Activatable antibodies and methods of making and using the same

Abstract

Provided herein are libraries containing synthetic polynucleotides encoding activatable binding polypeptides. Also provided herein are activatable binding polypeptides and polypeptide libraries containing the activatable binding polypeptides. Also provided herein are vectors, vector libraries, cells, kits, and methods for preparing and using activatable polypeptide libraries.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of international application No. PCT / CN2018 / 075065, filed on February 2, 2018, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing Submission Using ASCII Text Files

[0004] The following submission, made as an ASCII text file, is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 695402000641SEQLIST.TXT, Record Date: January 31, 2019, Size: 54KB). Technical Field

[0005] The present disclosure relates to polynucleotides and polynucleotide libraries that can be used to screen and / or identify one or more precision / context-dependent activatable binding polypeptides (e.g., activatable antibodies), as well as polypeptides and polypeptide libraries that can be used to screen and / or identify precision / context-dependent activatable binding polypeptides (e.g., activatable antibodies that can bind to CTLA4 or CD137 when in active form), cells, methods, and kits related thereto. Background Art

[0006] Activatable binding polypeptides exhibit an "activatable" conformation such that the antigen-binding portion contained in the activatable binding polypeptide is less susceptible to binding to its target when not cleaved 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 able to bind to their targets under certain circumstances (e.g., in a tumor microenvironment rich in proteases). Although many interesting activatable binding polypeptides have been developed, the process of developing such proteins is slow, labor-intensive, and expensive. Therefore, there is a need for improved methods and products that can be used to identify self-blocking peptides for activatable binding polypeptides.

[0007] 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 to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] To meet the above and other needs, disclosed herein are libraries of polynucleotides that can be used, for example, to screen and / or identify activatable binding polypeptides (i.e., activatable antibodies). The present disclosure is based, at least in part, on the discovery that the polypeptides described herein exhibit significantly improved masking efficiency prior to activation, thereby allowing for better design, screening, and / or identification of activatable binding polypeptides (i.e., activatable antibodies) with superior therapeutic index and safety profiles. The present disclosure is also based, at least in part, on the surprising discovery that polynucleotide libraries described herein can be successfully constructed and screened to identify activatable binding polypeptides (see Examples 1 and 2 below). Disclosed herein are precise / context-dependent activatable binding polypeptides that bind to human CTLA4 (see Example 3) or human CD137 (see Example 5) when in active form, but not in inactive form, i.e., they bind to their targets (when in active form) only after cleavage of the cleavable moiety (CM) to remove the first peptide (FP) (i.e., the masking moiety (MM) or self-blocking peptide). The first peptides (FP) discovered herein (e.g., masking moieties) are capable of efficiently masking antibody activity and / or reducing or completely inhibiting antigen binding, while, in some embodiments, lacking chemically labile residues methionine and / or tryptophan. Furthermore, the activatable antibodies identified using the polynucleotide libraries described herein are as effective as their parent antibodies in treating a variety of cancer types, while also having significantly reduced cytotoxicity even in susceptible animals (NOD mice, see Example 4).

[0009] Thus, in one aspect, provided herein is a library comprising polynucleotides, wherein at least one of the polynucleotides encodes a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the FP comprises a polypeptide according to formula (XIII): m CX n CX o (SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the polynucleotides in the library encode at least two, at least three, at least four, at least five, at least ten, at least 50, at least 100, at least 500, at least 1000 unique polypeptides, and each unique polypeptide comprises, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): X m CX n CXo (SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that may be combined with any of the previous 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), wherein the FP comprises an amino acid according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that may be combined with any of the previous embodiments, FP consists of a polypeptide comprising a polypeptide according to formula (XIV): (NNK) m TGY(NNK) n TGY(NNK) o The polynucleotide sequence encodes the nucleic acid sequence of (SEQ ID NO: 87), wherein each N is independently A, G, T or C, wherein each K is independently T or G, and wherein each Y is independently T or C.

[0010] 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) 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. In some embodiments that may be combined with any of the previous embodiments, X of formula (XIII) n 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. In some embodiments that may be combined with any of the previous embodiments, X of formula (XIII) oIn 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 that may be combined with any of the previous embodiments, m is 6. In some embodiments that may be combined with any of the previous embodiments, m is 2-5, e.g., 2, 3, 4, or 5. In some embodiments that may be combined with any of the previous embodiments, n is 6-8. In some embodiments that may be combined with any of the previous embodiments, n is 6. In some embodiments that may be combined with any of the previous embodiments, o is 1-2. In some embodiments that may be combined with any of the previous embodiments, o is 2. In some embodiments that may be combined with any of the previous embodiments, 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.

[0011] In some embodiments that may be combined with any of the preceding embodiments, the first cleavage site is a protease cleavage site of 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, In some embodiments, the CM further comprises a first linker (L1) at the C-terminus of 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, the CM further comprises a second cleavage site. In some embodiments, the second cleavage site is at the C-terminus of L1. In some embodiments, the second cleavage site is a protease cleavage site of 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, casperase-1, casperase-2, casperase-3, casperase-4, casperase-5, casperase-6, casperase-7, casperase-8, casperase-9, casperase-10, casperase-11, casperase-12, casperase-13, casperase-14, and TACE. In some embodiments, the first cleavage site and the second cleavage site are different. In some embodiments that may be combined with any of the previous embodiments, the CM further comprises a second linker (L2) at the C-terminus of 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 previous embodiments, the CM further comprises a linker N-terminal to the first cleavage site.

[0012] In some embodiments that may be combined with any of the preceding embodiments, the polypeptide encoded by one or more polynucleotides in the library comprises a polypeptide according to formula (III): EVGSYX1X2X3X4X5X6CX7X8X9X 10 X 11 X 12 CX 13 X 14 The first peptide (FP) and the cleavable moiety (CM) of the amino acid sequence of 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, X 10 Is L, P or Y, X 11 Is F, P or Y, X 12 A, P, S or Y, 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 previous embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-46.

[0013] In some embodiments that may be combined with any of the previous embodiments, the TBM comprises an antibody light chain variable region. In some embodiments, the TBM further comprises a heavy chain variable region at the C-terminus of 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 can bind to the target in the absence of a masking moiety (MM).

[0014] In some embodiments that may be combined with any of the previous embodiments, the TBM comprises an antibody heavy chain variable region. In some embodiments, the TBM further comprises a light chain variable region at the C-terminus of 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 can bind to the target in the absence of a masking moiety (MM).

[0015] In some embodiments that may be combined with any of the previous embodiments, at least one of the polypeptides encoded in the polynucleotide is in a vector. In some embodiments, the vector is an expression vector or a display vector. In some embodiments that may be combined with any of the previous embodiments, at least one of the polypeptides encoded in the polynucleotide is in a cell. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0016] Other aspects of the present disclosure relate to a method for producing an activatable antibody, the method comprising culturing any of the cells described herein under conditions suitable for producing the activatable antibody. In some embodiments, the method further comprises recovering the activatable antibody produced by the cell. In some embodiments, the method further comprises testing the ability of the activatable antibody to maintain an activatable phenotype while being soluble.

[0017] Other aspects of the present disclosure relate to a method for screening for activatable antibodies that bind to a target using any of the libraries described herein, the method comprising the steps of: a) contacting the expression products of the library with the target before cleaving the CM, b) contacting the expression products of the library with the target after cleaving the CM, and c) isolating one or more of the expression products that bind to the target after cleaving the CM, but do not bind to the target before cleaving the CM. Also provided herein is a method for screening for activatable antibodies that bind to a target using any of the libraries described herein, the method comprising the steps of: a) contacting the expression products of the library with the target before cleaving the CM, b) contacting the expression products of the library with the target after cleaving the CM, and c) isolating one or more of the expression products that bind to the target after cleaving the CM, but have a reduced binding affinity for the target before cleaving the CM compared to the binding affinity after cleaving the CM. In some embodiments, if the K of the expression product before cleaving the CM is D is the K of the expression product after cleavage of CM DThe expression product is isolated if the expression product is at least 2-fold (e.g., at least 5-fold, at least 10-fold, at least 15-fold or more) greater than that of the expression product. In some embodiments, the CM comprises at least a first protease cleavage site, which is a 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.

[0018] Other aspects of the present disclosure relate to a polypeptide encoded by one or more polynucleotides of any of the libraries described herein, or a library having polypeptides encoded by one or more polynucleotides of any of the libraries described herein.

[0019] Other aspects of the disclosure relate to a kit comprising any of the libraries described herein.

[0020] Other aspects of the present disclosure relate to a library comprising antigen binding domains, wherein at least one of the antigen binding domains comprises a polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the FP comprises a peptide according to formula (XIII): X m CX n CX o(SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, at least two, at least three, at least four, at least five, at least ten, at least 50, at least 100, at least 1000 of the antigen binding domains comprise a unique polypeptide comprising, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the FP comprises according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that may be combined with any of the previous embodiments, each of the antigen binding domains comprises, from N-terminus to C-terminus, a unique polypeptide comprising a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the FP comprises according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments that may be combined with any of the previous 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 may be combined with any of the previous 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 may be combined with any of the previous embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the previous embodiments, X in formula (XIII) 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. In some embodiments that may be combined with any of the previous embodiments, X of formula (XIII) n 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. In some embodiments that may be combined with any of the previous embodiments, X of formula (XIII) o 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, each of the antigen binding domains in the library is displayed on a phage or a cell (e.g., a yeast cell).

[0021] Other aspects of the present disclosure relate 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 the FP comprises a polypeptide according to 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, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region. Other aspects of the present disclosure relate to an antibody comprising a heavy chain and a light chain, wherein the light chain is any antibody light chain described herein. In some embodiments that may be combined with any of the previous embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the previous embodiments, X in formula (XIII) is not M, W, or C. 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.

[0022] Other aspects of the present disclosure relate 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 the FP comprises a polypeptide according to 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, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody heavy chain variable region. Other aspects of the present disclosure relate to an antibody comprising a heavy chain and a light chain, wherein the heavy chain is any antibody heavy chain described herein. In some embodiments that may be combined with any of the previous embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the previous embodiments, X in formula (XIII) is not M, W, or C. 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.

[0023] Other aspects of the present disclosure relate to a cell comprising at least one polypeptide displayed on its surface, wherein the at least polypeptide comprises, from N-terminus to C-terminus, a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the FP comprises a polypeptide according to 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, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region and / or an antibody heavy chain variable region. 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 previous embodiments, each X is not M, W, or C. In some embodiments that may be combined with any of the previous 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. In some embodiments that may be combined with any of the previous embodiments, X of formula (I) 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.

[0024] Other aspects of the present disclosure relate 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 comprises a polypeptide according to 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, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 through the VH and the VL. 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 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. In some embodiments that may be combined with any of the previous embodiments, X of formula (I) nEach X in MM 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 previous 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 previous 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 previous embodiments, VL comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments that may be combined with any of the previous 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 previous embodiments, VH comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, m is 3-10.

[0025] Other aspects of the present disclosure relate 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 comprises a polypeptide according to 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, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits binding of the activatable antibody to human CD137; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH); and wherein when the CM is cleaved, the activatable antibody binds to human CD137 through the VH and the VL. 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 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. In some embodiments that may be combined with any of the previous embodiments, X of formula (I) nwherein 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 previous 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 previous 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 previous embodiments, VL comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments that may be combined with any of the previous 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 previous embodiments, VH comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, m is 3-10.

[0026] Other aspects of the present disclosure relate to a polynucleotide encoding any activatable antibody described herein. In other aspects, the present disclosure relates to a vector comprising any polynucleotide described herein (e.g., a polynucleotide encoding an activatable antibody). In some embodiments, the vector is an expression vector and / or a display vector. In other aspects, the present disclosure relates to a host cell comprising any polynucleotide and / or vector described herein (e.g., a polynucleotide and / or vector encoding an activatable antibody). 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 present disclosure relates to a method for preparing an activatable antibody, the method comprising culturing any host cell described herein under conditions suitable for producing the antibody or activatable antibody. In some embodiments, the method further comprises recovering the antibody or activatable antibody produced by the cell.

[0027] Other aspects of the present disclosure relate to a method for treating a cancer of a subject in need or delaying the progress of the cancer, the method comprising administering to the subject an effective amount of a polypeptide encoded by one or more polynucleotides from any library described herein and / or any activatable antibody described herein (e.g., an activatable antibody for human CTLA4 or an activatable antibody for 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, at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiotherapy, 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 derivative 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-dose radiation, fractionated radiation, focal radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anticancer vaccines and oncolytic viruses.

[0028] It should be understood that one, some, or all of the properties 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 become readily apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram showing an exemplary selection process for self-blocking peptides using Fab fragments of target antibodies displayed on the surface of yeast.

[0030] Figure 2Schematic diagram showing an exemplary selection process for self-blocking peptides using scFv fragments of target antibodies displayed on the surface of yeast.

[0031] Figure 3A-3B Functional display of CTLA4-targeting Fab and scFv on yeast was shown as determined by flow cytometry. Figure 3A Functional display of CTLA4-targeting Fabs on the surface of yeast was demonstrated. Figure 3B Functional display of a scFv targeting CTLA4 on the surface of yeast was demonstrated.

[0032] Figure 4 An exemplary selection process for activatable antibodies targeting human CTLA4 is shown. A yeast library displaying the fusion protein was subjected to several rounds of FACS-based screening.

[0033] Figure 5A-5B Shown are the CTLA4 binding affinities of exemplary CTLA4-activatable antibody clones, as determined by flow cytometry. Figure 5A The binding affinity of the CTLA4-activatable antibody clones in scFv format is shown compared to the scFv fragment of the target antibody without the masking peptide, including cases where the masking peptide of the CTLA4-activatable antibody clone B13287 remains intact or where the masking peptide is cleaved by TEV protease. Figure 5B The CTLA4 binding affinity of the CTLA4-activatable antibody clones in Fab format is shown compared to the Fab fragment of the target antibody without the masking peptide, including the case where the masking peptide of the CTLA4-activatable antibody clone B13189 remains intact or the case where the masking peptide is cleaved by TEV protease.

[0034] Figure 6A-6B Shown are the masking efficiencies of exemplary CTLA4-activatable antibodies TY22401, TY22403, TY22402, and TY22404 compared to the parent antibody TY21580. Figure 6A Shown are association and dissociation curves for the indicated activatable antibodies compared to the parental antibody TY21580, as determined by the ForteBio system. Figure 6B Graph showing the relative ratio of bound activatable antibody compared to the parental antibody TY21580.

[0035] Figures 7A-7C Shown are the masking efficiencies of exemplary CTLA4-activatable antibodies directed against recombinant human CTLA4-Fc, as determined by ELISA. Figure 7AShown are the first ELISA data indicating binding of CTLA4-activatable antibodies TY22401, TY22402, TY22403, TY22404 to recombinant human CTLA4-Fc compared to the parent antibody TY21580. Figure 7B Shown are second batch ELISA data indicating binding of CTLA4-activatable antibodies TY22401, TY22402, TY22403, TY22404 to recombinant human CTLA4-Fc compared to the parent antibody TY21580. Figure 7C The results showed that compared with the parent antibody TY21580, the CTLA4-activating antibodies TY22563, TY22564, TY22565, and TY22566 bound to recombinant human CTLA4-Fc.

[0036] Figure 8A-8B It was shown that after removal of the masking peptide, CTLA4 could activate the activity of the antibody TY22404. Figure 8A Shown are the results of SDS-PAGE of the activatable antibody TY22404 left untreated, treated with the protease uPA, or treated with 5 or 10 units of the protease MMP-9. Figure 8B Shown is the binding of the activatable antibody TY22404 left untreated, treated with the protease uPA, or treated with the protease MMP-9 compared to the parental antibody TY21580, as measured by ELISA.

[0037] Figures 9A-9C Shown are size exclusion chromatography (SEC) profiles of exemplary activatable antibodies under accelerated stress conditions. Figure 9A Shown is the SEC profile of the activatable antibody TY22402 after six freeze and thaw cycles compared to control conditions. Figure 9B Shown is the SEC profile of the activatable antibody TY22402 after seven days at 50°C compared to control conditions. Figure 9C Shown are the percentages of the SEC main peak area for exemplary activatable antibodies after seven days at 50°C, after up to 28 days of storage at 40°C, or after six freeze and thaw cycles compared to control conditions.

[0038] Figure 10 Shown are the percentages of the main SEC peak areas for activatable antibodies TY22401 and TY22402 after storage at approximately 8 mg / mL or at >150 mg / mL.

[0039] Figure 11 Shown are the masking efficiencies of untreated activatable antibodies TY21580, TY22401, TY22402, and TY22566 incubated at pH 3.7 for 30 minutes or at pH 3.7 for 1 hour, as determined by the ForteBio system.

[0040] Figures 12A-12B Shown are human peripheral blood mononuclear cell (PBMC) activation by an isotype control antibody, parental antibody TY21580, or exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22404, as measured by ELISA. Figure 12A Shown are the effects on IL-2 secretion from CD3-primed human PBMCs stimulated with an isotype control antibody, parental antibody TY21580, and exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22404. Figure 12B Shown are the effects on IFNγ secretion from CD3-primed human PBMCs stimulated with an isotype control antibody, parental antibody TY21580, and exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22404.

[0041] Figure 13 Shown are antibody-dependent cell-mediated cytotoxicity (ADCC) activity of an isotype control antibody, parental 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.

[0042] Figures 14A-14B In vivo anti-tumor efficacy of parental antibody TY21580, an isotype control antibody, or exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22566 in the MC38 syngeneic mouse colorectal tumor model is shown. Figure 14A Figure 5 Tumor growth curves of different treatment groups of female C57BL / 6 mice bearing MC38-established tumors. Data points represent group means; error bars represent SEM. Figure 14B Individual tumor growth curves are shown for groups treated with TY21580, TY22401, TY22402, and TY22566.

[0043] Figure 15 In vivo antitumor efficacy of an isotype control antibody, the parental antibody TY21580, or one of three activatable antibodies in the CT26 syngeneic mouse colorectal tumor model is shown. Tumor growth curves for the different treatment groups of female C57BL / 6 mice bearing established CT26 tumors are shown. Data points represent group means; error bars represent SEM.

[0044] Figure 16In vivo antitumor efficacy of an isotype control antibody, the parental antibody TY21580, or one of three activatable antibodies in the H22 syngeneic mouse liver tumor model is shown. Tumor growth curves for the different treatment groups of female C57BL / 6 mice bearing H22-established tumors are shown. Data points represent group means; error bars represent SEM.

[0045] Figures 17A-17B In vivo anti-tumor efficacy of parental antibody TY21580, an isotype control antibody, and exemplary activatable antibodies TY22401, TY22402, or TY22566 in the 3LL syngeneic mouse lung tumor model is shown. Figure 17A Figure 5 Tumor growth curves of different treatment groups of female C57BL / 6 mice bearing 3LL-established tumors. Data points represent group means; error bars represent SEM. Figure 17B Individual tumor growth curves are shown for groups treated with TY21580, TY22401, TY22402, and TY22566.

