Matrix metalloproteinase substrates and other cleavable moieties and methods of their use

Abstract

To identify new substrates for proteases and use these substrates for a variety of therapeutic, diagnostic and prophylactic applications.SOLUTION: The invention relates generally to polypeptides that include a cleavable moiety that is a substrate for at least one matrix metalloprotease (MMP), to activatable antibodies and other large molecules that include the cleavable moiety that is a substrate for at least one MMP protease, and to methods of making these polypeptides that include a cleavable moiety that is a substrate for at least one MMP protease and using them for a variety of therapeutic, diagnostic and prophylactic applications.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 882,377, filed September 25, 2013, and U.S. Provisional Patent Application No. 61 / 971,332, filed March 27, 2014, the contents of which are incorporated herein by reference in their entireties.

[0002] The present invention relates generally to polypeptides that include a cleavable moiety that is a substrate for at least one matrix metalloprotease (MMP), activatable antibodies and other large molecules that include a cleavable moiety that is a substrate for at least one MMP protease, and methods for making and using these polypeptides that include a cleavable moiety that is a substrate for at least one MMP protease for various therapeutic, diagnostic, and prophylactic applications. [Background technology]

[0003] Proteases are enzymes that break down proteins by cleaving peptide bonds between amino acid residues. Proteases are naturally present in all living organisms and are involved in a variety of physiological reactions, from simple processes to highly regulated pathways. Some proteases are known to cleave specific peptide bonds based on the presence of specific amino acid sequences within the protein.

[0004] Thus, there is a need to identify novel groups for proteases and use those substrates for a variety of therapeutic, diagnostic and prophylactic applications. Summary of the Invention

[0005] The present disclosure provides amino acid sequences containing cleavable moieties (CMs) that are substrates for at least one matrix metalloproteinase (MMP), which are useful in a variety of therapeutic, diagnostic, and prophylactic applications.

[0006] According to some embodiments, the CM is a substrate for at least one matrix metalloproteinase (MMP). Examples of MMPs include: MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. According to some embodiments, the CM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. According to some embodiments, the CM is a substrate for MMP9. According to some embodiments, the CM is a substrate for MMP14. According to some embodiments, the CM is a substrate for multiple MMPs. According to some embodiments, the CM is a substrate for at least MMP9 and MMP14. According to some embodiments, the CM comprises multiple substrates for the same MMP. According to some embodiments, the CM comprises at least multiple MMP9 substrates. According to some embodiments, the CM comprises at least multiple MMP14 substrates.

[0007] According to some embodiments, the CM is a substrate for MMPs and includes the sequences ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32); PAGLWLDP (SEQ ID NO: 33); and / or ISSGLSS (SEQ ID NO: 159).

[0008] In some embodiments, the CM comprises the amino acid sequence ISSGLLSS (SEQ ID NO: 14). In some embodiments, the CM comprises the amino acid sequence QNQALRMA (SEQ ID NO: 15). In some embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ ID NO: 16). In some embodiments, the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 17). In some embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 18). In some embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ ID NO: 19). In some embodiments, the CM comprises the amino acid sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments, the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). In some embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ ID NO: 22). In some embodiments, the CM comprises the amino acid sequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO:25). In some embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQ ID NO:26). In some embodiments, the CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO:27). In some embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ ID NO:28). In some embodiments, the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO:29). In some embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO:30). In some embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ ID NO:31). In some embodiments, the CM comprises the amino acid sequence LLAPSHRA (SEQ ID NO:32). In some embodiments, the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO:33). In some embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ ID NO:159).

[0009] In some embodiments, the CM is linked or otherwise conjugated to an antibody. For example, a CM is used to link one or more agents to an antibody or antigen-binding fragment thereof (AB) that binds a predetermined target, such that when exposed to an MMP, the CM is cleaved and the agent is released from the AB. Exemplary targets include, but are not limited to, those shown in Table 1. Exemplary ABs include, but are not limited to, those shown in Table 2. In some embodiments, the antibody in its uncleaved state has the following structural configuration from N-terminus to C-terminus: agent-CM-AB or AB-M-agent. In some embodiments, the antibody comprises a linking peptide between the AB and the CM. In some embodiments, the antibody comprises a linking peptide between the CM and the conjugated agent.

[0010] In some embodiments, the antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: agent-LP1-CM-LP2-AB or AB-LP2-CM-LP1-agent. In some embodiments, each LP1 and LP2 is a peptide about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other.

[0011] According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO:2), where n is an integer of at least one.

[0012] According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8).

[0013] According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO: 157).

[0014] According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12), or GSSG (SEQ ID NO: 13).

[0015] According to some embodiments, the AB has an equilibrium dissociation constant for binding to the target of about 100 nM or less.

[0016] In some embodiments, the antibody comprises an antibody or antigen-binding fragment thereof that specifically binds a target. In some embodiments, the antibody or immunologically active fragment thereof that binds the target is a monoclonal antibody, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds the target is a murine, other rodent, humanized, or fully human monoclonal antibody.

[0017] According to some embodiments, the MMP protease is co-localized with the target in the tissue, and the MMP protease cleaves the CM in the antibody when the antibody is exposed to the protease.

[0018] According to some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0019] According to some embodiments, the CM is a substrate for at least one matrix metalloproteinase (MMP). Examples of MMPs include: MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. According to some embodiments, the CM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. According to some embodiments, the CM is a substrate for MMP9. According to some embodiments, the CM is a substrate for MMP14. According to some embodiments, the CM is a substrate for multiple MMPs. According to some embodiments, the CM is a substrate for at least MMP9 and MMP14. According to some embodiments, the CM comprises multiple substrates for the same MMP. According to some embodiments, the CM comprises at least multiple MMP9 substrates. According to some embodiments, the CM comprises at least multiple MMP14 substrates.

[0020] According to some embodiments, the CM is a substrate for MMPs and includes the sequences ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32); PAGLWLDP (SEQ ID NO: 33); and / or ISSGLSS (SEQ ID NO: 159).

[0021] In some embodiments, the CM comprises the amino acid sequence ISSGLLSS (SEQ ID NO: 14). In some embodiments, the CM comprises the amino acid sequence QNQALRMA (SEQ ID NO: 15). In some embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ ID NO: 16). In some embodiments, the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 17). In some embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 18). In some embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ ID NO: 19). In some embodiments, the CM comprises the amino acid sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments, the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). In some embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ ID NO: 22). In some embodiments, the CM comprises the amino acid sequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO:25). In some embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQ ID NO:26). In some embodiments, the CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO:27). In some embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ ID NO:28). In some embodiments, the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO:29). In some embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO:30). In some embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ ID NO:31). In some embodiments, the CM comprises the amino acid sequence LLAPSHRA (SEQ ID NO:32). In some embodiments, the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO:33). In some embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ ID NO:159).

[0022] According to some embodiments, the CM is a substrate for at least one matrix metalloproteinase (MMP) and includes a motif sequence recognized by MMP9. According to some embodiments, the CM is a substrate for at least one MMP and includes a motif sequence recognized by MMP14.

[0023] According to some embodiments, the CM is a substrate for at least one MMP, and the CM polypeptide, and / or the CM portion of any polypeptide comprising the CM, includes polypeptides having a length of less than 50 amino acids, a length of less than 40 amino acids, a length of less than 30 amino acids, a length of less than 25 amino acids, a length of less than 20 amino acids, a length of less than 19 amino acids, a length of less than 18 amino acids, a length of less than 17 amino acids, a length of less than 16 amino acids, a length of less than 15 amino acids, a length of less than 14 amino acids, a length of less than 13 amino acids, a length of less than 12 amino acids, a length of less than 11 amino acids, or a length of less than 10 amino acids.

[0024] According to some embodiments, the CM is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease. According to some embodiments, the CM is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease.

[0025] According to some embodiments, the motif sequence is a substrate for at least an MMP and comprises a core CM consensus sequence set forth in Tables 8A-8M below. According to some embodiments, the motif sequence comprises a subclass or subset of the core CM consensus sequences set forth in Tables 8A-8M below.

[0026] According to some embodiments, the motif sequence is a substrate for at least MMP9 and includes the core CM consensus sequences shown in Tables 8A-8D. According to some embodiments, the motif sequence is a substrate for at least MMP9 and includes subclasses or subsets of the core CM consensus sequences shown in Tables 8A-8D below.

[0027] According to some embodiments, the motif sequences are substrates for at least MMP14 and include the core CM consensus sequences shown in Tables 8E-8M. According to some embodiments, the motif sequences are substrates for at least MMP14 and include subclasses or subsets of the core CM consensus sequences shown in Tables 8E-8M below.

[0028] Table 8A. MMP9 Cleavable Core CM Consensus Sequence 1 [Table 1]

[0029] Table 8B. MMP9 Cleavable Core CM Consensus Sequence 2 [Table 2]

[0030] Table 8C. MMP9 Cleavable Core CM Consensus Sequence 3 [Table 3]

[0031] Table 8D. MMP9 Cleavable Core CM Consensus Sequence 4 [Table 4]

[0032] Table 8E. MMP14 Cleavable Core CM Consensus Sequence 5 [Table 5]

[0033] [Table 6]

[0034] Table 8F-1. MMP14 Cleavable Core CM Consensus Sequence 6 [Table 7]

[0035] Table 8F-2. MMP14 Cleavable Core CM Consensus Sequence 6A [Table 8]

[0036] Table 8G. MMP14 Cleavable Core CM Consensus Sequence 7 [Table 9]

[0037] Table 8H-1. MMP14 Cleavable Core CM Consensus Sequence 8 [Table 10]

[0038] Table 8H-2. MMP14 Cleavable Extended Core CM Consensus Sequence 8 [Table 11]

[0039] [Table 12]

[0040] Table 8I. MMP14 Cleavable Core CM Consensus Sequence 9 [Table 13]

[0041] Table 8J. MMP14 Cleavable Core CM Consensus Sequence 10 [Table 14]

[0042] [Table 15]

[0043] Table 8K.MMP14 Cleavable Core CM Consensus Sequence 11 [Table 16]

[0044] Table 8. MMP14 Cleavable Core CM Consensus Sequence 12 [Table 17]

[0045] [Table 18]

[0046] Table 8. M.MMP14 Cleavage Core CM Consensus Sequence 13 [Table 19]

[0047] According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 352, 371, 394, 410, 425, 436, 453, 458, 473, 485, and 486. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 353-363, 372-375, 376-378, 395-401, 411-419, 426-433, 437-449, 454-456, 459-469, 475-482, and 487-495. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 353-363. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 372-375. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 376-378. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 395-401. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 411-419. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 426-433. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 437-449. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 454-456. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 459-469. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 475-482. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 487-495.

[0048] According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 317, 324, 329, and 340. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 318-323, 325-327, 330-335, and 341-347. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 318-323. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 325-327. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 330-335. According to some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 341-347.

[0049] According to some embodiments, the CM comprises a core CM consensus 1 sequence comprising the amino acid sequence RPSPMWAY (SEQ ID NO: 21).

[0050] According to some embodiments, the CM comprises a core CM consensus 2 sequence comprising the amino acid sequence WDHPISLL (SEQ ID NO: 328). According to some embodiments, the CM comprises a core CM consensus 2 sequence comprising the amino acid sequence WATPRPMR (SEQ ID NO: 22).

[0051] According to some embodiments, the CM comprises a core CM consensus 3 sequence comprising the amino acid sequence LTFPTYIF (SEQ ID NO: 336). According to some embodiments, the CM comprises a core CM consensus 3 sequence comprising the amino acid sequence MTFPTYIF (SEQ ID NO: 337). According to some embodiments, the CM comprises a core CM consensus 3 sequence comprising the amino acid sequence LTFPTYWF (SEQ ID NO: 338). According to some embodiments, the CM comprises a core CM consensus 3 sequence comprising the amino acid sequence MTFPTYWF (SEQ ID NO: 339). According to some embodiments, the CM comprises a core CM consensus 3 sequence comprising the amino acid sequence STFPFGMF (SEQ ID NO: 17).

[0052] In some embodiments, the CM comprises a core CM consensus 4 sequence comprising the amino acid sequence DWLYWMGI (SEQ ID NO: 348). In some embodiments, the CM comprises a core CM consensus 4 sequence comprising the amino acid sequence DWLYWPGI (SEQ ID NO: 19). In some embodiments, the CM comprises a core CM consensus 4 sequence comprising the amino acid sequence DWLYWMSI (SEQ ID NO: 349). In some embodiments, the CM comprises a core CM consensus 4 sequence comprising the amino acid sequence DWLYWPSI (SEQ ID NO: 350). In some embodiments, the CM comprises a core CM consensus 4 sequence comprising the amino acid sequence HWHLGPPT (SEQ ID NO: 351).

[0053] In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence ISSGLLSS (SEQ ID NO: 14). In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence SVSGLLSH (SEQ ID NO: 364). In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence SVSGLLSS (SEQ ID NO: 365). In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence SVSGLRSH (SEQ ID NO: 366). In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence SVSGLRSS (SEQ ID NO: 367). In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence TLSGLRSP (SEQ ID NO: 368). In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence TSSGLRSP (SEQ ID NO: 369). In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence TVSGLRSP (SEQ ID NO: 370).

