptides) (e.g., from a library of the present disclosure) in these cell types, as well as screening for activatable binding polypeptides of interest, is described in more detail below.

[0103] Alternatively, the polynucleotides can be expressed in an E. coli expression system, such as that described by Pluckthun and Skerra. (Meth. Enzymol., 1989, 178:476; Biotechnology, 1991, 9:(273). Mutant proteins can be expressed for secretion in the medium and / or bacterial cytoplasm as described by Better and Horwitz, Meth. Enzymol., 1989, 178:476. In some embodiments, the polypeptide is linked to the 3' end of a sequence encoding a signal sequence, such as the ompA, phoA or pelB signal sequence (Lei et al., J. Bacteriol., 1987, 169:4379). These gene fusions are assembled into dicistronic constructs so that they can be expressed from a single vector and secreted into the periplasmic region of E. coli where they can be refolded and recovered in an active form (Skerra et al., 2002). (e.g., J. Immunol., 1991, 9:273). For example, a gene encoding a polypeptide comprising a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM) comprising an antibody light chain can be co-expressed with an antibody heavy chain gene to produce the polypeptide of interest.

[0104] In other embodiments, the polypeptide sequences of the disclosure are expressed on the membrane surface of prokaryotes, such as E. coli, using secretion signals and lipidation moieties described, for example, in US20040072740, US20030100023, and US20030036092.

[0105] Alternatively, the polypeptide sequences of the disclosure can be expressed and screened by immobilized periplasmic expression (APEx2-hybrid surface display) as described, for example, in Jeong et al., PNAS, 2007, 104:8247, or by other immobilized methods as described, for example, in Mazor et al., Nature Biotechnology, 2007, 25:563.

[0106] Higher eukaryotic cells, such as mammalian cells, e.g., myeloma cells (e.g., NS / 0 cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, may also be used for expression of the polypeptides of the present disclosure. Polypeptides (e.g., activatable binding polypeptides) expressed in mammalian cells may be designed to be secreted into the culture medium or expressed on the surface of the cell.

[0107] In other embodiments, the polypeptides (e.g., activatable binding polypeptides) can be selected using mammalian cell display (Ho et al., PNAS, 2006, 103:9637). In some embodiments, as described above and exemplified below, the polypeptides (e.g., activatable binding polypeptides) can be selected following production of some or all of the polypeptide sequence fused to all or a portion of a viral coat protein (i.e., creating a fusion protein) and displayed on the surface of a particle or cell using, for example, phage display.

[0108] Certain aspects of the present disclosure relate to non-human animals that comprise the polynucleotide or polynucleotide library of the present disclosure. For example, the non-human animals of the present disclosure can be modified such that their genome comprises a polynucleotide that encodes a polypeptide (e.g., an activatable binding polypeptide) of the present disclosure. In some embodiments, the transgenic animal (e.g., a mouse) expresses the polypeptide encoded by the polynucleotide. Techniques for modifying the genome of non-human animals are known in the art (e.g., the methods used to generate Xenomouse™).

[0109] Screening of activatable binding polypeptides from libraries of the disclosure can be performed by any suitable means, such as determining target binding before and after activation (such as treating the polypeptide with one or more proteases that cleave a sequence within the cleavable moiety (CM)). For example, binding activity can be assessed by standard immunoassays and / or affinity chromatography. Screening of polypeptides of the disclosure for catalytic function, e.g., proteolytic function, can be accomplished using standard assays, e.g., hemoglobin plaque assays. Determining the binding affinity of a polypeptide (e.g., an activatable binding polypeptide) to a target can be assayed in vitro using a variety of known techniques, such as ELISA, BIACORE™ instruments, which measure the binding rate of a protein to a particular target based on surface plasmon resonance or biolayer interferometry (BLI), as exemplified below using the ForteBioOctet® RED96 platform (Pall Life Sciences). In vivo assays can be performed using a number of animal models and then subsequently tested in humans, if desired. Cell-based biological assays are also contemplated. The polypeptides (e.g., activatable binding polypeptides) can be further selected for functional activity, e.g., antagonist or agonist activity. For example, in some embodiments, the affinity of binding between a polypeptide comprising a fab fragment(s) and one or more target(s) is measured using BLI by tagging the antigen with a human IgG1-Fc tag and capturing with an anti-hIgG-Fc capture (AHC) biosensor (e.g., before and after activation). The polypeptides can be tagged with a His6 tag at the C-terminus of the CH1 domain, overexpressed in a host cell such as E. coli, and purified using Ni-NTA resin, or the like. The affinity can be measured using an AHC sensor (anti-human IgG-Fc capture soak and read biosensor) immersed in wells containing purified polypeptides comprising Fab diluted, e.g., to 5-10 μg / mL in kinetic buffer (e.g., before and after activation).

[0110] After a binder is identified (e.g., by determining that the polypeptide is capable of binding to a target or antigen when "active" (e.g., after protease treatment) but not when "inactive" (e.g., before protease treatment)), the nucleic acid can be extracted. The extracted DNA can then be used to directly transform E. coli host cells, or the coding sequence can be amplified, for example, using PCR with suitable primers and sequenced by any typical sequencing method. The DNA sequence of the binder can be restriction enzyme digested and inserted into a vector for protein expression.

[0111] First peptide (FP) In some embodiments, the disclosure relates to polynucleotides and / or polynucleotide libraries encoding one or more polypeptides comprising a first peptide (FP). In some embodiments, the disclosure relates to polypeptides and / or polypeptide libraries comprising at least one polypeptide comprising a first peptide (FP). In some embodiments, the first peptide (FP) is represented by formula (XIII): m CX n CX o (SEQ ID NO: 86), where m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, o is 1 to 10, and 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. In some embodiments, m is 3 to 10. In some embodiments, each X is not W, M, and / or C. In some embodiments, X in Formula (XIII) m Each X 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. n each X in is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; and / or X in formula (XIII) owherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, FP has the formula (XIV): (NNK) m TGY(NNK) n TGY(NNK) o (SEQ ID NO:87), wherein each N is independently A, G, T, or C; each K is independently T or G; each Y is independently T or C; and each H is independently A, T, or C.

[0112] In some embodiments, the first peptide (FP) has the formula (I): m CX n CZ o (SEQ ID NO: 1), wherein m is 2-10 (e.g., 3-10), n is 3-10, o is 1-10, 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, and 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. In some embodiments, m is 3-10. In some embodiments, X is not W, M, and / or C. In some embodiments, X in formula (I) m Each X in 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. n wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, FP has the formula (II): (NNK) m TGY(NNK) n TGY(NHC) o (SEQ ID NO:2), wherein each N is independently A, G, T, or C; each K is independently T or G; each Y is independently T or C; and each H is independently A, T, or C.

[0113] In some embodiments, the first peptide (FP) has the formula (XII): m CZ n CZ o (SEQ ID NO:71), where m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, o is 1 to 10, and 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. In some embodiments, m is 3 to 10.

[0114] In some embodiments, m is 2 to 5, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10. In some embodiments, m is 6 to 8. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6.

[0115] In some embodiments, n is 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, n is 6-8. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 6. In some embodiments, n is 8.

[0116] In some embodiments, o is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, o is 1-2. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 2.

[0117] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (IV): Z6CX6CZ2 (SEQ ID NO:55), 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, and 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.

[0118] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (V): Z6CX8CZ2 (SEQ ID NO:56), 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, and 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.

[0119] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (VI):(Z6)C(Z6)C(Z2) (SEQ ID NO:57), 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.

[0120] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (VII):(Z6)C(Z8)C(Z2) (SEQ ID NO:58), 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.

[0121] In some embodiments, the first peptide (FP) is m CADAPNHCXX (SEQ ID NO:88), X m CHHSPANCXX (SEQ ID NO:89), X m CPILRHRCXX (SEQ ID NO: 90), X m CKWRPSRCXX (SEQ ID NO:91), X m CRVLPRRCXX, (SEQ ID NO:92), X m CLWRHRSCXX (SEQ ID NO: 93), and X mCPRLRRKCXX (SEQ ID NO:94), where m is 2 to 10 and 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. In some embodiments, each X is not M, W, or C. In some embodiments, each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, m is 2. In some embodiments, the first peptide (FP) comprises the amino acid sequence EVGSYPTDLDACADAPNHCHF (SEQ ID NO: 95), EVGSYSSTHAHCHHSPANCIS (SEQ ID NO: 96), EVGSYDTDYDFCPILRHRCDS (SEQ ID NO: 97), EVGSYNDYNYHCKWRPSRCHN (SEQ ID NO: 98), EVGSYYHDYDDCRVLPRRCFN (SEQ ID NO: 99), EVGSYSNNFASCLWRHRSCAD (SEQ ID NO: 100), or EVGSYTDNYDYCPRLRRKCYH (SEQ ID NO: 101). In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CD137 antibodies described herein, including the antibodies described with respect to the specific amino acid sequences of the HVRs, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding moiety (TBM) comprises the full-length antibody light chain of one or more of the anti-CD137 antibodies described herein.

[0122] In some embodiments, the first peptide (FP) is m CPDHPYPCXX (SEQ ID NO: 102), X n CDAFYPYCXX (SEQ ID NO: 103), X m CDSHYPYCXX (SEQ ID NO: 104), and X mCVPYYYACXX (SEQ ID NO:105), where m is 2 to 10 and 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. In some embodiments, the first peptide (FP) comprises the amino acid sequence EVGSYNFVADSCPDHPYPCSA (SEQ ID NO: 110), EVGSYIVHHSDCDAFYPYCDS (SEQ ID NO: 111), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO: 112), EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 113), EVGSYYSAYPACDSHYPYCQS (SEQ ID NO: 114), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO: 115), EVGSYPQPSSDCVPYYYACAY (SEQ ID NO: 116), or EVGSYPNPASDCVPYYYACAY (SEQ ID NO: 117). In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CTLA4 antibodies described herein, including the antibodies described with respect to the specific amino acid sequences of the HVRs, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding moiety (TBM) comprises the full-length antibody light chain of one or more of the anti-CTLA4 antibodies described herein.

[0123] In some embodiments, the first peptide (FP) comprises an amino acid sequence selected from SEQ ID NOs: 72-85.

[0124] In some embodiments, any of the first peptides (FPs) described herein may further comprise one or more additional amino acid sequences (e.g., one or more polypeptide tags). Examples of suitable additional amino acid sequences may include, but are not limited to, purification tags (e.g., his tags, flag tags, maltose binding protein, and glutathione-S-transferase tags), detection tags (e.g., tags that can be photometrically detected (e.g., red or green fluorescent protein, etc.), tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.), tags containing secretion sequences, leader sequences, and / or stabilizing sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), and the like. In some embodiments, the one or more additional amino acid sequences are at the N-terminus of the first peptide (FP). In some embodiments, the additional amino acid sequence comprises or consists of the sequence EVGSY (SEQ ID NO: 16).

[0125] In some embodiments, the first peptide binds to the target binding moiety (TBM) and inhibits the polypeptide from binding to its target prior to activation (e.g., prior to treatment with one or more proteases that cleave within the cleavable moiety (CM), prior to undergoing a (local) change in pH (increase or decrease), prior to a temperature change (increase or decrease), prior to contact with a second molecule (such as a small molecule or protein ligand), etc.), but does not bind to the TBM and / or inhibit the polypeptide from binding to its target following activation (e.g., after treatment with one or more proteases that cleave within the cleavable moiety (CM), after undergoing a (local) change in pH (increase or decrease), after a temperature change (increase or decrease), after contact with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, the first peptide (FP) (e.g., a masking moiety) inhibits binding of a polypeptide (e.g., an activatable binding polypeptide) to its target if the CM is not cleaved, but does not inhibit binding of a polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) to its target if the CM is cleaved. In some embodiments, the first peptide (FP) (e.g., a masking moiety) has a dissociation constant for binding to the TBM that is greater than the dissociation constant of the polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) for its target (e.g., at least about 1.5-fold greater, at least about 2-fold greater, at least about 2.5-fold greater, at least about 3-fold greater, at least about 3.5-fold greater, at least about 4-fold greater, at least about 4.5-fold greater, at least about 5-fold greater, at least about 10-fold greater, at least about 100-fold greater, at least about 500-fold greater, etc.).

[0126] Cuttable part (CM) In some embodiments, the disclosure relates to polynucleotides and / or polynucleotide libraries encoding one or more polypeptides that comprise a cleavable moiety (CM). In some embodiments, the disclosure relates to polypeptides and / or polypeptide libraries that comprise at least one polypeptide that comprises a cleavable moiety (CM).

[0127] In some embodiments, the cleavable moiety (CM) comprises at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the first cleavage site is a first protease cleavage site. Any suitable protease cleavage site that is recognized and / or cleaved by any protease known in the art (e.g., a protease known to coexist with the target of the activatable antibody that comprises the CM) can be used, including, for example, urokinase-type plasminogen activator (uPA), matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19, MMP-20, MMP-21, MMP-22, MMP-23, MMP-24, MMP-25, MMP-26, MMP-27, MMP-28, MMP-29, MMP-30, MMP-31, MMP-32, MMP-33, MMP-34, MMP-35, MMP-36, MMP-37, MMP-38, MMP-39, MMP-40, MMP-41, MMP-42, MMP-43, MMP-44, MMP-45, MMP-46, MMP-47, MMP-48, MMP-49, MMP-50, MMP-51, MMP-52, MMP-53, MMP-54, MMP-55, MMP-56, MMP-57, MMP-58, MMP-59, MMP-60, MMP-61, MMP P-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and / or MMP-27), tobacco etch virus (TEV) protease, plasmin, thrombin, PSA, PSMA, ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, and / or ADAMTS5), caspar caspase (e.g., 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, and / or caspase-14), aspartic proteases (e.g., RACE and / or renin), aspartic cathepsins (e.g., cathepsin D and / or cathepsin E), cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin I ... cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and / or cathepsin X / Z / P), cysteine ​​proteinases (e.g., cruzipain, legumain, and / or Otubain-2), KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14), metalloproteinases (e.g., meprin, neprilysin, PSMA, and / or BMP-1), serine proteases (e.g.,Included are protease cleavage sites recognized and / or cleaved by activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, tPA, tryptase, type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and / or TMPRSS4), and the like. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, 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. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from uPA, MMP-2, MMP-9, and / or TEV protease. In some embodiments, the protease cleavage comprises an amino acid sequence selected from SGRSA (SEQ ID NO: 13), PLGLAG (SEQ ID NO: 14), and ENLYFQG (SEQ ID NO: 15).

[0128] In some embodiments, the polypeptide comprising the first peptide (FP) and the cleavable moiety (CM) comprises an amino acid sequence according to formula (VIII): EVGSY(Z6)C(Z6)C(Z2)SGRSA (SEQ ID NO: 4), wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H, and P.

[0129] In some embodiments, the polypeptide comprising the first peptide (FP) and the cleavable moiety (CM) comprises an amino acid sequence according to formula (IX): EVGSY(Z6)C(X6)C(Z2)SGRSA (SEQ ID NO:5), where each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and 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.

[0130] In some embodiments, the polypeptide comprising the first peptide (FP) and the cleavable moiety (CM) comprises an amino acid sequence according to the formula (X): EVGSY(Z6)C(Z8)C(Z2)SGRSA (SEQ ID NO: 6), where each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H, and P.

[0131] In some embodiments, the polypeptide comprising the first peptide (FP) and the cleavable moiety (CM) comprises an amino acid sequence according to formula (XI): EVGSY(Z6)C(X8)C(Z2)SGRSA (SEQ ID NO:7), 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, and 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.

[0132] In some embodiments, the cleavable moiety (CM) further comprises a first linker (L1). In some embodiments, the first linker (L1) is C-terminal to the first cleavage site (CS1) (e.g., the first protease cleavage site). In some embodiments, the cleavable moiety (CM) comprises, from the N-terminus to the C-terminus, the structure (CS1)-L1.

[0133] Any suitable linker (e.g., flexible linker) known in the art may be used, including, for example, glycine polymers (G)n, where n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.); glycine-serine polymers (GS)n, where n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as GGGGS (SEQ ID NO:17), SGGS (SEQ ID NO:18), GGSG (SEQ ID NO:19), GGSGG (SEQ ID NO:20), GSGSG (SEQ ID NO:21), GSGGG (SEQ ID NO:22), GGGSG (SEQ ID NO:23), and / or GSSSG (SEQ ID NO:24); glycine alanine polymers; alanine-serine polymers, and the like. The linker sequence can be any length, such as, for example, about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20 amino acid glycine polymer or glycine-serine polymer), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NOs: 17 to 24. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 17 or 18.