[0046] Figures 18A-18C Shown is the time course of blood concentrations of the test article (TA) administered intravenously to female BALB / c mice at a concentration of 10 mg / kg, as determined by ELISA. Figure 18A Shown is the time course of blood concentrations of the activatable antibody TY22401 administered intravenously to female BALB / c mice at a concentration of 10 mg / kg compared to the parental antibody TY21580. Figure 18B Shown is the time course of blood concentrations of the activatable antibody TY22402 administered intravenously to female BALB / c mice at a concentration of 10 mg / kg compared to the parental antibody TY21580. Figure 18C Shown is the time course of blood concentrations of the activatable antibody TY22404 administered intravenously to female BALB / c mice at a concentration of 10 mg / kg compared to the parental antibody TY21580.

[0047] Figure 19 Repeated dose toxicity using the NOD mouse model is shown for an isotype control antibody, parental antibody TY21580, and exemplary activatable antibodies TY22566, TY22401, and TY22402. The percent survival over 20 days is shown for each treatment group.

[0048] Figure 20A-Figure 20B Functional display of Fab and scFv targeting human CD137 on yeast as determined by flow cytometry was shown. Figure 20A Functional display of a CD137-targeting scFv on the surface of yeast was demonstrated. Figure 20B Functional display of Fabs targeting CD137 on the surface of yeast is shown.

[0049] Figure 21 An exemplary selection process for activatable antibodies targeting human CD 137 is shown. A yeast library displaying fusion proteins was subjected to several rounds of FACS-based screening.

[0050] Figures 22A-22B Shown are the CD137 binding affinities of exemplary CD137-activatable antibody clones, as determined by flow cytometry. Figure 22A The binding affinity of the CD137-activatable antibody clones in scFv format is shown compared to the scFv fragment of the target antibody without the masking peptide, including cases where the masking peptide of the CD137-activatable antibody clone B13428 remains intact or where the masking peptide is cleaved by TEV protease. Figure 22B The CD137 binding affinity of the CD137-activatable antibody clones in scFv format is shown compared to the scFv fragment of the target antibody without the masking peptide, including cases where the masking peptide of the CD137-activatable antibody clone B13439 remains intact or where the masking peptide is cleaved by TEV protease.

[0051] Figure 23 Shown are the masking efficiencies of exemplary activatable antibodies against human CD137 compared to the parent antibody TY21242, as determined by flow cytometry.

[0052] Figure 24A and Figure 24B Depicted are the masking efficiencies of exemplary activatable antibodies containing masking peptides of variable length compared to the parent antibody TY21580. Masking efficiency was determined using an ELISA-based method. Figure 24A and Figure 24B Represents two experimental setups using the same experimental method to test various activatable anti-CTLA4 antibodies.

[0053] Figure 25 Depicted are the masking efficiencies of exemplary activatable antibodies containing cleavage peptides of varying lengths compared to the parent antibody TY21580. Masking efficiencies were determined using an ELISA-based method. DETAILED DESCRIPTION

[0054] I. General Technology

[0055] The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using routine methods, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003)); the book series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR 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 (edited by JECellis, 1998) Academic Press; Animal Cell Culture (edited by RIFreshney, 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) PlenumPress; Cell and Tissue Culture: Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (eds. DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 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 (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, 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 JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0056] II. Definitions

[0057] Before describing the present disclosure in detail, it is to be understood that the present 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.

[0058] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more of those molecules, and so on.

[0059] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments related to that value or parameter itself.

[0060] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0061] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used herein in phrases such as "A, B, and / or C" is intended to encompass 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 (alone); B (alone); and C (alone).

[0062] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, but the C-terminal carboxyl group, the N-terminal amino group, or a side chain functional group has been chemically modified to another functional group. The term "amino acid mimetic" refers to a compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to the naturally occurring amino acids.

[0063] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See, for example, Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0064] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

[0065] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. Nucleotides may 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. Polynucleotides may contain modified nucleotides such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may contain one or more modifications performed after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, 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 intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of one or more polynucleotides. In addition, any hydroxyl group typically present in a sugar can be replaced, for example, with a phosphonate group, a phosphate group, protected with standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping groups having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to obtain standard protecting groups. Polynucleotides may also contain analogs of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-ribose; 2'-O-allyl-ribose; 2'-fluoro-ribose or 2'-azido-ribose; carbocyclic sugar analogs; α-anomeric sugars; epimeric sugars such as arabinose, xylose, or lyxose; pyranose; furanose; sedoheptulose; acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("methylal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl group (1-20 C) optionally containing an ether (-O-) linkage, an aryl, an alkenyl, a cycloalkyl, a cycloalkenyl, or an aralkyl group. Not all linkages in a polynucleotide need be identical.The preceding description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0066] 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 present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes the separation of the nucleic acid molecule 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 target nucleic acid.

[0067] As used herein, a "library" refers to a collection of two or more entities that share a common class. For example, a library containing polynucleotides may refer to a collection of two or more polynucleotides. The term "library" is used herein in the broadest sense and explicitly encompasses sublibraries that may or may not be combined.

[0068] As used herein, "unique" refers to a member of a set that is different from the other members of the set. For example, a unique activatable antibody in a library may refer to an activatable antibody having a specific 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 implementation of a library may exist in more than one copy. For example, a library may contain a plurality of "unique" activatable antibodies, wherein one or more of the "unique" activatable antibody molecules are present in more than one copy.

[0069] As used herein, "diversity" refers to variety and / or heterogeneity. For example, the diversity of antibodies in a library can refer to the presence of multiple antibodies with unique sequences in the library.

[0070] 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.

[0071] In some embodiments, the term "antibody" refers to an antigen-binding protein (i.e., an immunoglobulin) having 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 an H chain by a covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain has a variable region (abbreviated herein as V at the N-terminus). H ), followed by the constant region. The heavy chain constant region contains three domains, namely C H1、C H2 and C H3 Each light chain has a variable region (abbreviated herein as V I ), followed by a constant region at its other end. The light chain constant region comprises one domain, the C L . V L With V H Align, and C L Aligned with the first constant domain (CH1) of the heavy chain. H and V L IgM antibodies are composed of five basic heterotetrameric units plus an additional polypeptide called a J chain and therefore contain 10 antigen-binding sites, while secretory IgA antibodies can polymerize to form multivalent aggregates containing 2-5 basic four-chain units plus a J chain.

[0072] V H Area and V L The V domains can be further subdivided based on structural and sequence analysis into regions of high variability, termed hypervariable regions (HVRs). HVRs are interspersed with more conserved regions, termed framework regions (FWs) (see, e.g., Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V domain has a high variability. H and V L It consists of three HVRs and four FWs arranged in the following order from amino terminus to carboxyl terminus: 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.

[0073] The variable region of heavy and light chains contains a binding domain that interacts with an antigen. The constant region of an antibody can mediate immunoglobulin binding 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 light and heavy chains, the variable region and constant region are engaged by a "J" region with about 12 or more amino acids, wherein the heavy chain also includes a "D" region with about 10 or more amino acids (see, e.g., Fundamental Immunology Chapter 7 (Paul, W. ed., 2nd edition Raven Press, NY). (1989)).

[0074] L chains from any vertebrate species can be assigned to one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, antibodies can be assigned to different classes or isotypes. There are five antibody classes: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG antibody class can be further classified into four subclasses based on the γ heavy chains Y1-Y4: IgG1, IgG2, IgG3, and IgG4, respectively.

[0075] 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 an antibody include (i) a Fab fragment, i.e., a fragment consisting of V L 、V H 、C L and C H1 (ii) F(ab')2 fragments, which are bivalent fragments consisting of two Fab fragments connected by a disulfide bridge at the hinge region; (iii) H and C H1 (iv) an Fd fragment consisting of a single-arm V L and V H Fv fragments composed of V and V domains, (v)dAb fragments (Ward et al., Nature 341: 544-546 (1989)), which are composed of V H domain composition; and (vi) isolated complementarity determining regions (CDRs).

[0076] The term "CTLA4" is used in this application and includes 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), dog 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, the binding molecule may be completely specific for human CTLA4 and may not exhibit species cross-reactivity or other types of cross-reactivity.

[0077] The term "CD137" is used in this application and includes human CD137 (e.g., GenBank accession numbers NM_001561; NP_001552) as well as variants, isoforms, and species homologs thereof (e.g., mouse CD137 (GenBank gene identifier 21942), rat CD137 (GenBank gene identifier 500590), dog CD137 (GenBank gene identifier 608274), cynomolgus monkey CTLA4 (GenBank gene identifier 102127961), etc.). Thus, binding molecules (e.g., activatable antibodies) can also bind to CD137 from species other than human. In other cases, the binding molecule may be completely specific for human CD137 and may not exhibit species cross-reactivity or other types of cross-reactivity.

[0078] The term "chimeric antibody" refers to antibodies comprising amino acid sequences derived from different animal species, such as those having variable regions derived from human antibodies and murine immunoglobulin constant regions.

[0079] The term "competitive binding" refers to the interaction between two antibodies when they bind to a binding target. A first antibody competes with a second antibody for binding if, in the presence of a second antibody, binding of a first antibody to its cognate epitope is detectably reduced compared to binding of the first antibody in the absence of the second antibody. An alternative scenario in which binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may be, but need not be, true. That is, a first antibody may inhibit binding of a second antibody to its epitope without the second antibody inhibiting binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits binding of the other antibody to its cognate epitope, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to one or more of their corresponding epitopes.

[0080] The term "epitope" refers to the portion of an antigen that is bound by an antibody (or its antigen-binding fragment). An epitope can be formed by adjacent amino acids or by the tertiary folding of a protein and adjacent non-adjacent amino acids. Epitopes formed by continuous amino acids are usually retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are usually lost when treated with a denaturing solvent. An epitope may include various numbers of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods combined with computational modeling of the composite structure of the antigen and its binding antibody and its variants (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, ed. (1996)). Once the desired epitope of an antigen is determined, antibodies against that epitope can be generated, for example, using technology as described herein. The generation and characterization of antibodies can also illustrate information about desirable epitopes. Based on this information, it is then possible to competitively screen for antibodies that bind to the same epitope. One method for achieving this is to conduct cross-competition studies to find antibodies that competitively bind to each other, i.e., antibodies compete for binding to the antigen. A high-throughput method for "classifying" antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0081] The term "germline" refers to the nucleotide sequence of antibody genes and gene segments as they are passed from parent to offspring through germ cells. Germline sequences differ from the nucleotide sequences encoding antibodies in mature B cells, which have been altered by recombination and hypermutation events during the process of B cell maturation.

[0082] The term "glycosylation site" refers to an amino acid residue recognized by eukaryotic cells as the attachment position for a sugar residue. The amino acids at which carbohydrates such as oligosaccharides are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. The specific attachment site is typically indicated by an amino acid sequence 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 the attachment site between a sugar molecule and an amino acid residue. N-linked sugars are attached via an amino group; O-linked sugars are attached via a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety attached to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on a polypeptide, none (0 glycan site occupancy), one (1 glycan site occupancy), or both (2 glycan site occupancy) sites may be occupied by a carbohydrate moiety.

[0083] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, for example, mammalian cultured cells such as CHO, BHK, NSO, SP2 / 0, YB2 / 0 derived from rodents (rat, mouse, guinea pig or hamster); human cells, such as HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (such as S2 cells), bacterial cells (such as Escherichia coli (E. coli) cells) and cells contained in transgenic animals or cultured tissues. The term not only covers specific subject cells, but also covers the progeny of such cells. Because certain modifications may occur in succession due to mutations or environmental influences, the progeny may not be identical to the parent cell, but is still included within the scope of the term "host cell".

[0084] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0085] The term "humanized antibody" refers to a chimeric antibody containing 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, while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0086] The term "illustrative antibody" refers to any of the antibodies described herein. These antibodies may be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above encompasses antibodies in all five classes with the same V L Area and V H Furthermore, antibodies in the IgG class may be in any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each of the antibodies identified above as being in the IgG subclass encompasses antibodies in all four subclasses having the same V L Area and V H The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes and in the four IgG subclasses are known in the art. The amino acid sequences of the full-length heavy and light chains of the IgG4 subclass for each of the illustrative antibodies shown in Table 1b are provided in this disclosure.

[0087] An "isolated" antibody or binding molecule is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0088] The term "k a ” refers to the association rate constant for a specific antibody-antigen interaction, while the term “k d ” refers to the dissociation rate constant for a specific antibody-antigen interaction.

[0089] The term "K D ” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. It is represented by k d With k a The ratio (i.e. k d / k a ) is obtained and expressed as molar concentration (M). K D Used as a measure of the affinity of the binding of an antibody to its binding partner. D The smaller the K, the tighter 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 more tightly to a specific antigen than an antibody with a micromolar (μM) dissociation constant. D The K value can be determined using methods well established in the art. D The method is by using ELISA. For example, the determination procedure using ELISA is described in at least Example 3 of the present disclosure.

[0090] The term "mammal" refers to any animal species of the class Mammalia. Examples of mammals include humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; domestic animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0091] The terms "prevent" or "preventing" with respect to a disease condition in a mammal refer to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms of the disease.

[0092] As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. The amino acid sequence identity of a polypeptide can be routinely 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 Bestfit or any other sequence alignment program is used to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are set so that the percentage identity is calculated over the full length of the reference amino acid sequence, and spaces in the homology are allowed to account for up to 5% of the total number of amino acid residues in the reference sequence. This method mentioned above when determining the percent identity between polypeptides can be applied to all proteins, fragments or variants thereof disclosed herein.

[0093] As used herein, the terms "bind," "specifically bind," 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 biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In one embodiment, the extent to which an antibody binds 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, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0094] The terms "treat", "treating", and "treatment" with respect to a disease condition in a mammal refer to causing a desirable or beneficial effect in the mammal having the disease condition. A desirable 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 the number and / or activity of immune cells, etc.), or the arrest or inhibition of further development of the disease, disorder, or condition. In the case of treating cancer in a mammal, a desirable or beneficial effect may include inhibiting the further growth or spread of cancer cells, causing cancer cell death, inhibiting the recurrence of cancer, alleviating pain associated with cancer, or improving the survival of the mammal. The effect may be subjective or objective. For example, if the mammal is a human, the human may notice improved energy or vitality or reduced pain as a subjective sign of improvement or response to therapy. Alternatively, a clinician may perceive a decrease in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or radiographic studies. Some laboratory signs of response to treatment that clinicians may observe include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme level tests. In addition, clinicians may observe a decrease in detectable tumor markers. Alternatively, other tests may be used to assess objective improvement, such as ultrasonography, magnetic resonance imaging, and positron emission tomography.

[0095] The term "vector" refers to a nucleic acid molecule capable of transporting an external nucleic acid molecule. The external nucleic acid molecule is connected to the carrier nucleic acid molecule by recombinant techniques such as connection or recombination. This allows breeding, selection, further manipulation or expression of the external nucleic acid molecule in a host cell or organism. The vector can be a plasmid, phage, transposon, cosmid, chromosome, virus or virion. One type of vector can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome (e.g., non-episomal mammalian vector). Another type of vector can replicate autonomously in the host cell it introduces (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Another specific type of vector capable of guiding the expression of the expressible external nucleic acid to which they are operatively connected is commonly referred to as an "expression vector." An expression vector typically has a control sequence that drives the expression of the expressible external nucleic acid. The simpler vectors referred to as "transcription vectors" can only be transcribed rather than translated: they can be replicated rather than expressed in the target cell. The term "vector" encompasses all types of vectors, regardless of their function. Vectors that are capable of directing the expression of expressible nucleic acids to which they are operatively linked are generally referred to as "expression vectors." Other examples of "vectors" may include display vectors (e.g., vectors that direct the expression and display of encoded polypeptides on the surface of viruses or cells (such as bacterial cells, yeast cells, insect cells, and / or mammalian cells)).

[0096] As used herein, "subject," "patient," or "individual" may refer to a human or non-human animal. "Non-human animal" may refer to any animal that is not classified as a human, such as domestic, farm, or zoo animals, sports animals, pet animals (such as dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer to, without limitation, 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.

[0097] "Effective amount" refers to at least the following quantity: at the necessary dose and for the necessary period, the quantity effectively achieves one or more desired or indicated effects including treatment or prevention results. The effective amount can be provided by one or more administrations. For the purposes of this disclosure, the effective amount of an antibody, drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment. As understood in clinical situations, the effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition (e.g., as an effective amount administered as a monotherapy or combination therapy). Therefore, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and if, in combination with one or more other agents, a desirable result can be achieved or achieved, then a single agent can be considered to be given in an effective amount.

[0098] III. Activatable Binding Polypeptide Libraries and Library Generation

[0099] 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, e.g., activatable binding polypeptides, including activatable antibodies, activatable antigen-binding fragments thereof, or derivatives of activatable antibodies, that can be used to screen and / or identify one or more activatable binding polypeptides (i.e., one or more activatable antibodies).

[0100] The terms "activatable binding polypeptide," "ABP," or "activatable antibody" include polypeptides 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 antibody fragment thereof. In some embodiments, the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH and the VL form a binding domain that binds to a target in the absence of a MM. In some embodiments, VH and VL are covalently linked, for example, in scFv. In some embodiments, VH and VL form a Fab fragment. In some embodiments, VH is connected to the antibody heavy chain constant region, and VL is connected to the antibody light chain constant region.

[0101] In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-cleavable moiety (CM)-VL, and the activatable antibody further comprises a second polypeptide comprising VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-cleavable moiety (CM)-VH, and the activatable antibody further comprises a second polypeptide comprising VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-cleavable moiety (CM)-VH-VL (e.g., scFv).

[0102] In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL, and the activatable antibody further comprises a second polypeptide comprising VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL-L3-VH (e.g., a scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-cleavable moiety (CM)-L1-VH, and the activatable antibody further comprises a second polypeptide comprising VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising the following structure from the N-terminus to the C-terminus: masking moiety (MM)-L1-cleavable moiety (CM)-L2-VH-L3-VL (e.g., a scFv). In some embodiments, L1, L2, and / or L3 are linkers. In some embodiments, each of L1, L2, and L3 is a linker that can have an independently selected length of 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.

[0103] CMs typically include amino acid sequences that are cleavable, e.g., serve as substrates for enzymes, and / or cysteine-cysteine pairs capable of forming reducible disulfide bonds. Thus, when the terms "cleavage," "cleavable," "cleaved," and the like are used in connection with CMs, the terms encompass enzymatic cleavage, e.g., by proteases, as well as disruption of disulfide bonds between cysteine-cysteine pairs by reduction of the disulfide bonds, which can be caused by exposure to a reducing agent.

[0104] The MM refers to the amino acid sequence of the MM that interferes with or inhibits the binding of the TBM to its target when the CM of the activatable antibody is intact (e.g., not cleaved by the corresponding enzyme and / or contains unreduced cysteine-cysteine disulfide bonds). In some embodiments, the MM interferes with or inhibits the binding of the TBM to its target so effectively that the binding of the TBM to its target is extremely low and / or below the limit of detection (e.g., binding cannot be detected in an ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with the MM or be included within the MM. It should be noted that, for convenience, "ABP" or "activatable antibody" is used herein to refer to the ABP or activatable antibody in its uncleaved (or "native") state as well as in its cleaved state. It will be apparent to one of ordinary skill that, in some embodiments, the cleaved ABP may lack the MM due to, for example, cleavage of the CM by a protease, which results in the release of at least the MM (e.g., when 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 more detail below.