[0054] In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence AFQALRM (SEQ ID NO: 379). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence AHQALRM (SEQ ID NO: 380). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence ANQALRM (SEQ ID NO: 381). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence ANQALRMA (SEQ ID NO: 382). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLEALRAL (SEQ ID NO: 383). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLNALRAL (SEQ ID NO: 384). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLQALRAL (SEQ ID NO: 385). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLSALRAL (SEQ ID NO: 386). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLESLRAL (SEQ ID NO: 387). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLNSLRAL (SEQ ID NO: 388). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLQSLRAL (SEQ ID NO: 389). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence LLSSLRAL (SEQ ID NO: 390). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence QFQALRM (SEQ ID NO: 391). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence QHQALRM (SEQ ID NO: 392). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence QNQALRM (SEQ ID NO: 393). In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence QNQALRMA (SEQ ID NO: 15).

[0055] In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LKAAPVWA (SEQ ID NO: 403). In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LKAAPRWF (SEQ ID NO: 404). In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LKAAPVWF (SEQ ID NO: 405). In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LYAAPRWA (SEQ ID NO: 406). In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LYAAPVWA (SEQ ID NO: 407). In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LYAAPRWF (SEQ ID NO: 408). According to some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence LYAAPVWF (SEQ ID NO: 409).

[0056] In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence GPSHLVLT (SEQ ID NO: 25). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence LPAGLLL (SEQ ID NO: 402). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence LPAGLLLR (SEQ ID NO: 420). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence LPAHLVLL (SEQ ID NO: 421). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence LPSHLVLL (SEQ ID NO: 422). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence LPAHLVLV (SEQ ID NO: 423). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence LPSHLVLV (SEQ ID NO: 424).

[0057] In some embodiments, the CM comprises a core CM consensus 9 sequence comprising the amino acid sequence RMHLRSLG (SEQ ID NO: 29). In some embodiments, the CM comprises a core CM consensus 9 sequence comprising the amino acid sequence RRHDGLRA (SEQ ID NO: 434). In some embodiments, the CM comprises a core CM consensus 9 sequence comprising the amino acid sequence RRHDGLRS (SEQ ID NO: 435).

[0058] According to some embodiments, the CM comprises a core CM consensus 10 sequence comprising the amino acid sequence AQNLLGMV (SEQ ID NO: 16). According to some embodiments, the CM comprises a core CM consensus 10 sequence comprising the amino acid sequence IANNLLSMV (SEQ ID NO: 450). According to some embodiments, the CM comprises a core CM consensus 10 sequence comprising the amino acid sequence ILNLLSMV (SEQ ID NO: 451). According to some embodiments, the CM comprises a core CM consensus 10 sequence comprising the amino acid sequence IQNLLSMV (SEQ ID NO: 452).

[0059] In some embodiments, the CM comprises a core CM consensus 11 sequence comprising the amino acid sequence PAGLWLDP (SEQ ID NO: 33). In some embodiments, the CM comprises a core CM consensus 11 sequence comprising the amino acid sequence PASLWYTQ (SEQ ID NO: 457).

[0060] In some embodiments, the CM comprises a core CM consensus 12 sequence comprising the amino acid sequence ALGLLRLP (SEQ ID NO: 470). In some embodiments, the CM comprises a core CM consensus 12 sequence comprising the amino acid sequence ALGLLSLP (SEQ ID NO: 471). In some embodiments, the CM comprises a core CM consensus 12 sequence comprising the amino acid sequence ASGLLRFP (SEQ ID NO: 472). In some embodiments, the CM comprises a core CM consensus 12 sequence comprising the amino acid sequence AVGLLAPP (SEQ ID NO: 31).

[0061] According to some embodiments, the CM comprises a core CM consensus 13 sequence comprising the amino acid sequence LAAPLGLL (SEQ ID NO: 30). According to some embodiments, the CM comprises a core CM consensus 13 sequence comprising the amino acid sequence LLAPSHRA (SEQ ID NO: 32).

[0062] According to some embodiments, the CM comprises a core CM consensus 13 sequence comprising the amino acid sequence LLLPAHGG (SEQ ID NO: 474). According to some embodiments, the CM comprises a core CM consensus 13 sequence comprising the amino acid sequence LLLPLLGS (SEQ ID NO: 483).

[0063] According to some embodiments, the CM is a substrate for at least two proteases, wherein at least one protease is an MMP and the at least one protease is selected from the group consisting of those enzymes shown in Table 7.

[0064] [Table 20]

[0065] According to some embodiments, the antibody comprises at least a first CM and a second CM. According to some embodiments, the first CM and second CM are each polypeptides 15 or less amino acids in length. According to some embodiments, the first CM and second CM in the antibody in an uncleaved state have the following structural arrangement from N-terminus to C-terminus: agent-CM1-CM2-AB, AB-CM2-CM1-agent, agent-CM2-CM1-AB, or AB-CM1-CM2-agent. According to some embodiments, the activatable antibody comprises a connecting peptide between the agent and CM1. According to some embodiments, the activatable antibody comprises a connecting peptide between CM1 and CM2. According to some embodiments, the activatable antibody comprises a connecting peptide between CM2 and AB. According to some embodiments, the activatable antibody comprises a connecting peptide between the agent and CM1 and a connecting peptide between CM2 and AB. According to some embodiments, the activatable antibody comprises a connecting peptide between the agent and CM1 and a connecting peptide between CM2 and AB. According to some embodiments, the activatable antibody comprises a connecting peptide between the agent and CM1 and a connecting peptide between CM1 and CM2. According to some embodiments, the activatable antibody comprises a connecting peptide between CM1 and CM2, and a connecting peptide between CM2 and AB. According to some embodiments, the activatable antibody comprises a connecting peptide between the agent and CM1, a connecting peptide between CM1 and CM2, and a connecting peptide between CM2 and AB.

[0066] In some embodiments, an activatable antibody comprises at least a first CM comprising a substrate for at least one matrix metalloproteinase (MMP) and a second CM comprising a substrate sequence. Exemplary substrates for the second CM (CM2) include, but are not limited to, substrates cleavable by one or more of the following enzymes or proteases listed in Table 7:

[0067] In some embodiments, CM2 is selected for use with a particular protease, such as a matrix metalloproteinase (MMP), neutrophil esterase, u-type plasminogen activator (uPA, also known as urokinase), legumain, matriptase (also referred to herein as MT-SP1 or MTSP1), thrombin, a cysteine ​​protease, such as a cathepsin, ADAM17, BMP-1, HtrA1, or a TMPRSS, such as TMPRSS3 or TMPRSS4.

[0068] According to some embodiments, CM2 is a substrate for neutrophil elastase. According to some embodiments, CM2 is a substrate for uPA. According to some embodiments, CM2 is a substrate for legumain. According to some embodiments, CM2 is a substrate for matriptase. According to some embodiments, CM2 is a substrate for thrombin. According to some embodiments, CM2 is a substrate for cysteine ​​proteases. According to some embodiments, CM2 is a substrate for cathepsins. According to some embodiments, CM2 is a substrate for ADAM17. According to some embodiments, CM2 is a substrate for BMP-1. According to some embodiments, CM2 is a substrate for HtrA1. According to some embodiments, CM2 is a substrate for TMPRSS. According to some embodiments, CM2 is a substrate for TMPRSS3. According to some embodiments, CM2 is a substrate for TMPRSS4.

[0069] According to some embodiments, suitable CM2 is cleaved by at least one protease and comprises the sequences TGRGPSWV (SEQ ID NO:34); SARGPSRW (SEQ ID NO:35); TARGPSFK (SEQ ID NO:36); LSGRSDNH (SEQ ID NO:37); GGWHTGRN (SEQ ID NO:38); HTGRSGAL (SEQ ID NO:39); PLTGRSGG (SEQ ID NO:40); AARGPAIH (SEQ ID NO:41); RGPAFNPM (SEQ ID NO:42); SSRGPAYL (SEQ ID NO:43); RGPATPIM (SEQ ID NO:44); RGPA (SEQ ID NO:45); GGQPSGMWGW (SEQ ID NO:46); FPRPLGITGL (SEQ ID NO:47); VHMPLGFLGP (SEQ ID NO:48); SPLTGRSG (SEQ ID NO:49); SAGFSLPA (SEQ ID NO:126); LAPLGLQRR (SEQ ID NO:50); SGGPLGVR (SEQ ID NO:51); PLGL (SEQ ID NO:52); GPRSFGL (SEQ ID NO:315) and / or GPRSFG (SEQ ID NO:316).

[0070] According to some embodiments, CM2 comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 34). According to some embodiments, CM2 comprises the amino acid sequence SARGPSRW (SEQ ID NO: 35). According to some embodiments, CM2 comprises the amino acid sequence TARGPSFK (SEQ ID NO: 36). According to some embodiments, CM2 comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 37). According to some embodiments, CM2 comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 38). According to some embodiments, CM2 comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 39). According to some embodiments, CM2 comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 40). According to some embodiments, CM2 comprises the amino acid sequence AARGPAIH (SEQ ID NO: 41). According to some embodiments, CM2 comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 42). According to some embodiments, CM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 43). According to some embodiments, CM2 comprises the amino acid sequence RGPATPIM (SEQ ID NO: 44). According to some embodiments, CM2 comprises the amino acid sequence RGPA (SEQ ID NO: 45). According to some embodiments, CM2 comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO: 46). According to some embodiments, CM2 comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 47). According to some embodiments, CM2 comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 48). According to some embodiments, CM2 comprises the amino acid sequence SPLTGRSG (SEQ ID NO: 49). According to some embodiments, CM2 comprises the amino acid sequence LAPLGLQRR (SEQ ID NO: 50). According to some embodiments, CM2 comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 51). According to some embodiments, CM2 comprises the amino acid sequence PLGL (SEQ ID NO: 52). According to some embodiments, CM2 comprises the amino acid sequence GPRSFGL (SEQ ID NO: 315). According to some embodiments, CM2 comprises the amino acid sequence GPRSFG (SEQ ID NO: 316).

[0071] According to some embodiments, CM2 is a substrate for at least one MMP. According to some embodiments, CM2 is a substrate for at least one MMP listed in Table 7. According to some embodiments, CM2 is a substrate for MMP9. According to some embodiments, CM2 is a substrate for an MMP. According to some embodiments, CM1 is a substrate for a first MMP and CM2 is a substrate for a second MMP, where the first MMP and the second MMP are different MMPs. According to some embodiments, CM1 is a first substrate sequence for an MMP and CM2 is a second substrate for the same MMP, where CM1 and CM2 have different substrate sequences. According to some embodiments, CM2 is a substrate for multiple MMPs. According to some embodiments, CM2 is a substrate for at least MMP9 or MMP14. According to some embodiments, CM2 is a substrate for multiple MMPs. According to some embodiments, CM2 is a substrate for at least MMP9 and MMP14. According to some embodiments, both CM1 and CM2 are substrates for MMP9. According to some embodiments, both CM1 and CM2 are substrates for MMP 14. According to some embodiments, CM1 is a substrate for MMP9 and CM2 is a substrate for MMP 14. According to some embodiments, CM1 is a substrate for MMP 14 and CM2 is a substrate for MMP 9.

[0072] According to some embodiments, at least one of CM1 and / or CM2 is a substrate for an MMP and comprises the sequence ISSGLLSS (SEQ ID NO:14); QNQALRMA (SEQ ID NO:15); AQNLLGMV (SEQ ID NO:16); STFPFGMF (SEQ ID NO:17); PVGYTSSL (SEQ ID NO:18);DWLYWPGI (SEQ ID NO:19); MIAPVAYR (SEQ ID NO:20); RPSPMWAY (SEQ ID NO:21); WATPRPMR (SEQ ID NO:22); FRLLDWQW (SEQ ID NO:23); LKAAPRWA (SEQ ID NO:24); GPSHLVLT (SEQ ID NO:25); LPGGLSPW (SEQ ID NO:26);MGLFSEAG (SEQ ID NO:27); SPLPLRVP (SEQ ID NO:28); RMHLRSLG (SEQ ID NO:29); LAAPLGLL (SEQ ID NO:30); AVGLLAPP (SEQ ID NO:31); LLAPSHRA (SEQ ID NO:32); PAGLWLDP (SEQ ID NO:33); and / or ISSGLSS (SEQ ID NO:159).

[0073] According to some embodiments, the first cleaving agent and the second cleaving agent are the same protease, and the first CM and the second CM are different substrates for the enzyme. According to some embodiments, the first cleaving agent and the second cleaving agent are different proteases. According to some embodiments, the first cleaving agent and the second cleaving agent are co-localized in the target tissue. According to some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

[0074] In some embodiments, the agent conjugated to AB is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. As used herein, a toxin fragment is a fragment that preserves toxic activity. In some embodiments, the agent is conjugated to AB via a cleavable linker. In some embodiments, the agent is conjugated to AB via a linker comprising at least one MMP-cleavable substrate sequence. In some embodiments, the agent is conjugated to AB via a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or DNA intercalator, or other DNA damaging agent. In some embodiments, the agent is an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a derivative thereof. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0075] According to some embodiments, the agent is an anti-inflammatory agent.

[0076] According to some embodiments, the antibody also comprises a detectable moiety. According to some embodiments, the detectable moiety is a diagnostic agent.