[0134] In some embodiments, the cleavable moiety (CM) further comprises at least a second cleavage site (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable moiety (CM) further comprises a second cleavage site (CS2). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site can be any suitable protease cleavage site recognized and / or cleaved by any of the proteases listed above. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites recognized and / or cleaved by the same protease. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites that are recognized and / or cleaved by different proteases (e.g., the first protease cleavage site is recognized and / or cleaved by uPA and the second protease cleavage site is recognized and / or cleaved by MMP-2; the first protease cleavage site is recognized and / or cleaved by uPA and the second protease cleavage site is recognized and / or cleaved by MMP-9; the first protease cleavage site is recognized and / or cleaved by uPA and the second protease cleavage site is recognized and / or cleaved by TEV protease, etc.). In some embodiments, at least the second cleavage site (CS2) is at the C-terminus of the first linker (L1). In some embodiments, the cleavable moiety (CM) comprises the structure (CS1)-L1-(CS2) from the N-terminus to the C-terminus.

[0135] In some embodiments, the cleavable moiety (CM) further comprises at least a second linker (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable moiety (CM) further comprises a second linker (L2). The second linker (L2) can be any suitable linker described above. In some embodiments, the second linker comprises an amino acid sequence selected from SEQ ID NOs: 17-24. In some embodiments, the first (L1) and second (L2) linkers are the same (e.g., both linkers comprise a sequence of SEQ ID NO: 17 or 18). In some embodiments, the first (L1) and second (L2) linkers are different (e.g., the first linker (L1) comprises an amino acid sequence of SEQ ID NO: 17 and the second linker (L2) comprises an amino acid sequence of SEQ ID NO: 18, etc.). In some embodiments, at least the second linker (L2) is C-terminal to the second cleavage site (CS2). In some embodiments, the cleavable moiety (CM) comprises the structure, from N-terminus to C-terminus, (CS1)-L1-(CS2)-L2.

[0136] Exemplary FP-CM Sequences In some embodiments, a polypeptide of the disclosure comprises, from N-terminus to C-terminus, the structure (FP)-(PCS1)-L1-(PCS2)-L2. In some embodiments, a polypeptide of the disclosure comprises the structure EVGSYDALHYACPPDYYACYYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 25), EVGSYNSYHAYCPHPLYPCTASGRSAGGGGGTENLYFQGSGGS (SEQ ID NO: 26), EVGSYASSAVLCVTAYFSCNSSGRSAGGGGGTENLYFQGSGGS (SEQ ID NO: 27), EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 28), EVGSY NFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 29), EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 30), EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGGTENLYFQGSGGS (SEQ ID NO: 31), EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 32), EVGSYYSAYPACDSHYPYCNSSGRSAGGGGGTENLYF QGSGGS (SEQ ID NO: 33), EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 34), EVGSYPNPSSDCVPYYYACAYSGRSAGGGGGTENLYFQGSGGS (SEQ ID NO: 35), EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 36), EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 37), EVGSYPQPSSDCVPYYY ACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 38), EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 39), EVGSYPTDLDACADAPNHCHFSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 40), EVGSYSSTHAHCHHSPANCISSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 41), EVGSYDTDYDFCPILRHRCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 42),EVGSYNDYNYHCKWRPSRCHNSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 43), EVGSYYHDYDDCRVLPRRCFNSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 44), EVGSYSNNFASCLWRHRSCADSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 45), and / or EVGSYTDNYDYCPRLRRKCYHSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 46). In some embodiments, the polypeptides of the disclosure comprise, from N-terminus to C-terminus, the structure: (FP)-(PCS1)-L1-(PCS2)-L2-(TBM).

[0137] Target binding moiety (TBM) In some embodiments, the disclosure relates to polynucleotides and / or polynucleotide libraries encoding one or more polypeptides comprising a target binding moiety (TBM). In some embodiments, the disclosure relates to polypeptides and / or polypeptide libraries comprising at least one polypeptide comprising a target binding moiety (TBM). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region and an antibody heavy chain variable region. In some embodiments, the antibody heavy chain variable region is C-terminal to the antibody light chain variable region. In some embodiments, the antibody light chain variable region is C-terminal to the antibody heavy chain variable region. In some embodiments, the target binding moiety (TBM) of the present disclosure comprises an antibody light chain variable region and / or an antibody heavy chain variable region with specificity for any target of interest, including, for example, CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, B7-H4.

[0138] In some embodiments, the target binding moiety (TBM) comprises a full-length antibody light chain and a full-length antibody heavy chain. The antibody light chain can be a kappa or lambda light chain. The antibody heavy chain can be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is of IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein can be converted from one class or subclass to another class or subclass using methods known in the art.

[0139] Any one or more of the target binding moieties (TBMs) described herein may comprise HVR sequences (e.g., one, two, or three heavy chain variable region HVR sequences, and / or one, two, or three light chain variable region HVR sequences), as described in PCT Application No. PCT / CN2017 / 098333, which is incorporated by reference in its entirety, PCT Application No. PCT / CN2017 / 098299, which is incorporated by reference in its entirety, PCT Application No. PCT / CN2017 / 098332, which is incorporated by reference in its entirety, and / or the concurrently filed application entitled "Compositions Comprising Cross-reactive Anti-CTLA4 Antibodies, and Methods of Making and Using the Antibodies," filed under Attorney Docket No. 69540-2000540, which is incorporated by reference in its entirety. The antibody may incorporate any of the heavy chain variable region sequences and / or any of the light chain variable region sequences of any of the antibodies described in the PCT application entitled "Same."

[0140] Any one or more of the target binding moieties (TBMs) described herein may incorporate HVR sequences (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the light chain variable region HVR sequences), heavy chain variable region sequences, and / or any of the light chain variable region sequences of any of the antibodies described herein (e.g., anti-CTLA4 antibodies, anti-CD137 antibodies).

[0141] In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CTLA4 antibodies described herein, including the antibodies described with respect to the specific amino acid sequences of the HVRs, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising an HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 62), an HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO: 63), and / or an HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 64). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and / or HVR-H3 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO:61). In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47. In some embodiments, the target binding moiety (TBM) comprises a) an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 62), HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO: 63), and / or HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 64), and b) an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQ ID NO: 59), HVR-H2 comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 60), and / or HVR-H3 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO: 61). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 48, and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47.

[0142] In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CD137 antibodies described herein, including the antibodies described with respect to the specific amino acid sequences of the HVRs, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising an HVR-L1 comprising the amino acid sequence RASQSIGSYLA (SEQ ID NO: 68), an HVR-L2 comprising the amino acid sequence DASNLETGV (SEQ ID NO: 69), and / or an HVR-L3 comprising the amino acid sequence YCQQGYYLWT (SEQ ID NO: 70). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region comprising an HVR-H1 comprising the amino acid sequence FSLSTGGVGVGWI (SEQ ID NO: 65), an HVR-H2 comprising the amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO: 66), and / or an HVR-H3 comprising the amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 67). In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49. In some embodiments, the target binding moiety (TBM) comprises a) an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence RASQSIGSYLA (SEQ ID NO: 68), HVR-L2 comprising the amino acid sequence DASNLETGV (SEQ ID NO: 69), and / or HVR-L3 comprising the amino acid sequence YCQQGYYLWT (SEQ ID NO: 70), and b) an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence FSLSTGGVGVGWI (SEQ ID NO: 65), HVR-H2 comprising the amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO: 66), and / or HVR-H3 comprising the amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 67). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 50, and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49.

[0143] V. Polypeptides and Polypeptide Libraries Other aspects of the disclosure relate to libraries of polypeptides useful for screening, identifying, and / or selecting one or more activatable binding polypeptides (i.e., one or more activatable antibodies), including polynucleotides (e.g., any of the polypeptides described herein) and / or activatable antibodies, activatable antigen-binding fragments thereof, or derivatives of activatable antibodies. The libraries of the disclosure may contain one or more of the polypeptides (e.g., one or more activatable binding polypeptides) described herein. In some embodiments, one or more of the polypeptides (i.e., one, some, or all) of the libraries described herein comprise an antigen-binding domain(s). In some embodiments, one or more of the polypeptides (i.e., one, some, or all) of the libraries described herein comprise a full-length antibody light chain and / or heavy chain(s). In some embodiments, one or more of the polypeptides (i.e., one, some, or all) of the libraries described herein comprise a light chain and / or heavy chain Fab fragment(s). In some embodiments, one or more (i.e., one, some, or all) of the polypeptides of the libraries described herein comprise a single chain variable fragment(s) (scFv). In some embodiments, the polypeptides are expressed on a cell surface (e.g., yeast or mammalian cell display).

[0144] In some embodiments, a polypeptide of the present disclosure (e.g., in a library) comprises (a) a first peptide (FP), (b) a cleavable moiety (CM), and (c) a target binding moiety (TBM). In some embodiments, the FP is any of the first peptides described herein (e.g., those of formula (XIII):XIII). m CX n CX o(SEQ ID NO: 86), where m is 2-10, n is 3-10, o is 1-10, and 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. In some embodiments, the FP is any of the first peptides described herein (e.g., Formula (I): X m CX n CZ o (SEQ ID NO:1), wherein m is 2-10, n is 3-10, o is 1-10, each X is independently an amino acid selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and 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. In some embodiments, m is 3-10. In some embodiments, the CM is any of the cleavable moieties described herein (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site). In some embodiments, the TBM is any of the target binding moieties described herein (e.g., a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region).

[0145] In some embodiments, provided herein is an antigen-binding domain and / or a library comprising antigen-binding domains, wherein at least one (e.g., one, some, or all) of the antigen-binding domains comprises a polypeptide of the present disclosure. In some embodiments, at least one (e.g., one, some, or all) of the antigen-binding domains comprises, from the N-terminus to the C-terminus, (a) a first peptide (FP) (e.g., a polypeptide of formula (XIII): m CX n CX o(a) a first peptide (FP) (e.g., a polypeptide comprising an amino acid sequence according to Formula (I): X (SEQ ID NO: 86), where m is 2-10, n is 3-10, and o is 1-10, and 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; (b) a cleavable portion (e.g., a cleavable portion (CM) comprising at least a first cleavage site); and (c) a target binding portion comprising an antibody light chain variable region. In some embodiments, m is 3-10. In some embodiments, at least one (e.g., one, some, or all) of the antigen-binding domains comprises an amino acid sequence according to Formula (I): X (SEQ ID NO: 86), where m is 2-10, n is 3-10, and o is 1-10, and 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. m CX n CZ o (b) a cleavable portion (e.g., a cleavable portion (CM) comprising at least a first cleavage site); and (c) a target binding portion comprising an antibody light chain variable region. In some embodiments, the antigen-binding domain further comprises an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, at least one of the antigen-binding domains (e.g., one, some, or all) comprises, from the N-terminus to the C-terminus, (a) a first peptide (FP) (e.g., a peptide represented by Formula (XIII): m CX n CX o(a) a first peptide (FP) (e.g., a polypeptide comprising an amino acid sequence according to Formula (I): X (SEQ ID NO: 86), where m is 2-10, n is 3-10, and o is 1-10, and 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; (b) a cleavable portion (e.g., a cleavable portion (CM) comprising at least a first cleavage site); and (c) a target binding portion comprising an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, at least one (e.g., one, some, or all) of the antigen-binding domains spans from the N-terminus to the C-terminus and comprises: (a) a first peptide (FP) (e.g., a polypeptide comprising an amino acid sequence according to Formula (I): X (SEQ ID NO: 86), where m is 2-10, n is 3-10, and o is 1-10, and 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. m CX n CZ o (b) a first peptide (FP) comprising an amino acid sequence according to (SEQ ID NO: 1), where m is 2-10, n is 3-10, and o is 1-10, 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, and each Z is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; (b) a cleavable portion (e.g., a cleavable portion (CM) comprising at least a first cleavage site); and (c) a target binding portion comprising an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, the antigen-binding domain further comprises an antibody light chain variable region.

[0146] Further provided herein in some embodiments is an antibody fragment or scFv comprising any of the polypeptides described herein. In some embodiments, the antibody fragment or scFv comprises a polypeptide comprising a target binding moiety (TBM) comprising an antibody light chain variable region. In some embodiments, the antibody fragment or scFv comprises a polypeptide comprising a target binding moiety (TBM) comprising an antibody heavy chain variable region. In some embodiments, provided herein is a library of antibody fragments or scFvs, at least one of the antibody fragments or scFvs comprises any of the polypeptides described herein. In some embodiments, at least one (e.g., one, some, or all) of the antibody fragments or scFvs comprises a polypeptide comprising a target binding moiety (TBM) comprising an antibody light chain variable region. In some embodiments, at least one (e.g., one, some, or all) of the antibody fragments or scFvs comprises a polypeptide comprising a target binding moiety (TBM) comprising an antibody heavy chain variable region. Further provided herein is a cell and / or library of cells expressing on their surface one or more of the antibody fragments and / or cFvs described herein.

[0147] In some embodiments, the present disclosure relates to an antibody light chain comprising a polypeptide of the present disclosure. In some embodiments, the antibody light chain comprises, from the N-terminus to the C-terminus, (a) a first peptide (FP) (e.g., a polypeptide of formula (XIII): m CX n CX o(SEQ ID NO: 86), where m is 2 to 10 (e.g., 3 to 10), n is 3 to 10, o is 1 to 10, and 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; and (b) a cleavable moiety (e.g., a cleavable moiety (CM) comprising at least a first cleavage site), and a target binding moiety (TBM) comprising an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the antibody light chain comprises a polypeptide comprising, from the N-terminus to the C-terminus, (a) a first peptide (FP) (e.g., a polypeptide according to Formula (I):X m CX n CZ o (b) a polypeptide comprising a cleavable portion (e.g., a cleavable portion (CM) comprising at least a first cleavage site); and a target binding portion (TBM) comprising an antibody light chain variable region. In some embodiments, m is 3 to 10. In some embodiments, the present disclosure relates to a library comprising an antibody light chain, wherein at least one (e.g., one, some, or all) of the antibody light chains in the library is an antibody light chain as described above. In some embodiments, the disclosure relates to libraries comprising an antibody light chain and an antibody heavy chain, wherein the antibody light chain is an antibody light chain as described above. In some embodiments, the antibody heavy chain is any antibody heavy chain known in the art (including any of the antibody heavy chains described herein). In some embodiments, the disclosure relates to libraries comprising antibodies, wherein at least one (e.g., one, some, or all) of the antibodies is an antibody as described above.

[0148] In some embodiments, the present disclosure relates to an antibody heavy chain comprising a polypeptide of the present disclosure. In some embodiments, the antibody heavy chain comprises, from the N-terminus to the C-terminus, (a) a first peptide (FP) (e.g., a polypeptide of formula (XIII): m CX n CX o (SEQ ID NO:86), where m is 2-10, n is 3-10, o is 1-10, and 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; and (b) a cleavable moiety (e.g., a cleavable moiety (CM) comprising at least a first cleavage site), and a target binding moiety (TBM) comprising an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, the antibody heavy chain comprises a polypeptide comprising, from the N-terminus to the C-terminus, (a) a first peptide (FP) (e.g., a polypeptide according to Formula (I):X m CX n CZ o(b) a polypeptide comprising an amino acid sequence according to (SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10, and 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, and each Z is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; and (b) a cleavable portion (e.g., a cleavable portion (CM) comprising at least a first cleavage site), and a target binding portion (TBM) comprising an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, the present disclosure relates to a library comprising antibody heavy chains, wherein at least one (e.g., one, some, or all) of the antibody heavy chains in the library is an antibody heavy chain as described above. In some embodiments, the disclosure relates to libraries comprising an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain is an antibody heavy chain as described above. In some embodiments, the antibody light chain is any antibody light chain known in the art (including any of the antibody light chains described herein). In some embodiments, the disclosure relates to libraries comprising antibodies, wherein at least one (e.g., one, some, or all) of the antibodies is an antibody as described above.