[0105] The libraries of the present disclosure may contain one or more polynucleotides encoding any polypeptide 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 the libraries described herein encode polypeptides comprising one or more full-length antibody light chains and / or heavy chains. In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of the libraries described herein encode polypeptides comprising one or more light and / or heavy chain Fab fragments. In some embodiments, one or more (i.e., one, some, or all) of the polynucleotides of the libraries described herein encode polypeptides comprising one or more single-chain variable fragments (scFv).

[0106] Other aspects of the present disclosure relate to libraries of polypeptides (e.g., any polypeptides described herein) and / or polypeptides that can be used to screen and / or identify one or more activatable binding polypeptides (i.e., one or more activatable antibodies), including activatable antibodies, their activatable antigen-binding fragments, or derivatives of activatable antibodies. The library of the present 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 (e.g., one, some, or all) of the polypeptides of the library described herein comprise one or more full-length antibody light chains and / or heavy chains. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the library described herein comprise one or more light and / or heavy chain Fab fragments. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the library described herein comprise one or more single-chain variable fragments (scFv). In some embodiments, the polypeptides are expressed on the surface of a cell (e.g., displayed by yeast or mammalian cells).

[0107] In some embodiments, the polypeptides of the present disclosure comprise: (a) a first peptide (FP); (b) a cleavable moiety (CM); and (c) a target binding moiety (TBM). In some embodiments, FP is any first peptide described herein (e.g., comprising a peptide according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and 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. In some embodiments, X is not W, M, and / or C. In some embodiments, X of formula (XIII) m Each X in the 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 the 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, and / or X o 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, m is 3-10. In some embodiments, FP is any first peptide described herein (e.g., comprising a peptide according to 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 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 the 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, and / or X 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, FP is any first peptide described herein (e.g., comprising a peptide according to formula (XII): Z m CZ n CZ o (SEQ ID NO: 71), wherein m is 2-10, n is 3-10, and 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, CM is any cleavable moiety described herein (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site). In some embodiments, CM is any cleavable moiety described herein (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site). In some embodiments, TBM is any target binding moiety described herein (e.g., a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region).

[0108] In some embodiments, the first peptide (FP) interferes with, hinders the target binding portion from binding to its target, reduces the ability of the target binding portion to bind to its target, prevents, inhibits the target binding portion from binding to its target, or competes with the target binding portion for binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the first peptide (FP) interferes with, hinders, reduces, prevents, inhibits the target binding portion from binding to its target, or competes with the target binding portion for binding to its target only when the polypeptide has not yet been activated (e.g., activated by a pH change (increase or decrease), activated by a temperature change (increase or decrease), 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 portion. In some embodiments, activation induces a conformational change in the polypeptide (e.g., a shift in the first peptide (FP)), resulting in the first peptide no longer preventing the activatable antibody from binding to its target. In some embodiments, the first peptide (FP) interferes with, hinders, reduces the ability of the target binding moiety to bind to its target, prevents, inhibits, or competes with the target binding moiety for binding to its target only when the cleavable moiety (CM) has not been cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, prior to activation, 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.). In some embodiments, masking efficiency is measured as the difference in affinity of an activatable antibody comprising a first peptide (FP) binding to its target (before activation) relative to the affinity of a polypeptide lacking the first peptide binding to its target (e.g., the difference in affinity of an activatable antibody comprising a first peptide (FP) for a target antigen (such as CTLA4) relative to a parent antibody lacking the first peptide (FP), or the difference in affinity of an activatable antibody comprising a first peptide (FP) for a target antigen (such as CTLA4) (before activation) relative to the affinity of the activatable antibody for the target antigen after activation). In some embodiments, masking efficiency is measured by measuring the affinity of an activatable antibody comprising a first peptide (FP) for binding to an activatable antibody comprising a first peptide (FP). 50 (before activation) divided by the EC of the parent antibody 50 to measure (e.g., by measuring EC using ELISA) 50; See, e.g., the method of Example 3). In some embodiments, the masking efficiency is measured as the difference in affinity of an activatable antibody comprising a first peptide (FP) before activation to bind to its target relative to the affinity of an activatable antibody comprising the first peptide (FP) after activation to bind to its target (e.g., the difference in affinity of an activatable antibody before activation relative to the activatable antibody after activation for a target antigen (such as CTLA4)). In some embodiments, the first peptide (FP) binds to the 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.

[0109] In some embodiments, the first peptide (FP) does not interfere with, hinder, reduce the ability of the target binding moiety (TBM) to bind to its target, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target after the polypeptide has been activated (e.g., by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a change in pH (increase or decrease), by a shift in temperature (increase or decrease), upon contact with a second molecule (such as an enzyme), etc.). In some embodiments, the first peptide (FP) does not interfere with, hinder, reduce the ability of the target binding moiety (TBM) to bind to its target, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target after the cleavable moiety (CM) has been cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, after activation, the first peptide (FP) has a masking efficiency (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.).

[0110] In some embodiments, the polypeptides of the present disclosure comprise the following structure from N-terminus to C-terminus: first peptide (FP)-cleavable moiety (CM)-target binding moiety (TBM). The libraries of the present disclosure can be used to screen for one or more activatable binding polypeptides (i.e., activatable antibodies) that bind to any target of interest when in active form, 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.

[0111] In some embodiments, the disclosed libraries contain genes encoding at least one, 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 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 The invention also provides a plurality of polynucleotides encoding a unique polypeptide as described herein, the polypeptide comprising: (a) a first peptide (FP); (b) a cleavable moiety (CM); and (c) a target binding moiety (TBM).

[0112] In some embodiments, the disclosed libraries contain genes encoding at least one, 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 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 1010 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 The invention provides a plurality of polynucleotides for a polypeptide as described herein, wherein the polypeptide comprises: (a) a unique first peptide (FP); (b) a cleavable moiety (CM); and (c) a target binding moiety (TBM).

[0113] In some embodiments, the libraries of the present disclosure: 1) encode and / or contain a number of unique peptides less than the number found in a typical random peptide library (e.g., comprising peptides according to formula (XIII): X m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o (FP of the amino acid sequence of SEQ ID NO: 1); 2) encoding and / or containing a peptide comprising a pair of cysteine residues at fixed positions to ensure that the displayed peptide has a constrained conformation (e.g., comprising a peptide according to formula (XIII): X m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o (SEQ ID NO: 1)); and / or 3) encoding and / or containing a peptide with few or no chemically labile residues (such as methionine or tryptophan) (e.g., comprising a peptide according to formula (XIII): X m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o Advantageously, the libraries disclosed herein have significantly reduced library sizes relative to random peptide libraries, thereby enabling the construction of peptides with much better characterization (e.g., comprising a peptide according to formula (XIII): X m CX n CX o (SEQ ID NO: 86) or formula (I): X m CX n CZ o(SEQ ID NO: 1)). In addition, the inclusion of a pair of cysteine residues at fixed positions ensures that the displayed peptides have a constrained conformation, thereby tending to exhibit increased binding affinity and / or specificity. In addition, the disclosed libraries have peptides (e.g., comprising peptides according to formula (XIII): XIII) that include little to no residues that are detrimental 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 of the amino acid sequence of SEQ ID NO: 1)).

[0114] In some embodiments, the libraries of the present disclosure contain a plurality of polynucleotides, wherein at least one of the polynucleotides in the library encodes a polypeptide comprising: (a) comprising a polypeptide according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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, 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, the library of the present disclosure contains a plurality of polynucleotides, wherein at least one of the polynucleotides in the library encodes a polypeptide comprising: (a) comprising according to 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, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; (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 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).

[0115] In some embodiments, at least one of the polynucleotides in the library encodes a sequence comprising a polypeptide according to formula (III): EVGSYX1X2X3X4X5X6CX7X8X9X 10 X 11 X 12 CX 13 X 14 A polypeptide having the amino acid sequence of 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, X 10 Is L, P or Y, X 11 Is F, P or Y, X 12 A, P, S or Y, 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).

[0116] 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).

[0117] In some embodiments, 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 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 The polypeptide comprising: (a) comprising a polypeptide according to formula (XIII): m CX n CX o(SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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, 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 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 The polypeptide encoding the following: (a) comprising according to 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, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; (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 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).

[0118] In some embodiments, 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 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 The encoding comprises according to formula (III) EVGSYX1X2X3X4X5X6CX7X8X9X 10 X 11 X 12 CX 13 X 14A polypeptide having the amino acid sequence of 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, X 10 Is L, P or Y, X 11 Is F, P or Y, X 12 A, P, S or Y, 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).

[0119] In some embodiments, 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 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 In some embodiments, the polynucleotides in the library encode 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).

[0120] In some embodiments, each of the polynucleotides in the library encodes a polypeptide comprising: (a) comprising a polypeptide according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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) comprising according to 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, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; (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 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).

[0121] In some embodiments, each of the polynucleotides in the library encodes a sequence comprising a polypeptide according to formula (III): EVGSYX1X2X3X4X5X6CX7X8X9X 10 X 11 X 12 CX 13 X 14A polypeptide having the amino acid sequence of SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 3), wherein X1 is A, D, I, N, P, or Y, X2 is A, F, N, S, or V, X3 is A, H, L, P, S, V, or Y, X4 is A, H, S, or Y, X5 is A, D, P, S, V, or Y, X6 is A, D, L, S, or Y, X7 is D, P, or V, X8 is A, D, H, P, S, or T, X9 is A, D, F, H, P, or Y, X10 is L, P, or Y, X11 is F, P, or Y, X12 is A, P, S, or Y, X13 is A, D, N, S, T, or Y, and X14 is A, S, or Y. 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).

[0122] 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).

[0123] In some embodiments, the disclosed libraries contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (a) a polynucleotide comprising: m CX n CX o(SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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); c) a target binding moiety (TBM) comprising an antibody light chain variable region; and d) an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, the libraries of the present disclosure contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (i) a polypeptide comprising: m CX n CZ o(SEQ ID NO: 1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from D, A, Y, S, T, N, I, L, F, V, H, and P; (b) a cleavable moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a target binding moiety (TBM) comprising an antibody light chain variable region; and d) an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, the polypeptide comprises the following structure from N-terminus to C-terminus: 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 VL and VH (i.e., the structure VL-linker-VH). The linker sequence can be any linker sequence known in the art, such as any linker sequence described herein. In some embodiments, the linker sequence is GGGGS (SEQ ID NO: 17) at any number of copies (e.g., repeated 2 times, repeated 3 times, etc.).

[0124] In some embodiments, the disclosed libraries contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (a) a polynucleotide comprising: m CX n CX o(SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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); c) a target binding moiety (TBM) comprising an antibody light chain variable region, and the library further comprises one or more polynucleotides encoding an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, the libraries of the present disclosure contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (i) a polypeptide comprising: m CX n CZ o(SEQ ID NO: 1) wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y amino acids, and wherein each Z is independently selected from D, A, Y, S, T, N, I, L, F, V, H and P amino acids; (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 the library further comprises one or more polynucleotides encoding an antibody heavy chain variable region. In some embodiments, m is 3-10. In some embodiments, the polynucleotide encoding the polypeptide comprising the target binding portion (TBM) comprising the 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 (ie, an activatable antibody) when coupled to an antibody heavy chain variable region.

[0125] In some embodiments, the disclosed libraries contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (a) a polynucleotide comprising: m CX n CX o(SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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); c) a target binding moiety (TBM) comprising an antibody heavy chain variable region; and d) an antibody light chain variable region. In some embodiments, m is 3-10. In some embodiments, the activatable binding polypeptide comprises a polypeptide comprising the following structure from N-terminus to C-terminus: first peptide (FP)-cleavable moiety (CM)-VH-VL. In some embodiments, the libraries of the present disclosure contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (i) a polypeptide comprising: m CX n CZ o(SEQ ID NO: 1), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; (b) a cleavable moiety (CM) comprising at least a first cleavage site (e.g., at least a first protease cleavage site); c) a target binding moiety (TBM) comprising an antibody heavy chain variable region; and d) an antibody light chain variable region. In some embodiments, m is 3-10. In some embodiments, the activatable binding polypeptide comprises a polypeptide comprising, from N-terminus to C-terminus, the following 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 VH and VL (i.e., the structure VH-linker-VL). The linker sequence can be any linker sequence known in the art, such as any linker sequence described herein. In some embodiments, the linker sequence is GGGGS (SEQ ID NO: 17) at any number of copies (e.g., repeated 2 times, repeated 3 times, etc.).

[0126] In some embodiments, the disclosed libraries contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (a) a polynucleotide comprising: m CX n CX o(SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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); c) a target binding moiety (TBM) comprising an antibody heavy chain variable region, and the library further comprises one or more polynucleotides encoding an antibody light chain variable region. In some embodiments, m is 3-10. In some embodiments, the libraries of the present disclosure contain at least one (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 (a) a polynucleotide encoding a polypeptide comprising: (i) a polypeptide comprising: m CX n CZ o(SEQ ID NO: 1) wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently 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 amino acids, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P amino acids; (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 the library further comprises one or more polynucleotides encoding an antibody light chain variable region. In some embodiments, m is 3-10. In some embodiments, the polynucleotide encoding the polypeptide comprising the target binding portion (TBM) comprising the 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 (ie, an activatable antibody) when coupled to an antibody light chain variable region.

[0127] The polynucleotides and / or polynucleotide libraries described herein may be combined with any HVR sequence (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) of any antibody described herein (e.g., an anti-CTLA4 antibody, an anti-CD137 antibody), a heavy chain variable region sequence, and / or a light chain variable region sequence. The polynucleotides and / or polynucleotide libraries described herein may also be combined with any HVR sequence (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) described in PCT Application No. PCT / CN2017 / 098333 (incorporated herein by reference in its entirety) and / or PCT Application No. PCT / CN2017 / 098299 (incorporated herein by reference in its entirety), a heavy chain variable region sequence, a light chain variable region sequence, a heavy chain, and / or a light chain.

[0128] In some embodiments, the libraries of the present disclosure include one or more vectors (eg, expression vectors and / or display vectors) encoding one or more polynucleotides (eg, synthetic polynucleotides) of the present disclosure.

[0129] Also provided herein is a method for preparing a library, for example, by providing and assembling polynucleotide sequences (e.g., one or more synthetic polynucleotides) of the library disclosed herein. Also provided herein is a method for preparing a library, for example, by selecting a plurality (e.g., at least one, at least two, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 105 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 or at least 10 19 In some embodiments, the present invention provides a library of polynucleotides encoding a plurality of polypeptides, comprising: 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 generate 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).

[0130] The polynucleotide encoding a polypeptide as described herein can be cloned into any suitable vector to express a part or the entire polypeptide sequence. In some embodiments, the polynucleotide is cloned into a vector, thereby allowing the production of all or a portion of (i.e., creating a fusion protein) fused to 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), and displayed on a part or the entire polypeptide on the surface of a particle or a cell. Several types of vectors are available and can be used to implement the present disclosure, such as phagemid vectors. Phagemid vectors typically contain multiple components, including promoters, signal sequences, phenotypic selection genes, replication origin sites, and other essential components as known to those of ordinary skill in the art. In some embodiments, the polynucleotide encoding polypeptide region can be cloned into a vector to express in bacterial cells to achieve bacterial display, or to express in yeast cells to achieve yeast display. Exemplary vectors are described in U.S. Publication No. US20160145604 before granting. In some embodiments, the vector is a display vector comprising a polynucleotide encoding an amino acid sequence to be displayed on a surface (e.g., a surface of a phage, bacteria, yeast, insect, or mammalian cell), a restriction site, a second polynucleotide encoding a surface peptide capable of being displayed on the surface, and a second restriction site from 5' to 3'. In some embodiments, the second polynucleotide encodes a phage coat protein, a yeast exine protein (such as Aga2), a bacterial outer membrane protein, a cell surface tethered domain, or a linker, or a truncated form or derivative thereof. In some embodiments, the surface peptide is used for phage display, yeast display, bacterial display, insect display, or mammalian display, or for shuttle display therebetween. In some embodiments, when expressed, the amino acid sequence and surface peptide are displayed on the surface as a fusion protein. In some embodiments, the vector further comprises a fusion tag at 5' of the first restriction site or at 3' of the second restriction site.

[0131] Certain aspects of the present disclosure relate to a cell colony containing one or more vectors as described herein. Polypeptides encoded by polynucleotides produced by any technology described herein or other suitable technologies can be expressed and screened to identify activatable binding polypeptides with desired structure and / or activity. Expression of 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, cells are bacterial cells, yeast cells, insect cells or mammalian cells (such as Chinese hamster ovary (CHO) cells). Methods for transfecting bacterial cells, yeast cells or mammalian cells are known in the art and are described in the references cited herein. Expression of polypeptides (e.g., one or more activatable binding polypeptides) and screening of target activatable binding polypeptides in these cell types (e.g., from a library disclosed herein) are described in more detail below.

[0132] Alternatively, the polynucleotide 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 into the culture medium and / or into the 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 in a bicistronic construct so that they can be expressed from a single vector and secreted into the periplasmic space of E. coli, where they are refolded and recovered in an active form (Skerra et al., Biotechnology, 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 expressed in parallel with an antibody heavy chain gene to produce a polypeptide of interest.

[0133] In other embodiments, the polypeptide sequences of the present disclosure are expressed on the membrane surface of prokaryotes, such as E. coli, using secretion signals and lipidation moieties as described in, for example, US20040072740; US20030100023; and US20030036092.

[0134] Alternatively, the polypeptide sequences of the present disclosure can be expressed and screened by anchored periplasmic expression (APEx two-hybrid surface display) as described, for example, in Jeong et al., PNAS, 2007, 104:8247, or by other anchored methods as described, for example, in Mazor et al., Nature Biotechnology, 2007, 25:563.

[0135] Higher eukaryotic cells, such as mammalian cells, for example myeloma cells (e.g., NS / 0 cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, can also be used to express the polypeptides of the present disclosure. Polypeptides (e.g., activatable binding polypeptides) expressed in mammalian cells can be designed to be secreted into the culture medium or expressed on the surface of the cells.

[0136] In other embodiments, 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, polypeptides (e.g., activatable binding polypeptides) can be selected after, for example, phage display is used to produce all or a portion of a viral coat protein (i.e., to produce a fusion protein) and displaying a portion or the entire polypeptide sequence on the surface of a particle or cell.

[0137] Certain aspects of the present disclosure relate to a non-human animal comprising a polynucleotide or polynucleotide library of the present disclosure. For example, the non-human animal of the present disclosure can be modified so that its genome includes a polynucleotide encoding a polypeptide of the present disclosure (e.g., an activatable binding polypeptide). In some embodiments, a transgenic animal (e.g., a mouse) expresses a polypeptide encoded by the polynucleotide. Techniques for modifying the genome of a non-human animal are known in the art (e.g., for producing a Xenomouse TM method).