[0077] According to some embodiments, the conjugated antibody and / or the conjugated activatable antibody comprises a detectable label. According to some embodiments, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. According to some embodiments, the imaging agent comprises a radioisotope. According to some embodiments, the radioisotope is indium or technetium. According to some embodiments, the contrast agent comprises iodine, gadolinium, or iron oxide. According to some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. According to some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. According to some embodiments, the luminescent label comprises an N-methylacridium derivative. According to some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. According to some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0078] In some embodiments, the AB in an antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0079] According to some embodiments, the antibodies and / or conjugated antibodies are monospecific. According to some embodiments, the antibodies and / or conjugated antibodies are multispecific, also referred to herein as multispecific antibodies and / or conjugated multispecific antibodies. According to some embodiments, the multispecific antibodies and / or conjugated multispecific antibodies are bispecific or trispecific. According to some embodiments, the antibodies and / or conjugated antibodies are formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. According to some embodiments, the antibodies and / or conjugated antibodies are formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor.

[0080] According to some embodiments, the activatable antibody and / or conjugated activatable antibody is monospecific. According to some embodiments, the activatable antibody and / or conjugated activatable antibody is multispecific and is also referred to herein as a multispecific activatable antibody and / or a conjugated multispecific activatable antibody. As used herein, the term "activatable antibody" and all grammatical variations thereof, unless otherwise specified, is intended to encompass, but is not limited to, embodiments in which the activatable antibody is a multispecific activatable antibody of the present disclosure. As used herein, the term "conjugated activatable antibody" and all grammatical variations thereof, unless otherwise specified, is intended to encompass, but is not limited to, embodiments in which the conjugated activatable antibody is a conjugated multispecific activatable antibody of the present disclosure. According to some embodiments, the multispecific activatable antibody and / or conjugated multispecific activatable antibody is bispecific or trispecific. According to some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. According to some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor.

[0081] According to some embodiments, the activatable antibodies, conjugated activatable antibodies, multispecific activatable antibodies, and / or conjugated multispecific activatable antibodies described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended use, e.g., cancer. For example, the activatable antibodies, conjugated activatable antibodies, multispecific activatable antibodies, and / or conjugated multispecific activatable antibodies may be used in conjunction with an additional chemotherapeutic or anti-tumor agent.

[0082] The activatable antibodies described herein, in their activated state, bind to a predetermined target and comprise (i) an antibody or antigen-binding fragment thereof (AB) that specifically binds to the target; (ii) a masking moiety (MM) that inhibits binding of the AB to the target under uncleaved conditions; and (iii) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that acts as a substrate for a matrix metalloproteinase.

[0083] According to some embodiments, the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-CM-AB and AB-CM-MM.

[0084] According to some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM.

[0085] According to some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB.

[0086] According to some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and in an uncleaved state, the activatable antibody has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.

[0087] According to some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

[0088] According to some embodiments, the two connecting peptides need not be identical to each other.

[0089] According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2), where n is an integer of at least one.

[0090] According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8).

[0091] According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO: 157).

[0092] According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12), or GSSG (SEQ ID NO: 13).

[0093] According to some embodiments, the AB has an equilibrium dissociation constant for binding to the target of about 100 nM or less.

[0094] In some embodiments, the activatable antibody comprises an antibody or antigen-binding fragment thereof that specifically binds a target. In some embodiments, the antibody or immunologically active fragment thereof that binds the target is a monoclonal antibody, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, such antibody or immunologically active fragment thereof that binds the target is a murine, other rodent, humanized, or fully human monoclonal antibody.

[0095] According to some embodiments, the activatable antibody is a multispecific activatable antibody. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize multiple antigens or epitopes and include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody, where coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. According to some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for at least one MMP protease. The multispecific activatable antibodies provided herein are stable in the circulation, are activated at intended sites of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit target binding at least comparable to that of the corresponding, unmodified multispecific antibody.

[0096] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind a Jagged target, such as Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence SYAMS (SEQ ID NO: 498); a VH CDR2 sequence comprising at least the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 499); a VH CDR3 sequence comprising the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500); and combinations thereof.

[0097] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind a Jagged target, such as Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence RASQSISSY (SEQ ID NO: 501); a VH CDR2 sequence comprising at least the amino acid sequence AASSLQS (SEQ ID NO: 502); a VL CDR3 sequence comprising the amino acid sequence QQTVVAPPL (SEQ ID NO: 503); and combinations thereof.

[0098] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind a Jagged target, such as Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SYAMS (SEQ ID NO: 498); a VH CDR2 sequence comprising a sequence that is 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500); a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500); and combinations thereof.

[0099] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind a Jagged target, such as Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSISSY (SEQ ID NO: 501); a VH CDR2 sequence comprising a sequence that is 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQTVVAPPL (SEQ ID NO: 503); a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQTVVAPPL (SEQ ID NO: 503); and combinations thereof.

[0100] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind a Jagged target, such as Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence at least comprises the amino acid sequence SYAMS (SEQ ID NO: 498); the VH CDR2 sequence at least comprises the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 499); the VH CDR3 sequence comprises the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500); the VL CDR1 sequence at least comprises the amino acid sequence RASQSISSY (SEQ ID NO: 501); The CDR2 sequence comprises at least the amino acid sequence AASSLQS (SEQ ID NO: 502); and the VL CDR3 sequence comprises the amino acid sequence QQTVVAPPL (SEQ ID NO: 503).

[0101] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind a Jagged target, such as Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SYAMS (SEQ ID NO: 498); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSISSY (SEQ ID NO: 501); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQ ID NO: 502). and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQTVVAPPL (SEQ ID NO: 503).

[0102] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind epidermal growth factor receptor (EGFR) and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence NYGVH (SEQ ID NO: 504); a VH CDR2 sequence comprising at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 505); a VH CDR3 sequence comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 506); and combinations thereof.

[0103] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507); a VH CDR2 sequence comprising at least the amino acid sequence KYASESIS (SEQ ID NO: 508); a VH CDR3 sequence comprising the amino acid sequence QQNNNWPTT (SEQ ID NO: 509); and combinations thereof.

[0104] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 504); a VH CDR2 sequence comprising a sequence that is 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 506); a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 506); and combinations thereof.

[0105] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507); a VH CDR2 sequence comprising a sequence that is 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 509); a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 509); and combinations thereof.

[0106] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence at least comprises the amino acid sequence NYGVH (SEQ ID NO: 504); the VH CDR2 sequence at least comprises the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 505); the VH CDR3 sequence comprises the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 506); the VL CDR1 sequence at least comprises the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507); The CDR2 sequence comprises at least the amino acid sequence KYASESIS (SEQ ID NO: 508); and the VL CDR3 sequence comprises the amino acid sequence QQNNNWPTT (SEQ ID NO: 509).

[0107] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 504); the VH CDR3 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 506); the VL CDR1 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507); and the VL CDR2 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO: 508). and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 509).

[0108] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, comprise at least a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114. According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, comprise at least a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 59, 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113. According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, include those selected from the group consisting of SEQ ID NOs: 54, 56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114, and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 59, 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113.

[0109] According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, comprise at least a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114. According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, comprise at least a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 59, 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113.According to some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 106%, 108, 110, 112, and 114 amino acid sequence identity. The antibody comprises at least an H-chain amino acid sequence that is 99% or more identical to, and an L-chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 59, 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113.

[0110] According to some embodiments, the MM has an equilibrium dissociation constant for binding to the AB that is higher than the equilibrium dissociation constant of the AB to the target.

[0111] According to some embodiments, the MM has an equilibrium dissociation constant for binding to the Ab that is less than or equal to the equilibrium dissociation constant of the AB to the target.

[0112] According to some embodiments, the MM, in its cleaved state, does not interfere with or compete with the AB for binding to the target.

[0113] According to some embodiments, the MM is a polypeptide of about 2-40 amino acids in length, for example, the MM is a polypeptide of up to about 40 amino acids in length.

[0114] In some embodiments, the MM polypeptide sequence differs from that of any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of AB.

[0115] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least twice as high.

[0116] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least three times higher than

[0117] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least five times higher than

[0118] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least 10 times higher than

[0119] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least 20 times higher than

[0120] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least 40 times higher than

[0121] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least 100 times higher than

[0122] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least 1000 times higher than

[0123] According to some embodiments, coupling of the MM to the AB reduces the ability of the AB to bind its target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the target MM d At least 10,000 times higher than

[0124] According to some embodiments, the MMP protease is co-localized with the target in the tissue, and the MMP cleaves the CM in the activatable antibody when the activatable antibody is exposed to the MMP.

[0125] According to some embodiments, in the presence of the target, the MM reduces the ability of the AB to bind the target by at least 90% when the CM is uncleaved compared to when the CM is cleaved, when assayed in vitro using a target displacement assay, such as the assays described in PCT Publication Nos. 2009 / 025846 and 2010 / 081173.

[0126] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least two-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0127] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least 5-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0128] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least 10-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0129] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least 20-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0130] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least 40-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0131] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least 50-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0132] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least 100-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0133] In some embodiments, the CM is positioned on the activatable antibody such that in the uncleaved state, binding of the activatable antibody to the target occurs at an equilibrium dissociation constant that is at least 200-fold higher than the equilibrium dissociation constant of unmodified AB binding to the target, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0134] According to some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0135] According to some embodiments, the CM is a substrate for at least one matrix metalloproteinase (MMP). Examples of MMPs include: MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. According to some embodiments, the CM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. According to some embodiments, the CM is a substrate for MMP9. According to some embodiments, the CM is a substrate for MMP14. According to some embodiments, the CM is a substrate for multiple MMPs. According to some embodiments, the CM is a substrate for at least MMP9 and MMP14. According to some embodiments, the CM comprises multiple substrates for the same MMP. According to some embodiments, the CM comprises at least multiple MMP9 substrates. According to some embodiments, the CM comprises at least multiple MMP14 substrates.

[0136] According to some embodiments, the CM is a substrate for MMPs and includes the sequences ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32); PAGLWLDP (SEQ ID NO: 33); and / or ISSGLSS (SEQ ID NO: 159).

[0137] In some embodiments, the CM comprises the amino acid sequence ISSGLLSS (SEQ ID NO: 14). In some embodiments, the CM comprises the amino acid sequence QNQALRMA (SEQ ID NO: 15). In some embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ ID NO: 16). In some embodiments, the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 17). In some embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 18). In some embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ ID NO: 19). In some embodiments, the CM comprises the amino acid sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments, the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). In some embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ ID NO: 22). In some embodiments, the CM comprises the amino acid sequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO:25). In some embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQ ID NO:26). In some embodiments, the CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO:27). In some embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ ID NO:28). In some embodiments, the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO:29). In some embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO:30). In some embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ ID NO:31). In some embodiments, the CM comprises the amino acid sequence LLAPSHRA (SEQ ID NO:32). In some embodiments, the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO:33). In some embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ ID NO:159).

[0138] According to some embodiments, the CM is a substrate for at least two proteases, wherein at least one protease is an MMP and the at least one protease is selected from the group consisting of those enzymes shown in Table 7.

[0139] According to some embodiments, an activatable antibody comprises at least a first CM and a second CM. According to some embodiments, the first CM and second CM are each polypeptides 15 or less amino acids in length. According to some embodiments, the first CM and second CM in an activatable antibody in an uncleaved state have the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM. According to some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1. According to some embodiments, an activatable antibody comprises a connecting peptide between CM1 and CM2. According to some embodiments, an activatable antibody comprises a connecting peptide between CM2 and AB. According to some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1 and a connecting peptide between CM2 and AB. According to some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1 and a connecting peptide between CM2 and AB. According to some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1 and a connecting peptide between CM1 and CM2. According to some embodiments, the activatable antibody comprises a connecting peptide between CM1 and CM2, and a connecting peptide between CM2 and AB. According to some embodiments, the activatable antibody comprises a connecting peptide between MM and CM1, a connecting peptide between CM1 and CM2, and a connecting peptide between CM2 and AB.

[0140] In some embodiments, an activatable antibody comprises at least a first CM comprising a substrate for at least one matrix metalloproteinase (MMP) and a second CM comprising a substrate sequence. Exemplary substrates for the second CM (CM2) include, but are not limited to, substrates cleavable by one or more of the following enzymes or proteases listed in Table 7:

[0141] In some embodiments, CM2 is selected for use with a particular protease, such as a matrix metalloproteinase (MMP), neutrophil esterase, u-type plasminogen activator (uPA, also known as urokinase), legumain, matriptase (MT-SP1), thrombin, a cysteine ​​protease, such as a cathepsin, ADAM17, BMP-1, HtrA1, or a TMPRSS, such as TMPRSS3 or TMPRSS4.

[0142] According to some embodiments, CM2 is a substrate for neutrophil elastase. According to some embodiments, CM2 is a substrate for uPA. According to some embodiments, CM2 is a substrate for legumain. According to some embodiments, CM2 is a substrate for matriptase. According to some embodiments, CM2 is a substrate for thrombin. According to some embodiments, CM2 is a substrate for cysteine ​​proteases. According to some embodiments, CM2 is a substrate for cathepsins. According to some embodiments, CM2 is a substrate for ADAM17. According to some embodiments, CM2 is a substrate for BMP-1. According to some embodiments, CM2 is a substrate for HtrA1. According to some embodiments, CM2 is a substrate for TMPRSS. According to some embodiments, CM2 is a substrate for TMPRSS3. According to some embodiments, CM2 is a substrate for TMPRSS4.