[0149] Polypeptides of the disclosure (e.g., any of the antibodies described above) can be produced using recombinant methods and compositions, such as those described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids encoding any of the polypeptides (e.g., any of the antibodies described above) are provided. Such nucleic acids may be used to encode the V and VD domains of an antibody (e.g., the light and / or heavy chains of the antibody). L and / or V H In some embodiments, provided herein is one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In some embodiments, provided herein is a host cell comprising such a nucleic acid. In one such embodiment, the host cell comprises: (1) a nucleic acid sequence comprising: Land / or V H (2) a vector comprising a nucleic acid encoding an amino acid sequence (e.g., an activatable binding polypeptide (i.e., an activatable antibody) comprising V H and / or V L (3) a vector comprising a nucleic acid encoding an amino acid sequence (e.g., an activatable binding polypeptide (i.e., an activatable antibody) comprising V L A first vector comprising a nucleic acid encoding an amino acid comprising a polypeptide of the present disclosure comprising H or (4) a second vector comprising a nucleic acid encoding an amino acid sequence comprising a polypeptide of the disclosure (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) comprising V H A first vector comprising a nucleic acid encoding an amino acid comprising a polypeptide of the present disclosure comprising L and a second vector comprising (e.g., transformed with) a nucleic acid encoding an amino acid sequence comprising a polypeptide of the present disclosure comprising (e.g., an activatable binding polypeptide (i.e., an activatable antibody)). In some embodiments, the host cell is a eukaryotic cell, such as a yeast cell, an insect cell, a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In some embodiments, a method of making a polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) is provided, the method comprising culturing an anther host cell comprising a nucleic acid encoding the polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) under conditions suitable for expression of the polypeptide, and, optionally, recovering the polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) from the host cell (or host cell culture medium).

[0150] For recombinant production of a polypeptide of the disclosure (e.g., an activatable binding polypeptide (i.e., an activatable antibody)), a nucleic acid encoding a polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody) as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to the gene encoding the polypeptide(s)).

[0151] Suitable host cells for cloning or expressing a polypeptide-encoding (e.g., activatable binding polypeptide (i.e., activatable antibody)-encoding vector include prokaryotic or eukaryotic cells. For example, a polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required (see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction or may be further purified.

[0152] In addition to prokaryotic microbes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding (e.g., activatable binding polypeptide (i.e., activatable antibody)-encoding) vectors, including fungal or yeast strains whose glycosylation pathways have been "humanized," resulting in the production of polypeptides having partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0153] Suitable host cells for the expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0154] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0155] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney cell lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals TRI cells, MRC5 cells, and FS4 cells described in NYAcad.Sci.383:44-68(1982). Other useful mammalian host cells include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc.Natl.Acad.Sci.USA 77:4216(1980)), and myeloma cells such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (BKC Lo,ed.,Humana Press,Totowa,NJ),pp.255-268(2003).

[0156] VI. ACTIVATABLE BINDING PEPTIDES AND THEIR PRODUCTION In some embodiments, provided herein are activatable binding polypeptides (e.g., activatable antibodies) screened, identified, and / or selected from any of the polynucleotide and / or polypeptide libraries described herein.

[0157] In some embodiments, the activatable antibodies of the present disclosure are context-dependent (e.g., are activated (only able to bind to their target) in certain contexts (such as a protease-rich tumor microenvironment). In some embodiments, the activatable antibodies of the present disclosure offer improved safety over more traditional non-activatable antibodies (e.g., exhibit reduced toxicity, do not induce significant changes in the weight of many organs, do not alter liver histopathology, hematology, and / or blood biochemistry, etc.). In some embodiments, the activatable antibodies of the present disclosure have improved pharmacokinetic properties (e.g., longer in vivo half-life) compared to more traditional non-activatable antibodies.

[0158] In some embodiments, an activatable binding polypeptide of the disclosure comprises (a) a first peptide (FP) (e.g., a masking moiety), (b) a cleavable moiety, and (c) a target binding moiety. In some embodiments, the first peptide (FP) binds to the target binding moiety (TBM) of the activatable binding domain and reduces or inhibits binding of the activatable binding moiety to its target (e.g., human CTLA4 or human CD137) compared to binding of a corresponding binding polypeptide lacking the masking moiety and / or compared to binding of a parent antibody. In some embodiments, the masking moiety (MM) has a masking efficiency of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.) prior to activation. In some embodiments, masking efficiency is measured by the difference in affinity of an activatable antibody comprising a masking moiety (before activation) for binding to its target compared to the affinity of a polypeptide lacking the masking moiety (MM) for binding to its target (e.g., the difference in affinity of an activatable antibody comprising a masking moiety (MM) (before activation) for a target antigen (such as CTLA4 or CD137) compared to a parent antibody lacking the masking moiety (MM), or the difference in affinity of an activatable antibody comprising a masking moiety (MM) (before activation) for a target antigen (such as CTLA4 or CD137) compared to the affinity of the activatable antibody after activation). In some embodiments, masking efficiency is measured by the EC of binding of an activatable antibody comprising a masking moiety (MM) (before activation). 50 , EC of the parent antibody 50 (e.g., EC by ELISA) 50(see, e.g., methods in Example 3). In some embodiments, masking efficiency is measured by the difference in affinity of an activatable antibody comprising a masking moiety (MM) for binding to its target before activation compared to the affinity of an activatable antibody comprising a masking moiety (MM) for binding to its target after activation (e.g., the difference in affinity of an activatable antibody for a target antigen before activation compared to the affinity of the activatable antibody after activation). In some embodiments, the masking moiety (MM) binds to a target binding moiety (TBM) and prevents the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody).

[0159] In some embodiments, an "activatable" binding polypeptide refers to a binding polypeptide that exhibits a first level of binding to a target when in an inhibited, masked, and / or uncleaved state, and exhibits a second level of binding to a target when in an uninhibited, unmasked, and / or cleaved state, the second level of target binding being greater than the first level of target binding. In some embodiments, access to a target by an activatable binding polypeptide is increased following cleavage within the cleavable moiety (e.g., by one or more proteases).

[0160] In some embodiments, the polypeptides of the disclosure generally exhibit at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 20-fold, at least about 25-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 40-fold, at least about 50-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 5 ... In some embodiments, a polypeptide of the present disclosure is generally considered to be an "activatable" binding polypeptide if the EC of the polypeptide is increased by at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold or more. 50 is reduced by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold or more) following "activation" (e.g., as measured by ELISA or FACS assay; see Examples below). In some embodiments, a polypeptide of the present disclosure is generally 50is considered "activatable" if it is decreased by at least about two-fold following treatment with a protease that cleaves within the cleavable moiety (e.g., as measured by ELISA or FACS assay; see Examples below).

[0161] In some embodiments, when a masking moiety is bound to a target binding portion of an activatable binding polypeptide, the K D is greater than when the masking moiety is not attached to the target binding moiety (e.g., after "activation" of the activatable binding polypeptide (e.g., after protease treatment to cleave within the cleavable moiety)) and / or the K D about 2-fold or more (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) greater than. Methods of measuring affinity are known in the art and include, for example, the methods described in Example 3 below.

[0162] In some embodiments, when a masking moiety is attached to a target binding portion of an activatable binding polypeptide, the K D is compared to when the masking moiety is not bound to the target binding moiety (e.g., after "activation" of the activatable binding polypeptide (e.g., after treatment with a protease that cleaves within the cleavable moiety)) and / or the K D is decreased by at least about 25% (e.g., at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%) compared to. Methods of measuring affinity are known in the art and include, for example, the methods described in Example 3 below.

[0163] In some embodiments, the masking moiety sterically masks the binding of the activatable binding polypeptide to its target and / or allosterically masks the binding of the activatable binding polypeptide to its target, hi some embodiments, the masking moiety does not comprise the amino acid sequence of the natural binding partner of the activatable binding polypeptide.

[0164] In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is greater than the dissociation constant of the activatable binding polypeptide for its target (when active, activated). In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is greater than about 2-fold or more (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000-fold or more) than the dissociation constant of the activatable binding polypeptide for its target (when activated). In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is approximately equal to the dissociation constant of the activatable binding polypeptide for its target (when activated). In some embodiments, the first peptide (FP) binds to the target binding moiety (TBM) and prevents the polypeptide from binding to its target only when the polypeptide is activated (e.g., activated by treatment with one or more proteases that cleave within the cleavable moiety (CM), activated by a change in pH (increasing or decreasing), activated by a change in temperature (increasing or decreasing), activated after contact with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces a conformational change in the polypeptide (e.g., dislocation of the first peptide (FP)) such that the first peptide no longer prevents the polypeptide from binding to its target.

[0165] The activatable binding polypeptides (i.e., activatable antibodies) described herein may be further modified. In some embodiments, the activatable binding polypeptides are linked to additional molecular entities. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or labels, and antibodies.

[0166] In some embodiments, the activatable binding polypeptides of the present disclosure are linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, corticine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclosporine, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include iodine. 131 ,indium 111 ,yttrium 90 , and lutetium 177Methods of linking a polypeptide to a pharmaceutical agent are known in the art, including, but not limited to, using a variety of linker technologies. Exemplary linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. For further discussion of linkers and methods for linking therapeutic agents to antibodies, see, e.g., Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail, et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0167] Activatable binding polypeptides targeting CTLA4 - Patent application In some embodiments, the present disclosure relates to activatable binding polypeptides (i.e., activatable antibodies) that bind to human CTLA4, including activatable anti-CTLA4 antibodies, antigen-binding fragments of activatable anti-CTLA4 antibodies, and / or derivatives of activatable anti-CTLA4 antibodies. In some embodiments, an activatable antibody is a polypeptide that includes, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is represented by the formula (I):X: m CX n CZ o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10; 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; 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; the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and the TBM comprises a polypeptide comprising an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH). In some embodiments, m is 3-10. In some embodiments, the MM inhibits binding of the activatable antibody to human CTLA4 if the CM is uncleaved. In some embodiments, the activatable antibody is capable of binding to human CTLA4 if the CM is cleaved. In some embodiments, the MM comprises an amino acid sequence selected from SEQ ID NOs: 72-78, as listed in Table A. TIFF2025037964000001.tif58170

[0168] In some embodiments, the activatable binding polypeptide comprises any of the anti-CTLA4 antibodies described herein, including those antibodies described with respect to the HVRs, variable regions (VL, VH), and / or specific amino acid sequences of the light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the anti-CTLA4 antibody is a human antibody. In some embodiments, the anti-CTLA4 antibody is a humanized antibody and / or a chimeric antibody.

[0169] In some embodiments, the activatable binding polypeptide comprises a) an HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQ ID NO:59), an HVR-H2 comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and an HVR-H3 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO:61), and / or b) an HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO:62), an HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO:63), and an HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO:64). In some embodiments, the activatable binding polypeptide comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47, and / or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:48.

[0170] In some embodiments, the disclosure provides activatable binding polypeptides that, when in an active form, bind to human CTLA4 (e.g., an activatable binding polypeptide is active after cleavage (e.g., by one or more proteases) at the cleavable moiety but is inactive prior to cleavage (e.g., by one or more proteases) at the cleavable moiety) and have the following functional properties: (a) a K of 500 nM or less for human, cynomolgus monkey, mouse, rat, and / or dog CTLA4; D(b) has antagonist activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, and / or B7-H4 at concentrations up to 100 nM; (d) is cross-reactive with monkey, mouse, rat, and / or dog CTLA4; (e) induces an ADCC effect (e.g., against Tregs); (f) activates human PBMCs ( For example, activatable binding polypeptides that have at least one of the following properties (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine): (i) stimulate secretion of IL-2 and / or IFNγ, (ii) are capable of inhibiting tumor cell proliferation, (iii) have a therapeutic effect against cancer, and (iv) inhibit binding of human CTLA4 to human CD80 and / or human CD86. Also provided herein is one or more activatable binding polypeptides that cross-compete with one or more of the CTLA4-targeted activatable binding polypeptides and / or anti-CTLA4 antibodies described herein for binding to human CTLA4.

[0171] In some embodiments, the activatable binding polypeptide, when in an inactive form, has a K of about 500 nM or greater for human, cynomolgus monkey, mouse, rat, and / or dog CTLA4. D In some embodiments, the activatable binding polypeptide, when in its active form, binds to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4 with a K of about 500 nM or less (e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). DIn some embodiments, the activatable binding polypeptide, when in an activated form, binds to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4 with a K of about 350 nM or less. D In some embodiments, the activatable binding polypeptide, when in an activated form, binds to human CTLA4 with a K of about 100 nM or less. D In some embodiments, the activatable binding polypeptide, when in an activated form, binds to human CTLA4 with a K of about 50 nM or less. D In some embodiments, the activatable binding polypeptide, when in an activated form, binds to human CTLA4 with a K of about 10 nM or less. D The K of the activatable binding polypeptide D Methods for measuring K can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc. In some embodiments, K D is measured by ELISA (see, for example, Example 3 below).

[0172] In some embodiments, the activatable binding polypeptide, when in an inactive form, has no antagonist activity against human CTLA4. In some embodiments, the activatable binding polypeptide, when in an active form, has antagonist activity against human CTLA4 (e.g., induces an ADCC effect (e.g., against Tregs), activates PBMCs (e.g., by activating, inducing, and / or stimulating IL-2 and / or IFNγ secretion), blocks binding of human CTLA4 to human CD80 and / or human CD86, etc.). In some embodiments, the activatable binding polypeptide, when in an active form, inhibits one or more activities of human CTLA4 (e.g., inhibits one or more activities of human CTLA4 when a cell (e.g., a human cell) expressing human CTLA4 is contacted by an activated activatable binding polypeptide).

[0173] In some embodiments, the activatable binding polypeptide, when in an inactive form, is not cross-reactive with monkey (e.g., cynomolgus), mouse, rat, and / or dog CTLA4. In some embodiments, the activatable binding polypeptide, when in an active form, is cross-reactive with monkey (e.g., cynomolgus), mouse, rat, and / or dog CTLA4. In some embodiments, the activatable binding polypeptide, when in an active form, is cross-reactive with monkey CTLA4. In some embodiments, the activatable binding polypeptide, when in an active form, is cross-reactive with mouse CTLA4. In some embodiments, the activatable binding polypeptide, when in an active form, is cross-reactive with rat CTLA4. In some embodiments, the activatable binding polypeptide, when in an active form, is cross-reactive with dog CTLA4. In some embodiments, the activatable binding polypeptide, when in its active form, is cross-reactive with monkey and mouse CTLA4; monkey and rat CTLA4; monkey and dog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4; monkey, mouse, and rat CTLA4; monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4. In some embodiments, the activatable binding polypeptide, when in its active form, is cross-reactive at about 350 nM (e.g., about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM). Methods for measuring cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like.

[0174] In some embodiments, the activatable binding polypeptide, when in an inactive form, does not induce an ADCC effect (e.g., on human cells such as Tregs). In some embodiments, the activatable binding polypeptide, when in an inactive form, has a reduced ADCC effect (e.g., on human cells such as Tregs) compared to a control binding polypeptide (e.g., a parent antibody lacking the first peptide (FP) and the cleavable moiety (CM). In some embodiments, the activatable antibody, when in an activated form, induces an ADCC effect (e.g., on human cells such as Tregs). Methods (e.g., in vitro methods) for measuring an ADCC effect are known in the art, including, but not limited to, by the methods described in Example 4 below. In some embodiments, the activatable binding polypeptide, when in an inactive form, induces less than about 10% of the ADCC effect (e.g., less than about 10%, less than about 5%, less than about 1%, etc.) compared to a control (e.g., a parent antibody lacking the first peptide (FP) and the cleavable moiety (CM). In some embodiments, the activatable binding polypeptide, when in an active form, induces an ADCC effect of greater than about 10% compared to a control (e.g., an isotype control) (e.g., induces an ADCC effect of greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, etc.).

[0175] In some embodiments, the activatable binding polypeptide is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, tumor cell growth and / or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with an activatable binding polypeptide compared to corresponding tumor cells not contacted with the activatable binding polypeptide (or compared to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, the activatable binding polypeptide is capable of reducing tumor volume in a subject when the activatable binding polypeptide is administered to the subject. In some embodiments, the activatable binding polypeptide is capable of reducing tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) compared to the initial tumor volume in the subject (e.g., compared to a corresponding tumor in a subject administered an isotype control antibody prior to administration of the activatable binding polypeptide). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art, including, for example, by the methods described in Example 4 below.