[0138] Screening of activatable binding polypeptides obtained from the libraries of the present disclosure can be performed by any appropriate means (e.g., assaying target binding before and after activation, such as treatment of the polypeptide with one or more proteases that cleave sequences within the cleavable moiety (CM)). For example, binding activity can be assessed by standard immunoassays and / or affinity chromatography. Screening of the polypeptides of the present disclosure for catalytic function, such as proteolytic function, can be accomplished using standard assays, such as hemoglobin plaque assays. Determination of the binding affinity of a polypeptide (e.g., an activatable binding polypeptide) for a target can be determined in vitro using a variety of well-known techniques, such as ELISA, BIACORE, which measures the binding rate of a protein to a given target based on surface plasmon resonance. TM Instruments, or if using ForteBio The RED96 platform (Pall Life Sciences) uses biolayer interferometry (BLI) as exemplified below. In vivo assays can be performed using any of a number of animal models, followed by subsequent testing in humans when appropriate. Cell-based bioassays are also contemplated. Polypeptides (e.g., activatable binding polypeptides) can be further selected for functional activity, such as antagonistic or agonistic activity. For example, in some embodiments, BLI is used to measure the binding affinity between a polypeptide comprising one or more fab fragments and one or more targets by tagging the antigen with a human IgG1-Fc tag and capturing it with an anti-hIgG-Fc capture (AHC) biosensor (e.g., before and after activation). The polypeptide can be tagged with a His6 tag at the C-terminus of the CH1 domain, overexpressed in host cells such as E. coli, and purified, for example, using Ni-NTA resin. Affinity can then be measured (e.g., before and after activation) using an AHC sensor (anti-human IgG-Fc capture dip and read biosensor) dipped into wells containing purified Fab-containing polypeptide diluted with kinetic buffer, e.g., to 5-10 μg / mL.

[0139] After the binding agent is identified (e.g., by determining that the polypeptide is able to bind to the target or antigen when it is "active" (e.g., after treatment with a protease) but not when it is "inactive" (e.g., before treatment with a protease), nucleic acid can be extracted. The extracted DNA can then be used directly to transform E. coli host cells, or the coding sequence can be amplified, for example, using PCR with appropriate primers and sequenced by any typical sequencing method. The DNA sequence of the binding agent can be subjected to restriction enzyme digestion and then inserted into a vector for protein expression.

[0140] First peptide (FP)

[0141] In some embodiments, the present disclosure relates to polynucleotides and / or polynucleotide libraries encoding one or more polypeptides comprising a first peptide (FP). In some embodiments, the present 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) comprises a polypeptide according to formula (XIII): m CX n CX o(SEQ ID NO: 86), wherein m is 2-10 (e.g., 3-10), n is 3-10, and o is 1-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In some embodiments, m is 3-10. In some embodiments, X is not W, M, and / or C. In some embodiments, X of formula (XIII) m Each X in the 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 the 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, and / or X o Each X in the formula (XIV) 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 comprises a molecule according to formula (XIV): (NNK) m TGY(NNK) n TGY(NNK) o (SEQ ID NO: 87), wherein each N is independently A, G, T or C, wherein each K is independently T or G, and wherein each Y is independently T or C, and wherein each H is independently A, T or C.

[0142] In some embodiments, the first peptide (FP) comprises a peptide according to 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, and o is 1-10, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. 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 the 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, and X 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, FP comprises a compound according to formula (II): (NNK)m TGY(NNK) n TGY(NHC) o (SEQ ID NO: 2), wherein each N is independently A, G, T or C, wherein each K is independently T or G, wherein each Y is independently T or C, and wherein each H is independently A, T or C.

[0143] In some embodiments, the first peptide (FP) comprises according to formula (XII): Z m CZ n CZ o (SEQ ID NO: 71), wherein m is 2-10 (e.g., 3-10), n is 3-10, and 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.

[0144] In some embodiments, m is 2-5, 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, m is 6-8. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6.

[0145] 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.

[0146] 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.

[0147] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (IV): Z6CX6CZ2 (SEQ ID NO: 55), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0148] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (V): Z6CX8CZ2 (SEQ ID NO: 56), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0149] 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.

[0150] 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.

[0151] In some embodiments, the first peptide (FP) comprises an amino acid sequence selected from the group consisting of: X mCADAPNHCXX (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 m CPRLRRKCXX (SEQ ID NO: 94), wherein m is 2-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 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 portion (TBM) comprises the sequence of one or more of the anti-CD137 antibodies described herein, including antibodies described with respect to specific amino acid sequences of 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 a full-length antibody light chain of one or more of the anti-CD137 antibodies described herein.

[0152] In some embodiments, the first peptide (FP) comprises an amino acid sequence selected from the group consisting of: X m CPDHPYPCXX (SEQ ID NO: 102), X n CDAFYPYCXX (SEQ ID NO: 103), Xm CDSHYPYCXX (SEQ ID NO: 104) and X m CVPYYYACXX (SEQ ID NO: 105), wherein m is 2-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 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.

[0153] In some embodiments, the first peptide (FP) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 72-85.

[0154] In some embodiments, any first peptide (FP) 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 (such as his tags, flag tags, maltose binding protein, and glutathione-S-transferase tags), detection tags (such as tags detectable photometrically (e.g., red or green fluorescent proteins, etc.), tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.), tags containing secretion sequences, leader sequences, and / or stabilization 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).

[0155] In some embodiments, the first peptide is a masking peptide that binds to the target binding moiety (TBM) and inhibits the polypeptide from binding to its target before activation (e.g., before treatment with one or more proteases that cleave within the cleavable moiety (CM), before being subjected to a (local) pH change (increase or decrease), before being exposed to a temperature shift (increase or decrease), before being contacted with a second molecule (such as a small molecule or protein ligand), etc.), but does not bind to the TBM and / or inhibits the polypeptide from binding to its target after activation (e.g., after treatment with one or more proteases that cleave within the cleavable moiety (CM), after being subjected to a (local) pH change (increase or decrease), after being exposed to a temperature shift (increase or decrease), after being contacted 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 (i.e., an activatable antibody)) to its target when the CM is not cleaved, but does not inhibit binding of the polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) to its target when 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 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.).

[0156] Cleavable portion (CM)

[0157] In some embodiments, the present disclosure relates to polynucleotides and / or polynucleotide libraries encoding one or more polypeptides comprising a cleavable moiety (CM). In some embodiments, the present disclosure relates to polypeptides and / or polypeptide libraries comprising at least one polypeptide comprising a cleavable moiety (CM).

[0158] In some embodiments, the cleavable moiety (CM) comprises at least a first cleavage site (CS1) (eg, 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 co-localize with the target of the CM-containing polypeptide) can be used, including, for example, protease cleavage sites recognized and / or cleaved by: 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-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., ADAM 8, ADAM 9, ADAM 10, ADAM 11, ADAM 12, ADAM 13, ADAM 14, ADAM 15, ADAM 16, ADAM 17, ADAM 19, ADAM 18, ADAM 19, ADAM 20, ADAM 21, ADAM 22, ADAM 23, ADAM 24, ADAM 25, ADAM 26, ADAM 27, ADAM 28, ADAM 29, ADAM 30, ADAM 31, ADAM 32, ADAM 33, ADAM 34, ADAM 35, ADAM 36, ADAM 37, ADAM 38, ADAM 39, ADAM 31, ADAM 32, ADAM 39, ADAM 31, ADAM 32, ADAM 33, ADAM 34, ADAM 3 9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 and / or ADAMTS5); caspases (e.g., caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13 and / or caspases-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 K, cathepsin L, cathepsin S, cathepsin V / L2 and / or cathepsins X / Z / P); cysteine proteases (e.g., Cruzipain, Legumain, and / or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14); metalloproteases (e.g., Meprin, Neprilysin, PSMA, and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; granzyme B; guanidinobenzoic acidase; HtrA1; human neutrophil elastase;Lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine protease (TTSP) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3 and / or TMPRSS4); etc. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the group consisting of 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 of a protease selected from the group consisting of uPA, MMP-2, MMP-9, and / or TEV protease. In some embodiments, the protease cleavage site comprises an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 13), PLGLAG (SEQ ID NO: 14), and ENLYFQG (SEQ ID NO: 15).

[0159] In some embodiments, the polypeptide comprising a first peptide (FP) and a 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.

[0160] In some embodiments, the polypeptide comprising a first peptide (FP) and a cleavable moiety (CM) comprises an amino acid sequence according to Formula (IX): EVGSY(Z6)C(X6)C(Z2)SGRSA (SEQ ID NO:5), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0161] In some embodiments, the polypeptide comprising a first peptide (FP) and a cleavable moiety (CM) comprises an amino acid sequence according to formula (X): EVGSY(Z6)C(Z8)C(Z2)SGRSA (SEQ ID NO:6), wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P.

[0162] In some embodiments, the polypeptide comprising a first peptide (FP) and a cleavable moiety (CM) comprises an amino acid sequence according to Formula (XI): EVGSY(Z6)C(X8)C(Z2)SGRSA (SEQ ID NO:7), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0163] 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., a first protease cleavage site). In some embodiments, the cleavable moiety (CM) comprises the following structure from N-terminus to C-terminus: (CS1)-L1.

[0164] Any suitable linker known in the art (e.g., a flexible linker) can be used, including, for example: a glycine polymer (G)n, wherein n is an integer of at least 1 (e.g., at least one, 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.); a glycine-serine polymer (GS)n, wherein n is an integer of at least 1 (e.g., at least one, 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); a glycine-alanine polymer; an alanine-serine polymer; etc. The linker sequence can be of any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20 amino acid glycine polymer or a glycine-serine polymer), from about 1 amino acid to about 15 amino acids, from about 3 amino acids to about 12 amino acids, from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, from about 6 amino acids to about 8 amino acids, etc. In some embodiments, the length of 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 some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NOs: 17-24. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NOs: 17 or 18.

[0165] In some embodiments, the cleavable portion (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 portion (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 protease described 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 recognized and / or cleaved by different proteases (e.g., the first protease cleavage site is recognized and / or cleaved by uPA, while 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, while 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, while 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 following structure from N-terminus to C-terminus: (CS1)-L1-(CS2).

[0166] In some embodiments, the cleavable portion (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 portion (CM) further comprises a second linker (L2). The second linker (L2) can be any suitable linker as 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 identical (e.g., both linkers comprise a sequence of SEQ ID NOs: 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, while 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 at the C-terminus of the second cleavage site (CS2). In some embodiments, the cleavable portion (CM) comprises the following structure from N-terminus to C-terminus: (CS1)-L1-(CS2)-L2.

[0167] Exemplary FP-CM sequences

[0168] In some embodiments, the polypeptides of the present disclosure comprise the following structure from N-terminus to C-terminus: (FP)-(PCS1)-L1-(PCS2)-L2. In some embodiments, the polypeptides of the present disclosure comprise the following amino acid sequences: EVGSYDALHYACPPDYYACYYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 25); EVGSYNSYHAYCPHPLYPCTASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 26); EVGSYASSAVLCVTAYFSCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 27); EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 28); EVGSYNFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 29); EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 30); EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 31). NO: 31); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 32); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 33); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 34); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 35); EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 36); EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO:37); EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS(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 present disclosure comprise the following structure from N-terminus to C-terminus: (FP)-(PCS1)-L1-(PCS2)-L2-(TBM). ;

[0169] Target Binding Moiety (TBM)

[0170] In some embodiments, the present disclosure relates to polynucleotides and / or polynucleotide libraries encoding one or more polypeptides comprising a target binding portion (TBM). In some embodiments, the present disclosure relates to polypeptides and / or polypeptide libraries comprising at least one polypeptide comprising a target binding portion (TBM). In some embodiments, the target binding portion (TBM) comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the target binding portion (TBM) comprises an antibody light chain variable region. In some embodiments, the target binding portion (TBM) comprises an antibody heavy chain variable region. In some embodiments, the target binding portion (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 at the C-terminus of the antibody light chain variable region. In some embodiments, the antibody light chain variable region is at the C-terminus of 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 that is specific 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 and / or B7-H4.

[0171] In some embodiments, the target binding moiety (TBM) comprises a full-length antibody light chain and / or a full-length antibody heavy chain. The antibody light chain may be a kappa or lambda light chain. The antibody heavy chain may be in any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is in the 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.

[0172] Any one or more of the target binding moieties (TBMs) described herein may be combined with any HVR sequence (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 sequence, and / or light chain variable region sequence of any antibody described in PCT Application No. PCT / CN2017 / 098333 (incorporated herein by reference in its entirety), PCT Application No. PCT / CN2017 / 098299 (incorporated herein by reference in its entirety), PCT Application No. PCT / CN2017 / 098332 (incorporated herein by reference in its entirety), and / or PCT Application No. 69540-2000540, entitled “Compositions Comprising Cross-reactive Anti-CTLA4 Antibodies, and Methods of Making and Using the Same,” filed concurrently herewith, and incorporated herein by reference in its entirety.

[0173] Any one or more of the target binding moieties (TBMs) described herein may be combined with any HVR sequence (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 sequence, and / or light chain variable region sequence of any antibody described herein (e.g., anti-CTLA4 antibody, anti-CD137 antibody).

[0174] In some embodiments, the target binding portion (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 portion (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 portion (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 an HVR-H1 comprising the amino acid sequence of YSISSGYHWSWI (SEQ ID NO: 59), an HVR-H2 comprising the amino acid sequence of LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 60), and / or an HVR-H3 comprising the amino acid sequence of 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 of RASQSVRGRFLA (SEQ ID NO:62), HVR-L2 comprising the amino acid sequence of DASNRATGI (SEQ ID NO:63), and / or HVR-L3 comprising the amino acid sequence of YCQQSSSWPPT (SEQ ID NO:64); and b) an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising the amino acid sequence of LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and / or HVR-H3 comprising the amino acid sequence of 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.

[0175] In some embodiments, the target binding portion (TBM) comprises the sequence of one or more of the anti-CD137 antibodies described herein, including antibodies described with respect to specific amino acid sequences of HVRs, variable regions (VL, VH) and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding portion (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 portion (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 of FSLSTGGVGVGWI (SEQ ID NO: 65), an HVR-H2 comprising the amino acid sequence of LALIDWADDKYYSPSLKSRL (SEQ ID NO: 66), and / or an HVR-H3 comprising the amino acid sequence of 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 of RASQSIGSYLA (SEQ ID NO:68), HVR-L2 comprising the amino acid sequence of DASNLETGV (SEQ ID NO:69), and / or HVR-L3 comprising the amino acid sequence of YCQQGYYLWT (SEQ ID NO:70); and b) an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of FSLSTGGVGVGWI (SEQ ID NO:65), HVR-H2 comprising the amino acid sequence of LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and / or HVR-H3 comprising the amino acid sequence of 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.

[0176] V. Peptides and Peptide Libraries

[0177] Other aspects of the present disclosure relate to libraries of polypeptides (e.g., any polypeptides described herein) and / or polypeptides that can be used to screen, identify, and / or select one or more activatable binding polypeptides (i.e., one or more activatable antibodies), including activatable antibodies, their activatable antigen-binding fragments, or derivatives of activatable antibodies. The library of the present 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 (e.g., one, some, or all) of the polypeptides of the library described herein comprise one or more antigen-binding domains. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the library described herein comprise one or more full-length antibody light chains and / or heavy chains. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the library described herein comprise one or more light and / or heavy chain Fab fragments. In some embodiments, one or more (e.g., one, some, or all) of the polypeptides of the library described herein comprise one or more single-chain variable fragments (scFv). In some embodiments, the polypeptide is expressed on the surface of a cell (eg, yeast or mammalian cell display).

[0178] In some embodiments, the polypeptides of the present disclosure (e.g., in a library) comprise: (a) a first peptide (FP); (b) a cleavable moiety (CM); and (c) a target binding moiety (TBM). In some embodiments, FP is any first peptide described herein (e.g., comprising a peptide according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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. In some embodiments, FP is any first peptide described herein (e.g., comprising a peptide according to 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 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 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, CM is any cleavable moiety described herein (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site). In some embodiments, TBM is any target binding moiety described herein (e.g., a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region).

[0179] 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 a polypeptide comprising, from N-terminus to C-terminus: (a) a first peptide (FP) (e.g., comprising according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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 (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety 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, from N-terminus to C-terminus, a polypeptide comprising: (a) a first peptide (FP) (e.g., comprising according to 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 selected from 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 selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P); (b) a cleavable moiety (e.g., a cleavable moiety (CM) comprising at least a first protease 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 (e.g., one, some, or all) of the antigen binding domains comprises, from N-terminus to C-terminus, a polypeptide comprising: (a) a first peptide (FP) (e.g., comprising according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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 (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety 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 comprises, from N-terminus to C-terminus, a polypeptide comprising: (a) a first peptide (FP) (e.g., comprising according to 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 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 selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P); (b) a cleavable moiety (e.g., a cleavable moiety (CM) comprising at least a first protease 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.

[0180] In some embodiments, there is also provided herein an antibody fragment or scFv comprising any polypeptide described herein. In some embodiments, the antibody fragment or scFv comprises a polypeptide containing a target binding moiety (TBM) comprising an antibody light chain variable region. In some embodiments, the antibody fragment or scFv comprises a polypeptide containing a target binding moiety (TBM) comprising an antibody heavy chain variable region. In some embodiments, there is provided herein a library with an antibody fragment or scFv, wherein at least one of the antibody fragments or scFv comprises any polypeptide described herein. In some embodiments, at least one (such as one, some or all) of the antibody fragments or scFv in the library comprises a polypeptide containing a target binding moiety (TBM) comprising an antibody light chain variable region. In some embodiments, at least one (such as one, some or all) of the antibody fragments or scFv in the library comprises a polypeptide containing a target binding moiety (TBM) comprising an antibody heavy chain variable region. Also provided herein is a library of cells and / or cells expressing one or more of the antibody fragments and / or scFv described herein on their surfaces.

[0181] 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 a polypeptide comprising, from N-terminus to C-terminus: (a) a first peptide (FP) (e.g., comprising according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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 (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety (TBM) comprising an antibody light chain variable region. In some embodiments, m is 3-10. In some embodiments, the antibody light chain comprises, from N-terminus to C-terminus, a polypeptide comprising: (a) a first peptide (FP) (e.g., comprising according to 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 selected from 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 selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P); (b) a cleavable moiety (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety (TBM) comprising an antibody light chain variable region. In some embodiments, m is 3-10. In some embodiments, the present disclosure relates to a library comprising antibody light chains, wherein at least one (e.g., one, some, or all) of the antibody light chains in the library are antibody light chains as described above. In some embodiments, the present disclosure relates to an antibody 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 antibody heavy chain described herein). In some embodiments, the present disclosure relates to a library comprising antibodies, wherein at least one (e.g., one, some, or all) of the antibodies is an antibody as described above.

[0182] 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 a polypeptide comprising, from N-terminus to C-terminus: (a) a first peptide (FP) (e.g., comprising according to formula (XIII): X m CX n CX o (SEQ ID NO: 86), wherein m is 2-10, n is 3-10, and o is 1-10, and wherein 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 (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site); and (c) 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, from N-terminus to C-terminus, a polypeptide comprising: (a) a first peptide (FP) (e.g., comprising according to 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 selected from 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 selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P); (b) a cleavable moiety (e.g., a cleavable moiety (CM) comprising at least a first protease cleavage site); and (c) a target binding moiety (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 are antibody heavy chains as described above. In some embodiments, the present disclosure relates to an antibody 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 antibody light chain described herein). In some embodiments, the present disclosure relates to a library comprising antibodies, wherein at least one (e.g., one, some, or all) of the antibodies is an antibody as described above.