[0143] According to some embodiments, suitable CM2 is cleaved by at least one protease and comprises the sequences TGRGPSWV (SEQ ID NO:34); SARGPSRW (SEQ ID NO:35); TARGPSFK (SEQ ID NO:36); LSGRSDNH (SEQ ID NO:37); GGWHTGRN (SEQ ID NO:38); HTGRSGAL (SEQ ID NO:39); PLTGRSGG (SEQ ID NO:40); AARGPAIH (SEQ ID NO:41); RGPAFNPM (SEQ ID NO:42); SSRGPAYL (SEQ ID NO:43); RGPATPIM (SEQ ID NO:44); RGPA (SEQ ID NO:45); GGQPSGMWGW (SEQ ID NO:46); FPRPLGITGL (SEQ ID NO:47); VHMPLGFLGP (SEQ ID NO:48); SPLTGRSG (SEQ ID NO:49); SAGFSLPA (SEQ ID NO:126); LAPLGLQRR (SEQ ID NO:50); SGGPLGVR (SEQ ID NO:51); PLGL (SEQ ID NO:52); GPRSFGL (SEQ ID NO:315) and / or GPRSFG (SEQ ID NO:316).

[0144] According to some embodiments, CM2 comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 34). According to some embodiments, CM2 comprises the amino acid sequence SARGPSRW (SEQ ID NO: 35). According to some embodiments, CM2 comprises the amino acid sequence TARGPSFK (SEQ ID NO: 36). According to some embodiments, CM2 comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 37). According to some embodiments, CM2 comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 38). According to some embodiments, CM2 comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 39). According to some embodiments, CM2 comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 40). According to some embodiments, CM2 comprises the amino acid sequence AARGPAIH (SEQ ID NO: 41). According to some embodiments, CM2 comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 42). According to some embodiments, CM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 43). According to some embodiments, CM2 comprises the amino acid sequence RGPATPIM (SEQ ID NO: 44). According to some embodiments, CM2 comprises the amino acid sequence RGPA (SEQ ID NO: 45). According to some embodiments, CM2 comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO: 46). According to some embodiments, CM2 comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 47). According to some embodiments, CM2 comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 48). According to some embodiments, CM2 comprises the amino acid sequence SPLTGRSG (SEQ ID NO: 49). According to some embodiments, CM2 comprises the amino acid sequence LAPLGLQRR (SEQ ID NO: 50). According to some embodiments, CM2 comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 51). According to some embodiments, CM2 comprises the amino acid sequence PLGL (SEQ ID NO: 52). According to some embodiments, CM2 comprises the amino acid sequence GPRSFGL (SEQ ID NO: 315). According to some embodiments, CM2 comprises the amino acid sequence GPRSFG (SEQ ID NO: 316).

[0145] According to some embodiments, CM2 is a substrate for at least one MMP. According to some embodiments, CM2 is a substrate for at least one MMP listed in Table 7. According to some embodiments, CM2 is a substrate for MMP9. According to some embodiments, CM2 is a substrate for an MMP. According to some embodiments, CM1 is a substrate for a first MMP and CM2 is a substrate for a second MMP, where the first MMP and the second MMP are different MMPs. According to some embodiments, CM1 is a first substrate sequence for an MMP and CM2 is a second substrate for the same MMP, where CM1 and CM2 have different substrate sequences. According to some embodiments, CM2 is a substrate for multiple MMPs. According to some embodiments, CM2 is a substrate for at least MMP9 or MMP14. According to some embodiments, CM2 is a substrate for multiple MMPs. According to some embodiments, CM2 is a substrate for at least MMP9 and MMP14. According to some embodiments, both CM1 and CM2 are substrates for MMP9. According to some embodiments, both CM1 and CM2 are substrates for MMP 14. According to some embodiments, CM1 is a substrate for MMP9 and CM2 is a substrate for MMP 14. According to some embodiments, CM1 is a substrate for MMP 14 and CM2 is a substrate for MMP 9.

[0146] According to some embodiments, at least one of CM1 and / or CM2 is a substrate for an MMP and comprises the sequence ISSGLLSS (SEQ ID NO:14); QNQALRMA (SEQ ID NO:15); AQNLLGMV (SEQ ID NO:16); STFPFGMF (SEQ ID NO:17); PVGYTSSL (SEQ ID NO:18);DWLYWPGI (SEQ ID NO:19); MIAPVAYR (SEQ ID NO:20); RPSPMWAY (SEQ ID NO:21); WATPRPMR (SEQ ID NO:22); FRLLDWQW (SEQ ID NO:23); LKAAPRWA (SEQ ID NO:24); GPSHLVLT (SEQ ID NO:25); LPGGLSPW (SEQ ID NO:26);MGLFSEAG (SEQ ID NO:27); SPLPLRVP (SEQ ID NO:28); RMHLRSLG (SEQ ID NO:29); LAAPLGLL (SEQ ID NO:30); AVGLLAPP (SEQ ID NO:31); LLAPSHRA (SEQ ID NO:32); PAGLWLDP (SEQ ID NO:33); and / or ISSGLSS (SEQ ID NO:159).

[0147] According to some embodiments, the first cleaving agent and the second cleaving agent are the same matrix metalloprotease, and the first CM and the second CM are different substrates for the enzyme. According to some embodiments, the first cleaving agent and the second cleaving agent are different proteases, wherein at least one protease is an MMP. According to some embodiments, the first cleaving agent and the second cleaving agent are co-localized in the target tissue. According to some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

[0148] In some embodiments, the activatable antibody is exposed to and cleaved by an MMP, and in the resulting activated or cleaved state, the activated antibody comprises an L chain amino acid sequence that includes at least a portion of the LP2 and / or CM sequence after the MMP cleaves the CM.

[0149] According to some embodiments, the CM comprises the non-prime side of a protease cleavage site; i.e., the CM comprises at least the P1 and P2 amino acids, and according to some embodiments, the P1, P2, and P3 amino acids, and according to some embodiments, the P1, P2, P3, and P4 amino acids. According to some embodiments, the CM comprises the non-prime side and the prime side of a protease cleavage site. According to some embodiments, the CM comprises the non-prime side of a protease cleavage site but lacks at least a portion of the prime side. According to some embodiments, the CM comprises the non-prime side of a protease cleavage site but lacks the prime side. Such a CM can be linked directly or via a linker to an antibody or other molecule as disclosed herein, such as, but not limited to, a detection moiety.

[0150] According to some embodiments, the activatable antibody is an anti-EGFR activatable antibody comprising at least an AB that is or is derived from cetuximab or panitumumab; a MM that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 167-200, and 497; and a CM that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33, and 159. According to some embodiments, the activatable antibody is an anti-EGFR activatable antibody comprising at least an AB that is or is derived from cetuximab or panitumumab; a MM that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 167-200, and 497; and a CM that comprises an amino acid sequence selected from the group consisting of the sequences provided in Tables 8A-8M. According to some embodiments, the anti-EGFR activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. According to some embodiments, each of LP1 and LP2 is a peptide about 1-20 amino acids in length. According to some embodiments, the two connecting peptides need not be identical to each other. According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0151] According to some embodiments, the anti-EGFR activatable antibody comprises at least: AB comprising an H chain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR3 sequence of SEQ ID NO: 509; MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 167-200, and 497; and CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33, and 159. According to some embodiments, the anti-EGFR activatable antibody comprises at least: AB, which comprises a heavy chain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR3 sequence of SEQ ID NO: 509; MM, which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 167-200, and 497; and CM, which comprises an amino acid sequence selected from the group consisting of the sequences provided in Tables 8A-8M. According to some embodiments, the anti-EGFR activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide of about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, at least one of LP1 or LP2 is a peptide of the formula (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0152] According to some embodiments, the activatable antibody is an anti-EGFR activatable antibody comprising at least an AB comprising a heavy chain amino acid sequence of SEQ ID NO: 56, 57, or 58, and a light chain amino acid sequence of SEQ ID NO: 59; a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 167-200, and 497; and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33, and 159. According to some embodiments, the activatable antibody is an anti-EGFR activatable antibody comprising at least an AB comprising a heavy chain amino acid sequence of SEQ ID NO: 56, 57, or 58, and a light chain amino acid sequence of SEQ ID NO: 59; a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 167-200, and 497; and a CM comprising an amino acid sequence selected from the group consisting of the sequences provided in Tables 8A-8M. According to some embodiments, the anti-EGFR activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. According to some embodiments, each of LP1 and LP2 is a peptide about 1-20 amino acids in length. According to some embodiments, the two connecting peptides need not be identical to each other. According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0153] According to some embodiments, the activatable antibody is an anti-EGFR activatable antibody comprising at least an AB comprising a heavy chain amino acid sequence of SEQ ID NO: 56 and a light chain amino acid sequence of SEQ ID NO: 59; a MM comprising the amino acid sequence of SEQ ID NO: 160; and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33 and 159. According to some embodiments, the activatable antibody is an anti-EGFR activatable antibody comprising at least an AB comprising a heavy chain amino acid sequence of SEQ ID NO: 56 and a light chain amino acid sequence of SEQ ID NO: 59; a MM comprising the amino acid sequence of SEQ ID NO: 160; and a CM comprising an amino acid sequence selected from the group consisting of the sequences provided in Tables 8A-8M. According to some embodiments, the anti-EGFR activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide of about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, at least one of LP1 or LP2 is a peptide of the formula (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0154] According to some embodiments, the anti-EGFR activatable antibody comprises at least: AB comprising an H chain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR3 sequence of SEQ ID NO: 509; MM comprising the amino acid sequence of SEQ ID NO: 160; and CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33 and 159. According to some embodiments, the anti-EGFR activatable antibody comprises at least an AB comprising a heavy chain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR3 sequence of SEQ ID NO: 509; an MM comprising the amino acid sequence of SEQ ID NO: 160; and a CM comprising an amino acid sequence selected from the group consisting of the sequences provided in Tables 8A-8M. According to some embodiments, the anti-EGFR activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. According to some embodiments, each of LP1 and LP2 is a peptide of about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, at least one of LP1 or LP2 comprises the following: (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0155] According to some embodiments, the activatable antibody is selected from the group consisting of SEQ ID NOs: 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112 or and an AB comprising an H chain amino acid sequence of SEQ ID NO: 114 and an L chain amino acid sequence of SEQ ID NO: 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111 or 113; an MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-263 and 497; and an CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33 and 159. According to some embodiments, the activatable antibody is selected from the group consisting of SEQ ID NOs: 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112 or and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-263 and 497. According to some embodiments, the anti-Jagged activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide of about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, at least one of LP1 or LP2 is a peptide of the formula (GS) n , (GGS) n , (GSGGS)n (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0156] According to some embodiments, the activatable antibody is an anti-Jagged activatable antibody comprising at least an AB comprising a heavy chain amino acid sequence of SEQ ID NO: 112 and a light chain amino acid sequence of SEQ ID NO: 111; a MM comprising the amino acid sequence of SEQ ID NO: 217, and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33 and 159. According to some embodiments, the activatable antibody is an anti-Jagged activatable antibody comprising at least an AB comprising a heavy chain amino acid sequence of SEQ ID NO: 112 and a light chain amino acid sequence of SEQ ID NO: 111; a MM comprising the amino acid sequence of SEQ ID NO: 217, and a CM comprising an amino acid sequence selected from the group consisting of the sequences provided in Tables 8A-8M. According to some embodiments, the anti-Jagged activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide of about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, at least one of LP1 or LP2 is a peptide of the formula (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0157] According to some embodiments, the anti-Jagged activatable antibody comprises at least an AB comprising an H chain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO: 498, the VH CDR2 sequence of SEQ ID NO: 499, the VH CDR3 sequence of SEQ ID NO: 500, the VL CDR1 sequence of SEQ ID NO: 501, the VL CDR2 sequence of SEQ ID NO: 502, and the VL CDR3 sequence of SEQ ID NO: 503; a MM comprising the amino acid sequence of SEQ ID NO: 217; and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33 and 159. According to some embodiments, the anti-Jagged activatable antibody comprises at least an AB comprising a heavy chain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO: 498, the VH CDR2 sequence of SEQ ID NO: 499, the VH CDR3 sequence of SEQ ID NO: 500, the VL CDR1 sequence of SEQ ID NO: 501, the VL CDR2 sequence of SEQ ID NO: 502, and the VL CDR3 sequence of SEQ ID NO: 503; an MM comprising the amino acid sequence of SEQ ID NO: 217; and a CM comprising an amino acid sequence selected from the group consisting of the sequences provided in Tables 8A-8M. According to some embodiments, the anti-Jagged activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. According to some embodiments, each of LP1 and LP2 is a peptide of about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, at least one of LP1 or LP2 comprises the following: (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n(SEQ ID NO:2) (n is at least one integer). According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:9), GSSGGSGGSGG (SEQ ID NO:10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO:155), GSSGGSGGSG (SEQ ID NO:156), or GSSGGSGGSGS (SEQ ID NO:157). According to some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:158), GSSGT (SEQ ID NO:12), or GSSG (SEQ ID NO:13).

[0158] In some embodiments, the activatable antibody also includes an agent conjugated to AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is conjugated to AB via a linker comprising at least one MMP-cleavable substrate sequence. In some embodiments, the agent is an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a derivative thereof. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0159] According to some embodiments, the agent is an anti-inflammatory agent.