[0176] In some embodiments, the activatable binding polypeptide has a therapeutic effect against cancer. In some embodiments, the activatable binding polypeptide alleviates one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer goes into partial or complete remission when administered the activatable binding polypeptide.

[0177] In some embodiments, the disclosure provides isolated activatable binding polypeptides that, when in an activated form, compete or cross-compete for binding to human CTLA4 with an antibody comprising a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:59, HVR-H2 comprising the amino acid sequence of SEQ ID NO:60, HVR-H3 comprising the amino acid sequence of SEQ ID NO:61, and / or b) HVR-L1 comprising the amino acid sequence of SEQ ID NO:62, HVR-L2 comprising the amino acid sequence of SEQ ID NO:63, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the disclosure provides isolated activatable binding polypeptides that, when in an activated form, compete or cross-compete for binding to human CTLA4 with an antibody comprising a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47, and / or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:48. The ability of an activatable binding polypeptide to compete or cross-compete for binding to an antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, one can allow an antibody (e.g., as described above) to bind to human CTLA4 under saturating conditions, and then measure the ability of a test activatable binding polypeptide (when in an activated form) to bind to CTLA4. If the test activatable binding polypeptide can bind to CTLA4 at the same time as the antibody, the test activatable binding polypeptide binds to a different epitope than the antibody. However, if the test activatable binding polypeptide cannot bind to CTLA4 at the same time, the test activatable binding polypeptide binds to the same epitope, an overlapping epitope, or an epitope that is very close to the epitope bound by the antibody. This experiment can be carried out using a variety of methods, such as ELISA, RIA, FACS, or surface plasmon resonance.

[0178] In some embodiments, the activatable binding polypeptide (when in an inactive form) does not inhibit binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the activatable binding polypeptide (when in an active form) inhibits binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the activatable binding polypeptide inhibits binding between CTLA4 and its ligand in vitro. In some embodiments, the activatable binding polypeptide has a half maximal inhibitory concentration (IC) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for inhibiting binding of CTLA4 to CD80 and / or CD86. 50 In some embodiments, the activatable binding polypeptide has a half maximal inhibitory concentration (IC) of about 100 nM or less for inhibiting binding of CTLA4 to CD80 and / or CD86. 50 ). In some embodiments, the activatable binding polypeptide completely inhibits binding of human CTLA4 to CD80 and / or CD86 when provided at a concentration of about 100 nM or more (e.g., about 100 nM or more, about 500 nM or more, about 1 μM or more, about 10 μM or more, etc.). As used herein, the term "complete inhibition" or "completely inhibits" refers to the ability of an activatable binding polypeptide to reduce binding between a first protein and a second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods for measuring the ability of a polypeptide to inhibit binding of a first protein (e.g., human CTLA4) and a second protein (e.g., human CD80 or human CD86) are known in the art and include, but are not limited to, by BIAcore analysis, ELISA assays, and flow cytometry.

[0179] Activatable binding polypeptides targeting CD137 In some embodiments, the present disclosure relates to activatable binding polypeptides (i.e., activatable antibodies) that bind to human CD137, including activatable anti-CD137 antibodies, antigen-binding fragments of activatable anti-CD137 antibodies, and / or derivatives of activatable anti-CD137 antibodies. In some embodiments, an activatable antibody is a polypeptide that comprises, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is represented by the formula (I):X: m CX n CZ o (SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10; 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; and 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; the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH). In some embodiments, the activatable antibody is a polypeptide comprising, from N-terminus to C-terminus, (a) a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is represented by the formula (I): X: m CX n CZ o(SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10; 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; and 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; the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and the TBM comprises a polypeptide comprising an antibody light chain variable region (VH); and (b) an antibody heavy chain variable region (VL). In some embodiments, the activatable antibody is a polypeptide comprising, from N-terminus to C-terminus, (a) a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is represented by the formula (I): X m CX n CZ o (SEQ ID NO:1), wherein m is 2-10, n is 3-10, and o is 1-10; 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; 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; the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and the TBM comprises a polypeptide comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0180] In some embodiments, the MM inhibits binding of an activatable antibody to human CD137 when the CM is not cleaved. In some embodiments, the activatable antibody is capable of binding to human CD137 when the CM is cleaved. In some embodiments, the MM comprises an amino acid sequence selected from SEQ ID NOs: 79-85 and 88-94, as listed in Table B. TIFF2025037964000002.tif119170

[0181] In some embodiments, the activatable binding polypeptide comprises any of the anti-CD137 antibodies described herein, including those antibodies described with respect to the HVRs, variable regions (VL, VH), and / or specific amino acid sequences of the light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the anti-CD137 antibody is a human antibody. In some embodiments, the anti-CD137 antibody is a humanized antibody and / or a chimeric antibody.

[0182] In some embodiments, the activatable binding polypeptide comprises a) an HVR-H1 comprising the amino acid sequence FSLSTGGVGVGWI (SEQ ID NO:65), an HVR-H2 comprising the amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and an HVR-H3 comprising the amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO:67), and / or b) an HVR-L1 comprising the amino acid sequence RASQSIGSYLA (SEQ ID NO:68), an HVR-L2 comprising the amino acid sequence DASNLETGV (SEQ ID NO:69), and an HVR-L3 comprising the amino acid sequence YCQQGYYLWT (SEQ ID NO:70). In some embodiments, the activatable binding polypeptide comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:49 or a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequence of SEQ ID NO:49; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:50 or a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequence of SEQ ID NO:50.

[0183] In some embodiments, the disclosure provides activatable binding polypeptides that, when in an activated form, bind to human CD137 (e.g., an activatable binding polypeptide is active after cleavage at the cleavable moiety (e.g., by one or more proteases) but is inactive prior to cleavage at the cleavable moiety (e.g., by one or more proteases)) and have the following functional properties: (a) a K DThe present invention relates to activatable binding polypeptides having at least one of the following properties (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight): (a) binds to human CD137 at 1000 nM, (b) has antagonist activity against human CD137, (c) does not bind to human OX40, CD40, GITR, and / or CD27 receptors at concentrations up to 1000 nM, (d) is cross-reactive with monkey, mouse, rat, and / or dog CD137, (e) does not induce ADCC effects, (f) can inhibit tumor cell proliferation, (g) has a therapeutic effect against cancer, and (h) inhibits binding between CD137 and CD137L. In some embodiments, the activatable binding polypeptides disclosed herein can also inhibit, e.g., completely inhibit, the binding between CD137 and its ligand, CD137L. Also provided herein are one or more activatable binding polypeptide anti-CD137 antibodies or antigen-binding fragments that cross-compete with one or more of the CD137-targeted activatable binding polypeptide and / or anti-CD137 antibodies described herein for binding to human CD137.

[0184] In some embodiments, the activatable binding polypeptide (when in an inactive form) has a K D In some embodiments, the activatable binding polypeptide (when in its active form) binds to human CD137 with a K of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). D In some embodiments, the activatable binding polypeptide binds to human CD137 with a K of about 100 nM or less. D In some embodiments, the activatable binding polypeptide binds to human CD137 with a K of about 50 nM or less. DThe K of the activatable binding polypeptide D Methods for measuring K can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc. In some embodiments, K D is measured by ELISA (see, for example, Example 5 below).

[0185] In some embodiments, the activatable binding polypeptides described herein (when in an activated form) have agonistic activity against human CD137. In some embodiments, the activatable binding polypeptide induces one or more (e.g., one or more, two or more, three or more, etc.) activities of human CD137 when a cell expressing human CD137 (e.g., a human cell) is contacted by the (active) activatable binding polypeptide. Various CD137 activities are known in the art, which may include, but are not limited to, induction of NF-κB-dependent transcription, induction of T cell proliferation, prolongation of T cell viability, costimulation of activated T cells, induction of cytokine secretion (e.g., IL-2), and induction of monocyte activation. In some embodiments, the one or more activities of CD137 is not binding of CD137 to its ligand. Methods for measuring CD137 activities (e.g., induction of NF-κB-dependent transcription and / or T cell proliferation) are known in the art. In some embodiments, the activatable binding polypeptide increases NF-κB-dependent transcription in cells (e.g., human cells) expressing human CD137. In some embodiments, NF-κB-dependent transcription is increased by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 99% or more in cells (e.g., human cells) expressing human CD137 that have been contacted with an (active) activatable binding polypeptide compared to corresponding cells that have not been contacted with the activatable binding polypeptide (e.g., corresponding cells contacted with an isotype control antibody) or corresponding cells that have been contacted with the activatable binding polypeptide when in an inactive form. In some embodiments, NF-κB-dependent transcription is increased by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold or more in cells (e.g., human cells) expressing human CD137 that have been contacted with an activatable binding polypeptide compared to corresponding cells that have not been contacted with the activatable binding polypeptide (e.g., corresponding cells contacted with an isotype control antibody) or corresponding cells that have been contacted with the activatable binding polypeptide when in an inactive form.

[0186] In some embodiments, the activatable binding polypeptide (when in an inactive form) is not cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CD137. In some embodiments, the activatable binding polypeptide (when in an active form) is cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CD137. In some embodiments, the activatable binding polypeptide is cross-reactive with monkey CD137. In some embodiments, the activatable binding polypeptide is cross-reactive with mouse CD137. In some embodiments, the activatable binding polypeptide is cross-reactive with rat CD137. In some embodiments, the activatable binding polypeptide is cross-reactive with dog CD137. In some embodiments, the activatable binding polypeptide is cross-reactive with monkey and mouse CD137; monkey and rat CD137; monkey and dog CD137; mouse and rat CD137; mouse and dog CD137; rat and dog CD137; monkey, mouse, and rat CD137; monkey, rat, and dog CD137; mouse, rat, and dog CD137; or monkey, mouse, rat, and dog CD137. In some embodiments, the activatable binding polypeptide is cross-reactive at about 100 nM (e.g., about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM). Methods for measuring cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, and the like.

[0187] In some embodiments, the activatable binding polypeptide does not induce ADCC effect. Methods for measuring ADCC effect are known in the art. In some embodiments, the activatable binding polypeptide (when activated) does not induce ADCC by more than about 10% compared to the control (does not induce ADCC by more than about 10%, more than about 5%, more than about 1%, more than about 0.1%, more than about 0.01%).

[0188] In some embodiments, the activatable binding polypeptide is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, tumor cell growth and / or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with an activatable binding polypeptide compared to corresponding tumor cells not contacted with the activatable binding polypeptide (or compared to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, the activatable binding polypeptide is capable of reducing tumor volume in a subject when the activatable binding polypeptide is administered to the subject. In some embodiments, the activatable binding polypeptide is capable of reducing tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) as compared to the initial tumor volume in the subject (e.g., as compared to a corresponding tumor in a subject administered an isotype control antibody prior to administration of the activatable binding polypeptide). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art.

[0189] In some embodiments, the activatable binding polypeptide has a therapeutic effect against cancer. In some embodiments, the activatable binding polypeptide alleviates one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer goes into partial or complete remission when administered the activatable binding polypeptide.

[0190] In some embodiments, the disclosure provides isolated activatable binding polypeptides that, when in an activated form, compete or cross-compete for binding to human CD137 with an antibody comprising a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 65, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 66, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 67, and / or b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the disclosure provides isolated activatable binding polypeptides that, when in an activated form, compete or cross-compete for binding to human CD137 with an antibody comprising a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and / or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50. The ability of an activatable binding polypeptide to compete or cross-compete for binding to an antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, one can allow an antibody (e.g., as described above) to bind to human CD137 under saturating conditions, and then measure the ability of a test activatable binding polypeptide (when in an activated form) to bind to CD137. If the test activatable binding polypeptide can bind to CD137 at the same time as the antibody, the test activatable binding polypeptide binds to a different epitope than the antibody. However, if the test activatable binding polypeptide cannot bind to CD137 at the same time, the test activatable binding polypeptide binds to the same epitope, an overlapping epitope, or an epitope that is very close to the epitope bound by the antibody. This experiment can be carried out using various methods, such as ELISA, RIA, FACS, or surface plasmon resonance.

[0191] In some embodiments, the activatable binding polypeptide (when in inactive form) does not inhibit binding between CD137 and its ligand (e.g., human CD137 and human CD137L). In some embodiments, the activatable binding polypeptide (when active form) inhibits binding between CD137 and its ligand (e.g., human CD137 and human CD137L). In some embodiments, the activatable binding polypeptide inhibits binding between CD137 and its ligand in vitro. In some embodiments, the activatable binding polypeptide (when active form) has a half maximal inhibitory concentration (IC) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for inhibiting binding of CD137 to its ligand. 50 In some embodiments, the activatable binding polypeptide has a half maximal inhibitory concentration (IC) of about 100 nM or less for inhibiting binding of CD137 to its ligand. 50 ). In some embodiments, the activatable binding polypeptide completely inhibits binding of human CD137 to its ligand when provided at a concentration of about 100 nM or more (e.g., about 100 nM or more, about 500 nM or more, about 1 μM or more, about 10 μM or more, etc.). Methods for measuring the ability of a polypeptide to inhibit binding of a first protein (e.g., CD137) and a second protein (e.g., CD137L) are known in the art and include, but are not limited to, by BIAcore analysis, ELISA assays, and flow cytometry.

[0192] antibody In some embodiments, the disclosure relates to activatable binding polypeptides comprising an antibody described herein (e.g., a CTLA4 or CD137 antibody described above). The antibody described herein (e.g., a CTLA4 or CD137 antibody) can be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody described herein (e.g., a CTLA4 or CD137 antibody) is of the IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass. The antibody described herein (e.g., a CTLA4 or CD137 antibody) can be converted from one class or subclass to another class or subclass using methods known in the art. An exemplary method for producing an antibody of a desired class or subclass includes isolating nucleic acid encoding a heavy chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody) and nucleic acid encoding a light chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody), and isolating the nucleic acid encoding the V-chain of the antibody. H The coding sequence for the region was isolated and H The steps include linking the sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in a cell, and recovering the antibody.

[0193] antigen binding fragment In some embodiments, the disclosure relates to activatable binding polypeptides that include an antigen-binding fragment (eg, an antigen-binding fragment of an antibody (eg, a CTLA4 or CD137 antibody) described herein).

[0194] The antigen-binding fragment may comprise any sequence of any of the antibodies described herein. In some embodiments, the antigen-binding fragment comprises the amino acid sequence of (1) a light chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody), (2) a heavy chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody), (3) a variable region from a light chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody), (4) a variable region from a heavy chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody), (5) one or more HVRs (e.g., one, two, three, four, five, or six HVRs) of an antibody described herein (e.g., a CTLA4 or CD137 antibody), or (6) three HVRs from the light chain and three HVRs from the heavy chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody).

[0195] In some embodiments, the disclosure provides an antigen-binding fragment of an antibody (that binds human CTLA4) comprising a heavy chain variable region comprising HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and HVR-H3 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO:61), and / or a light chain variable region comprising HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO:62), HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO:63), and HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO:64). In some embodiments, the disclosure provides an antigen-binding fragment of an antibody comprising a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47, and / or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:48.

[0196] In some embodiments, the disclosure provides an antigen-binding fragment of an antibody (that binds human CD137) comprising a heavy chain variable region comprising HVR-H1 comprising the amino acid sequence FSLSTGGVGVGWI (SEQ ID NO:65), HVR-H2 comprising the amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and HVR-H3 comprising the amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO:67), and / or a light chain variable region comprising HVR-L1 comprising the amino acid sequence RASQSIGSYLA (SEQ ID NO:68), HVR-L2 comprising the amino acid sequence DASNLETGV (SEQ ID NO:69), and HVR-L3 comprising the amino acid sequence YCQQGYYLWT (SEQ ID NO:70). In some embodiments, the disclosure provides an antigen-binding fragment of an antibody comprising a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:49, and / or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:50.