[0183] The polypeptides of the present disclosure (e.g., any of the antibodies described above) can be produced using, for example, recombinant methods and compositions as described in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids encoding any polypeptide (e.g., any of the antibodies described above) are provided. The nucleic acids may encode V sequences comprising the antibody. L The amino acid sequence and / or V comprising the antibody H In some embodiments, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided herein. In some embodiments, a host cell comprising the nucleic acid is provided. In one embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding a V L The amino acid sequence of the polypeptide of the present disclosure and the amino acid sequence of the polypeptide comprising V H (e.g., an activatable binding polypeptide (i.e., an activatable antibody)), (2) a vector comprising a nucleic acid encoding a vector comprising V H The amino acid sequence of the polypeptide of the present disclosure and the amino acid sequence of the polypeptide comprising V L The amino acid sequence of (eg, an activatable binding polypeptide (ie, an activatable antibody)), (3) comprises a sequence encoding a V L The first vector of the nucleic acid encoding the amino acid sequence of the polypeptide of the present disclosure, and the first vector of the nucleic acid encoding the polypeptide of the present disclosure Hor (4) a second vector comprising a nucleic acid encoding a V H The first vector of the nucleic acid encoding the amino acid sequence of the polypeptide of the present disclosure, and the first vector of the nucleic acid encoding the polypeptide of the present disclosure L In some embodiments, the host cell is eukaryotic, 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 for preparing a polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the polypeptide (e.g., an activatable binding polypeptide (i.e., an activatable antibody)) as provided above 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).

[0184] For recombinant production of polypeptides of the present disclosure (e.g., activatable binding polypeptides (i.e., activatable antibodies)), nucleic acids encoding polypeptides, e.g., as described above (e.g., activatable binding polypeptides (i.e., activatable antibodies)), are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the one or more polypeptides).

[0185] Suitable host cells for cloning or expressing polypeptide-encoding vectors (e.g., encoding an activatable binding polypeptide (i.e., an activatable antibody)) include prokaryotic or eukaryotic cells. For example, polypeptides (e.g., activatable binding polypeptides (i.e., activatable antibodies)) can be produced in bacteria, particularly when glycosylation and Fc effector functions are not desired (see, e.g., U.S. Patent 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, describing expression of antibody fragments in E. coli). Following expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0186] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for polypeptide encoding vectors (e.g., encoding activatable binding polypeptides (i.e., activatable antibodies)), including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0187] Suitable host cells for expressing glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified for use in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0188] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0189] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 strain (COS-7) transformed by SV40; human embryonic kidney strain (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, 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 liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT) 060562); TRI cells, as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for producing antibodies, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0190] VI. Activatable Binding Polypeptides and Their Production

[0191] In some embodiments, provided herein are activatable binding polypeptides (eg, activatable antibodies) screened, identified, and / or selected from any of the polynucleotide and / or polypeptide libraries described herein.

[0192] In some embodiments, the activatable antibodies of the present disclosure are context-dependent (e.g., they are activated (only able to bind to their targets) under certain circumstances, such as in a protease-rich tumor microenvironment). In some embodiments, the activatable antibodies of the present disclosure provide 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 tissue pathology, hematology, and / or blood biochemistry, etc.). In some embodiments, the activatable antibodies of the present disclosure have improved pharmacokinetic properties (e.g., have a longer in vivo half-life) compared to more traditional non-activatable antibodies.

[0193] In some embodiments, the activatable binding polypeptides of the present disclosure comprise: (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 to the target, and / or compared to binding of a parent antibody to the target. In some embodiments, prior to activation, 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.). In some embodiments, masking efficiency is measured as the difference in affinity with which an activatable antibody comprising a masking moiety (MM) binds to its target (before activation) relative to the affinity with which a polypeptide lacking the masking moiety binds to its target (e.g., the difference in affinity for a target antigen (such as CTLA4 or CD137) of an activatable antibody comprising a masking moiety (MM) relative to a parent antibody lacking the masking moiety (MM), or the difference in affinity for a target antigen (such as CTLA4 or CD137) of an activatable antibody comprising a masking moiety (MM) relative to the affinity of the activatable antibody for the target antigen after activation). In some embodiments, masking efficiency is measured by measuring the affinity of an activatable antibody comprising a masking moiety (MM) with the bound EC of the activatable antibody. 50 (before activation) divided by the EC of the parent antibody 50 to measure (e.g., by measuring EC using ELISA) 50 ; See, e.g., the methods of Example 3). In some embodiments, masking efficiency is measured as the difference in affinity with which an activatable antibody comprising a masking moiety (MM) binds to its target prior to activation relative to the affinity with which an activatable antibody comprising a masking moiety (MM) binds to its target after activation (e.g., the difference in affinity for the target antigen before activation relative to the activatable antibody after activation). In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM) and prevents the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody).

[0194] 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 the target when in an uninhibited, unmasked, and / or cleaved state, wherein the second target binding level is greater than the first target binding level. In some embodiments, upon cleavage within the cleavable moiety (e.g., by one or more proteases), the activatable binding polypeptide has increased access to the target.

[0195] In some embodiments, the binding affinity of the polypeptide for its target (e.g., human CTLA4 or CD137) is increased by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold) after activation of the polypeptide (e.g., after activation by treatment with one or more proteases that cleave within the cleavable moiety (CM), after activation by a pH change (increase or decrease), after activation by a temperature shift (increase or decrease), after activation by contact with a second molecule (such as a small molecule or protein ligand), etc.) as compared to before activation of the polypeptide. fold, at least about 3, 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). In some embodiments, if, after "activation," the EC of the polypeptide 50 A polypeptide of the present disclosure is generally considered "activatable" if its expression is reduced by at least about 2-fold (e.g., 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 less) (e.g., as measured by an ELISA or FACS assay; see the Examples below). In some embodiments, if the EC of the polypeptide is 50A polypeptide of the present disclosure is generally considered "activatable" if its activity is reduced by at least about 1 / 2 (e.g., as measured by ELISA or FACS assay; see Examples below).

[0196] In some embodiments, when the masking moiety is bound to the target binding portion of the activatable binding polypeptide, the K of the activatable binding polypeptide with respect to its target is D is the K of the parent antibody with respect to the target when the masking moiety is not bound to the target binding moiety (e.g., after "activation" of the activatable binding polypeptide (such as after protease treatment to cause cleavage within the cleavable moiety)) and / or D Methods of measuring affinity are known in the art and include, for example, the methods described in Example 3 below.

[0197] In some embodiments, the K of the parent antibody for the target is relative to when the masking moiety is not bound to the target binding moiety (e.g., after "activation" of the activatable binding polypeptide (such as after protease treatment to cleave within the cleavable moiety)), and / or relative to the K of the parent antibody for the target. D When the masking moiety is bound to the target binding portion of the activatable binding polypeptide, the K of the activatable binding polypeptide with respect to its target is D A decrease of 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%). Methods of measuring affinity are known in the art and include, for example, the methods described in Example 3 below.

[0198] In some embodiments, the masking moiety sterically hinders the binding of the activatable binding polypeptide to its target and / or allosterically hinders the binding of the activatable binding polypeptide to its target. In some embodiments, the masking moiety does not comprise the amino acid sequence of the natural binding partner of the activatable binding polypeptide.

[0199] In some embodiments, the dissociation constant of the masking moiety with respect to the target binding moiety is greater than the dissociation constant of the activatable binding polypeptide with respect to the target (when in the active activated form). In some embodiments, the dissociation constant of the masking moiety with respect to the target binding moiety is about 2 (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) times greater than the dissociation constant of the activatable binding polypeptide with respect to the target (when in the activated form). In some embodiments, the dissociation constant of the masking moiety with respect to the target binding moiety is about equal to the dissociation constant of the activatable binding polypeptide with respect to the target (when in the activated form). 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 has not yet been activated (e.g., by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a change in pH (increase or decrease), by a shift in temperature (increase or decrease), 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 cleavable moiety. In some embodiments, activation induces a conformational change in the polypeptide (e.g., a shift in the first peptide (FP)) such that the first peptide no longer prevents the polypeptide from binding to its target.

[0200] The activatable binding polypeptides described herein (i.e., activatable antibodies) can 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.

[0201] In some embodiments, the activatable binding polypeptides of the present disclosure are linked to pharmaceutical agents. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutics and radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, d-1, d-1, d-2, d-3, d-4, d-5, d-7, d-8, d-9, d-10, d-11, d-12, d-13, d-14, d-15, d-16, d-17, d-18, d-19, d-20, d-21, d-22, d-23, d-24, d-35, d-36, d-37, d-38, d-40, d-5 D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and their analogs or homologs.Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, 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 radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 and lutetium 177Methods for linking polypeptides to pharmaceutical agents are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfide, and peptide-containing linkers. For further discussion of linkers and methods for attaching 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.

[0202] Activatable binding peptide targeting CTLA4

[0203] 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, the activatable antibody comprises: (a) 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 comprises a polypeptide according to 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, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein the TBM comprises 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, when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4. In some embodiments, when the CM is cleaved, the activatable antibody is able to bind to human CTLA4. In some embodiments, the MM comprises an amino acid sequence selected from SEQ ID NOs: 72-78, as listed in Table A.

[0204] Table A. Masking peptide sequences that can activate antibodies

[0205]

[0206]

[0207] In some embodiments, the activatable binding polypeptide comprises any of the anti-CTLA4 antibodies described herein, including 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 anti-CTLA4 antibody is a human antibody. In some embodiments, the anti-CTLA4 antibody is a humanized antibody and / or a chimeric antibody.

[0208] In some embodiments, the activatable binding polypeptide comprises: a) HVR-H1 comprising the amino acid sequence of YSISSGYHWSWI (SEQ ID NO: 59), HVR-H2 comprising the amino acid sequence of LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 60), and HVR-H3 comprising the amino acid sequence of ARSYVYFDY (SEQ ID NO: 61); and / or b) HVR-L1 comprising the amino acid sequence of RASQSVRGRFLA (SEQ ID NO: 62), HVR-L2 comprising the amino acid sequence of DASNRATGI (SEQ ID NO: 63), and HVR-L3 comprising the amino acid sequence of 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.

[0209] In some embodiments, the present disclosure relates to activatable binding polypeptides that, when in active form (e.g., the activatable binding polypeptide is active after cleavage in the cleavable moiety (e.g., with one or more proteases), but is inactive before cleavage in the cleavable moiety (e.g., with one or more proteases)), bind to human CTLA4 and have at least one (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) of the following functional properties: (a) binds to human CTLA4 with a K of 500 nM or less; DBinds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4; (b) has antagonistic 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) has cross-reactivity with monkey, mouse, rat and / or dog CTLA4; (e) induces ADCC effects (e.g., on Tregs); (f) activates human PBMCs (e.g., stimulates the secretion of IL-2 and / or IFNγ); (g) is capable of inhibiting tumor cell growth; (h) has a therapeutic effect on cancer; and (i) inhibits the binding of human CTLA4 to human CD80 and / or human CD86. Also provided herein are one or more activatable binding polypeptides that cross-compete for binding to human CTLA4 with one or more of the activatable binding polypeptides targeting CTLA4 and / or anti-CTLA4 antibodies described herein.

[0210] In some embodiments, when in an inactive form, the activatable binding polypeptide can bind to a polypeptide with a K of about 500 nM or greater. D Binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4. In some embodiments, when in active form, the activatable binding polypeptide has 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.). D Binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4. In some embodiments, when in active form, the activatable binding polypeptide has a K of about 350 nM or less. D Binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4. In some embodiments, when in active form, the activatable binding polypeptide has a K of about 100 nM or less. D Binds to human CTLA4. In some embodiments, when in active form, the activatable binding polypeptide binds to human CTLA4 with a K of about 50 nM or less. D Binds to human CTLA4. In some embodiments, when in active form, the activatable binding polypeptide binds to human CTLA4 with a K of about 10 nM or less. D Binds to human CTLA4. Measure K of the activated binding peptide DThe method can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc. In some embodiments, K D Measured by ELISA (see, e.g., Example 3 below).

[0211] In some embodiments, when in an inactive form, the activatable binding polypeptide does not have antagonistic activity against human CTLA4. In some embodiments, when in an active form, the activatable binding polypeptide has antagonistic activity against human CTLA4 (e.g., inducing ADCC effects (such as against Treg), activating PBMC (such as by activating, inducing and / or stimulating IL-2 and / or IFNγ secretion), blocking the binding of human CTLA4 to human CD80 and / or human CD86, etc.). In some embodiments, when in an active form, the activatable binding polypeptide suppresses one or more activities of human CTLA4 (e.g., when a cell expressing human CTLA4 (such as a human cell) is contacted by an activated activatable binding polypeptide, one or more activities of human CTLA4 are suppressed).

[0212] In some embodiments, when in an inactive form, the activatable binding polypeptide does not cross-react with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activatable binding polypeptide has cross-reactivity with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activatable binding polypeptide has cross-reactivity with monkey CTLA4. In some embodiments, when in an active form, the activatable binding polypeptide has cross-reactivity with mouse CTLA4. In some embodiments, when in an active form, the activatable binding polypeptide has cross-reactivity with rat CTLA4. In some embodiments, when in an active form, the activatable binding polypeptide has cross-reactivity with dog CTLA4. In some embodiments, when in active form, the activatable binding polypeptide has cross-reactivity 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, mouse, and dog CTLA4; monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4. In some embodiments, when in active form, the activatable binding polypeptide has cross-reactivity at about 350 nM (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about 100 nM, at about 150 nM, at about 200 nM, at about 250 nM, at about 300 nM, at 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.

[0213] In some embodiments, when in an inactive form, the activatable binding polypeptide does not induce an ADCC effect (e.g., on human cells such as Treg). In some embodiments, compared to a control binding polypeptide (e.g., a parent antibody lacking a first peptide (FP) and a cleavable portion (CM)), when in an inactive form, the activatable binding polypeptide has a reduced ADCC effect (e.g., on human cells such as Treg). In some embodiments, when in an active form, the activatable antibody induces an ADCC effect (e.g., on human cells such as Treg). Methods for measuring ADCC effects (e.g., in vitro methods) are known in the art, including but not limited to the methods described in Example 4 below. In some embodiments, relative to a control (e.g., a parent antibody lacking a first peptide (FP) and a cleavable portion (CM)), when in an inactive form, the activatable binding polypeptide induces an ADCC effect of less than about 10% (e.g., induces ADCC less than about 10%, less than about 5%, less than about 1%, etc.). In some embodiments, the activatable binding polypeptide induces an ADCC effect greater than about 10% (e.g., induces ADCC 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.) when in active form relative to a control (e.g., an isotype control).

[0214] In some embodiments, the activatable binding polypeptide is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, when contacted with the activatable binding polypeptide, 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%) relative to corresponding tumor cells not contacted with the activatable binding polypeptide (or relative to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, when the activatable binding polypeptide is administered to a subject, the activatable binding polypeptide is capable of reducing the tumor volume in the subject. In some embodiments, the activatable binding polypeptide is capable of reducing the volume of a tumor 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%) relative to the initial tumor volume in the subject (e.g., before administration of the activatable binding polypeptide; compared to a corresponding tumor in a subject administered an isotype control antibody). 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.

[0215] In some embodiments, the activatable binding polypeptide has a therapeutic effect on cancer. In some embodiments, the activatable binding polypeptide alleviates one or more signs or symptoms of cancer. In some embodiments, when administered with the activatable binding polypeptide, a subject suffering from cancer experiences partial or complete remission.

[0216] In some embodiments, the present disclosure provides isolated activatable binding polypeptides that, when in active 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; and 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 present disclosure provides isolated activatable binding polypeptides that, when in active 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 the activatable binding polypeptide to compete or cross-compete for binding with the antibody can be determined using standard binding assays known in the art such as BIAcore analysis, ELISA assays, or flow cytometry. For example, the antibody (e.g., as described above) can be allowed to bind to human CTLA4 under saturation conditions, followed by measuring the ability of the test activatable binding polypeptide (when in active form) to bind to CTLA4. If the test activatable binding polypeptide is able to bind to CTLA4 simultaneously with the antibody, then the test activatable binding polypeptide is tested for binding to different epitopes of the antibody. However, if the test activatable binding polypeptide cannot bind to CTLA4 simultaneously, then the test activatable binding polypeptide is tested for binding to the same epitope, overlapping epitopes, or an epitope that is closely adjacent to the epitope bound by the antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS, or surface plasmon resonance.

[0217] In some embodiments, the activatable binding polypeptide (when in an inactive form) does not inhibit the 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 the 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 the 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 the 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 the binding of CTLA4 to CD80 and / or CD86. 50 ). In some embodiments, when provided at a concentration of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.), the activatable binding polypeptide completely inhibits the binding of human CTLA4 to CD80 and / or CD86. As used herein, the term "completely inhibiting / completely inhibits" means that the activatable binding polypeptide is able to reduce the binding between the first protein and the 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 the 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, including but not limited to BIAcore analysis, ELISA assays, and flow cytometry.

[0218] Activatable binding peptide targeting CD137

[0219] 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, the activatable antibody comprises: (a) 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 comprises a polypeptide according to 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, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein 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 comprises: (a) 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 comprises an amino acid according to 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, wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein the TBM comprises an antibody light chain variable region (VH); and (b) an antibody heavy chain variable region (VL). In some embodiments, the activatable antibody comprises: (a) 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 comprises an amino acid according to 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, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the CM comprises at least a first cleavage site (e.g., at least a first protease cleavage site); and wherein the TBM comprises 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.

[0220] In some embodiments, when the CM is not cleaved, the MM inhibits binding of the activatable antibody to human CD 137. In some embodiments, when the CM is cleaved, the activatable antibody is able to bind to human CD 137. 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.

[0221] Table B. Masking peptide sequences that can activate antibodies

[0222]

[0223]

[0224] m is 2-10; 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.

[0225] In some embodiments, the activatable binding polypeptide comprises any anti-CD137 antibody described herein, including antibodies described in relation to the specific amino acid sequences of HVRs, variable regions (VL, VH) and / or 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.

[0226] In some embodiments, the activatable binding polypeptide comprises: a) HVR-H1 comprising the amino acid sequence of FSLSTGGVGVGWI (SEQ ID NO:65), HVR-H2 comprising the amino acid sequence of LALIDWADDKYYSPSLKSRL (SEQ ID NO:66), and HVR-H3 comprising the amino acid sequence of ARGGSDTVIGDWFAY (SEQ ID NO:67); and / or b) HVR-L1 comprising the amino acid sequence of RASQSIGSYLA (SEQ ID NO:68), HVR-L2 comprising the amino acid sequence of DASNLETGV (SEQ ID NO:69), and HVR-L3 comprising the amino acid sequence of 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 SEQ ID NO: 49; and / or b) 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 SEQ ID NO: 50.

[0227] In some embodiments, the present disclosure relates to activatable binding polypeptides that, when in active form (e.g., the activatable binding polypeptide is active after cleavage in a cleavable moiety (e.g., with one or more proteases), but is inactive before cleavage in a cleavable moiety (e.g., with one or more proteases)), bind to human CD137 and have at least one (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) of the following functional properties: (a) binding to human CD137 with a K of 500 nM or less; DBinding to human CD137; (b) having agonist activity on human CD137; (c) not binding to human OX40, CD40, GITR and / or CD27 receptors at concentrations up to 1000nM; (d) having cross-reactivity with monkey, mouse, rat and / or dog CD137; (e) not inducing ADCC effect; (f) being able to inhibit tumor cell growth; (g) having a therapeutic effect on cancer; and (h) inhibiting the binding between CD137 and CD137L. In some embodiments, the activatable binding polypeptides disclosed herein can also inhibit, for example, completely inhibit the binding between CD137 and its ligand CD137L. Also provided herein is one or more activatable binding polypeptides and / or anti-CD137 antibodies targeting CD137 described herein that cross-compete for binding to human CD137 or antigen-binding fragments thereof.