[0160] According to some embodiments, the activatable antibody also comprises a detectable moiety. According to some embodiments, the detectable moiety is a diagnostic agent.

[0161] According to some embodiments, the conjugated antibody comprises a detectable label. According to some embodiments, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. According to some embodiments, the imaging agent comprises a radioisotope. According to some embodiments, the radioisotope is indium or technetium. According to some embodiments, the imaging agent comprises iodine, gadolinium, or iron oxide. According to some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. According to some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. According to some embodiments, the luminescent label comprises an N-methylacridium derivative. According to some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. According to some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0162] According to some embodiments, the activatable antibody also comprises a signal peptide. According to some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. According to some embodiments, the spacer is conjugated to the activatable antibody in the absence of an immediately following peptide. According to some embodiments, the spacer is directly linked to the MM of the activatable antibody. According to some embodiments, the spacer is directly linked to the MM of the activatable antibody in an N- to C-terminal structural arrangement of spacer-MM-CM-A. An example of a spacer directly linked to the N-terminus of the MM of an activatable antibody is QGQSGQ (SEQ ID NO: 53). According to some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 53).

[0163] In some embodiments, the AB in the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0164] According to some embodiments, the serum half-life of an activatable antibody is longer than the half-life of its corresponding antibody; for example, the pK of an activatable antibody is longer than the pK of its corresponding antibody. According to some embodiments, the serum half-life of an activatable antibody is similar to the half-life of its corresponding antibody. According to some embodiments, the serum half-life of an activatable antibody is at least 15 days when administered to an organism. According to some embodiments, the serum half-life of an activatable antibody is at least 12 days when administered to an organism. According to some embodiments, the serum half-life of an activatable antibody is at least 11 days when administered to an organism. According to some embodiments, the serum half-life of an activatable antibody is at least 10 days when administered to an organism. According to some embodiments, the serum half-life of an activatable antibody is at least 9 days when administered to an organism. According to some embodiments, the serum half-life of an activatable antibody is at least 8 days when administered to an organism. According to some embodiments, the serum half-life of an activatable antibody is at least 7 days when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism.According to some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. According to some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism.

[0165] According to some embodiments, the activatable antibody and / or conjugated activatable antibody is monospecific. According to some embodiments, the activatable antibody and / or conjugated activatable antibody is multispecific, including but not limited to, bispecific or trispecific. According to some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. According to some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor.

[0166] The present disclosure also provides compositions and methods comprising activatable antibodies, including an antibody or antibody fragment (AB) that specifically binds a predetermined target, wherein the AB is coupled to a masking moiety (MM) that reduces the ability of the AB to bind its target. According to some embodiments, the activatable antibody further comprises a cleavable moiety (CM) that is a substrate for at least one MMP. The compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues in the AB without impairing the activity (e.g., masking, activation, or binding activity) of the activatable antibody. According to some embodiments, the compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues in the AB without reducing or otherwise interfering with one or more disulfide bonds in the MM. The compositions and methods provided herein produce activatable antibodies conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic, and / or prophylactic agents; for example, according to some embodiments, none of the agents are conjugated to any of the MMs of the activatable antibody. The compositions and methods provided herein produce conjugated activatable antibodies that retain the ability to effectively and efficiently mask the AB of the activatable antibody when the MM is not cleaved. The compositions and methods provided herein produce conjugated activatable antibodies in which the activatable antibody is still activated, i.e., cleaved, in the presence of an MMP capable of cleaving the CM.

[0167] Although an activatable antibody has at least one attachment point for an agent, in the methods and compositions provided herein, all possible attachment points are available for conjugation to an agent. According to some embodiments, one or more attachment points are sulfur atoms involved in disulfide bonds. According to some embodiments, one or more attachment points are sulfur atoms involved in interchain disulfide bonds. According to some embodiments, one or more attachment points are sulfur atoms involved in interchain sulfide bonds, but not intrachain disulfide bonds. According to some embodiments, one or more attachment points are sulfur atoms of cysteine ​​or other amino acid residues that contain sulfur atoms. Such residues can occur naturally in antibody structures or can be incorporated into antibodies by site-directed mutagenesis, chemical conversion, or misincorporation of unnatural amino acids.

[0168] Also provided are methods for preparing conjugates of activatable antibodies having one or more interchain disulfide bonds in one or more ABs and one or more intrachain disulfide bonds in the MM, and for which drugs reactive with free thiols are provided. The methods generally involve partially reducing the interchain disulfide bonds in the activatable antibody with a reducing agent, e.g., TCEP; and conjugating a drug reactive with free thiols to the partially reduced activatable antibody. As used herein, the term "partial reduction" refers to a situation in which a multispecific activatable antibody is contacted with a reducing agent and fewer than all disulfide bonds, e.g., fewer than all possible conjugation sites, are reduced. According to some embodiments, less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of all possible conjugation sites are reduced.

[0169] According to some embodiments, a method is provided for reducing an agent, e.g., a drug, and conjugating it to an activatable antibody that results in selection under the displacement of the agent. The method involves reducing the activatable antibody with a reducing agent, thereby unreducing any conjugation sites on either the masking portion or other non-AB portions of the activatable antibody, and then conjugating the agent to one or more interchain thiols in the AB of the activatable antibody. The conjugation site is selected to allow for the desired displacement of the agent, allowing conjugation to occur at the desired site. For example, the reducing agent is TCEP. The reduction reaction conditions, such as the ratio of reducing agent to activatable antibody, the length of incubation, the temperature during incubation, the pH of the reduction reaction solution, etc., are determined by identifying conditions that produce a conjugated activatable antibody that retains the ability to effectively and efficiently mask the AB of the activatable antibody under conditions where the MM is not cleaved. The ratio of reducing agent to activatable antibody will vary depending on the activatable antibody. According to some embodiments, the ratio of reducing agent:activatable antibody is about 20:1-1:1, about 10:1-1:1, about 9:1-1:1, about 8:1-1:1, about 7:1-1:1, about 6:1-1:1, about 5:1-1:1, about 4:1-1:1, about 3:1-1:1, about 2:1-1:1, about 20:1-1:1.5, about 10:1-1:1.5, about 9:1-1:1.5, about 8:1-1:1.5, about 7:1-1:1.5, about 6:1-1:1.5, about 5:1-1:1.5, about 4:1-1:1.5, about 3:1-1:1.5, about 2:1-1:1.5, or The ratio will be in the range of about 1:1 to 1:1.5. According to some embodiments, the ratio is in the range of about 5:1 to 1.5:1. According to some embodiments, the ratio is in the range of about 4:1 to 1:1. According to some embodiments, the ratio is in the range of about 4:1 to 1:1. According to some embodiments, the ratio is in the range of about 4:1 to 1.4:1. According to some embodiments, the ratio is in the range of about 8:1 to about 1:1. According to some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0170] According to some embodiments, methods are provided for reducing interchain disulfide bonds in one or more ABs of an activatable antibody and conjugating an agent, e.g., a thiol-containing agent, e.g., a drug, to the resulting interchain thiols to selectively locate the agent on the ABs. The methods generally involve partially reducing one or more ABs with a reducing agent to form at least two interchain thiols, but not all possible interchain thiols, in the activatable antibody; and conjugating the agent to the interchain thiols of the partially reduced ABs. For example, one or more ABs of an activatable antibody are partially reduced at a desired ratio of reducing agent:activatable antibody at about 37°C for about 1 hour. According to some embodiments, the ratio of reducing agent:activatable antibody is about 20:1-1:1, about 10:1-1:1, about 9:1-1:1, about 8:1-1:1, about 7:1-1:1, about 6:1-1:1, about 5:1-1:1, about 4:1-1:1, about 3:1-1:1, about 2:1-1:1, about 20:1-1:1.5, about 10:1-1:1.5, about 9:1-1:1.5, about 8:1-1:1.5, about 7:1-1:1.5, about 6:1-1:1.5, about 5:1-1:1.5, about 4:1-1:1.5, about 3:1-1:1.5, about 2:1-1:1.5, or The ratio will be in the range of about 1:1 to 1:1.5. According to some embodiments, the ratio is in the range of about 5:1 to 1.5:1. According to some embodiments, the ratio is in the range of about 4:1 to 1:1. According to some embodiments, the ratio is in the range of about 4:1 to 1:1. According to some embodiments, the ratio is in the range of about 4:1 to 1.4:1. According to some embodiments, the ratio is in the range of about 8:1 to about 1:1. According to some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0171] The thiol-containing reagent can be, for example, cysteine ​​or N-acetylcysteine. The reducing agent can be, for example, TCEP. According to some embodiments, the reduced activatable antibody can be purified prior to conjugation, for example, using column chromatography, dialysis, or diafiltration. Alternatively, the reduced antibody is not purified after partial reduction and prior to conjugation.

[0172] The present disclosure also provides a partially reduced activatable antibody in which at least one interchain disulfide bond in the activatable antibody has been reduced by a reducing agent without disrupting any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that inhibits binding of the activatable antibody to the target under uncleaved conditions, and a cleavable moiety (CM) (a polypeptide that functions as a substrate for at least one MMP) coupled to the AB. According to some embodiments, the MM is coupled to the AB via the CM. According to some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disrupted by the reducing agent. According to some embodiments, one or more intrachain disulfide bonds of the MM in the activatable antibody are not disrupted by the reducing agent. According to some embodiments, the activatable antibody under uncleaved conditions has the following structural configuration from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM. According to some embodiments, the reducing agent is TCEP.

[0173] The present disclosure also provides partially reduced activatable antibodies (e.g., including, but not limited to, the multispecific activatable antibodies of the present disclosure) in which at least one intrachain disulfide bond in the activatable antibody has been reduced with a reducing agent without disrupting or otherwise impairing the activity and / or efficacy of the activatable antibody, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that inhibits binding of the activatable antibody to the target under uncleaved conditions, and a cleavable moiety (CM) (which is a polypeptide that functions as a substrate for a protease) coupled to the AB. The activity and / or efficacy of the activatable antibody, by way of non-limiting example, is the masking activity, the activation of the activatable antibody, and / or the binding activity of the activated activatable antibody. According to some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disrupted by a reducing agent. In some embodiments, one or more intrachain disulfide bonds of MM in the activatable antibody are not disrupted by the reducing agent. In some embodiments, the activatable antibody under uncleaved conditions has the following structural configuration from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM. In some embodiments, the reducing agent is TCEP.

[0174] The present disclosure also provides a conjugated activatable antibody comprising an activatable antibody linked to a monomethyl auristatin D (MMAD) payload, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that inhibits binding of the AB of the activatable antibody in its uncleaved state to the target, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for at least one MMP protease.

[0175] According to some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any of several methods for linking an agent to the AB, such as: (a) conjugation to the carbohydrate moiety of the AB, or (b) binding to a sulfhydryl group of the AB, or (c) binding to an amino group of the AB, or (d) binding to a carboxylate group of the AB.

[0176] In some embodiments, the MMAD payload is conjugated to AB via a linker. In some embodiments, the MMAD payload is conjugated to a cysteine ​​via a linker. In some embodiments, the MMAD payload is conjugated to a lysine in AB via a linker. In some embodiments, the MMAD is conjugated to another residue in AB via a linker, such as those residues disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is selected from the group consisting of linkers shown in Tables 5 and 6. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidoPEG-valine-citrulline linker. According to some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaprolide-valine-citrulline-para-aminobenzyloxycarbonyl linker. According to some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidoPEG-valine-citrulline-para-aminobenzyloxycarbonyl linker. According to some embodiments, the MMAD payload is conjugated to AB using the partial reduction and conjugation techniques disclosed herein.

[0177] In some embodiments, the target is selected from the group of targets listed in Table 1. In some embodiments, the target is EGFR. In some embodiments, the target is a Jagged protein, e.g., Jagged1 and / or Jagged2. In some embodiments, the target is interleukin-6 receptor (IL-6R). In some embodiments, the AB is or is derived from an antibody selected from the group of antibodies listed in Table 2. In some embodiments, the antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an scFv, a scab, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the AB has an equilibrium dissociation constant for binding to the target of about 100 nM or less. In some embodiments, the MM has an equilibrium dissociation constant for binding to the AB that is higher than the equilibrium dissociation constant for binding of the AB to the target. In some embodiments, the MM, in its cleaved state, does not interfere with or compete with the AB of the activatable antibody for binding to the target. According to some embodiments, the MM is a polypeptide 40 amino acids or less in length. According to some embodiments, the MM polypeptide sequence is different from that of the target, and the MM polypeptide sequence is at most 50% identical to any natural binding partner of the AB. According to some embodiments, the MM does not have 25% or more amino acid sequence identity to the target. According to some embodiments, the MM does not have 10% or more amino acid sequence identity to the target. According to some embodiments, the CM is a polypeptide up to 15 amino acids in length. According to some embodiments, the MMP protease co-localizes with the target in a tissue, and the MMP protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the MMP protease. According to some embodiments, the MMP protease is an MMP9 protease. According to some embodiments, the MMP protease is an MMP14 protease. According to some embodiments, the activatable antibody comprises a linking peptide between the MM and CM.In some embodiments, the activatable antibody comprises a connecting peptide between CM and AB. In some embodiments, the activatable antibody also comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and in its uncleaved state, the activatable antibody has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two connecting peptides need not be identical to each other. In some embodiments, each of LP1 and LP2 is a peptide about 1-20 amino acids in length. In some embodiments, at least one of LP1 or LP2 is (GS). n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO:2), where n is at least one integer. According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8). According to some embodiments, the activatable antibody comprises a second CM; according to some embodiments, the second CM is a substrate for an enzyme selected from the group consisting of those enzymes set forth in Table 7.