[0197] In some embodiments, an antigen-binding fragment of an antibody (e.g., a CTLA4 or CD137 antibody) described herein comprises: (i) V L , V H , C L , and C H (ii) a Fab fragment, which is a monovalent fragment consisting of one domain; (iii) a F(ab′)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iv) a V H and C H (iv) a V of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a V H (vi) isolated CDRs, and (vii) antibody V domains (Ward et al., (1989) Nature 341:544-546). H V of antibody linked to domain L and single chain antibodies (scFv), which are polypeptides comprising a region (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0198] antibody derivative In some embodiments, the disclosure provides activatable binding polypeptides that comprise derivatives of the antibodies described herein (eg, CTLA4 or CD137 antibodies).

[0199] In some embodiments, antibody derivatives are derived from the amino acid sequence of a parent antibody by modification, preserving the overall molecular structure of the amino acid sequence of the parent antibody. The amino acid sequence of any region of the parent antibody chain can be modified, such as the framework region, the HVR region, or the constant region. The types of modifications include substitution, insertion, deletion, or combinations of one or more amino acids of the parent antibody.

[0200] In some embodiments, the antibody derivative has a V sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence set forth in any of SEQ ID NOs: 47-50. L or V HIn some embodiments, the antibody derivative comprises an HVR-H1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in either SEQ ID NO:59 or 65. In some embodiments, the antibody derivative comprises an HVR-H2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in either SEQ ID NO:60 or 66. In some embodiments, the antibody derivative comprises an HVR-H3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in either SEQ ID NO: 61 or 67. In some embodiments, the antibody derivative comprises an HVR-L1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in either SEQ ID NO: 62 or 68. In some embodiments, the antibody derivative comprises an HVR-L2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in either SEQ ID NO:63 or 69.In some embodiments, the antibody derivative comprises an HVR-L3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in either SEQ ID NO:64 or 70.

[0201] In some particular embodiments, the derivatives include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions to the amino acid sequence of an antibody described herein (e.g., a CTLA4 or CD137 antibody).

[0202] Amino acid substitution includes both conservative and non-conservative substitution. The term "conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid when the two amino acids have similarities in certain physicochemical properties such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the related residues. For example, typically, substitutions can be made within each of the following groups: (a) non-polar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids such as aspartic acid and glutamic acid.

[0203] Modifications may be made anywhere in the amino acid sequence of the antibody, including in the HVRs, framework regions, or constant regions. In one embodiment, the present disclosure provides a method for the preparation of a VVR of an antibody (e.g., a CTLA4 or CD137 antibody) described herein. H and V LThe present invention provides antibody derivatives that contain HVR sequences of the present disclosure, but contain framework sequences that differ from those of the exemplary antibodies. Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" human germline sequence database (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson et al., J. Mol. Biol. 227:776-798 (1992); and Cox et al., Eur J. Immunol. 24:827-836 (1994)). Framework sequences that can be used in the construction of antibody derivatives include those that are structurally similar to the framework sequences used by the exemplary antibodies of the present disclosure. For example, the HVR-H1, HVR-H2, and HVR-H3 sequences, and the HVR-L1, HVR-L2, and HVR-L3 sequences of an exemplary antibody can be grafted into framework regions having sequences identical to those found in the germline immunoglobulin gene from which the framework sequences are derived, or the HVR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequences.

[0204] In some embodiments, the antibody derivative is a chimeric antibody that comprises the amino acid sequence of an exemplary antibody (e.g., a CTLA4 or CD137 antibody) described herein. In one example, one or more HVRs from one or more exemplary antibodies are combined with an HVR from an antibody from a non-human animal, such as a mouse or rat. In another example, all HVRs of the chimeric antibody are derived from one or more exemplary antibodies. In some specific embodiments, the chimeric antibody comprises one, two, or three HVRs from the heavy chain variable region and / or one, two, or three HVRs from the light chain variable region of an exemplary antibody. Chimeric antibodies can be generated using conventional methods known in the art.

[0205] Another type of modification is the V H and / or V L The preferred method is to mutate amino acid residues in the HVR regions of the heavy chain. Site-directed mutagenesis or PCR-mediated mutagenesis can be used to introduce the mutation(s) and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutations can be amino acid additions and / or deletions. Furthermore, typically no more than one, two, three, four, or five residues in the HVR regions are altered. In some embodiments, the antibody derivative comprises one, two, three, or four amino acid substitutions in the heavy chain HVR and / or light chain HVR. In another embodiment, the amino acid substitution is to change one or more cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. The cysteines can be standard or non-standard cysteines. In one embodiment, the antibody derivative has one, two, three, or four conservative amino acid substitutions in the heavy chain HVR regions compared to the amino acid sequence of the exemplary antibody.

[0206] Also, V H and / or V LModifications can also be made to framework residues within the region. Typically, such framework variants are made to reduce the immunogenicity of antibodies. One approach is to "back-mutate" one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic mutations may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "back-mutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis.

[0207] In addition, modifications can also be made within the Fc region of an exemplary antibody, typically altering one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified to change, e.g., increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Pat. No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another case, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody.

[0208] Additionally, the antibodies of the present disclosure may be modified to alter their potential glycosylation sites or patterns, according to routine experimentation known in the art. In another aspect, the present disclosure provides derivatives of the antibodies described herein (e.g., CTLA4 or CD137 antibodies) that contain at least one mutation in the variable region of the light or heavy chain that alters the pattern of glycosylation in the variable region. Such antibody derivatives may have increased affinity and / or altered specificity for binding the antigen. The mutations may add new glycosylation sites to the V region, change the position of one or more V region glycosylation site(s), or remove existing V region glycosylation sites. In one embodiment, the present disclosure provides derivatives of the antibodies described herein (e.g., CTLA4 or CD137 antibodies) that have a potential N-linked glycosylation site at an asparagine in the heavy chain variable region, such that one potential N-linked glycosylation site in the heavy chain variable region is removed. In another embodiment, the present disclosure provides derivatives of the antibodies described herein (e.g., CTLA4 or CD137 antibodies) that have potential N-linked glycosylation sites at asparagines in the heavy chain variable regions, such that potential N-linked glycosylation sites in both heavy chain variable regions are eliminated. Methods for altering the glycosylation pattern of antibodies are known in the art, such as those described in U.S. Patent No. 6,933,368, the disclosure of which is incorporated herein by reference.

[0209] Other examples of antibody derivatives provided by the present disclosure include single chain antibodies, diabodies, domain antibodies, nanobodies, and unibodies. A "single chain antibody" (scFv) is a V H V linked to domain L It consists of a single polypeptide chain containing the V L Domain and V HThe domains pair to form a monovalent molecule. Single-chain antibodies can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). "Diabodies" are composed of two chains, each of which comprises a heavy chain variable region connected to a light chain variable region on the same polypeptide chain, connected by a short peptide linker, and the two regions on the same chain do not pair with each other, but have complementary domains on the other chain, forming a bispecific molecule. Methods for preparing diabodies are known in the art (see, for example, Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, and Poljak RJ et al., (1994) Structure 2:1121-1123). Domain antibodies (dAbs) are small functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies. Domain antibodies are well expressed in bacteria, yeast, and mammalian cell systems. Further details of domain antibodies and methods for their production are known in the art (see, for example, U.S. Pat. Nos. 6,291,158, 6,582,915, 6,593,081, 6,172,197, 6,696,245, European Patent Nos. 0368684 and 0616640, WO05 / 035572, WO04 / 101790, WO04 / 081026, WO04 / 058821, WO04 / 003019, and WO03 / 002609). Nanobodies are derived from the heavy chain of an antibody. Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Patent No. 6,765,087, U.S. Patent No. 6,838,254, WO06 / 079372). Unibodies consist of one light chain and one heavy chain of an IgG4 antibody. Unibodies can be made by removing the hinge region of an IgG4 antibody.Further details of unibodies and methods for preparing them can be found in WO2007 / 059782.

[0210] VII. Composition In other aspects, the disclosure provides compositions comprising one or more of the polypeptides (e.g., activatable binding polypeptides) described herein. In some embodiments, the composition is a pharmaceutical composition comprising a polypeptide (e.g., an activatable binding polypeptide) and a pharma- ceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0211] The term "pharmaceutical acceptable carrier" refers to any inert substance suitable for use in a formulation for delivery of a polypeptide (e.g., an activatable binding polypeptide). Carriers can be anti-adherents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption retardants, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutical acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), glucose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0212] The composition may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of the composition suitable for delivering a polypeptide (e.g., an activatable binding polypeptide) is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. A sterile solution can be prepared by incorporating the required amount of the polypeptide (e.g., an activatable binding polypeptide) in a suitable carrier, followed by sterile microfiltration. A dispersion is prepared by incorporating the polypeptide (e.g., an activatable binding polypeptide) in a sterile vehicle containing a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquids, the preparation method includes vacuum drying and freeze-drying (lyophilization) to obtain a powder containing the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. Various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0213] The relative amount of polypeptide (e.g., activatable binding polypeptide) included in a composition will vary depending on many factors, such as the particular polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of polypeptide (e.g., activatable binding polypeptide) in a single dosage form will generally be that amount that provides a therapeutic effect, but may be a lesser amount. Generally, this amount will range from about 0.01 percent to about 99 percent, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent, based on the total weight of the dosage form.

[0214] In addition to the polypeptide (e.g., activatable binding polypeptide), one or more additional therapeutic agents can be included in the composition. Examples of additional therapeutic agents are described herein below. The suitable amount of additional therapeutic agent to be included in the composition can be easily selected by those skilled in the art and will vary depending on many factors, such as the specific drug and carrier used, dosage form, and desired release and pharmacodynamic properties. The amount of additional therapeutic agent included in a single dosage form is generally the amount of drug that produces a therapeutic effect, but may be a smaller amount.

[0215] Any of the polypeptides (e.g., activatable binding polypeptides) and / or compositions (e.g., pharmaceutical compositions) described herein can be used in the preparation of a medicament (e.g., a medicament for use in treating cancer or delaying the progression of cancer in a subject in need thereof).

[0216] VIII. Uses of Activatable Binding Polypeptides and Pharmaceutical Compositions The polypeptides (e.g., activatable binding polypeptides) and pharmaceutical compositions provided by the present disclosure are useful for therapeutic, diagnostic, or other purposes, such as modulating immune responses, treating cancer, enhancing the effectiveness of other cancer therapies, enhancing the effectiveness of vaccines, or treating autoimmune diseases. Thus, in another aspect, the present disclosure provides a method of using a polypeptide (e.g., activatable binding polypeptide) or pharmaceutical composition thereof. In one aspect, the present disclosure provides a method of treating a disorder in a mammal, comprising administering to a mammal in need of treatment an effective amount of a polypeptide (e.g., activatable binding polypeptide) or composition thereof provided by the present disclosure. In some embodiments, the polypeptide is an activatable binding polypeptide that binds to CTLA4 (e.g., human CTLA4) or CD137 (e.g., human CD137), and the mammal is a human. In some embodiments, the mammal is a human.

[0217] In some embodiments, the disorder is cancer. The methods, uses, or medicaments provided by the present disclosure can be used to treat or prevent various cancers.Examples of such cancers include lung cancers such as bronchogenic carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondroitin hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, gliomas (e.g., glioblastoma multiforme), ... brain cancers such as cyst, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germinoma, teratoma, dermoid cyst, and hemangioma; cancers in the digestive system such as leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colon polyp, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and hemangiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, hypernephropathy, and transitional cell carcinoma of the renal pelvis; bladder cancer; acute lymphoblastic blood cancers such as acute myeloid (lymphoblastic) leukemia, acute myeloid (myelocytic, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sézary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloid, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myelogenous leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancer; retinoblastoma cancers related to the eye, such as melanoma and intraocular melanoma; cancers of the male reproductive system, such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system, such as uterine cancer (endometrial cancer), cervical cancer (cervical carcinoma), ovarian cancer (ovarian carcinoma), vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); noncancerous growths of the parathyroid glands; pancreatic cancer; and blood cancers, such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

[0218] In another aspect, the present disclosure provides a method of enhancing an immune response in a mammal, comprising administering to the mammal an effective amount of a polypeptide (e.g., an activatable binding polypeptide) provided by the present disclosure or a composition thereof. In some embodiments, the polypeptide is an activatable binding polypeptide that binds to CTLA4 (e.g., human CTLA4) or CD137 (e.g., human CD137), and the mammal is a human. The term "enhancing an immune response" or grammatical variations thereof means stimulating, eliciting, increasing, improving, or enhancing any response of the immune system of a mammal. The immune response may be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated), or a humoral response (i.e., antibody-mediated), and may be a primary or secondary immune response. Examples of enhancing an immune response include activation of PBMCs and / or T cells, including increased secretion of one or more cytokines, such as IL-2 and / or IFNγ. The enhanced immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release, tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxic activity. Typically, the methods of the present disclosure enhance the immune response by a mammal as compared to an immune response by an untreated mammal or a mammal that has not been treated using the cited methods.

[0219] In carrying out the treatment, the polypeptide (e.g., activatable binding polypeptide) may be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Thus, in another aspect, the present disclosure provides a combination therapy comprising a polypeptide (e.g., activatable binding polypeptide) in combination with one or more additional therapies or therapeutic agents for separate, sequential, or simultaneous administration. The term "additional therapeutic agent" may refer to any therapeutic agent other than the polypeptide (e.g., activatable binding polypeptide) provided by the present disclosure. In certain aspects, the present disclosure provides a combination therapy for treating cancer in a mammal, comprising administering to the mammal an effective amount of a polypeptide (e.g., activatable binding polypeptide) provided herein in combination with one or more additional therapeutic agents. In further embodiments, the mammal is a human.

[0220] A wide variety of cancer therapeutic agents may be used in combination with the polypeptides (e.g., activatable binding polypeptides) provided by the present disclosure. Those skilled in the art will recognize the existence and development of other cancer therapies that can be used in combination with the methods and polypeptides of the present disclosure and will not be limited to the forms of therapy described herein. Examples of categories of additional therapeutic agents that can be used in combination therapy to treat cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents. In some embodiments, the additional therapeutic agent is a viral gene therapy, an immune checkpoint inhibitor, a targeted therapy, radiation therapy, and / or chemotherapy.

[0221] The term "chemotherapeutic agent" refers to a chemical or biological substance that can cause the death of cancer cells or interfere with the growth, division, repair, and / or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO2006 / 129163 and US20060153808, the disclosures of which are incorporated herein by reference. Examples of specific chemotherapeutic agents include: (1) alkylating agents such as chlorambucil (LEUKERAN), cyclophosphamide (CYTOXAN), ifosfamide (IFEX), mechlorethamine hydrochloride (MUSTARGEN), thiotepa (THIOPLEX), streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine (CEENU), and dacarbazine (DTIC-DOME); (2) doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN), and the like. (3) alkaloids or plant vinca alkaloids, including cytotoxic antibiotics such as capecitabine (XELODA), cytarabine (CYTOSAR-U), mitoxantrone (DHAD, NOVANTRONE), dactinomycin (ACTINOMYCIN D, COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin (BLENOXANE), vinorelbine tartrate (NAVELBINE), vinblastine (VELBAN), vincristine (ONCOVIN), and vindesine (ELDISINE); (4) antimetabolites such as 5-fluorouracil (5-FU), capecitabine (XELODA), raltitrexed (TOMUDEX), tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5) docetaxel (TAXOTERE);(6) platinum agents such as cisplatin (PLATINOL), carboplatin (PARAPLATIN), and oxaliplatin (ELOXATIN); (7) topoisomerase inhibitors such as irinotecan (CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), and teniposide (VUMON); (8) etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), and cyclosporine; (AR) and other epipodophyllotoxins (podophyllotoxin derivatives), (9) folic acid derivatives such as leucovorin (WELLCOVORIN), (10) nitrosoureas such as carmustine (BiCNU) and lomustine (CeeNU), (11) gefitinib (IRESSA), erlotinib (TARCEVA), bortezomib (VELCADE), imatinib mesylate (GLEEVEC), genefitinib, lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib, rituximab, etc. Epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocyte growth factor, such as tuximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN), and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), antibodies against insulin-like growth factor-1 receptor (IGF-1R) described in WO2002 / 053596 Receptor tyrosine kinases, including the HGFR and platelet-derived growth factor receptors (PDGFR), (12) angiogenesis inhibitors such as bevacizumab (AVASTIN), suramin (GERMANIN), angiostatin, SU5416, thalidomide, and matrix metalloproteinase inhibitors (such as batimastat and marimastat), and those described in WO2002055106, and (13) proteasome inhibitors such as bortezomib (VELCADE).