[0228] In some embodiments, the activatable binding polypeptide (when in an inactive form) has a K of about 500 nM or greater. D Binds to human CD137. In some embodiments, the activatable binding polypeptide (when in active form) has 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 Binds to human CD137. In some embodiments, the activatable binding polypeptide binds to human CD137 with a K of about 100 nM or less. D Binds to human CD137. In some embodiments, the activatable binding polypeptide binds to human CD137 with a K of about 50 nM or less. D Binds to human CD137. Measure K of the activatable binding polypeptide D The method can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc. In some embodiments, K D Measured by ELISA (see, e.g., Example 5 below).

[0229] In some embodiments, the activatable binding polypeptides described herein (when in active form) have agonist activity to human CD137. In some embodiments, when cells expressing human CD137 (e.g., human cells) are contacted by (active) activatable binding polypeptides, the activatable binding polypeptides induce one or more (e.g., one or more, two or more, three or more, etc.) activities of human CD137. Various CD137 activities are known in the art and may include but are not limited to inducing NF-κB-dependent transcription, inducing T cell proliferation, prolonging T cell survival, costimulating activated T cells, inducing cytokine secretion (such as IL-2), and inducing monocyte activation. In some embodiments, one or more CD137 activities are not the combination of CD137 and its ligand. Methods for measuring CD137 activity (e.g., induction of NF-κB-dependent transcription and / or T cell proliferation, etc.) are known in the art. In some embodiments, activatable binding polypeptides increase NF-κB-dependent transcription in cells expressing human CD137 (e.g., human cells). 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 expressing CD137 (e.g., human cells) contacted with an (active) activatable binding polypeptide relative to corresponding cells not contacted with the activatable binding polypeptide (e.g., corresponding cells contacted with an isotype control antibody) or 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 expressing CD137 (e.g., human cells) contacted with the activatable binding polypeptide (when in active form) relative to corresponding cells not contacted with the activatable binding polypeptide (e.g., corresponding cells contacted with an isotype control antibody) or contacted with the activatable binding polypeptide when in an inactive form.

[0230] In some embodiments, the activatable binding polypeptide (when in an inactive form) does not cross-react with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CD137. In some embodiments, the activatable binding polypeptide (when in an active form) has cross-reactivity with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CD137. In some embodiments, the activatable binding polypeptide has cross-reactivity with monkey CD137. In some embodiments, the activatable binding polypeptide has cross-reactivity with mouse CD137. In some embodiments, the activatable binding polypeptide has cross-reactivity with rat CD137. In some embodiments, the activatable binding polypeptide has cross-reactivity with dog CD137. In some embodiments, the activatable binding polypeptide has cross-reactivity 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, mouse and dog 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 has cross-reactivity at about 100 nM (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about 100 nM). Methods for measuring cross-reactivity are known in the art, including but not limited to surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc.

[0231] In some embodiments, the activatable binding polypeptide does not induce an ADCC effect. Methods for measuring ADCC effects are known in the art. In some embodiments, the activatable binding polypeptide (when in active form or inactive form) does not induce an ADCC effect greater than about 10% (does not induce ADCC greater than about 10%, greater than about 5%, greater than about 1%, greater than about 0.1%, greater than about 0.01%) relative to a control.

[0232] In some embodiments, the activatable binding polypeptide is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, when contacted with the activatable binding polypeptide, 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%) relative to corresponding tumor cells not contacted with the activatable binding polypeptide (or relative to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, when the activatable binding polypeptide is administered to a subject, the activatable binding polypeptide is capable of reducing the tumor volume in the subject. In some embodiments, the activatable binding polypeptide is capable of reducing the volume of a tumor 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%) relative to the initial tumor volume in the subject (e.g., before administration of the activatable binding polypeptide; compared to a corresponding tumor in a subject administered an isotype control antibody). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art.

[0233] In some embodiments, the activatable binding polypeptide has a therapeutic effect on cancer. In some embodiments, the activatable binding polypeptide alleviates one or more signs or symptoms of cancer. In some embodiments, when administered with the activatable binding polypeptide, a subject suffering from cancer experiences partial or complete remission.

[0234] In some embodiments, the present disclosure provides isolated activatable binding polypeptides that, when in active 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; and 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 present disclosure provides isolated activatable binding polypeptides that, when in active 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 activatable binding polypeptides to compete with antibodies or cross-competitive binding can be determined using standard binding assays known in the art such as BIAcore analysis, ELISA assays or flow cytometry. For example, antibodies (such as described above) can be made to bind to human CD137 under saturation conditions, followed by measurement of the ability of the test activatable binding polypeptides (when in active form) to bind to CD137. If the test activatable binding polypeptides can bind to CD137 simultaneously with the antibody, then the test activatable binding polypeptides bind to different epitopes with the antibody. However, if the test activatable binding polypeptides cannot bind to CD137 simultaneously, then the test activatable binding polypeptides bind to the same epitope, overlapping epitopes, or an epitope that is closely adjacent to the epitope bound by the antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS or surface plasma resonance.

[0235] In some embodiments, the activatable binding polypeptide (when in an inactive form) does not inhibit the binding between CD137 and its ligand (e.g., human CD137 and human CD137L). In some embodiments, the activatable binding polypeptide (when in an active form) inhibits the binding between CD137 and its ligand (e.g., human CD137 and human CD137L). In some embodiments, the activatable binding polypeptide inhibits the binding between CD137 and its ligand in vitro. In some embodiments, the activatable binding polypeptide (when in an 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 the binding of CD137 to its ligand. 50In some embodiments, the activatable binding polypeptide has a half-maximal inhibitory concentration (IC) of about 100 nM or less for inhibiting the binding of CD137 to its ligand. 50 ). In some embodiments, when provided at a concentration of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.), the activatable binding polypeptide completely inhibits the binding of human CD137 to its ligand. Methods for measuring the ability of a polypeptide to inhibit the binding of a first protein (e.g., CD137) and a second protein (e.g., CD137L) are known in the art, including but not limited to BIAcore analysis, ELISA assays, and flow cytometry.

[0236] Antibody

[0237] In some embodiments, the present disclosure relates to an activatable binding polypeptide comprising an antibody as described herein (e.g., the CTLA4 or CD137 antibody described above). Antibodies as described herein (e.g., CTLA4 or CD137 antibodies) may be in any category, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, antibodies as described herein (e.g., CTLA4 or CD137 antibodies) are in IgG categories, such as IgG1, IgG2, IgG3, or IgG4 subclasses. Using methods known in the art, antibodies as described herein (e.g., CTLA4 or CD137 antibodies) may be converted from one category or subclass to another category or subclass. An exemplary method for producing an antibody in a desired category or subclass comprises the following steps: separating nucleic acids encoding the heavy chain of an antibody as described herein (e.g., CTLA4 or CD137 antibodies) and nucleic acids encoding the light chain of an antibody as described herein (e.g., CTLA4 or CD137 antibodies), separating nucleic acids encoding V H The sequence of the region makes V H The sequence is linked to a sequence encoding a heavy chain constant region of the desired class or subclass, the light chain gene and heavy chain construct are expressed in cells, and the antibodies are collected.

[0238] antigen-binding fragment

[0239] In some embodiments, the disclosure relates to an activatable binding polypeptide comprising an antigen-binding fragment, eg, an antigen-binding fragment of an antibody described herein (eg, a CTLA4 or CD137 antibody).

[0240] The antigen-binding fragment may comprise any sequence of any antibody described herein. In some embodiments, the antigen-binding fragment comprises (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) the amino acid sequences of three HVRs from a light chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody) and three HVRs from a heavy chain of an antibody described herein (e.g., a CTLA4 or CD137 antibody).

[0241] In some embodiments, the present disclosure provides an antigen-binding fragment of an antibody (which binds to human CTLA4), comprising a heavy chain variable region comprising: HVR-H1 comprising the amino acid sequence of YSISSGYHWSWI (SEQ ID NO:59), HVR-H2 comprising the amino acid sequence of LARIDWDDDKYYSTSLKSRL (SEQ ID NO:60), and HVR-H3 comprising the amino acid sequence of ARSYVYFDY (SEQ ID NO:61); and / or a light chain variable region comprising: HVR-L1 comprising the amino acid sequence of RASQSVRGRFLA (SEQ ID NO:62), HVR-L2 comprising the amino acid sequence of DASNRATGI (SEQ ID NO:63), and HVR-L3 comprising the amino acid sequence of YCQQSSSWPPT (SEQ ID NO:64). In some embodiments, the present 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.

[0242] In some embodiments, the present disclosure provides an antigen-binding fragment of an antibody that binds to human CD137, comprising a heavy chain variable region comprising: HVR-H1 comprising the amino acid sequence of FSLSTGGVGVGWI (SEQ ID NO: 65), HVR-H2 comprising the amino acid sequence of LALIDWADDKYYSPSLKSRL (SEQ ID NO: 66), and HVR-H3 comprising the amino acid sequence of ARGGSDTVIGDWFAY (SEQ ID NO: 67); and / or a light chain variable region comprising: HVR-L1 comprising the amino acid sequence of RASQSIGSYLA (SEQ ID NO: 68), HVR-L2 comprising the amino acid sequence of DASNLETGV (SEQ ID NO: 69), and HVR-L3 comprising the amino acid sequence of YCQQGYYLWT (SEQ ID NO: 70). In some embodiments, the present 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.

[0243] In some embodiments, the antigen-binding fragments of the antibodies described herein (eg, CTLA4 or CD137 antibodies) include: (i) a Fab fragment, which is a fragment consisting of a V L 、V H 、C L and C H 1 domain; (ii) F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) H and C H (iv) an Fd fragment consisting of a single-arm V domain of an antibody; L and V H Fv fragments composed of V domains; (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which are composed of V H Domain composition; (vi) isolated CDRs, and (vii) single chain antibodies (scFv), which are V L The V region is connected to the antibody H Regions of interest include polypeptides (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0244] Antibody derivatives

[0245] In some embodiments, the present disclosure provides an activatable binding polypeptide comprising a derivative of an antibody described herein (eg, a CTLA4 or CD137 antibody).

[0246] In some embodiments, antibody derivatives are obtained by modifying the amino acid sequence of a parent antibody while maintaining the overall molecular structure of the parent antibody amino acid sequence. The amino acid sequence of any region of the parent antibody chain can be modified, such as the framework region, HVR region, or constant region. The type of modification includes substitution, insertion, deletion, or a combination thereof of one or more amino acids of the parent antibody.

[0247] In some embodiments, the antibody derivative comprises 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 as set forth in any one 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 as set forth in any one of SEQ ID NOs: 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 as set forth in any one of SEQ ID NOs: 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 any one of SEQ ID NOs: 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 any one of SEQ ID NOs: 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 any one of SEQ ID NOs: 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 any one of SEQ ID NOs: 64 or 70.

[0248] In some specific embodiments, the derivative comprises 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).

[0249] Amino acid substitutions encompass both conservative and non-conservative substitutions. The term "conservative amino acid substitution" means that one amino acid is replaced by another amino acid where the two amino acids are similar in certain physicochemical properties, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. For example, substitutions can generally 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.

[0250] Modifications can be made at any position in the amino acid sequence of the antibody, including HVRs, framework regions, or constant regions. In one embodiment, the disclosure provides a V sequence comprising an illustrative antibody described herein (e.g., a CTLA4 or CD137 antibody). H and V LHVR sequences, but antibody derivatives containing framework sequences different from those of the illustrative antibodies. The framework sequences can be obtained from public DNA databases or public references including germline antibody gene sequences. For example, the 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, US Department 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 to construct antibody derivatives include those that are structurally similar to the framework sequences used by the illustrative antibodies of the present disclosure. For example, the HVR-H1, HVR-H2, and HVR-H3 sequences and HVR-L1, HVR-L2, and HVR-L3 sequences of the illustrative antibodies may be grafted onto framework regions that have identical sequences to the framework regions found in the germline immunoglobulin gene from which the framework sequences are derived, or the HVR sequences may be grafted onto framework regions that contain one or more mutations compared to the germline sequences.

[0251] In some embodiments, antibody derivatives are chimeric antibodies comprising the amino acid sequence of an illustrative antibody as described herein (e.g., CTLA4 or CD137 antibody). In one example, one or more HVRs from one or more illustrative antibodies are combined with HVRs from antibodies of non-human animals such as mice or rats. In another example, the HVRs of the chimeric antibodies are all derived from one or more illustrative antibodies. In some specific embodiments, the chimeric antibodies comprise one, two, or three HVRs from the heavy chain variable region of an illustrative antibody and / or one, two, or three HVRs from the light chain variable region of an illustrative antibody. Chimeric antibodies can be produced using conventional methods known in the art.

[0252] Another type of modification is to make V H and / or V LThe amino acid residues in the HVR region of the heavy chain are mutated. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effect on antibody binding or other target functional properties can be evaluated in in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. Mutations can be amino acid additions and / or deletions. In addition, typically one, two, three, four, or five residues in the HVR region are changed. In some embodiments, the antibody derivatives comprise 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVR and / or in the 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. Cysteine can be typical or atypical cysteine. In one embodiment, the antibody derivatives have 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of the illustrative antibody.

[0253] You can also V H and / or V L In some embodiments, the framework residues in the region of the antibody are modified. Generally, the framework variants are prepared to reduce the immunogenicity of the antibody. One method is to "reverse mutation" of one or more framework residues to the corresponding germline sequence. An antibody that has been mutated by a recipient cell may contain framework residues that are different from the germline sequence from which the antibody is derived. The residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to return the framework region sequences to their germline configuration, somatic mutations can be "reverse mutation" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0254] In addition, modifications can also be made in the Fc region of the illustrative antibodies, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified so that the number of cysteine residues in the hinge region is altered, for example, increased or decreased. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered to, for example, 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 the antibody is mutated to reduce the biological half-life of the antibody.

[0255] In addition, the antibodies of the present disclosure can be modified to change its potential glycosylation site or pattern according to routine experiments known in the art. On the other hand, the present disclosure provides derivatives of antibodies described herein (e.g., CTLA4 or CD137 antibodies) containing at least one mutation that changes the glycosylation pattern in the variable region of the light chain or heavy chain. Such antibody derivatives can have increased affinity and / or changed specificity in terms of binding to antigens. The mutation can add new glycosylation sites in the V region, change the position of one or more V region glycosylation sites, or remove pre-existing V region glycosylation sites. In one embodiment, the present disclosure provides derivatives of antibodies described herein (e.g., CTLA4 or CD137 antibodies) having a potential N-connected glycosylation site at asparagine in the heavy chain variable region, wherein the potential N-connected glycosylation site in one heavy chain variable region is removed. In another embodiment, the disclosure provides derivatives of antibodies described herein (e.g., CTLA4 or CD137 antibodies) having potential N-linked glycosylation sites at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in both heavy chain variable regions are removed. Methods for changing 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.

[0256] Examples of other antibody derivatives provided by the present disclosure include single-chain antibodies, mini-diabodies, domain antibodies, nanobodies, and monobodies. "Single-chain antibodies" (scFv) consist of a V L Domain connected to V H The single polypeptide chain of the domain, in which V L domain and V HDomain pairing 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). A "mini bifunctional antibody" consists of two chains, each chain comprising a heavy chain variable region connected to a light chain variable region on the same polypeptide chain, connected by a short peptide linker, wherein the two regions on the same chain are not paired with each other, but paired with complementary domains on the other chain to form a bispecific molecule. Methods for preparing mini bifunctional antibodies 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 the heavy or light chains of antibodies. Domain antibodies are well expressed in bacteria, yeast, and mammalian cell systems. Other details of domain antibodies and their production methods are known in the art (see, for example, U.S. Patent 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 antibodies. Nanobodies typically comprise 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, WO 06 / 079372). Monoclonal antibodies are composed of a light chain and a heavy chain of an IgG4 antibody. Monoclonal antibodies can be prepared by removing the hinge region of an IgG4 antibody. Other details of monoclonal antibodies and methods for preparing them can be found in WO2007 / 059782.

[0257] VII. Composition

[0258] In other aspects, the present disclosure provides a composition comprising one or more of the polypeptides described herein (e.g., activatable binding polypeptides). In some embodiments, the composition is a pharmaceutical composition comprising a polypeptide (e.g., activatable binding polypeptide) and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0259] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering a polypeptide (e.g., an activatable binding polypeptide). The carrier can be an anti-adhesive, adhesive, coating agent, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Suitable examples of pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0260] The composition can be in any suitable form, such as liquid, semisolid and solid dosage form. 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 a 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. Sterile solutions can be prepared by incorporating polypeptide (e.g., an activatable binding polypeptide) into a suitable carrier in the desired amount, followed by sterilization microfiltration. Dispersion can be prepared by incorporating polypeptide (e.g., an activatable binding polypeptide) into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of a sterile powder for the preparation of a sterile liquid, the preparation method includes vacuum drying and freeze drying (lyophilization) to produce a powder of any additional desired ingredients of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution. The various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0261] The relative amount of polypeptide (e.g., activatable binding polypeptide) included in the composition will vary depending on many factors, such as the specific polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of polypeptide (e.g., activatable binding polypeptide) in a single dosage form will generally be that amount that produces a therapeutic effect, but may also be a smaller amount. Typically, this amount will be in the range of about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30%, relative to the total weight of the dosage form.

[0262] In addition to the polypeptide (e.g., activatable binding polypeptide), one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described herein below. The appropriate amount of the additional therapeutic agent to be included in the composition can be readily selected by one skilled in the art and will vary depending on many factors, such as the specific agent and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of the additional therapeutic agent included in a single dosage form will generally be that amount of the agent that produces a therapeutic effect, but may also be a smaller amount.

[0263] Any polypeptide (eg, activatable binding polypeptide) and / or composition (eg, pharmaceutical composition) described herein can be used to prepare a medicament (eg, an agent for treating cancer or delaying progression of the cancer in a subject in need thereof).

[0264] VIII. Uses of Activatable Binding Polypeptides and Pharmaceutical Compositions

[0265] The polypeptides provided by the present disclosure (e.g., activatable binding polypeptides) and pharmaceutical compositions thereof can be used for treatment, diagnosis or other purposes, such as regulating immune responses, treating cancer, enhancing the efficacy of other cancer therapies, enhancing vaccine efficacy, or treating autoimmune diseases. Therefore, in other aspects, the present disclosure provides methods of using polypeptides (e.g., activatable binding polypeptides) or pharmaceutical compositions thereof. In one aspect, the present disclosure provides a method for treating a mammalian condition, comprising administering an effective amount of a polypeptide provided by the present disclosure (e.g., activatable binding polypeptides) or a composition thereof to the mammal in need of treatment. In some embodiments, the polypeptide is an activatable binding polypeptide that binds to CTLA4 (e.g., human CTLA4) or CD137 (e.g., human CD137) when in active form. In some embodiments, the mammal is a human.