[0178] The present disclosure also provides polypeptides and other large molecules comprising one or more MMP-cleavable substrate sequences provided herein. By way of non-limiting example, the MMP-cleavable substrate sequences provided herein are useful in prodrug compositions and methods of use thereof. The MMP-cleavable substrate sequences provided herein are also useful in probes and other detection agents and methods of use thereof. For example, the MMP-cleavable substrate sequences provided herein can be used in combination with a flower and a quencher to produce a detection agent, such as an imaging agent and / or other diagnostic agent. Those skilled in the art will understand that the MMP-cleavable substrate sequences provided herein are useful in any composition and / or method in the art that employs a substrate that can be cleaved by one or more MMPs, such as MMP9 and / or MMP14.

[0179] The present disclosure also provides isolated nucleic acid molecules encoding the antibodies and / or activatable antibodies described herein, and vectors comprising those isolated nucleic acid sequences. The present disclosure provides methods for producing the antibodies and / or activatable antibodies by culturing cells containing the vectors under conditions conducive to expression of the antibodies and / or activatable antibodies.

[0180] The present disclosure provides a method for producing a conjugated antibody of the present disclosure that binds a predetermined target by (a) culturing cells containing a nucleic acid construct encoding the antibody under conditions conducive to expression of the antibody, where (i) the antibody comprises a cleavable moiety (CH), and (ii) the CM is a polypeptide that functions as a substrate for a matrix metalloproteinase; (b) recovering the antibody; and (c) conjugating the recovered antibody to one or more additional agents.

[0181] The present disclosure also provides a method for producing an activatable antibody of the present disclosure that binds a predetermined target under activated conditions by: (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (i) the CM is a polypeptide that functions as a substrate for an MMP; and (ii) the CM is positioned on the activatable antibody such that, under uncleaved conditions, the MM interferes with specific binding of the AB to the target, and under cleaved conditions, the MM does not interfere with or compete with specific binding of the AB to the target; and (b) recovering the activatable antibody.

[0182] The present disclosure provides methods of preventing, delaying the progression of, treating, alleviating the symptoms of, or otherwise ameliorating a target-related disease in a subject by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody and / or conjugated activatable antibody described herein.

[0183] The present disclosure provides methods of preventing, delaying the progression of, treating, alleviating the symptoms of, or otherwise ameliorating inflammation and / or inflammatory diseases in a subject in need thereof by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody described herein. The present disclosure provides methods of preventing, delaying the progression of, treating, alleviating the symptoms of, or otherwise ameliorating cancer in a subject in need thereof by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody described herein. The present disclosure provides methods of preventing, delaying the progression of, treating, alleviating the symptoms of, or otherwise ameliorating autoimmune diseases in a subject in need thereof by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody described herein.

[0184] The conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies used in any of these method and use embodiments can be administered at any stage of disease. For example, such conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be administered to patients with cancer at any stage from early stage to metastatic. The terms subject and patient are used interchangeably herein.

[0185] In some embodiments, the subject is a mammal, such as a human, a non-human primate, a companion animal (e.g., a cat, dog, horse), a farm animal, a work animal, or a zoo animal. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal in veterinary care.

[0186] The conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies, and therapeutic formulations thereof, are administered to a subject having or susceptible to a disease or disorder associated with aberrant target expression and / or activity. Subjects having or susceptible to a disease or disorder associated with aberrant target expression and / or activity are identified using any of a variety of methods known in the art. For example, subjects having cancer or other neoplastic conditions are identified using any of a variety of clinical and / or laboratory tests, such as physical examinations and blood, urine, and / or stool analyses to assess health status. For example, subjects having inflammation and / or inflammatory disorders are identified using any of a variety of clinical and / or laboratory tests, such as physical examinations and / or body fluid analyses, such as blood, urine, and / or stool analyses to assess health status.

[0187] Administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody to a patient having a disease or disorder associated with aberrant target expression and / or activity is considered successful if any of a variety of laboratory or clinical objectives are achieved. For example, administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody is considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, inhibited, or do not progress to a further worsening state. Administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody is considered successful if the patient or disorder goes into remission or does not progress to a further worsening state.

[0188] In some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody are administered during and / or after treatment in combination with one or more additional agents, for example, by non-limiting example, an anti-inflammatory agent, an immunosuppressant, a chemotherapeutic agent, such as an alkylating agent, an antimetabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and / or any other nucleic acid damaging agent. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapeutic agent, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, such as a kinase inhibitor, such as sorafenib or erlotinib. In some embodiments, the additional agent is a targeted agent, such as another antibody, such as a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteasome inhibitor, such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulatory agent, such as lenolidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art.

[0189] According to some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody. According to some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent are administered simultaneously. For example, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent may be formulated into a single composition or administered as multiple separate compositions. According to some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent are administered sequentially, or the antibody and / or conjugated antibody and the additional agent are administered at different times during a treatment regimen. For example, the antibody and / or conjugated antibody is administered before the administration of the additional agent, the antibody and / or conjugated antibody is administered subsequent to the administration of the additional agent, or the antibody and / or conjugated antibody and the additional agent are administered in alternation. As described herein, the antibody and / or conjugated antibody and the additional agent are administered in a single dose or in multiple doses.

[0190] According to some embodiments, the CM is linked or otherwise attached to an activatable antibody, which comprises an antibody or antigen-binding fragment thereof that specifically binds a predetermined target, which is coupled to a masking moiety (MM), such that coupling of the MM to the AB reduces the ability of the antibody or antigen-binding fragment thereof to bind the target. According to some embodiments, the MM is coupled via a CM. Exemplary targets include, but are not limited to, those shown in Table 1. Exemplary ABs include, but are not limited to, those shown in Table 2. The activatable antibodies provided herein are stable in circulation, are activated in the intended therapeutic and / or diagnostic setting, but are not activated in normal, e.g., healthy, tissues or other tissues not targeted for therapeutic and diagnostic use, and, when activated, exhibit binding to the target that is at least equivalent to that of its corresponding unmodified antibody.

[0191] The present disclosure also provides methods and kits for using the conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies for various diagnostic and / or prophylactic applications.

[0192] According to some embodiments, the disclosure provides a method for producing a cytosine-binding protein comprising: (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavage moiety (CM) that is cleaved by a cleavage agent, and an antigen binding domain or fragment thereof (AB) that specifically binds a target of interest, wherein the activatable antibody, in its uncleaved, non-activated state, has the following structural arrangement from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM; wherein (a) MM is a peptide that inhibits binding of AB to the target, and MM does not have the amino acid sequence of a naturally occurring binding partner of AB and is not a denatured form of the natural binding partner of AB; and (b) in its uncleaved, non-activated state, The MM interferes with the specific binding of the AB to the target, and in the cleaved, activated state, the MM does not interfere with or compete with the specific binding of the AB to the target; and (ii) methods and kits are provided for detecting the presence or absence of a cleaving agent and a target of interest in a subject or sample by measuring the level of activated activatable antibody in the subject or sample, wherein a detected level of activated activatable antibody in the subject or sample indicates that the cleaving agent and the target are present in the subject or sample, and an absence of a detected level of activated activatable antibody in the subject or sample indicates that the cleaving agent, the target, or both the cleaving agent and the target are not present in the subject or sample.

[0193] According to some embodiments, the activatable antibody is an activatable antibody conjugated to a therapeutic agent. According to some embodiments, the activatable antibody is not conjugated to an agent. According to some embodiments, the activatable antibody comprises a detectable label. According to some embodiments, the detectable label is located on the AB. According to some embodiments, measuring the level of the activatable antibody in a subject or sample is achieved using a secondary reagent that specifically binds to the activated activatable antibody and comprises a detectable label. According to some embodiments, the secondary reagent is an antibody that comprises a detectable label.

[0194] According to some embodiments of the methods and kits, the activatable antibody comprises a detectable label. According to some embodiments of the methods and kits, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. According to some embodiments of the methods and kits, the imaging agent comprises a radioisotope. According to some embodiments of the methods and kits, the radioisotope is indium or technetium. According to some embodiments of the methods and kits, the contrast agent comprises iodine, gadolinium, or iron oxide. According to some embodiments of the methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. According to some embodiments of the methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (MRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. According to some embodiments of the methods and kits, the luminescent label comprises an N-methylacridinium derivative. According to some embodiments of the methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. According to some embodiments of the methods and kits, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0195] In some embodiments of the methods and kits, the subject is a mammal. In some embodiments of the methods and kits, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), an agricultural animal, a work animal, or a zoo animal. In some embodiments, the subject is a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal in veterinary care.

[0196] According to some embodiments of these methods, the method is an in vivo method. According to some embodiments of these methods, the method is an in situ method. According to some embodiments of these methods, the method is an ex vivo method. According to some embodiments of these methods, the method is an in vitro method.

[0197] According to some embodiments of these methods, the methods are used to identify or otherwise further narrow a patient population appropriate for treatment with an activatable antibody of the present disclosure, followed by treatment by administering the activatable antibody and / or conjugated activatable antibody to a subject in need thereof. For example, a patient who tests positive for both the target and at least one MMP that cleaves a substrate at the cleavable moiety (CM) in the activatable antibody tested in these methods is identified as an appropriate candidate for treatment with an activatable antibody containing such a CM, and the patient is then administered a therapeutically effective amount of the activatable antibody and / or conjugated activatable antibody tested. Similarly, a patient who tests negative for both the target and an MMP that cleaves a substrate at the CM in the activatable antibody tested using these methods can be identified as an appropriate candidate for another form of treatment. According to some embodiments, such patients can be tested with other activatable antibodies until an appropriate activatable antibody for treatment (e.g., an activatable antibody containing a CM that is cleaved by the patient at the site of disease) is identified. According to some embodiments, the patient is then administered a therapeutically effective amount of an activatable antibody and / or a conjugated activatable antibody, and the patient is tested positive.

[0198] The pharmaceutical composition of the present disclosure can include an antibody of the present disclosure and a carrier. The pharmaceutical composition can be included in a kit, such as a diagnostic kit. [Brief explanation of the drawings]

[0199] [Figure 1A] FIG. 1A is a series of graphs showing the ability of activatable anti-EGFR antibodies, including a masking moiety comprising the amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 160), a truncation moiety comprising MMP14 substrate 520 (also referred to herein as MN520) ISSGLLSS (SEQ ID NO: 14), and the anti-EGFR antibody C225v5 (wherein the complete activatable antibody construct is also referred to herein as Pb-MN520), to inhibit tumor growth in an H292 xenograft lung cancer model. [Figure 1B] FIG. 1B is a series of graphs showing the ability of activatable anti-EGFR antibodies, including a masking moiety comprising the amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 160), a truncation moiety comprising MMP14 substrate 520 (also referred to herein as MN520) ISSGLLSS (SEQ ID NO: 14), and the anti-EGFR antibody C225v5 (wherein the complete activatable antibody construct is also referred to herein as Pb-MN520), to inhibit tumor growth in an H292 xenograft lung cancer model. [Figure 2A] FIG. 2A is a series of graphs showing cleavage by 5 nM MMP9 of a substrate pool referred to herein as SMP87. [Figure 2B] FIG. 2B is a series of graphs showing cleavage by 5 nM MMP9 of a substrate pool referred to herein as SMP87. [Figure 3A] FIG. 3A is a series of graphs showing cleavage of the substrate sequence VAGRSMRP (SEQ ID NO: 484) by 5 nM MMP9. [Figure 3B] FIG. 3B is a series of graphs showing cleavage of the substrate sequence VAGRSMRP (SEQ ID NO: 484) by 5 nM MMP9. [Figure 4] FIG. 4 is a graph showing the correlation between substrate sequence frequency and function. [Figure 5A] FIG. 5A is a series of graphs showing cleavage of the substrate pool SMP39 by 60 nM MMP14. [Figure 5B] FIG. 5B is a series of graphs showing cleavage of the substrate pool SMP39 by 60 nM MMP14. [Figure 6A] FIG. 6A is a series of graphs showing cleavage of the substrate sequence QNQALRMA (SEQ ID NO: 15) by 30 nM MMP14. [Figure 6B] FIG. 6B is a series of graphs showing cleavage of the substrate sequence QNQALRMA (SEQ ID NO: 15) by 30 nM MMP14. [Figure 7A]Figure 7A is a series of schematic diagrams of the peptide display platform used in the examples provided herein. Figure 7A is a schematic diagram of the sequence of the display platform referred to herein as "display platform CYTX-DP-XXXXXXXX" or "CYTX-DP-XXXXXXXX" (SEQ ID NO: 512). [Figure 7B] Figure 7B is a series of schematic diagrams of the peptide display platform used in the examples provided herein. Figure 7B is a schematic diagram of the sequence of the display platform referred to herein as "display platform SP-CYTX-DP-XXXXXXXX" or "SP-CYTX-DP-XXXXXXXX" (SEQ ID NO: 513), where SP-CYTX-DP-XXXXXXXXX (SEQ ID NO: 513) is the CYTX-DP-XXXXXXXX (SEQ ID NO: 512) platform with a signal peptide. DETAILED DESCRIPTION OF THE INVENTION

[0200] The present disclosure provides amino acid sequences containing cleavable moieties (CMs) that are substrates for at least one matrix metalloproteinase (MMP), which are useful in a variety of therapeutic, diagnostic, and prophylactic applications.