[0222] The term "immunotherapeutic agent" refers to a chemical or biological substance capable of enhancing the immune response of a mammal. Examples of immunotherapeutic agents include bacillus Calmette-Guerin (BCG); cytokines such as interferons; vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX, and NeuVax; and vaccines such as alemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX), gemtuzumab ozogamicin (MYLOTARG), ibritumomab tiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), tositumomab (BEXXAR ... These include antibodies such as limumab (YERVOY), tremelimumab, CAT-3888, agonist antibodies against the OX40 receptor (e.g., those disclosed in WO2009 / 079335), agonist antibodies against the CD40 receptor (e.g., those disclosed in WO2003 / 040170), and TLR-9 agonists (e.g., those disclosed in WO2003 / 015711, WO2004 / 016805, and WO2009 / 022215).

[0223] The term "hormonal therapy" refers to a chemical or biological substance that inhibits or eliminates the production of hormones or inhibits or interferes with the effect of hormones on the growth and / or survival of cancerous cells. Examples of such agents suitable for the methods herein include those disclosed in US20070117809. Examples of specific hormonal therapy agents include tamoxifen (NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole (ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The binding molecules of the disclosure may also be used in combination with non-drug hormone therapies such as (1) surgical procedures to remove all or part of the organs or glands involved in the production of hormones, such as the ovaries, testes, adrenal glands, and pituitary gland, and (2) radiation therapy, in which a patient's organs or glands are irradiated in an amount sufficient to inhibit or eliminate production of the targeted hormone.

[0224] In some embodiments, the additional therapeutic agent is one or more of pomalyst, Revlimid, lenalidomide, pomalidomide, thalidomide, DNA alkylating platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-cTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anti-cancer vaccine, and oncolytic virus.

[0225] Combination therapies for treating cancer also include the use of binding molecules in combination with surgery to remove tumors. The binding molecules can be administered to the mammal before, during, or after surgery.

[0226] Combination therapies for treating cancer also include the use of a polypeptide (e.g., an activatable binding polypeptide) in combination with radiation therapy, such as ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle radiation therapy (e.g., high linear energy radiation). The source of radiation can be external or internal to the mammal. The polypeptide can be administered to the mammal before, during, or after radiation therapy.

[0227] Polypeptides (e.g., activatable binding polypeptides) and compositions provided by the present disclosure can be administered via any suitable enteral or parenteral route of administration. The term "enteral route" of administration refers to administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal, and rectal routes, or intragastric routes. A "parenteral route" of administration refers to a route of administration other than the enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumor, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or topical administration. Polypeptides (e.g., activatable binding polypeptides) and compositions of the present disclosure can be administered using any suitable method, such as oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. Suitable routes and methods of administration can vary depending on a number of factors, such as the polypeptide used, the desired rate of absorption, the particular formulation or dosage form used, the type or severity of the disorder being treated, the particular site of action, and the condition of the patient, and can be readily selected by one of skill in the art.

[0228] The term "effective amount" of a binding molecule may refer to an amount effective for an intended therapeutic purpose. For example, in the context of enhancing an immune response, an "effective amount" may be any amount that is effective to stimulate, induce, increase, improve, or enhance any response of the immune system of a mammal. In the context of treating a disease, an "effective amount" may be any amount sufficient to cause any desired or beneficial effect in the treated mammal. Specifically, in the treatment of cancer, examples of desired or beneficial effects include inhibiting further growth or metastasis of cancer cells, killing cancer cells, inhibiting recurrence of cancer, reducing pain associated with cancer, or improving survival of the mammal. A therapeutically effective amount of a polypeptide (e.g., an activatable binding polypeptide) described herein may range from about 0.001 to about 500 mg / kg, or from about 0.01 to about 100 mg / kg of the mammal's body weight. For example, the amount may be about 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, or 100 mg / kg of the mammal's body weight. In some embodiments, a therapeutically effective amount of a polypeptide (e.g., an activatable binding polypeptide) of the present disclosure ranges from about 0.01 to 30 mg / kg of mammalian body weight. In some other embodiments, a therapeutically effective amount of a polypeptide (e.g., an activatable binding polypeptide) of the present disclosure ranges from about 0.05 to 15 mg / kg of mammalian body weight. The exact dosage level to be administered can be readily determined by one of skill in the art and will depend on numerous factors, such as, for example, the type and severity of the disorder being treated, the particular polypeptide being administered, the route of administration, the time of administration, the duration of treatment, any particular additional therapy being administered, the age, sex, weight, condition, general health and prior medical history of the patient being treated, as well as factors well known in the medical arts.

[0229] The polypeptide (e.g., activatable binding polypeptide) or composition thereof may be administered multiple times. The interval between single doses may be, for example, daily, weekly, monthly, every three months, or yearly. Exemplary treatment regimens involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Drug administration of the polypeptide (e.g., activatable binding polypeptide) of the present disclosure may include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration using one of the following dosing schedules: (i) six doses every four weeks, then every three months, (ii) every three weeks, (iii) one dose of 3 mg / kg body weight, then 1 mg / kg body weight every three weeks.

[0230] IX. Kit In another aspect, a kit is provided herein that includes a library of polynucleotides of the present disclosure. In some embodiments, the kit further includes a package insert that includes instructions for expressing, modifying, screening, or otherwise using the library, e.g., to identify an activatable binding polypeptide of interest. In some embodiments, the kit further includes one or more buffers, e.g., for storing, transferring, administering, or otherwise using one or more of the polynucleotides (e.g., synthetic polynucleotides). In some embodiments, the kit further includes one or more containers for storing one or more of the polynucleotides. In some embodiments, the kit further includes one or more vectors, e.g., for transfecting a host cell with one or more of the polynucleotides.

[0231] In another aspect, provided herein are kits comprising the activatable binding polypeptides and / or compositions described herein. In some embodiments, the kits further comprise a package insert containing instructions for use of the activatable binding polypeptides and / or compositions. In some embodiments, the kits further comprise one or more buffers, e.g., for storing, transferring, administering, or otherwise using the activatable binding polypeptides and / or compositions. In some embodiments, the kits further comprise one or more containers for storing or administering the activatable binding polypeptides and / or compositions.

[0232] The above description is deemed sufficient to enable those skilled in the art to practice the present disclosure. The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, in addition to the modifications shown and described herein, various modifications of the present disclosure will become apparent to those skilled in the art from the above description and will fall within the scope of the appended claims. EXAMPLES

[0233] Example 1: Methods for identifying self-blocking peptides that are activatable binding polypeptides As mentioned above, there exists a need for improved methods and products useful for identifying self-blocking peptides for activatable binding polypeptides. Thus, a new system is described herein that has been designed and implemented to efficiently discover masking moieties with good developability. In this system, a target antibody fragment, either Fab (Figure 1) or scFv (Figure 2), was first displayed on the yeast surface and confirmed to be functional in binding to its antigen. The improved peptide library was then fused directly to the N-terminus of the light chain to construct a yeast library that displayed the fusion protein on the yeast surface. The yeast library was then subjected to several rounds of FACS-based screening: first, yeast clones that had low binding to the antigen were enriched, and then the enriched yeast clones were treated with protease to remove the N-terminal peptides and clones with high binding to the antigen were selected (Figures 1 and 2). After 4-5 rounds of selection, plasmids were extracted from these clones and the masking peptide sequences were confirmed by DNA sequencing.

[0234] There are several unique features built into this new system that make it highly developable and powerful in identifying masking peptides for target antibodies: 1) The peptide library was directly fused to the N-terminus of the target antibody instead of any foreign scaffold protein, and the masking peptides were discovered in the same context as the final product, which eliminated contamination with false-positive peptide sequences and dramatically reduced the workload of their downstream characterization. 2) A protease cleavage-mediated activation mechanism was incorporated into the screening process, which ensured that the discovered peptides not only masked antigen binding before activation, but also no longer blocked antigen binding after protease cleavage, which were prerequisites considered to qualify as good masking peptides for any activatable antibody. 3) Improved design of peptide libraries was utilized. In contrast to commonly used random peptide libraries, a pair of cysteine ​​residues was introduced at fixed positions in the peptide library to ensure that the presented peptides have a constrained conformation. It was observed that constrained peptides tend to exhibit increased binding affinity and specificity (Uchiyama et al. (2005) 99(5):448-56). In contrast to the widely used NNK (or NNS) codon, which codes for all 20 residues, including chemically labile residues such as M and W, the NHC codon was utilized in some or all of the peptide libraries. The NHC codon codes for 12 amino acid residues (D, A, Y, S, T, N, I, L, F, V, H, and P) and does not include residues that are unfavorable to the manufacturing process, such as methionine, tryptophan, or cysteine. In addition, the use of the NHC codon also dramatically reduced the theoretical peptide library size compared to the NNK (or NNS) codon, thus allowing the construction of libraries with significantly improved coverage. These libraries performed well when tested against different target antibodies.

[0235] Example 2: Design of Constrained Peptide Libraries (CPLs) Four exemplary constrained peptide libraries (CPLs) were designed (Table 1). TIFF2025037964000003.tif142170

[0236] Their cores have the sequence Z6CX6CZ2 (SEQ ID NO:55) or Z6CX8CZ2 (SEQ ID NO:56), with two fixed cysteine ​​residues forming disulfide bonds to constrain the conformation of the peptide. In the synthesized oligonucleotides, the degenerate codon NHC was applied everywhere except inside the loop, and the NNK codon was also employed in CPL011 and CPL013. In contrast to the NNK or NNS codons, the NHC codon codes for 12 residues (Table 2), encompassing significant diversity but lacking the chemically labile residues methionine, tryptophan, and cysteine. Moreover, the reduced theoretical diversity compared to the NNK or NNS codons allowed the construction of libraries with a broader coverage. TIFF2025037964000004.tif33170

[0237] Following these masking peptide sequences was an invariant cleavage peptide sequence (SGRSAGGGGSPLGLAGSGGS, SEQ ID NO: 12) containing two protease recognition sites: SGRSA (SEQ ID NO: 13) for the protease urokinase-type plasminogen activator (uPA) and PLGLAG (SEQ ID NO: 14) for the proteases matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). These recognition sites have been used by many groups in in vivo tumor cell-specific activation of targeting agents (see, for example, Ke et al. (1997) J Biol Chem 272(33):20456-62, Gerspach et al. (2006) Cancer Immunol Immunother 55(12):1590-600, and Jiang et al. (2004) Proc Natl Acad Sci USA 101(51):17867-72). During yeast-based screening, the MMP-9 recognition sequence was replaced with the tobacco etch virus (TEV) protease recognition sequence (ENLYFQG, SEQ ID NO: 15) due to the availability and specificity of TEV protease.

[0238] The CPL and invariant cleavage peptides were fused to the N-terminus of the light chain of the target antibody in the form of either an scFv or Fab connected to the Aga2 protein displayed on the yeast surface. The inclusion of a surrogate TEV protease recognition site was important in identifying the correct type of masking peptide sequence; that is, antigen binding is blocked before protease cleavage and antigen binding is permitted after protease cleavage. The examples described below show that the cleavage activation mechanism of an activatable antibody first demonstrated in yeast is replicated in a complete IgG molecule expressed in mammalian cells.

[0239] Example 3: Construction and validation of an activatable antibody targeting CTLA4 Display of functional targeting antibodies on the yeast surface A low copy number CEN / ARS-based vector was used to express a target antibody (antibody TY21580 targeting human CTLA4) under the control of the inducible GAL1-10 promoter in the yeast S. cerevisiae. Surface display of the scFv was achieved via the Aga2 protein fused to its C-terminus under the control of the GAL1 promoter, similar to a previously published arrangement (Boder and Wittrup (1997) Nat Biotechnol 15(6):553-7). For the Fabs, their surface display was achieved by the Aga2 protein fused to the N-terminus of the heavy chain (fusion of VH and CH1) under the control of the GAL1 promoter, whereas the light chain (fusion of VL and CL) was under the control of the GAL10 promoter. The Fabs were displayed on the yeast surface by association with the membrane-anchored heavy chain.

[0240] Surface display of Fab or scFv was verified by staining with an antibody recognizing the fusion affinity tag, and the functionality of Fab or scFv displayed on yeast was investigated using biotinylated human CTLA4. Briefly, 48 hours after induction in galactose medium, yeast cells (1x10^6) were harvested, washed once with PBSA buffer, and then incubated with 10 nM biotinylated antigen for 1 hour at room temperature. Yeast cells were then washed twice with PBSA buffer and incubated with PE-conjugated streptavidin (1:500 dilution) (eBioscience#2-4317-87) for 30 minutes at 4°C. Yeast cells were then analyzed by flow cytometry. As shown in Figure 3A-B, both Fab (Figure 3A) and scFv (Figure 3B) targeting CTLA4 were successfully displayed on the yeast surface, and both of them were able to strongly bind to their antigens.

[0241] Construction of a yeast library containing CPLs The synthesized oligonucleotides encoding CPL were fused with oligonucleotides encoding the cleavage peptide by five cycles of PCR. The primers used (F and R primers) are listed in Table 3. The PCR reaction consisted of 1x PrimeSTAR buffer, 2.5 mM dNTPs, 100 μM each of F and R primers, and 100 μM each of template 1 (CPL oligonucleotide) and template 2 (oligonucleotide encoding the cleavage peptide), and 2.5 U of PrimeSTAR HS DNA polymerase. The PCR program used was a) 1 cycle at 96°C for 5 min, 2) 5 cycles of 96°C (15 s), 60°C (15 s), 72°C (6 s), and 3) 1 cycle at 72°C for 3 min. Exonuclease I was used to degrade single-stranded DNA before purification of the PCR products by gel electrophoresis. The purified PCR products were then digested with BamHI and KpnI and cloned into a bacterial filter vector digested with the same two restriction enzymes. In the filter vector, the CPL and cleavage peptide were placed downstream of a bacterial secretion signal peptide and upstream of a beta-lactamase lacking a signal sequence. Functional beta-lactamase selected on ampicillin plates indicated an in-frame fusion of the CPL and cleavage peptide, thereby eliminating any out-of-frame errors (N-1 or N-2) introduced into the synthesized degenerate oligonucleotides. In addition, some incompletely folded sequences were also reduced from the pool. The ligation products were transformed into electrocompetent bacterial cells, and the diversity of the CPL libraries was typically 5x10^9 to 1x10^10. Sequencing of individual clones showed that very high in-frame ratios (often nearly 100%) were achieved by this approach. TIFF2025037964000005.tif82170

[0242] To generate yeast libraries containing CPLs, plasmids were extracted from the bacterial library and used as templates for PCR amplification of DNA fragments encoding CPLs and truncated peptides. The primers used (PL0009_F and BL1024_R) are listed in Table 3. The amplified PCR fragments were purified by gel electrophoresis and transformed into electrocompetent yeast cells together with linearized plasmids that expressed the target antibodies fused to Aga2. Homologous sequences at both ends of the PCR fragments and plasmids ensured efficient homologous recombination in yeast cells. The diversity of the constructed yeast libraries was generally 1×10^9 to 2×10^9.

[0243] FACS-based screening of masking peptides for CTLA4 antibodies A total of 1x10^8 yeast cells from the CPL yeast library were used to screen masking peptides against the target antibody. For each round of sorting with MoFlo XDP, yeast cells induced in galactose medium were harvested, washed once with PBSA buffer, and then incubated with 10nM (reduced to 1nM in later rounds) biotinylated antigen for 1 hour at room temperature. The yeast cells were then washed twice with PBSA buffer and incubated with PE-conjugated streptavidin (1:500 dilution) (eBioscience#2-4317-87) for 30 minutes at 4°C. After two more washes with PBSA buffer, the yeast cells were adjusted to 2-3OD / mL and subjected to sorting. As shown in Figure 4, in round 1, 10nM biotinylated CTLA4-Fc was used to enrich for weak binders. Yeast cells from round 1, after growth in glucose medium, were induced in galactose medium and treated with AcTEV protease (6U / OD cells) (Thermo Fisher Scientific #12575015) for 2 hours at 30 °C to purify strong binders. Starting from the third selection round, the concentration of biotinylated CTLA4-Fc was reduced to 1 nM to collect weak binders. In the fourth round, a fraction of yeast cells was also treated with AcTEV in parallel to verify protease cleavage-mediated activation of the target antibody. As shown in Figure 4, it was clear that AcTEV cleavage led to a dramatic increase in the cell population that strongly bound to the antigen, suggesting that the screening strategy was effective. Single clones from the fifth selection round were plated in selective medium and grown individually for further confirmation of cleavage-mediated activated antigen binding.