[0266] In some embodiments, the disorder is cancer.A variety of cancers can be treated or prevented using the methods, uses, or medicaments provided by the present disclosure.Examples of such cancers include lung cancers such as bronchogenic carcinomas (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, enchondromatous hamartoma (non-cancerous), and sarcoma (cancerous); heart cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondroma, enchondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and fibrosarcoma. sarcoma) and reticulum cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, astrocytomas, oligodendrogliomas, medulloblastomas, chordomas, schwannomas, ependymomas, meningiomas, pituitary adenomas, pinealomas, osteomas, hemangioblastomas, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermoid cysts, and hemangiomas; cancers of the digestive system such as leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipomas , intestinal neurofibromas, intestinal fibromas, colorectal polyps, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, bile duct carcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, adrenal tumor, and renal pelvic transitional cell carcinoma; bladder cancer; blood cancers such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myelocytic, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome syndrome and hairy cell leukemia), chronic myeloid (myelogenous, bone marrow-derived, 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; disease); head and neck cancer; eye-related cancers such as retinoblastoma and intraocular melanoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer (cervical cancer), 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); non-cancerous growths of the parathyroid glands; pancreatic cancer; and blood cancers such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

[0267] On the other hand, the present disclosure provides a method for enhancing the immune response in a mammal, the method comprising administering to the mammal an effective amount of a polypeptide (e.g., activatable binding polypeptide) provided by the present disclosure or a composition thereof. In some embodiments, the polypeptide is an activatable binding polypeptide in conjunction with CTLA4 (e.g., human CTLA4) or CD137 (e.g., human CD137), and the mammal is a human. The term "enhance the immune response" or its grammatical variations means any response that stimulates, provokes, increases, improves, or strengthens the immune system of a mammal. The immune response can be a cellular response (i.e., mediated by cells, such as mediated by cytotoxic T lymphocytes) or a humoral response (i.e., mediated by antibodies), and can be a primary or secondary immune response. Examples of enhancing the immune response include activating PBMC and / or T cells (including increasing the secretion of one or more cytokines such as IL-2 and / or IFNγ). The enhancement of the 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 cytotoxicity. Generally, the methods of the present disclosure enhance the immune response achieved by a mammal when compared to the immune response achieved by an untreated mammal or a mammal not treated using the described methods.

[0268] When implementing the treatment method, the polypeptide (e.g., activatable binding polypeptide) can be administered alone as a monotherapy, or in combination with one or more additional therapeutic agents or therapies. Therefore, on the other hand, the present disclosure provides a combination therapy comprising a polypeptide (e.g., activatable binding polypeptide) and one or more additional therapies or therapeutic combinations 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 a specific aspect, the present disclosure provides a combination therapy for treating cancer in a mammal, the combination therapy 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 another embodiment, the mammal is a human.

[0269] A wide variety of cancer therapeutics can be used in combination with the polypeptides provided by the present disclosure (e.g., activatable binding polypeptides). One of ordinary skill in the art will recognize the existence and development of other cancer therapies that can be used in combination with the methods and polypeptides disclosed herein, and will not be limited to those forms of therapy set forth herein. Examples of the types of additional therapeutic agents that can be used in combination therapies for the treatment of cancer include (1) chemotherapeutics, (2) immunotherapeutics, and (3) hormone therapy agents. In some embodiments, the additional therapeutic agent is a viral gene therapy, an immune checkpoint inhibitor, a targeted therapy, a radiotherapy, and / or a chemotherapeutic agent.

[0270] The term "chemotherapeutic agent" refers to a chemical or biological substance that can cause cancer cell death, or interfere with the growth, division, repair and / or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO 2006 / 129163 and US 20060153808, 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) Alkaloids or vinca alkaloids, including cytotoxic antibiotics such as doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN PFS, ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE), dactinomycin (actinomycin D), (D), COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN) and bleomycin (BLENOXANE), vinorelbine tartrate (NAVELBINE), vinblastine (VELBAN), vincristine (ONCOVIN) and vindesine (ELDISINE); (3) antimetabolites such as capecitabine (XELODA), Cytarabine (CYTOSAR-U), fludarabine (FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate (FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate (NEUTREXIN), and pemetrexed (ALIMTA); (4) pyrimidine antagonists, such as 5-fluorouracil (5-FU);Capecitabine (Xeloda), raltitrexed (TOMUDEX), tegafur-uracil (UFTORAL), and gemcitabine (Gemzar); (5) taxanes, such as docetaxel (TAXOTERE), paclitaxel (TAXOL); (6) platinum drugs, such as cisplatin (PLATINOL) and carboplatin (PARAPLATIN), and oxaliplatin (ELOXATIN); (7) topoisomerase inhibitors, such as irinotecan (Caplastin), (CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR) and teniposide (VUMON); (8) epipodophyllotoxins (podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID, TOPOSAR); (9) folic acid derivatives, such as leucovorin (WELLCOVORIN); (10) nitrosoureas, such as carmustine (Bicknew), lomustine (Syseu);(11) Inhibitors of receptor tyrosine kinases including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA), bortezomib (VELCADE), imatinib mesylate (VELCADE), and telotropinib (TARCEVA). mesylate (Gleevec), gefitinib, lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib, rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN), )) and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), antibodies against insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596; (12) angiogenesis inhibitors such as bevacizumab (Acrivastine), suramin (GERMANIN), angiostatin, SU5416, thalidomide and matrix metalloproteinase inhibitors (such as batimastat and marimastat) and those disclosed in WO2002055106; and (13) proteasome inhibitors such as bortezomib (Vilcord). ;

[0271] The term "immunotherapeutic agent" refers to a chemical or biological substance that can enhance the immune response of a mammal. Examples of immunotherapeutic agents include: Bacillus Calmette-Guérin (BCG); cytokines such as interferon; vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX, and NeuVax; and antibodies such as alemtuzumab (CAMPATH), bevacizumab (Acrivastine), cetuximab (Erbitux), gemtuzunab ozogamicin (MYLOTARG), ibritumomab (Ibritumomab), and sirolimus.

[00145] The present invention relates to an antibody directed against adenomatous polymorphism (Abcam), a monoclonal antibody directed against aviator-activated protein kinase ...

[0272] The term "hormonal therapeutic agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones, or inhibits or counteracts the effects 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 therapeutic 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 present disclosure may also be used in combination with non-drug hormone therapies, such as (1) surgical procedures to remove all or part of an organ or gland involved in hormone production, such as the ovaries, testicles, adrenal glands, and pituitary gland, and (2) radiation therapy, in which the patient's organ or gland is subjected to an amount of radiation sufficient to suppress or eliminate the production of the targeted hormone.

[0273] In some embodiments, the additional therapeutic agent is one or more of: pomalidomide, levitra, lenalidomide, pomalidomide, thalidomide, DNA alkylating platinum-containing derivative 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 dose 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.

[0274] Combination therapy for treating cancer also encompasses the combination of binding molecules with surgery to remove the tumor. The binding molecules can be administered to the mammal before, during, or after surgery.

[0275] Combination therapies for treating cancer also encompass combinations of polypeptides (e.g., activatable binding polypeptides) with radiation therapy, such as ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high linear energy radiation). The radiation source can be external or internal to the mammal. The polypeptide can be administered to the mammal before, during, or after radiation therapy.

[0276] The polypeptides provided by the present disclosure (e.g., activatable binding polypeptides) and compositions thereof can be administered by any suitable enteral route of administration or parenteral route of administration. The term "enteral route of administration" refers to administration by any part of the gastrointestinal tract. Examples of enteral routes include oral, transmucosal, buccal and rectal routes, or intragastric routes. "Parenteral route of administration" refers to routes of administration other than enteral routes. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or surface administration. Polypeptides (e.g., activatable binding polypeptides) and compositions of the present disclosure can be administered using any suitable method, such as by oral ingestion, nasogastric tubes, gastrostomy tubes, injections, infusions, implantable infusion pumps, and osmotic pumps. Suitable routes and methods of administration may vary depending on many factors, such as the particular polypeptide employed, the desired rate of absorption, the particular formulation or dosage form employed, the type or severity of the condition being treated, the particular site of action, and the condition of the patient, and can be readily selected by one skilled in the art.

[0277] The term "effective amount" of a binding molecule may refer to an amount that effectively achieves a predetermined therapeutic purpose. For example, in the case of enhancing an immune response, an "effective amount" may be any amount that effectively stimulates, provokes, increases, improves, or strengthens any response of the immune system of a mammal. In the case of treating a disease, an "effective amount" may be any amount sufficient to cause any desirable or beneficial effect in the treated mammal. In particular, in the case of treating cancer, examples of desirable or beneficial effects include inhibiting further growth or spread of cancer cells, causing cancer cell death, inhibiting the recurrence of cancer, alleviating pain associated with cancer, or improving the survival of a mammal. The effective amount of a polypeptide as described herein (e.g., an activatable binding polypeptide) may be in the range of about 0.001 to about 500 mg per kg body weight of a mammal, or in the range of about 0.01 to about 100 mg / kg. For example, an amount may be about 0.3 mg, 1 mg, 3 mg, 5 mg, 10 mg, 50 mg, or 100 mg per kg body weight of a mammal. In some embodiments, the effective amount of a polypeptide (e.g., an activatable binding polypeptide) of the present disclosure is within the range of about 0.01-30 mg per kg body weight of a mammal. In some other embodiments, the effective amount of a polypeptide (e.g., an activatable binding polypeptide) of the present disclosure is within the range of about 0.05-15 mg per kg body weight of a mammal. The precise dosage level to be administered can be readily determined by those skilled in the art and will depend on many factors, such as the type and severity of the condition to be treated, the specific polypeptide employed, the route of administration, the time of administration, the duration of treatment, the specific additional therapy employed, the age, sex, weight, condition, overall health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0278] The polypeptide (e.g., activatable binding polypeptide) or its composition can be administered at multiple times. The interval between single doses can be, for example, daily, weekly, monthly, every three months, or annually. An exemplary treatment regimen requires 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. The dosage regimen for a polypeptide (e.g., activatable binding polypeptide) of the present disclosure may include intravenous administration of 1 mg per kg body weight or 3 mg per kg body weight, using one of the following dosing schedules: (i) every four weeks for six doses, followed by every three months; (ii) every three weeks; (iii) 3 mg per kg body weight, 1 time, followed by 1 mg per kg body weight every three weeks.

[0279] IX. Kit

[0280] On the other hand, a kind of kit of the library including the polynucleotide of the present disclosure is provided herein.In some embodiments, the kit also includes a package insert, and the package insert includes instructions about expressing, modifying, screening or otherwise using the library, for example, to identify the target activatable binding polypeptide.In some embodiments, the kit also includes one or more buffers such as for storing, transferring, transfecting or otherwise using one or more of the polynucleotides (such as synthetic polynucleotides).In some embodiments, the kit also includes one or more containers for storing one or more of the polynucleotides.In some embodiments, the kit also includes one or more vectors such as for transfecting host cells with one or more of the polynucleotides.

[0281] In another aspect, the present invention provides a kit comprising an activatable binding polypeptide and / or composition as described herein. In some embodiments, the kit further comprises a package insert containing instructions for using the activatable binding polypeptide and / or composition. In some embodiments, the kit further comprises one or more buffers, such as for storing, transferring, administering, or otherwise using the activatable binding polypeptide and / or composition. In some embodiments, the kit further comprises one or more containers (e.g., syringes, etc.) for storing or administering the activatable binding polypeptide and / or composition.

[0282] The foregoing written description is considered sufficient to enable those skilled in the art to practice the present disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, various modifications of the present disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art based on the foregoing description and fall within the scope of the appended claims.

[0283] Example

[0284] Example 1: Methods for Identifying Self-Blocking Peptides for Activating Binding Polypeptides

[0285] As described above, there is a need for improved methods and products for identifying self-blocking peptides for activatable binding polypeptides. Therefore, a new system has been designed and implemented to efficiently discover masking moieties with good developability. In this system, a target antibody fragment Fab ( Figure 1 ) or scFv( Figure 2) were displayed on the yeast surface and confirmed to be functional in binding to its antigen. Next, the improved peptide library was directly fused to the N-terminus of the light chain, and a yeast library of fusion proteins displayed on the yeast surface was constructed. The yeast library was then subjected to several rounds of FACS-based screening: first, yeast clones with low binding to the antigen were enriched, then the enriched yeast clones were treated with protease to remove the N-terminal peptide, and clones with high binding to the antigen were selected ( Figure 1 and Figure 2 After 4-5 rounds of sorting, plasmids were extracted from these clones and the masking peptide sequence was confirmed by DNA sequencing.

[0286] There are several unique features built into this new system that make it powerful in identifying masking peptides for target antibodies with good developability:

[0287] 1) The peptide library is directly fused to the N-terminus of the target antibody fragment rather than any external scaffold protein, and the masked peptides are discovered in the same context as the final product. This eliminates contamination by false positive peptide sequences and significantly reduces the workload for their downstream characterization.

[0288] 2) A protease-mediated activation mechanism is integrated into the screening process. This ensures that the discovered peptides not only mask antigen binding prior to activation, but also no longer block antigen binding after protease cleavage. These are prerequisites for being considered a good masking peptide for any activatable antibody.

[0289] 3) An improved design of the peptide library was adopted. Unlike the commonly used random peptide library, a pair of cysteine residues were introduced into a fixed position in the peptide library to ensure that the displayed peptide had 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). Unlike the widely used NNK (or NNS) codons that encode all 20 residues including chemically unstable residues such as M and W, NHC codons were adopted in part or all of the peptide library. NHC codons encode 12 kinds of amino acid residues (D, A, Y, S, T, N, I, L, F, V, H and P) and do not include residues that are unfavorable for the manufacturing process, such as methionine, tryptophan or cysteine. In addition, relative to NNK (or NNS) codons, the use of NHC codons also significantly reduces the theoretical peptide library size, therefore, making it possible to construct a library with much better coverage. When tested for different target antibodies, these libraries performed well.

[0290] Example 2: Design of a constrained peptide library (CPL)

[0291] Four exemplary constrained peptide libraries (CPLs) were designed (Table 1).

[0292] Table 1: Designed CPL

[0293]

[0294] 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

[0295] In their core is sequence Z6CX6CZ2 (SEQ ID NO:55) or Z6CX8CZ2 (SEQ ID NO:56), and two fixed cysteine residues form disulfide bond so that peptide conformation is constrained. In the synthesized oligonucleotides, all positions except the inner ring adopt degenerate codon NHC, wherein in CPL011 and CPL013, also adopt NNK codon. Compared with NNK or NNS codon, NHC codon encodes 12 kinds of residues (table 2), thus covering significant diversity, but lacks chemically unstable residues methionine, tryptophan and cysteine. In addition, compared to NNK or NNS codon, theoretical diversity reduction makes it possible to build a library with better coverage.

[0296] Table 2: NHC codons

[0297]

[0298] Following these masking peptide sequences is 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 for in vivo tumor cell-specific activation of targeting agents (see, e.g., 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 the yeast-based screen, 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.

[0299] CPL and an invariant cleavage peptide are fused to the N-terminus of the light chain of a target antibody in scFv or Fab format, linked to the Aga2 protein displayed on the yeast surface. The inclusion of an alternative TEV protease recognition site is crucial in identifying the correct type of masking peptide sequence, one that blocks antigen binding before and enables antigen binding after protease cleavage. The examples described below demonstrate that the cleavage-activation mechanism of activatable antibodies, initially demonstrated in yeast, can be reproduced in full IgG molecules expressed in mammalian cells.

[0300] Example 3: Construction and validation of activatable antibodies targeting CTLA4

[0301] Displaying functional target antibodies on the yeast surface

[0302] A low-copy number CEN / ARS-based vector was used to express the target antibody (antibody TY21580 targeting human CTLA4) in yeast Saccharomyces cerevisiae under the control of the inducible GAL1-10 promoter. Surface display of scFv was achieved by fusion of the Aga2 protein at its C-terminus under the control of the GAL1 promoter, similar to a previously disclosed arrangement (Boder and Wittrup (1997) Nat Biotechnol 15(6):553-7). For Fab, their surface display was achieved by fusion of the Aga2 protein at the N-terminus of the heavy chain (fusion of VH and CH1) under the control of the GAL1 promoter, while the light chain (fusion of VL and CL) was under the control of the GAL10 promoter. Fab was displayed on the yeast surface by association of the light chain with the membrane-anchored heavy chain.

[0303] The surface display of Fab or scFv was verified by staining with an antibody that recognizes the fusion affinity tag, and the functionality of the Fab or scFv displayed on yeast was examined using biotinylated human CTLA4. Briefly, 48 hours after induction in galactose medium, yeast cells (1x10^6) were collected, washed once with PBSA buffer, and then incubated with 10nM biotinylated antigen at room temperature for 1 hour. The yeast cells were then washed twice with PBSA buffer and incubated with PE-conjugated streptavidin (1:500 dilution) (eBioscience#2-4317-87) at 4°C for 30 minutes. The yeast cells were then analyzed by flow cytometry. Figure 3A-3B As shown in , Fab targeting CTLA4 ( Figure 3A ) and scFv( Figure 3B ) were both successfully displayed on the yeast surface and were able to strongly bind their antigens.

[0304] Construction of a yeast library containing CPL

[0305] The synthesized oligonucleotide encoding CPL was fused to the oligonucleotide encoding the lytic peptide via five cycles of PCR. The primers used (F and R primers) are listed in Table 3. The PCR reaction composition was: 1X PrimeSTAR buffer, 2.5 mM dNTPs, 100 μM each of the F and R primers, 100 μM each of template 1 (CPL oligonucleotide) and template 2 (oligonucleotide encoding the lytic peptide), and 2.5 U of PrimeSTAR HS DNA polymerase. The PCR program used was: a) 1 cycle of 96°C for 5 minutes; 2) 5 cycles of 96°C (15 seconds), 60°C (15 seconds), and 72°C (6 seconds); and 3) 1 cycle of 72°C for 3 minutes. Exonuclease I was used to digest the single-stranded DNA, and the PCR product was subsequently purified by gel electrophoresis. The purified PCR product was then digested with BamHI and KpnI and cloned into a bacterial-filtered vector digested with the same two restriction enzymes. In the filter vector, the CPL and lytic peptide are placed downstream of the bacterial secretion signal peptide and upstream of the β-lactamase lacking a signal sequence. Functional β-lactamase selected on ampicillin plates indicates in-frame fusion of the CPL and lytic peptide, thereby eliminating any out-of-frame errors (N-1 or N-2) introduced into the synthesized degenerate oligonucleotide. In addition, some poorly folded sequences are also reduced from the pool. The ligation products are transformed into electrocompetent bacterial cells, and the diversity of the CPL library is typically between 5x10^9 and 1x10^10. Sequencing of individual clones indicates that this method achieves extremely high in-frame rates (in many cases, almost 100%).

[0306] Table 3: PCR primers

[0307]

[0308] To prepare a yeast library containing CPL, plasmids were extracted from a bacterial library and used as templates for PCR amplification of DNA fragments encoding CPL and the lytic peptide. 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 along with a linearized plasmid expressing the target antibody fused to Aga2. Homologous sequences at both ends of the PCR fragment and the plasmid ensured efficient homologous recombination within the yeast cells. The diversity of the constructed yeast libraries typically ranged from 1x10^9 to 2x10^9.