[0201] The examples provided herein demonstrate that these CMs, when displayed on a peptide display platform, exhibit many desirable cleavage characteristics when exposed to MMP proteases under specific conditions. For example, Table 9 shows: (a) the percentage of MMP9-selected substrates tested on the CYTX-DP display platform that showed at least a 20% cleavage rate when incubated with 50 nM human MMP9 in 50 mM Tris-HCl (pH 7.4) supplemented with 150 mM NaCl, 10 mM CaCl, and 0.05% (w / v) Brij-35 for 1 hour at 37°C (greater than 20% cleavage rate by 50 nM MMP9); (b) the percentage of MMP9-selected substrates tested on the CYTX-DP display platform that showed at least a 20% cleavage rate when incubated with 50 nM human MMP9 in 50 mM HEPES (pH 6.8) supplemented with 10 mM CaCl and 0.5 mM MgCl for 1 hour at 37°C (greater than 20% cleavage rate by 50 nM MMP9); (c) the percentage of MMP9-selected or MMP14-selected substrates tested on the CYTX-DP display platform that showed at least a 20% cleavage rate when incubated with 500 pM human plasmin in 50 mM Tris-HCl (pH 7.4) supplemented with 100 mM NaCl, 0.01% Tween 20, and 1 mM EDTA for 1 hour at 37°C (20% or less cleavage rate with 500 pM plasmin).

[0202] According to some embodiments, when displayed on the CYTX-DP platform, the MMP9 substrate exhibits at least a 20% non-cleavage rate when incubated with 50 nM human MMP9 in 50 mM Tris-HCl (pH 7.4) supplemented with 150 mM NaCl, 10 mM CaCl, and 0.05% (w / v) Brij-35 for 1 hour at 37° C. According to some embodiments, when displayed on the CYTX-DP platform, the MMP9 substrate exhibits no more than a 20% cleavage rate when incubated with 500 pM human plasmin in 50 mM Tris-HCl (pH 7.4) supplemented with 100 mM NaCl, 0.01% Tween 20, and 1 mM EDTA for 1 hour at 37° C. According to some embodiments, when displayed on the CYTX-DP platform, the MMP9 substrate exhibits at least a 20% non-cleavage rate when incubated with 50 nM human MMP9 in 50 mM Tris-HCl (pH 7.4) supplemented with 150 mM NaCl, 10 mM CaCl2, and 0.05% (w / v) Brij-35 for 1 hour at 37°C, and exhibits a cleavage rate of 20% or less when incubated with 500 pM human plasmin in 50 mM Tris-HCl (pH 7.4) supplemented with 100 mM NaCl, 0.01% Tween 20, and 1 mM EDTA for 1 hour at 37°C.

[0203] According to some embodiments, the MMP14 substrate exhibits at least a 20% cleavage rate when incubated with 50 mM human MMP14 in 50 mM HEPES (pH 6.8) supplemented with 10 mM CaCl2 and 0.5 mM MaCl2 for 1 hour at 37° C. According to some embodiments, the MMP14 substrate exhibits no more than a 20% cleavage rate when incubated with 500 pM human plasmin in 50 mM Tris-HCl (pH 7.4) supplemented with 100 mM NaCl, 0.01% Tween 20, and 1 mM EDTA for 1 hour at 37° C., as demonstrated by the CYTX-DP assay platform. According to some embodiments, the MMP14 substrate exhibits at least a 20% cleavage rate when incubated with 50 pM human MMP14 in 50 mM HEPES (pH 6.8) supplemented with 10 mM CaCl2 and 0.5 mM MaCl2 for 1 hour at 37°C, and exhibits a cleavage rate of 20% or less when incubated with 500 pM human plasmin in 50 mM Tris-HCl (pH 7.4) supplemented with 100 mM NaCl, 0.01% Tween 20, and 1 mM EDTA for 1 hour at 37°C.

[0204] According to some embodiments, the observed K of a substrate for an activatable antibody for MMP9 cat / K M The value is 100M -1 s -1 According to some embodiments, the observed K of the substrate for an activatable antibody for MMP9 cat / K M The value is 1,000M -1 s -1 According to some embodiments, the observed K of the substrate for an activatable antibody for MMP9 cat / K M The value is 10,000M -1 s -1 That's all.

[0205] According to some embodiments, the observed K of a substrate for an activatable antibody for MMP14 cat / K M The value is 100M -1 s -1 According to some embodiments, the observed K of the substrate for an activatable antibody for MMP14 cat / K M The value is 1,000M -1 s -1 According to some embodiments, the observed K of the substrate for an activatable antibody for MMP14 cat / K M The value is 10,000M -1 s -1 That's all.

[0206] The present disclosure also provides antibodies comprising one or more of these MMP-cleavable substrates. For example, these MMP-cleavable substrates are useful when conjugating an antibody to one or more additional agents to generate a conjugated antibody. These MMP-cleavable substrates are useful in activatable antibody constructs.

[0207] The conjugated and / or activatable antibody comprises an antibody or antigen-binding fragment (AB) thereof that specifically binds a target. Typical types of targets for ABs include, but are not limited to, cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), and the like. According to some embodiments, the conjugated and / or activatable antibody has an AB that binds an extracellular target, typically an extracellular protein target. According to some embodiments, the conjugated and / or activatable antibody is designed for cellular uptake and is switchable intracellularly.

[0208] As a non-limiting example, AB is a conjugation partner for any of the targets listed in Table 1.

[0209] [Table 21]

[0210] [Table 22]

[0211] As a non-limiting example, AB is or is derived from an antibody listed in Table 2.

[0212] [Table 23]

[0213] [Table 24]

[0214] Exemplary conjugated and / or activatable antibodies of the present disclosure include, for example, antibodies that bind the interleukin-6 receptor (IL-6R) and that are, or include heavy and light chains derived from, the antibody referred to herein as the "Av1" antibody. The amino acid sequences for the Av1 heavy chain and Av1 light chain are set forth below as SEQ ID NO:54 and SEQ ID NO:55, respectively.

[0215] Av1 antibody H chain amino acid sequence: [ka]

[0216] Av1 antibody L chain amino acid sequence: [ka]

[0217] Exemplary conjugated and / or activatable antibodies of the present disclosure include, for example, antibodies that bind interleukin-6 receptor (IL-6R) and comprise heavy and light chains and a masking moiety that are or are derived from Av1 antibodies. Exemplary conjugated and / or activatable antibodies of the present disclosure comprise an amino acid sequence attached to the N-terminus of the AV1 light chain. These N-terminal amino acid sequences include, for example, YGSCSWNYVHIFMDC (SEQ ID NO: 161); QGDFDIPFPAHWVPIT (SEQ ID NO: 162); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 163); QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 164); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 165); or QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 166). It should also be understood that such amino acid sequences can be attached to the N-terminus of the AV1 heavy chain or to the C-terminus of the AV1 heavy or light chain.

[0218] Exemplary activatable antibodies of the disclosure include, for example, antibodies that bind epidermal growth factor receptor (EGFR) and are selected from the group consisting of the antibodies referred to herein as the "c22v5" antibody, the antibodies referred to herein as the "c225v4" antibody, and the antibodies referred to herein as the "c225v6" antibody (each of which binds EGFR), or antibodies comprising heavy and light chains derived therefrom. The c225v5, c225v4, and c225v6 antibodies share the same light chain sequence, referred to herein as the "c225 light chain." The amino acid sequences for the c225v5 heavy chain, c225v4 antibody, c225v6 antibody, and c225 light chain are set forth below.

[0219] c225v5 antibody H chain amino acid sequence: [ka]

[0220] c225v4 antibody H chain amino acid sequence: [ka]

[0221] c225v6 antibody H chain amino acid sequence: [ka]

[0222] c225 antibody L chain amino acid sequence: [ka]

[0223] Exemplary conjugated and / or activatable antibodies of the present disclosure include, for example, antibodies that bind a Jagged target, e.g., Jagged-1, Jagged-2, and / or both Jagged-1 and Jagged-2, and that include combinations of variable heavy and light chain regions that are, or are derived from, the variable heavy and light chain sequences shown below.

[0224] Variable L chain amino acid sequence Lc4 [ka]

[0225] Variable H chain amino acid sequence Hc4 [ka]

[0226] Variable L chain amino acid sequence Lc5 [ka]

[0227] Variable H chain amino acid sequence Hc5 [ka]

[0228] Variable light chain amino acid sequence Lc7 [ka]

[0229] Variable H chain amino acid sequence Hc7 [ka]

[0230] Variable light chain amino acid sequence Lc8 [ka]

[0231] Variable H chain amino acid sequence Hc8 [ka]

[0232] Variable light chain amino acid sequence Lcl3 [ka]

[0233] Variable H chain amino acid sequence Hcl3 [ka]

[0234] Variable light chain amino acid sequence Lcl6 [ka]

[0235] Variable H chain amino acid sequence Hcl6 [ka]

[0236] Variable light chain amino acid sequence Lcl9 [ka]

[0237] Variable H chain amino acid sequence Hcl9 [ka]

[0238] Variable L chain amino acid sequence Lc21 [ka]

[0239] Variable H chain amino acid sequence Hc21 [ka]

[0240] Variable light chain amino acid sequence Lc24 [ka]

[0241] Variable H chain amino acid sequence Hc24 [ka]

[0242] Variable L chain amino acid sequence Lc26 [ka]

[0243] Variable H chain amino acid sequence Hc26 [ka]

[0244] Variable light chain amino acid sequence Lc27 [ka]

[0245] Variable H chain amino acid sequence Hc27 [ka]

[0246] Variable light chain amino acid sequence Lc28 [ka]

[0247] Variable H chain amino acid sequence Hc28 [ka]

[0248] Variable L chain amino acid sequence Lc30 [ka]

[0249] Variable H chain amino acid sequence Hc30 [ka]

[0250] Variable light chain amino acid sequence Lc31 [ka]

[0251] Variable H chain amino acid sequence Hc31 [ka]

[0252] Variable light chain amino acid sequence Lc32 [ka]

[0253] Variable H chain amino acid sequence Hc32 [ka]

[0254] Variable light chain amino acid sequence Lc37 [ka]

[0255] Variable H chain amino acid sequence Hc37 [ka]

[0256] Variable light chain amino acid sequence Lc39 [ka]

[0257] Variable H chain amino acid sequence Hc39 [ka]

[0258] Variable light chain amino acid sequence Lc40 [ka]

[0259] H chain amino acid sequence Hc40 [ka]

[0260] Variable light chain amino acid sequence Lc47 [ka]

[0261] Variable H chain amino acid sequence Hc47 [ka]

[0262] Variable 4B2 light chain [ka]

[0263] Variable 4B2H chain [ka]

[0264] Variable 4D11 light chain [ka]

[0265] Variable 4D11 H chain [ka]

[0266] Variable 4E7L chain [ka]

[0267] Variable 4E7 heavy chain [ka]

[0268] Variable 4E11L chain [ka]

[0269] Variable 4E11 heavy chain [ka]

[0270] Variable 6B7L chain [ka]

[0271] Variable 6B7 heavy chain [ka]

[0272] Variable 6F8 light chain [ka]

[0273] Variable 6F8 heavy chain [ka]

[0274] Exemplary conjugated and / or activatable antibodies of the present disclosure include, for example, antibodies that bind a Jagged target, such as Jagged-1, Jagged-2, and / or both Jagged-1 and Jagged-2, and that include combinations of heavy and light chain regions that are or are derived from the heavy and light chain sequences shown below.

[0275] 4D11 light chain sequence: [ka]

[0276] 4D11 heavy chain sequence: [ka]

[0277] 4Dllv2 heavy chain sequence: [ka]

[0278] 4Dllv2 light chain sequence: [ka]

[0279] The activatable antibodies and activatable antibody compositions provided herein include at least an antibody or antibody fragment thereof (collectively referred to throughout this disclosure as an AB) that specifically binds a target, e.g., a human target, wherein the AB is modified with a masking moiety (MM).