[0244] As shown in Figure 5A-B, the selected CTLA4 activatable antibody clones, in either scFv (Figure 5A) or Fab (Figure 5B) format, showed little binding to the antigen in the presence of the masking peptide. However, when the yeast cells were treated with TEV protease to remove the masking peptide, binding to the antigen increased dramatically. Incorporating a TEV recognition site into the cleavage peptide, together with the application of TEV protease to validate the selected clones, significantly increased the success rate of masking peptide selection.

[0245] To identify the masking peptide sequences, shuttle plasmids were extracted from selected yeast clones (Generay #GK2002-200) and transformed into competent E. coli cells. Plasmids were prepared and the regions encoding the masking peptides were sequenced and aligned. As expected, these sequences could be divided into several groups and showed clear enrichment by the selection rounds. The four groups of masking peptide sequences are listed in Table 4 together with the invariant cleavage peptide sequences. TIFF2025037964000006.tif94170

[0246] IgG conversion and expression Four groups of masking peptides listed in Table 4 to eliminate potential glycosylation sites, as well as four additional masking peptide sequences derived from two of them (B13192 and B13197) (Table 5), were converted to IgG1. TIFF2025037964000007.tif89170

[0247] The heavy and light chains were cloned separately into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific, Cat. No. K830001) and the masking peptide and invariant cleavage peptide were fused to the N-terminus of the light chain in the same manner as displayed on the yeast surface. The VH and VL sequences for the parent CTLA4 antibody (TY21580) are listed below (see also PCT International Application entitled "Compositions Comprising Cross-reactive Anti-CTLA4 Antibodies, and Methods of Making and Using the Same", filed concurrently herewith under Attorney Docket No. 69540-2000540, which is incorporated herein by reference in its entirety): Anti-CTLA4 heavy chain variable region (SEQ ID NO:47): EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS Anti-CTLA4 light chain variable region (SEQ ID NO:48): DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR.

[0248] The plasmid pairs were transiently transfected into HEK293F cells. After 6 days, the supernatants were harvested, clarified by centrifugation and filtration, and IgG was purified using standard Protein A affinity chromatography (MabSelect SuRe, GE Healthcare). IgG was eluted and neutralized, and buffer exchanged into PB buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). Protein concentration was determined by UV spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions. Importantly, the expression levels of the activatable antibodies in HEK293 cells were similar to the parental antibodies, and their purification yields after Protein A resin were also similar, suggesting that the presence of masking and cleavage peptides does not adversely affect antibody expression in mammalian cells.

[0249] Measurement of masking efficiency The efficiency of the masking peptides was rapidly evaluated using a ForteBio Octet RED96 system (Pall, USA). Briefly, the activatable antibodies (and their parent antibody, TY21580) were diluted to 30 μg / mL in KB buffer (PBS buffer supplemented with 0.02% Tween 20 and 0.1% BSA) and captured in parallel by an anti-human IgG capture (AHC) biosensor (Pall, USA). The sensor was then allowed to associate with His-tagged CTLA4 protein (25 nM) for 300 s and then dissociated in KB buffer for another 300 s. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 7.1 (Pall, USA) according to the manufacturer's guidelines. As shown in Figure 6A-B, the responses achieved with the activatable antibodies were significantly lower than those of the parent antibodies, suggesting that the masking peptide effectively blocked the binding of the antibody to its antigen. However, of the four activatable antibodies, TY22401 was the least effective, consistent with the results of the ELISA assay discussed below.

[0250] Recombinant human CTLA4-Fc was diluted to 1 μg / mL in PBS and coated on Maxisorp plates overnight at 4°C. The plates were blocked with PBS supplemented with 3% non-fat milk for 1 h at 37°C. After washing, 100 μL of 3-fold serially diluted antibody was added to each well. After 1 h incubation at 37°C, the plates were washed 4 times and 100 μL of HRP-conjugated anti-human IgG (Fab specific) (1:6000 dilution) was added to each well. The plates were incubated for 1 h at 37°C, washed 4 times, then 50 μL of TMB substrate solution was added to each well and the plates were incubated at room temperature. The reaction was stopped with 50 μL of H2SO4 per well before measuring absorbance at 450 nm. EC 50 was assessed by fitting the ELISA data using an asymmetric sigmoidal (five parameter logistic equation) model in GraphPad Prism 6 software. Experiments for activatable antibodies TY22401, TY22402, and TY22404 were performed in duplicate, resulting in two calculated masking efficiencies for each of these activatable antibodies. The masking efficiency of each activatable antibody was calculated as the EC 50 , and the EC 50 As shown in Figures 7A-C and Table 6, all activatable antibodies showed a dramatic reduction in binding to their antigens compared to the parental antibodies, with calculated masking efficiencies ranging from 48 to 2213. The differences in masking efficiency were calculated using the EC 50 The masking efficiency of each activatable antibody is likely within the calculated range (e.g., the masking efficiency of activatable antibody TY22402 is 377-2213). These results showed that multiple masking peptides identified from CPL maintained their masking efficiency when expressed in mammalian cells and as part of a complete IgG molecule. JPEG2025037964000008.jpg212170

[0251] Removal of the masking peptide restores antibody activity. The purified activatable antibodies were treated with a protease that recognizes the cleavage sequence and then tested to determine whether removal of the masking peptide restores their activity. As an example, 20 μg of TY22404 (0.5 mg / mL) was treated with 1 μg of recombinant human uPA (Acrobiosystems, #PLU-H5229) in reaction buffer (50 mM Tris HCl, 0.01% Tween 20, pH 8.5) or TY22404 was treated with 1 μg of recombinant human uPA (Acrobiosystems, #PLU-H5229) in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, 20 μ The cells were treated with 5 or 10 units of recombinant human MMP-9 (BioVision, #7867-500) in 100 mM ZnCl2, pH 7.5. The reaction was carried out at 37° C. for 21 hours. The masking peptide was confirmed to be removed from the light chain by SDS-PAGE analysis (FIG. 8A). Masking efficiency was then measured by ELISA as described above. As shown in FIG. 8B and Table 7, after removal of the masking peptide, the activatable antibodies were indistinguishable from the parent antibodies in binding to antigen. TIFF2025037964000009.tif47170

[0252] Developability profile of activatable antibodies For manufacturing purposes, it is important that the activatable antibodies discovered have a good developability profile. Several different tests were performed with purified activatable antibodies expressed in mammalian cells. The activatable antibodies were adjusted to 1 mg / mL in 20 mM histidine at pH 5.5 and antibody quality analysis was performed using analytical size exclusion chromatography using a Waters 2695 with a Waters 2996 UV detector and a TSKgel g3000 SWXL column (300 mm x 7.8 mm) (Tosoh Bioscience). For each assay, 10 μg of antibody was injected and fractions were performed in buffer (200 mM sodium phosphate at pH 7.0) at a flow rate of 0.5 mL / min.

[0253] Three accelerated stress tests were performed: incubation of the activatable antibodies at 50°C for 7 days, incubation of the activatable antibodies at 40°C for 28 days, and six cycles of freeze-thaw. The freeze-thaw tests were performed by freezing 100 μL of the samples (1 mg / mL in 20 mM histidine, pH 5.5) at -80°C for 30 min, followed by thawing at room temperature for 60 min. As shown in Figure 9A-C, all the activatable antibodies remained stable and showed almost no aggregation after storage at 50°C for 7 days or at 40°C for 28 days. Although they showed slight degradation after six cycles of freeze-thaw, the monomer main peak remained near 95%, indicating that these activatable antibodies were very stable under these accelerated stress tests. Without wishing to be bound by theory, it is worth noting that the activatable antibodies have not yet undergone an extensive buffer optimization process, and therefore, the stability of the activatable antibodies may be further improved with optimized buffers and excipients.

[0254] The activatable antibodies were then concentrated to over 150 mg / mL in 20 mM histidine at pH 5.5 (Table 8). No precipitation of the activatable antibodies was observed and the viscosity of the samples was very manageable. The concentrated activatable antibodies were then diluted to either 20 mg / mL or 1 mg / mL for analysis of high molecular weight (HMW) species. As shown in FIG. 10 and Table 8, no obvious increase in HMW species was observed, suggesting that these activatable antibodies were highly soluble and stable in the buffers tested up to high concentrations. TIFF2025037964000010.tif28170

[0255] To study the stability of activatable antibodies at low pH, purified activatable antibodies (10 mg / mL in 20 mM histidine, pH 5.5) were titrated to 1 mg / mL with citric acid, adjusted to pH 3.7, and held at room temperature for 30 and 60 minutes. Samples were then neutralized to pH 7.0 with 1 M Tris base. Masking efficiency of the activatable antibodies was measured on the ForteBio as described above. As shown in FIG. 11, the masking efficiency remained unchanged after 30 or 60 minutes of low pH incubation, suggesting that the masking peptide retains its blocking effect after low pH incubation.

[0256] Taken together, the data indicate that the discovered activatable antibodies remain stable under various stress conditions and therefore, they have a good developability profile.

[0257] Example 4: In vitro and in vivo characterization of activatable antibodies targeting CTLA4 It has been previously shown that the parent antibody TY21580 alone does not stimulate human T cell activation or activation of human PBMC cells (see also PCT International Application entitled "Compositions Comprising Cross-reactive Anti-CTLA4 Antibodies, and Methods of Making and Using the Same" filed concurrently herewith under Attorney Docket No. 69540-2000540, which is incorporated herein by reference in its entirety). CTLA-4 activity in T cells is known to be associated with a first (TCR / CD3) signal and a second signal involving B7-CD28 / CTLA-4. Consistently, at low concentrations of anti-CD3, the parent antibody TY21580 was shown to significantly enhance activation of human PBMC cells.

[0258] In vitro functional characterization Here, the activity of activatable antibodies targeting CTLA4 was evaluated in the presence of low concentrations of anti-CD3 antibodies on human PBMC activation. Human PBMCs were freshly isolated from the blood of a healthy donor (#44) by density gradient centrifugation using Histopaque-1077 (Sigma). Anti-CD3 (OKT-3) antibodies were coated overnight at 4°C on 96-well plates. After washing, 1x10^5 freshly isolated human PBMCs were added to each well, followed by the addition of test articles at different concentrations. Induction of IL-2 was measured 48 hours after stimulation using a human IL-2 ELISA Ready-SET-Go (Invitrogen) kit. IFN-γ in the supernatants was measured using a human IFN-γ ELISA Ready-SET-Go (Invitrogen) kit. As shown in Figure 12A-B, at high concentrations, TY22404 induced IL-2 production and TY22401 induced IFN-γ production. Nevertheless, the activity of the activatable antibody was significantly lower than that of the parent antibody TY21580.

[0259] The antibody-dependent cellular cytotoxicity of the activatable antibodies was then tested and compared to that of the parent antibody TY21580. The ADCC activity of the activatable antibodies was evaluated using an ADCC reporter gene assay. HEK293F cells overexpressing human CTLA4 (HEK293F / hCTLA-4 cells) were used as target cells, and Jurkat cell line overexpressing CD16a and NFAT-Luc (Jurkat / CD16a cells) were used as effector cells. 1×10^5 Jurkat / CD16a cells and 1×10^4 HEK293F / hCTLA-4 cells (E:T ratio 10:1) were mixed with different concentrations of antibodies. After 6 hours of incubation, 100 μL of One-Glo reagent was added to the cells, and the cells were lysed for 10 minutes. The supernatant was removed for luminescence measurement using a SpectraMax i3x plate reader. As shown in Figure 13, the activatable antibodies exhibited several logs less ADCC activity than the parent antibody TY21580. The ADCC activity of TY22401 was greater than that of TY22402 and TY22404. Taken together, the in vitro data indicate that the better masked activatable antibodies had lower ADCC activity.

[0260] The anti-tumor activity of the activatable antibodies was then evaluated and compared to that of the parental antibody TY21580 in multiple syngeneic mouse tumor models, including the MC38 colorectal tumor model, the CT26 colorectal tumor model, the H22 liver tumor model, and the 3LL lung tumor model.

[0261] Antitumor effects in MC38 colorectal tumor model C57BL / 6 mice (n=8 per group, female, 6-8 weeks old) were inoculated subcutaneously with MC38 (NTCC-MC38) murine colon carcinoma cells. When tumors were established (70 mm3), treatment was initiated with isotype control antibody, parental antibody TY21580, or one of the three activatable antibodies by intraperitoneal injection twice weekly. Tumor growth was monitored twice weekly to assess mean tumor volume ± sem over time (Figure 14A) and individual tumor growth curves (Figure 14B). As shown in Figures 14A-B, all three activatable antibodies demonstrated potent antitumor activity comparable to the parental antibody TY21580 in the MC38 syngeneic mouse tumor model.

[0262] Antitumor effects in CT26 colorectal tumor model BALB / c mice (n=8 per group, female, 7-8 weeks old) were inoculated subcutaneously with CT26 (Shanghai Institutes for Biological Sciences) mouse colon carcinoma cells. When tumors were established (100 mm3), treatment was initiated with isotype control antibody, parental antibody TY21580, or one of the three activatable antibodies at 5 mg / kg by intraperitoneal injection twice weekly. Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. As shown in Figure 15, all three activatable antibodies demonstrated potent antitumor activity comparable to the parental antibody TY21580 in the CT26 syngeneic mouse tumor model.

[0263] Antitumor effect in H22 liver tumor model BALB / c mice (n=8 per group, female, 7-8 weeks old) were inoculated subcutaneously with H22 (China Center for Type Culture Collection) mouse hepatoma cells. When tumors were established (100 mm3), treatment was initiated with isotype control antibody, parent antibody TY21580, or one of the three activatable antibodies at 5 mg / kg by intraperitoneal injection twice weekly. Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. As shown in Figure 16, all three activatable antibodies demonstrated potent antitumor activity comparable to the parent antibody TY21580 in the H22 syngeneic mouse tumor model.

[0264] Antitumor effect in 3LL lung cancer model C57BL / 6 mice (n=10 per group, female, 6-8 weeks old) were inoculated subcutaneously with 3LL (JCRB) murine lung carcinoma cells. When tumors were established (75 mm3), treatment was initiated with isotype control antibody, parental antibody TY21580, or one of the three activatable antibodies by intraperitoneal injection twice weekly. Tumor growth was monitored twice weekly to assess mean tumor volume ± sem over time (Figure 17A) and individual tumor growth curves (Figure 17B). As shown in Figures 17A-B, all three activatable antibodies demonstrated potent antitumor activity comparable to the parental antibody TY21580 in the 3LL syngeneic mouse tumor model.

[0265] Pharmacokinetic analysis Pharmacokinetic studies were performed in female BALB / c mice approximately 8 weeks old. Three mice per group were injected intraperitoneally with the test article at 10 mg / kg. Blood samples (approximately 50 ul per sample) were collected at 3, 6, 24, 48, 96, and 168 hours after dosing. Blank control blood was collected from three naive female mice that did not receive any antibody. Serum concentrations of each test antibody were measured by ELISA, with anti-human IgG Fc used for capture and HRP-labeled anti-human IgG (Fab specific) antibody (Sigma) used for detection (Figure 18A-C). Compared to previous data collected for the parent antibody TY21580, the activatable antibodies TY22401 (Figure 18A), TY22402 (Figure 18B), and TY22404 (Figure 18C) had much slower clearance times and much longer half-lives. TY22401 had a half-life of 196 hours, with a drug concentration of approximately 55 μg / mL at 168 hours. TY22402 had a half-life of 134 hours, with a drug concentration of approximately 40 μg / mL at 168 hours. TY22404 had a half-life of 254 hours, with a drug concentration of approximately 45 μg / mL at 168 hours. In comparison, the parent antibody TY21580 had a half-life of 107 hours, with a drug concentration of approximately 17 μg / mL at 168 hours.