[0309] FACS-based screening of masking peptides for CTLA4 antibodies

[0310] A total of 1x10^8 yeast cells from the CPL yeast library were used to screen for masked peptides against target antibodies. For each round of sorting by MoFlo XDP, yeast cells induced in galactose medium were collected, 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) at 4°C for 30 minutes. After washing twice more with PBSA buffer, the yeast cells were adjusted to 2-3OD / mL and subjected to sorting. Figure 4 As shown in , in round 1, 10 nM biotinylated CTLA4-Fc was used and weak binders were enriched. Yeast cells from round 1, after growth in glucose medium, were induced in galactose medium and treated with AcTEV protease (6 U / OD cell) (Thermo Fisher Scientific #12575015) at 30°C for 2 hours, and strong binders were purified. Starting from the third round of sorting, the concentration of biotinylated CTLA4-Fc was reduced to 1 nM and weak binders were collected. In round 4, each yeast cell fraction was also treated in parallel with AcTEV to verify protease cleavage-mediated activation of the target antibody. As shown in Figure 4 As shown in , it is clear that AcTEV cleavage leads to a significant increase in the population of cells that strongly bind to the antigen, indicating that the screening strategy is effective. Single clones from round 5 sorting were plated on selective medium and grown individually to further confirm cleavage-mediated activation of antigen binding.

[0311] like Figure 5A-5B As shown in Figure 5A ) or Fab( Figure 5B ) form of selected CTLA4-activatable antibody clones exhibited little binding to the antigen in the presence of a masking peptide. However, when yeast cells were treated with TEV protease to remove the masking peptide, binding to the antigen increased significantly. The combination of incorporating a TEV recognition site into the cleavage peptide and validating the selected clones using TEV protease significantly increased the success rate of selecting masking peptides.

[0312] To identify the masking peptide sequence, shuttle plasmids (Generay # GK2002-200) were extracted from the selected yeast clones 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, indicating the clear enrichment achieved by each round of sorting. Four groups of masking peptide sequences are listed in Table 4 together with the unchanged cleavage peptide sequences.

[0313] Table 4: Masked peptide sequences

[0314]

[0315] IgG conversion and expression

[0316] The four sets of masking peptides listed in Table 4, as well as four additional masking peptide sequences derived from two of them (B13192 and B13197) to eliminate potential glycosylation sites (Table 5), were converted into IgG1.

[0317] Table 5: Additional masked peptide sequences

[0318]

[0319]

[0320] The heavy and light chains were cloned separately into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific, catalog number K830001), and the masking peptide and the invariant cleavage peptide were fused to the N-terminus of the light chain in the same manner as for display on the yeast surface. The VH and VL sequences of 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 with this application under attorney docket number 69540-2000540, which is incorporated herein by reference in its entirety):

[0321] Anti-CTLA4 heavy chain variable region (SEQ ID NO: 47):

[0322] EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS

[0323] Anti-CTLA4 light chain variable region (SEQ ID NO: 48):

[0324] DIQLTQSPSSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR.

[0325] The plasmids are transfected into HEK293F cells for a short time. After six days, supernatants are collected and clarified by centrifugation and filtration, and IgG is purified by standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). IgG is eluted and neutralized, and buffer-exchanged to enter PB buffer (20mM sodium phosphate, 150mM NaCl, pH 7.0). Protein concentration is measured by ultraviolet spectrophotometry, and IgG purity is analyzed by SDS-PAGE or SEC-HPLC under denaturation, reducing and non-reducing conditions. Importantly, the expression level of activatable antibodies in HEK293 cells is similar to their parental antibodies, and their purification yields after protein A resin are also similar, thus showing that the presence of masking peptides and cleavage peptides does not have a negative impact on the expression of antibodies in mammalian cells.

[0326] Measurement of masking efficiency

[0327] The ForteBio Octet RED96 system (Pall, USA) was used to quickly evaluate the efficiency of the masked peptides. 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) in parallel and captured by an anti-human IgG capture (AHC) biosensor (Pall, USA). The sensor was then associated with His-tagged CTLA4 protein (25 nM) for 300 seconds, followed by dissociation in KB buffer for another 300 seconds. The 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. Figure 6A-6B As shown in , the response achieved with the activatable antibody was significantly lower than that achieved with the parent antibody, indicating that the masking peptide effectively blocked the binding of the antibody to its antigen. However, among the four activatable antibodies, TY22401 was less effective, which is consistent with the results from the ELISA assay discussed below.

[0328] Recombinant human CTLA4-Fc was diluted to 1 μg / mL in PBS and coated overnight on Maxisorp plates at 4°C. The plates were blocked at 37°C for 1 hour with PBS supplemented with 3% skim milk. After washing, 100 μL of 3-fold serial dilutions of the antibody were added to each well. After incubation at 37°C for 1 hour, the plates were washed four times, and 100 μL of HRP-conjugated anti-human IgG (Fab specific) antibody (1:6000 dilution) was added to each well. The plates were incubated at 37°C for 1 hour, washed four times, and then 50 μL of TMB substrate solution was added to each well, and the plates were incubated at room temperature. The absorbance at 450 nm was measured after terminating the reaction with 50 μL of H2SO4 per well. EC values were estimated by fitting the ELISA data using the asymmetric sigmoidal (five-parameter logistic equation) model of GraphPad Prism 6 software. 50 Two experiments were performed for the activatable antibodies TY22401, TY22402, and TY22404, resulting in two calculated masking efficiencies for each of these activatable antibodies. The masking efficiency of each activatable antibody was calculated by using the EC for binding of the activatable antibody. 50 Divided by the EC of the parent antibody (TY21580) 50 To calculate. Figures 7A-7C As shown in Table 6, all activatable antibodies showed significantly reduced binding to their antigens compared to the parent antibody, and the calculated masking efficiencies ranged from 48 to 2213. The differences in masking efficiencies may be due to the differences in EC 50 The results are due to variations in the measurement and data fitting of the values, and the masking efficiency of each activatable antibody may fall within the calculated range (e.g., the masking efficiency of the activatable antibody TY22402 is between 377 and 2213). These results indicate that the various masking peptides identified from CPL maintain their masking efficiency when expressed in mammalian cells and as part of a complete IgG molecule.

[0329] Table 6: Activatable Antibody ELISA before Protease Cleavage

[0330]

[0331] Removal of the masking peptide restored antibody activity

[0332] The activatable antibodies were purified by treatment with a protease that recognizes the cleavage sequence and then tested to determine whether removal of the masking peptide restored 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 5 or 10 units of recombinant human MMP-9 (BioVision, #7867-500) in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, 20 μM ZnCl2, pH 7.5). The reaction was carried out at 37°C for 21 hours. Removal of the masking peptide from the light chain was confirmed by SDS-PAGE analysis. Figure 8A The masking efficiency was then measured by ELISA as described above. Figure 8B As shown in Table 7, after removal of the masking peptide, the activatable antibody became indistinguishable from the parent antibody in its binding to the antigen.

[0333] Table 7: Activatable Antibody ELISA after Protease Cleavage

[0334]

[0335] Developability Overview of Activatable Antibodies

[0336] For manufacturing purposes, it is crucial that the activatable antibodies found 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 (pH 5.5), and antibody quality analysis was performed using analytical size exclusion chromatography using Waters 2695 and Waters 2996 UV detectors and TSKgelg3000SWXL columns (300 mm × 7.8 mm) (Tosoh Bioscience). For each assay, 10 μg of antibody was injected, and fractionation was performed at a flow rate of 0.5 mL / min using buffer (200 mM sodium phosphate, at pH 7.0).

[0337] Three accelerated stress tests were performed: incubation of the activatable antibody at 50°C for 7 days, incubation of the activatable antibody at 40°C for 28 days, and six freeze-thaw cycles. The freeze-thaw test was performed by freezing 100 μL of sample (1 mg / mL in 20 mM histidine (pH 5.5)) at -80°C for 30 minutes and then thawing at room temperature for 60 minutes. Figures 9A-9CAs shown in , after storage for 7 days at 50°C or 28 days at 40°C, all activatable antibodies remained stable and exhibited little aggregation. After six freeze-thaw cycles, they showed slight deterioration; however, the main monomer peak remained at approximately 95%, indicating that these activatable antibodies were extremely stable under these accelerated stress tests. Without wishing to be bound by theory, it is noteworthy that the activatable antibodies have not undergone extensive buffer optimization processes, and therefore, the stability of the activatable antibodies may be further improved with optimized buffers and excipients.

[0338] Next, the activatable antibody was concentrated to over 150 mg / mL in 20 mM histidine (pH 5.5) (Table 8). No precipitation of the activatable antibody was observed, and the viscosity of the sample was very manageable. The concentrated activatable antibody was then diluted to 20 mg / mL or 1 mg / mL for analysis of high molecular weight (HMW) species. Figure 10 As shown in Table 8, no significant increase in HMW species was observed, indicating that these activatable antibodies were extremely soluble and stable in the assay buffer up to high concentrations.

[0339] Table 8: Concentrations of activatable antibodies >150 mg / mL

[0340] Sample Identifier: Starting concentration (mg / mL): High concentration (mg / mL): TY22401 10.9 187.2 TY22402 8.4 160.0

[0341] To investigate 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, and the pH was adjusted to 3.7 and maintained at room temperature for 30 and 60 minutes. The samples were then neutralized to pH 7.0 with 1 M Tris base. The masking efficiency of the activatable antibodies was measured using ForteBio as described above. Figure 11 As shown in , the masking efficiency remained unchanged after 30 or 60 minutes of low pH incubation, indicating that the masking peptides retained their blocking efficacy after low pH incubation.

[0342] In summary, the data indicate that the discovered activatable antibodies remain stable under various stress conditions and, therefore, they have a good developability profile.

[0343] Example 4: In vitro and in vivo characterization of activatable antibodies targeting CTLA4

[0344] It was previously shown that the parent antibody TY21580 alone does not stimulate human T cell activation or human PBMC cell activation (see PCT International Application No. 69540-2000540, filed concurrently with this application and entitled "Compositions Comprising Cross-reactive Anti-CTLA4 Antibodies, and Methods of Making and Using the Same," which is incorporated herein by reference in its entirety). It is known that CTLA-4 activity on T cells is associated with a primary (TCR / CD3) signal and a secondary signal involving B7-CD28 / CTLA-4. Consistently, it was shown that the parent antibody TY21580 significantly enhanced human PBMC cell activation at low concentrations of anti-CD3 antibody.

[0345] In vitro functional characterization

[0346] Here, the activity of activatable antibodies targeting CTLA4 on human PBMC activation was evaluated in the presence of low concentrations of anti-CD3 antibodies. 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 on a 96-well plate at 4°C. After washing, 1x10^5 freshly isolated human PBMCs were added to each well, followed by addition of test articles at different concentrations. 48 hours after stimulation, the induction of IL-2 was measured using a human IL-2 ELISA Ready-SET-Go (Invitrogen) kit. IFN-γ in the supernatant was measured using a human IFN-γ ELISA Ready-SET-Go (Invitrogen) kit. As Figures 12A-12B As shown in , at high concentrations, TY22404 induced IL-2 production and TY22401 induced IFN-γ production. However, the activity of the activatable antibodies was significantly lower than that of the parental TY21580 antibody.

[0347] Next, the antibody-dependent cellular cytotoxicity activity of the activatable antibodies was tested and compared with the antibody-dependent cellular cytotoxicity activity of the parent antibody TY21580. The ADCC reporter gene assay was used to evaluate the ADCC activity of the activatable antibodies. HEK293F cells overexpressing human CTLA4 (HEK293F / hCTLA-4 cells) were used as target cells; Jurkat cell lines overexpressing CD16a and NFAT-Luc (Jurkat / CD16a cells) were used as effector cells. 1x10^5 Jurkat / CD16a cells and 1x10^4 HEK293F / hCTLA-4 cells (E:T ratio 10:1) were mixed with different concentrations of antibodies. After incubation for 6 hours, 100 μL 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. Figure 13 As shown in , the activatable antibodies exhibited ADCC activity that was several logs lower than that of the parent antibody, TY21580. The ADCC activity of TY22401 was higher than that of TY22402 and TY22404. In summary, the in vitro data indicate that better masked activatable antibodies have less A...

Claims

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), wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-85; wherein the CM comprises at least a first cleavage site and a first flexible linker (L1); and in: a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); b) the TBM comprises an antibody heavy chain variable region (VH), and the activatable antibody further comprises a second polypeptide comprising an antibody light chain variable region (VL); c) the TBM comprises, from 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 an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) from the N-terminus to the C-terminus; And among them (i) the VH comprises HVR-H1 as shown in the amino acid sequence of SEQ ID NO: 65, HVR-H2 as shown in the amino acid sequence of SEQ ID NO: 66, and HVR-H3 as shown in the amino acid sequence of SEQ ID NO: 67; and (ii) the VL comprises HVR-L1 as set forth in the amino acid sequence of SEQ ID NO: 68, HVR-L2 as set forth in the amino acid sequence of SEQ ID NO: 69, and HVR-L3 as set forth in the amino acid sequence of SEQ ID NO: 70; and Wherein when the CM is cleaved, the activatable antibody binds to human CD137 through the VH and the VL.

2. The activatable antibody of claim 1, 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).

3. The activatable antibody according to claim 1, wherein: a) the MM further comprises an additional amino acid sequence at its N-terminus; b) the first cleavage site is a protease cleavage site of 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; c) L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24; d) the CM further comprises a second cleavage site; and / or e) The CM further comprises a second linker (L2).

4. The activatable antibody of claim 3, wherein the MM further comprises an additional amino acid sequence at its N-terminus, wherein the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO:

16.

5. The activatable antibody of claim 3, wherein the CM further comprises L2, wherein the L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24.

6. The activatable antibody of claim 3, wherein the CM further comprises a second cleavage site, wherein a) the second cleavage site is at the C-terminus of L1; b) the second cleavage site is a protease cleavage site of 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; c) the first cleavage site and the second cleavage site are different; and / or d) the CM comprises L2 at the C-terminus of the second cleavage site.

7. The activatable antibody according to claim 1, wherein the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-46 and the TBM from the N-terminus to the C-terminus, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 40-46 comprises the MM and the CM.

8. The activatable antibody according to claim 1, wherein (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO:

50.

9. The activatable antibody of any one of claims 1 to 8, wherein the CM comprises the amino acid sequence of SEQ ID NO:

12.

10. The activatable antibody of any one of claims 1 to 8, wherein the MM comprises the amino acid sequence of SEQ ID NO:

79.

11. The activatable antibody according to claim 10, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO:

50.

12. The activatable antibody of claim 10, wherein the CM comprises the amino acid sequence of SEQ ID NO:

12.

13. The activatable antibody according to any one of claims 1 to 8, wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the MM comprises the amino acid sequence of SEQ ID NO: 79; and (c) the CM comprises the amino acid sequence of SEQ ID NO:

12.

14. The activatable antibody according to any one of claims 1 to 8, wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the MM comprises the amino acid sequence of SEQ ID NO: 79; (c) the CM comprises the amino acid sequence of SEQ ID NO: 12; (d) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (e) the VL comprises the amino acid sequence of SEQ ID NO:

50.

15. The activatable antibody of any one of claims 1 to 8, wherein the first polypeptide comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 40 and the TBM, and wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (c) the VL comprises the amino acid sequence of SEQ ID NO:

50.

16. The activatable antibody of any one of claims 1 to 8, wherein the MM comprises the amino acid sequence of SEQ ID NO:

82.

17. The activatable antibody of claim 16, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO:

50.

18. The activatable antibody of claim 16, wherein the CM comprises the amino acid sequence of SEQ ID NO:

12.

19. The activatable antibody of any one of claims 1 to 8, wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the MM comprises the amino acid sequence of SEQ ID NO: 82; and (c) the CM comprises the amino acid sequence of SEQ ID NO:

12.

20. The activatable antibody of any one of claims 1 to 8, wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the MM comprises the amino acid sequence of SEQ ID NO: 82; (c) the CM comprises the amino acid sequence of SEQ ID NO: 12; (d) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (e) the VL comprises the amino acid sequence of SEQ ID NO:

50.

21. The activatable antibody of any one of claims 1 to 8, wherein the first polypeptide comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 43 and the TBM, and wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (c) the VL comprises the amino acid sequence of SEQ ID NO:

50.

22. The activatable antibody of any one of claims 1 to 8, wherein the MM comprises the amino acid sequence of SEQ ID NO:

85.

23. The activatable antibody of claim 22, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO:

50.

24. The activatable antibody of claim 22, wherein the CM comprises the amino acid sequence of SEQ ID NO:

12.

25. The activatable antibody of any one of claims 1 to 8, wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the MM comprises the amino acid sequence of SEQ ID NO: 85; and (c) the CM comprises the amino acid sequence of SEQ ID NO:

12.

26. The activatable antibody of any one of claims 1 to 8, wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the MM comprises the amino acid sequence of SEQ ID NO: 85; (c) the CM comprises the amino acid sequence of SEQ ID NO: 12; (d) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (e) the VL comprises the amino acid sequence of SEQ ID NO:

50.

27. The activatable antibody of any one of claims 1 to 8, wherein the first polypeptide comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 46 and the TBM, and wherein: (a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain variable region (VH); (b) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (c) the VL comprises the amino acid sequence of SEQ ID NO:

50.

28. An activatable antibody, comprising: a first polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and an antibody light chain variable region (VL), and a second polypeptide comprising an antibody heavy chain variable region (VH); wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-85; wherein the CM comprises at least a first cleavage site and a first flexible linker (L1); in: (i) the VH comprises HVR-H1 as shown in the amino acid sequence of SEQ ID NO: 65, HVR-H2 as shown in the amino acid sequence of SEQ ID NO: 66, and HVR-H3 as shown in the amino acid sequence of SEQ ID NO: 67; and (ii) the VL comprises HVR-L1 as set forth in the amino acid sequence of SEQ ID NO: 68, HVR-L2 as set forth in the amino acid sequence of SEQ ID NO: 69, and HVR-L3 as set forth in the amino acid sequence of SEQ ID NO: 70; and Wherein when the CM is cleaved, the activatable antibody binds to human CD137 through the VH and the VL.

29. The activatable antibody of claim 28, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO:

50.

30. The activatable antibody of claim 28, wherein the MM comprises the amino acid sequence of SEQ ID NO:

79.

31. The activatable antibody of claim 28, wherein: (a) the MM comprises the amino acid sequence of SEQ ID NO: 79; and (b) the CM comprises the amino acid sequence of SEQ ID NO: 12; (c) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (d) the VL comprises the amino acid sequence of SEQ ID NO:

50.

32. The activatable antibody of claim 28, wherein the first polypeptide comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 40 and the VL, and wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO:

50.

33. The activatable antibody of claim 28, wherein the MM comprises the amino acid sequence of SEQ ID NO:

82.

34. The activatable antibody of claim 28, wherein: (a) the MM comprises the amino acid sequence of SEQ ID NO: 82; (b) the CM comprises the amino acid sequence of SEQ ID NO: 12; (c) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (d) the VL comprises the amino acid sequence of SEQ ID NO:

50.

35. The activatable antibody of claim 28, wherein the first polypeptide comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 43 and the VL, and wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO:

50.

36. The activatable antibody of claim 28, wherein the MM comprises the amino acid sequence of SEQ ID NO:

85.

37. The activatable antibody of claim 28, wherein: (a) the MM comprises the amino acid sequence of SEQ ID NO: 85; (b) the CM comprises the amino acid sequence of SEQ ID NO: 12; (c) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (d) the VL comprises the amino acid sequence of SEQ ID NO:

50.

38. The activatable antibody of claim 28, wherein the first polypeptide comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 46 and the VL, and wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 49; and (b) the VL comprises the amino acid sequence of SEQ ID NO: 50.

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