[0266] Repeated dose toxicity study While evaluating the effect of TY21580 on age at onset of diabetes in NOD mice, we found that high doses of TY21580 could lead to death of NOD animals, but not in normal BALB / c mice. Here, we used the NOD mouse model to evaluate the safety of activatable antibodies compared to that of TY21580. NOD mice (n=5 per group, female, 6 weeks old) were treated with isotype control antibody, parent antibody TY21580, or one of the three activatable antibodies at 50 mg / kg by intraperitoneal injection on days 0, 3, 7, and 12. In the TY21580-treated group, one animal died after the third dose and three died after the fourth dose. As shown in Figure 19, all animals treated with isotype control or any of the three activatable antibodies were alive and healthy at the end of the study. These data demonstrated that the activatable antibodies have an acceptable safety / toxicity profile in mice, and are much safer than the parent antibody TY21580 in NOD mice.

[0267] Example 5: Construction and validation of activatable antibodies targeting CD137 An activatable antibody targeting human CD137 was developed similar to the scheme used in the development of the anti-CTLA4 activatable antibody described in Example 3 above. Fab fragments (FIG. 20A) or scFvs (FIG. 20B) of the parent CD137 antibody were displayed on the surface of yeast via fusion to the Aga2 protein, and their ability to bind CD137 was confirmed by flow cytometry. The VH and VL sequences for the parent CD137 antibody (TY21242) are listed below (see also PCT International Application No. PCT / CN2017 / 098332, which is incorporated herein by reference in its entirety): Anti-CD137 heavy chain variable region (SEQ ID NO:49): EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSS Anti-CD137 light chain variable region (SEQ ID NO:50): DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIK.

[0268] Yeast libraries were also constructed with CPL fused to the N-terminus of the light chain and subjected to a FACS-based screening process. Single clones from the fourth or fifth selection round (FIG. 21) were plated on selective medium and grown individually for confirmation of cleavage-mediated activation antigen binding. As shown in FIG. 22A-B, selected CD137-activatable antibody clones showed little binding to antigen in the presence of masking peptide, but binding to antigen was dramatically increased when yeast cells were treated with TEV ...

Claims

1. A library comprising polynucleotides, at least one of the polynucleotides encoding a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM); The FP is represented by formula (XIII): ｍ C.X. ｎ C.X. ｏ (SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and 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; the CM comprises at least a first cleavage site; The library, wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region.

2. the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten unique polypeptides, each unique polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM); The FP is represented by formula (XIII): ｍ C.X. ｎ C.X. ｏ (SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and 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; the CM comprises at least a first cleavage site; The library of claim 1 , wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region.

3. each of the polynucleotides in the library encodes a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM); The FP is represented by formula (XIII): ｍ C.X. ｎ C.X. ｏ (SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and 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; the CM comprises at least a first cleavage site; The library of claim 1 or claim 2, wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region.

4. The FP is represented by the formula (XIV): (NNK) ｍ TGY (NNK) ｎ TGY (NNK) ｏ The library of any one of claims 1 to 3, wherein the library is encoded by a polynucleotide sequence comprising a nucleic acid sequence according to (SEQ ID NO:87), wherein each N is independently A, G, T, or C, each K is independently T or G, and each Y is independently T or C.

5. The library of any one of claims 1 to 4, wherein each X is not M, W, or C.

6. X in formula (XIII) ｍ wherein each X 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. X in formula (XIII) ｎ wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

8. X in formula (XIII) ｏ wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

9. The library according to any one of claims 1 to 8, wherein m is 2 or 3 to 6.

10. The library according to any one of claims 1 to 9, wherein n is 6 to 8.

11. The library according to any one of claims 1 to 9, wherein n is 6.

12. The library according to any one of claims 1 to 11, wherein o is 1 to 2.

13. The library according to any one of claims 1 to 11, wherein o is 2.

14. The library of any one of claims 1 to 13, wherein the FP further comprises an additional amino acid sequence at its N-terminus.

15. The library of claim 14 , wherein the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO:

16.

16. The first cleavage site is 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, and ADAMT. The library according to any one of claims 1 to 15, wherein the protease cleavage site is a protease cleavage site of a protease selected from the group consisting of 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 CM has a first linker (L １ The library according to any one of claims 1 to 16, further comprising:

18. Said L １ The library of claim 17, wherein said amino acid sequence is selected from the group consisting of SEQ ID NOs: 17 to 24.

19. The library of any one of claims 1 to 18, wherein the CM further comprises a second cleavage site.

20. The second cleavage site is １ The library of claim 19, wherein the C-terminus of

21. The second cleavage site is 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, and ADAMT. The library according to claim 19 or 20, wherein the protease cleavage site is a protease cleavage site of a protease selected from the group consisting of 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 according to any one of claims 19 to 21, wherein the first and second cleavage sites are different.

23. The CM has a second linker (L ２ The library according to any one of claims 19 to 22, further comprising:

24. Said L ２ The library of claim 23, wherein said amino acid sequence is selected from the group consisting of SEQ ID NOs: 17 to 24.

25. The polypeptide has the formula (III): EVGSYX １ X ２ X ３ X ４ X ５ X ６ C.X. ７ X ８ X ９ X １０ X １１ X １２ C.X. １３ X １４ SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:3), １ is A, D, I, N, P, or Y; X ２ is A, F, N, S, or V; X ３ is A, H, L, P, S, V, or Y; X ４ is A, H, S, or Y; X ５ is A, D, P, S, V, or Y; X ６ is A, D, L, S, or Y; X ７ is D, P, or V; X ８ is A, D, H, P, S, or T; X ９ is A, D, F, H, P, or Y; X １０ is L, P, or Y; X １１ is F, P, or Y; X １２ is A, P, S, or Y; X １３ is A, D, N, S, T, or Y; X １４ The library according to any one of claims 1 to 24, wherein is A, S, or Y.

26. 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 any one of claims 1 to 26, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 46.

28. The library of any one of claims 1 to 27, wherein the TBM comprises an antibody light chain variable region.

29. 29. The library of claim 28, wherein the polypeptides further comprise a heavy chain variable region C-terminal to the light chain variable region.

30. 29. The library of claim 28, further comprising polynucleotides encoding one or more antibody heavy chain variable regions.

31. The library of any one of claims 1 to 27, wherein the TBM comprises an antibody heavy chain variable region.

32. 32. The library of claim 31 , wherein the polypeptides further comprise a light chain variable region C-terminal to the heavy chain variable region.

33. The library of claim 31 , further comprising a polynucleotide encoding one or more antibody light chain variable regions.

34. The library of any one of claims 1 to 33, wherein at least one of the polynucleotides encoding the polypeptides is in a vector.

35. 35. The library of claim 34, wherein the vector is an expression vector or a display vector.

36. The library of any one of claims 1 to 35, wherein at least one of the polynucleotides encoding the polypeptides is in a cell.

37. 37. The library of claim 36, wherein the cells are bacterial cells, yeast cells, insect cells, or mammalian cells.

38. 38. A method for producing an activatable antibody, comprising culturing a cell according to claim 36 or claim 37 under conditions suitable for the production of said activatable antibody.

39. 39. The method of claim 38, further comprising recovering the activatable antibody produced by the cells.

40. 40. The method of claim 39, further comprising testing the activatable antibody for its ability to remain soluble while maintaining an activatable phenotype.

41. A method for screening for activatable antibodies that bind to a target using a library according to any one of claims 1 to 37, comprising the steps of: 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 has been cleaved; and c) isolating one or more of the expression products that bind to the target after the CM is cleaved, but have reduced binding to the target before the CM is cleaved.

42. 42. The method of claim 41, wherein an expression product is isolated if the binding affinity of the expression product after the CM is cleaved is at least twice the binding affinity of the expression product before the CM is cleaved.

43. The CM is 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, cass.

43. The method of claim 41 or 42, comprising at least one first protease cleavage site of a protease selected from the group consisting of 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.

44. The method of any one of claims 41 to 43, wherein the target is CTLA4 or CD137.

45. A polypeptide encoded by one or more polynucleotides from the library of any one of claims 1 to 37.

46. A kit comprising the library according to any one of claims 1 to 37.

47. 1. A library comprising antigen-binding domains, at least one of the antigen-binding domains comprising a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM); The FP is represented by formula (XIII): ｍ C.X. ｎ C.X. ｏ (SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and 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; the CM comprises at least a first cleavage site; The library, wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region.

48. 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); The FP is represented by formula (XIII): ｍ C.X. ｎ C.X. ｏ (SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and 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; the CM comprises at least a first cleavage site; 48. The library of claim 47, wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region.

49. each of said antigen binding domains comprises, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM); The FP is represented by formula (XIII): ｍ C.X. ｎ C.X. ｏ (SEQ ID NO:86), wherein m is 2-10, n is 3-10, o is 1-10, and 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; the CM comprises at least a first cleavage site; 49. The library of claim 47 or claim 48, wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region.

50. 50. The library of any one of claims 47 to 49, wherein the TBM comprises an antibody light chain variable region and the antigen binding domain further comprises an antibody heavy chain variable region.

51. 50. The library of any one of claims 47 to 49, wherein the TBM comprises an antibody heavy chain variable region and the antigen binding domain further comprises an antibody light chain variable region.

52. 52. The library of any one of claims 47 to 51, wherein each X is not M, W, or C.

53. X in formula (XIII) ｍ wherein each X 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. X in formula (XIII) ｎ wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

55. X in formula (XIII) ｏ wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

56. The library of any one of claims 47 to 55, wherein each of the antigen-binding domains is displayed on a cell surface or on a phage.

57. 1. 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), The FP is represented by the formula (I): X ｍ C.X. ｎ CZ ｏ wherein m is 2-10, n is 3-10, o is 1-10, 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, and 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; the CM comprises at least a first cleavage site; The antibody light chain, wherein the TBM comprises an antibody light chain variable region.

58. 58. An antibody comprising a heavy chain and a light chain, wherein the light chain is the light chain described in claim 57.

59. 1. 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), The FP is represented by the formula (I): X ｍ C.X. ｎ CZ ｏ wherein m is 2-10, n is 3-10, o is 1-10, 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, and 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; the CM comprises at least a first cleavage site; The antibody heavy chain, wherein the TBM comprises an antibody heavy chain variable region.

60. 60. An antibody comprising a heavy chain and a light chain, wherein the heavy chain is the heavy chain described in claim 59.

61. A cell comprising at least one polypeptide displayed on a surface, said at least one polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM); The FP is represented by the formula (I): X ｍ C.X. ｎ CZ ｏ wherein m is 2-10, n is 3-10, o is 1-10, 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, and 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; the CM comprises at least a first cleavage site; The cell, wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region.

62. 62. The cell of claim 61, wherein the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

63. 63. The cell of claim 61 or claim 62, wherein each X is not M, W, or C.

64. X in formula (I) ｍ wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

65. X in formula (I) ｎ wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

66. 1. 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); The MM is represented by the formula (I): X ｍ C.X. ｎ CZ ｏ wherein m is 2-10, n is 3-10, o is 1-10, 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, and 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, and wherein the MM inhibits binding of the activatable antibody to human CD137 when the CM is uncleaved; the CM comprises at least a first cleavage site; 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); or 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); or c) the TBM comprises, from N-terminus to C-terminus, an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); or d) the TBM comprises, from N-terminus to C-terminus, an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL); The activatable antibody binds to human CD137 via the VH and VL when the CM is cleaved.

67. 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. 68. The activatable antibody of claim 66 or claim 67, wherein m is 2 or 3-6.

69. 69. The activatable antibody of any one of claims 66 to 68, wherein n is 6 to 8.

70. 70. The activatable antibody of claim 69, wherein n is 6.

71. 70. The activatable antibody of any one of claims 66-69, wherein o is 1 or 2.

72. 72. The activatable antibody of any one of claims 66-71, wherein each X is not M, W, or C.

73. X in formula (I) ｍ 73. The activatable antibody of any one of claims 66-72, wherein each X 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. X in formula (I) ｎ 74. The activatable antibody of any one of claims 66-73, wherein each X is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

75. The MM is selected from the group consisting of SEQ ID NOs: 79 to 85, and X ｍ CADAPNHCXX (SEQ ID NO: 88), X ｍ CHHSPANCXX (SEQ ID NO: 89), X ｍ CPILRHRCXX (SEQ ID NO: 90), X ｍ CKWRPSRCXX (SEQ ID NO:91), X ｍ CRVLPRRCCXX (SEQ ID NO:92), X ｍ CLWRHRSCXX (SEQ ID NO: 93), and X ｍ 75. The activatable antibody of any one of claims 66-74, comprising an amino acid sequence selected from the group consisting of CPRLRRKCXX (SEQ ID NO:94), where m is 2 to 10 and 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 any one of claims 66-75, wherein the MM further comprises an additional amino acid sequence at its N-terminus.

77. 77. The activatable antibody of claim 76, wherein the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO:

16.

78. The first cleavage site is 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, and 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 CM has a first linker (L １ 79. The activatable antibody of any one of claims 66-78, further comprising:

80. Said L １ The activatable antibody of claim 79, wherein said antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24.

81. The activatable antibody of any one of claims 66-80, wherein the CM further comprises a second cleavage site.

82. The second cleavage site is １ The activatable antibody of claim 81, wherein the C-terminus of

83. The second cleavage site is 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, and ADAMT.

83. The activatable antibody of claim 81 or 82, wherein the cleavage site is a protease cleavage site for a protease selected from the group consisting of 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. 84. The activatable antibody of any one of claims 81 to 83, wherein the first and second cleavage sites are different.

85. The CM has a second linker (L ２ 85. The activatable antibody of any one of claims 81-84, further comprising:

86. Said L ２ The activatable antibody of claim 85, wherein said antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24.

87. The CM comprises at least a first protease cleavage site and is 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, ADA, 87. The activatable antibody of any one of claims 66 to 86, which is cleaved by one or more proteases selected from the group consisting of M12, 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.

88. The activatable antibody has the formula (III): EVGSYX １ X ２ X ３ X ４ X ５ X ６ C.X. ７ X ８ X ９ X １０ X １１ X １２ C.X. １３ X １４ SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:3), １ is A, D, I, N, P, or Y; X ２ is A, F, N, S, or V; X ３ is A, H, L, P, S, V, or Y; X ４ is A, H, S, or Y; X ５ is A, D, P, S, V, or Y; X ６ is A, D, L, S, or Y; X ７ is D, P, or V; X ８ is A, D, H, P, S, or T; X ９ is A, D, F, H, P, or Y; X １０ is L, P, or Y; X １１ is F, P, or Y; X １２ is A, P, S, or Y; X １３ is A, D, N, S, T, or Y; X １４ The activatable antibody of any one of claims 66-87, wherein is A, S, or Y.

89. The activatable antibody of any one of claims 66-88, 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 any one of claims 66 to 89, wherein the VL comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:

70.

91. The activatable antibody of any one of claims 66 to 90, wherein the VL comprises the amino acid sequence of SEQ ID NO:

50.

92. The activatable antibody of any one of claims 66 to 91, wherein the VH comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 65, HVR-H2 having the amino acid sequence of SEQ ID NO: 66, and HVR-H3 having the amino acid sequence of SEQ ID NO:

67.

93. The activatable antibody of any one of claims 66 to 92, wherein the VH comprises the amino acid sequence of SEQ ID NO:

49.

94. A method of treating cancer or delaying the progression of cancer in a subject in need thereof, said method comprising administering to the subject an effective amount of a polypeptide encoded by one or more polynucleotides from a library described in any one of claims 1-37, or an activatable antibody described in any one of claims 66-93.

95. 95. The method of claim 94, further comprising administering to the subject an effective amount of at least one additional therapeutic agent.

96. 96. The method of claim 95, wherein the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, and chemotherapy.

97. The at least one additional therapeutic agent is selected from the group consisting of pomalyst, revlimid, lenalidomide, pomalidomide, thalidomide, DNA alkylating platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, and anti-KIR receptor.

97. The method of claim 95 or claim 96, wherein the therapeutic agent is selected from the group consisting of an antibody, an anti-OX40 antibody, an anti-HER2 antibody, an anti-ErbB-2 antibody, an anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFNα, GM-CSF, a chimeric antigen receptor, adoptively transferred T cells, an anti-cancer vaccine, and an oncolytic virus.

Images