[0280] In some embodiments, a masking moiety is selected for use with a particular antibody or antibody fragment. For example, a suitable masking moiety for use with an antibody that binds EGFR includes a MM comprising the sequence CISPRG (SEQ ID NO: 167). By way of non-limiting example, a MM can include the sequence, e.g., CISPRGC (SEQ ID NO: 497); CISPRGCG (SEQ ID NO: 168); CISPRGCPDGPYVMY (SEQ ID NO: 160); CISPRGCPDGPYVM (SEQ ID NO: 169); CISPRGCEPGTYVPT (SEQ ID NO: 170) and CISPRGCPGQIWHPP (SEQ ID NO: 171). Other suitable masking moieties include any of the EGFR-specific masks disclosed in PCT Publication No. WO 2010 / 081173, for example, by non-limiting example, GSHCLIPINMGAPSC (SEQ ID NO: 172); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 173); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 174); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 175); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 176); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 177); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 178); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 179); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 180); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 181); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 182); CHHFTLTTCGCISPRGCPG (SEQ ID NO: 183); YNPCATPMCCISPRGCPG (SEQ ID NO: 184); CNHHYFYTCGCISPRGCG (SEQ ID NO: 185); CNHHYHYYCGCISPRGCG (SEQ ID NO: 186);CNHVYFGTCGCISPRGCG (SEQ ID NO: 187); CHHVYWGHCGCISPRGCG (SEQ ID NO: 188); CPHFTTTSCGCISPRGCG (SEQ ID NO: 189); CNHFTLTTCGCISPRGCG (SEQ ID NO: 190); CHHFTLTTCGCISPRGCG (SEQ ID NO: 191);CPYYTLSYCGCISPRGCG (SEQ ID NO: 192); CPHVSFGSCGCISPRGCG (SEQ ID NO: 193); ADHVFWGSYGCISPRGCG (SEQ ID NO: 194); YNPCATPMCCISPRGCG (SEQ ID NO: 195); CHHVYWGHCGCISPRGCG (SEQ ID NO: 196); C(N / P)H(H / V / F)(Y / T)(F / W / T / L)(Y / G / T / S)(T / S / Y / H)CGCISPRGCG (SEQ ID NO: 197); CISPRGCGQPIPSVK ​​(SEQ ID NO: 198); CISPRGCTQPYHVSR (SEQ ID NO: 199); and / or CISPRGCNAVSGLGS (SEQ ID NO: 200).

[0281] Suitable masking moieties for use with antibodies that bind Jagged targets, such as Jagged1 and / or Jagged2, include, by non-limiting example, sequences such as QGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 496); QGQSGQGQQQWCNIWINGGDCRGWNG (SEQ ID NO: 201); PWCMQRQDFLRCPQP (SEQ ID NO: 202); QLGLPAYMCTFECLR (SEQ ID NO: 203); CNLWVSGGDCGGLQG (SEQ ID NO: 204); SCSLWTSGSCLPHSP (SEQ ID NO: 205); YCLQLPHYMQAMCGR (SEQ ID NO: 206); CFLYSCTDVSYWNNT (SEQ ID NO: 207); PWCMQRQDYLRCPQP (SEQ ID NO: 208); CNLWISGGDCRGLAG (SEQ ID NO: 209); CNLWVSGGDCRGVQG (SEQ ID NO: 210); CNLWVSGGDCRGLRG (SEQ ID NO: 211); CNLWISGGDCRGLPG (SEQ ID NO: 212); CNLWVSGGDCRDAPW (SEQ ID NO: 213); CNLWVSGGDCRDLLG (SEQ ID NO: 214); CNLWVSGGDCRGLQG (SEQ ID NO: 215); CNLWLHGGDCRGWQG (SEQ ID NO: 216); CNIWLVGGDCRGWQG (SEQ ID NO: 217); CTTWFCGGDCGVMRG (SEQ ID NO: 218); CNIWGPSVDCGALLG (SEQ ID NO: 219); CNIWVNGGDCRSFEG (SEQ ID NO: 220); YCLNLPRYMQDMCWA (SEQ ID NO: 221); YCLALPHYMQADCAR (SEQ ID NO: 222); CFLYSCGDVSYWGSA (SEQ ID NO: 223); CYLYSCTDSAFWNNR (SEQ ID NO: 224); CYLYSCNDVSYWSNT (SEQ ID NO: 225); CFLYSCTDVSYW (SEQ ID NO: 226); CFLYSCTDVAYWNSA (SEQ ID NO: 227); CFLYSCTDVSYWGDT (SEQ ID NO: 228); CFLYSCTDVSYWGNS (SEQ ID NO: 229); CFLYSCTDVAYWNNT (SEQ ID NO: 230); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 231);CFLYSCTDVAYWSGL (SEQ ID NO: 232); CYLYSCTDGSYWNST (SEQ ID NO: 233); CFLYSCSDVSYWGNI (SEQ ID NO: 234); CFLYSCTDVAYW (SEQ ID NO: 235); CFLYSCTDVSYWGST (SEQ ID NO: 236); CFLYSCTDVAYWGDT (SEQ ID NO: 237);GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 238); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 239); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 240);GCNIWANGGDCRGWIDNIDG (SEQ ID NO: 241); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 242); GCNIWLVGGDCRGWLEEAVG (SEQ ID NO: 243);GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 244); GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 245); GQQQWCNIWINGGDCRGWNG (SEQ ID NO: 246);GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 247);GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 248);GASQYCNLWINGGDCRGWRG (SEQ ID NO: 249);GCNIWLVGGDCRPWVEGG (SEQ ID NO: 250);GCNIWAVGGDCRPFVDGG (SEQ ID NO: 251);GCNIWLNGGDCRAWVDTG (SEQ ID NO: 252);GCNIWIVGGDCRPFINDG (SEQ ID NO: 253);GCNIWLNGGDCRPVVFGG (SEQ ID NO: 254);GCNIWLSGGDCRMFMNEG (SEQ ID NO: 255);GCNIWVNGGDCRSFVYSG (SEQ ID NO: 256);GCNIWLNGGDCRGWEASG (SEQ ID NO: 257);GCNIWAHGGDCRGFIEPG (SEQ ID NO: 258); GCNIWLNGGDCRTFVASG (SEQ ID NO: 259); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 260); GFLENCNIWLNGGDCRTG (SEQ ID NO: 261); GIYENCNIWLNGGDCRMG (SEQ ID NO: 262);and / or a masking moiety comprising GIPDNCNIWINGGDCRYG (SEQ ID NO: 263);

[0282] Masking moieties for use with antibodies that bind an interleukin-6 target, such as the interleukin-6 receptor (IL-6R), include, by non-limiting example, sequences such as QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO:264); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO:265); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO:266); YRSCNWNYVSIFLDC (SEQ ID NO:267); PGAFDIPFPAHWVPNT (SEQ ID NO:268); ESSCVWNYVHIYMDC (SEQ ID NO:269); YPGCKWNYDRIFLDC (SEQ ID NO:270); YRTCSWNYVGIFLDC (SEQ ID NO:271); YGSCSWNYVHIFMDC (SEQ ID NO:161); YGSCSWNYVHIFLDC (SEQ ID NO:272); YGSCNWNYVHIFLDC (SEQ ID NO:273); YTSCNWNYVHIFMDC (SEQ ID NO:274); YPGCKWNYDRIFLDC (SEQ ID NO:275);WRSCNWNYAHIFLDC (SEQ ID NO:276); WSNCHWNYVHIFLDC (SEQ ID NO:277); DRSCTWNYVRISYDC (SEQ ID NO:278); SGSCKWDYVHIFLDC (SEQ ID NO:279); SRSCIWNYAHIHLDC (SEQ ID NO:280); SMSCYWQYERIFLDC (SEQ ID NO:281); YRSCNWNYVSIFLDC (SEQ ID NO:282); SGSCKWDYVHIFLDC (SEQ ID NO:283); YKSCHWDYVHIFLDC (SEQ ID NO:284); YGSCTWNYVHIFMEC (SEQ ID NO:285); FSSCNWNYVHIFLDC (SEQ ID NO:286); WRSCNWNYAHIFLDC (SEQ ID NO:287); YGSCQWNYVHIFLDC (SEQ ID NO:288); YRSCNWNYVHIFLDC (SEQ ID NO:289); NMSCHWDYVHIFLDC (SEQ ID NO:290); FGPCTWNYARISWDC (SEQ ID NO: 291); XXsCXWXYvhlfXdC (SEQ ID NO: 292); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 163); RDTGGQCRWDYVHIFMDC (SEQ ID NO: 293);AGVPAGCTWNYVHIFMEC (SEQ ID NO: 294);VGVPNGCVWNYAHIFMEC (SEQ ID NO: 295); DGGPAGCSWNYVHIFMEC (SEQ ID NO: 296); AVGPAGCWWNYVHIFMEC (SEQ ID NO: 297); CTWNYVHIFMDCGEGEGP (SEQ ID NO: 298); GGVPEGCTWNYAHIFMEC (SEQ ID NO: 299); AEVPAGCWWNYVHIFMEC (SEQ ID NO: 300); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 301); SGASGGCKWNYVHIFMDC (SEQ ID NO: 302); TPGCRWNYVHIFMECEAL (SEQ ID NO: 303); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 304); PGAFDIPFPAHWVPNT (SEQ ID NO: 305); RGACDIPFPAHWIPNT (SEQ ID NO: 306); QGDFDIPFPAHWVPIT (SEQ ID NO: 162); XGafDIPFPAHWvPnT (SEQ ID NO: 307); RGDGNDSDIPFPAHWVPRT (SEQ ID NO: 308); SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 309); WAGGNDCDIPFPAHWIPNT (SEQ ID NO: 310); WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 311); AGSGNDSDIPFPAHWVPRT (SEQ ID NO: 312); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 313); and / or RECGRCGDIPFPAHWVPRT (SEQ ID NO: 314).

[0283] When an AB is modified with a MM and in the presence of a target, the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to the target.

[0284] K of MM-modified AB against target d is the K of the AB unmodified by MM or the parent AB against the target dor more than at least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of an AB modified with a MM to a target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times lower.

[0285] The dissociation constant of MM with AB (K d ) is generally the K of AB against the target d Higher than AB. d is the K of AB against the target d The binding affinity of the MM to the AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times higher than the binding affinity of the MM to the AB. Conversely, the binding affinity of the MM to the AB is generally lower than the binding affinity of the AB to the target. The binding affinity of the MM to the AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times lower than the binding affinity of the MM to the target.

[0286] When an AB is modified with a MM and in the presence of its target, the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to its target. The ability of the AB to bind a target when modified with a MM, when compared to the target binding of an AB not modified with a MM or the target binding of the parent AB, is at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, as measured in an in vivo or in vitro assay. It can be reduced to 99% and even 100%.

[0287] The MM inhibits the binding of AB to its target. The MM binds the antigen-binding domain of AB and inhibits the binding of AB to its target. The MM sterically inhibits the binding of AB to its target. The MM can allosterically inhibit the binding of AB to its target. According to these embodiments, when the AB is modified or coupled to a MM and in the presence of a target, there is no, substantially no, or only 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, or more than 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more of binding of the AB to the target when measured in an in vivo or in vitro assay compared to binding of the AB unmodified with the MM, the parent AB, or the AB not coupled to the MM. There is 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% binding of AB to the target.

[0288] When an AB is coupled to or modified by an MM, the MM "masks" or reduces or otherwise inhibits the specific binding of the AB to its target. When an AB is coupled to or modified by an MM, such coupling or modification can affect a conformational change that reduces or inhibits the ability of the AB to specifically bind to its target.

[0289] An AB coupled to or modified with a MM can be represented by the following formula (in the order from the amino (N)-terminal region to the carboxyl (C)-terminal region):

[0290] [ka]

[0291] where MM is a masking moiety, AB is an antibody or antibody fragment thereof, and L is a linker. In many embodiments, it is desirable to insert one or more linkers, e.g., flexible linkers, into the composition to provide flexibility.

[0292] According to certain embodiments, the MM is not a natural binding partner of AB. According to some embodiments, the MM does not include or contain homology to any natural binding partner of AB. According to other embodiments, the MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0293] According to some embodiments, an activatable antibody comprises an AB modified with a MM and also comprising one or more cleavable moieties (CM). Such an activatable antibody exhibits activatable / switchable binding of the AB to a target. An activatable antibody generally comprises at least one antibody or antibody fragment (AB) modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). According to some embodiments, the CM comprises an amino acid sequence that acts as a substrate for a protease of interest.

[0294] The elements in an activatable antibody are positioned such that the MM and CM are positioned such that, under cleaved (or relatively active) conditions and in the presence of a target, the AB binds the target, whereas under uncleaved (or relatively inactive) conditions in the presence of a target, specific binding of the AB to its target is reduced or inhibited. Specific binding of the AB to its target can be reduced due to inhibition or masking of the ability of the AB to specifically bind its target by the MM.

[0295] K of AB modified with MM and CM against target d is the K of the AB unmodified by MM and CM, or the parent AB, against the target. dor more than at least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of an AB modified with MM and CM for a target may be at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times lower.

[0296] When an AB is modified with a MM and a CM and in the presence of its target, but not in the presence of a modifying agent (e.g., an MMP), the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM and a CM or the specific binding of the parent AB to its target. The ability of the AB when modified with MM and CM to bind a target when compared to the binding of the parent AB or the binding of an AB not modified with MM and CM to a target is at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 11109, 11209, 112109, 11309, 11409, 11509, 11609, 11709, 11809, 11909, 12009, 12109, 12209, 12309, 12409, 12509, 12609, 12709, 12809, 12909, 13009, 13109, 13209, 13309, 13409, 13509, 13609, 13709, 13809, 13909, 14009, 14109, 14209, 14309, 14409, 14509, 14609, 14709, 14809, 14909, 14910, 149209, 149309, 149409, 149509, 149609, 149709, 149809, 149809, 1 It can be reduced to 98%, 99% and even 100%.

[0297] As used herein, the term "cleaved state" refers to the state of an activatable antibody following modification of the CM by at least one matrix metalloprotease. The term "uncleaved state" refers to the state of an activatable antibody in the absence of cleavage of the CM by MMPs. As discussed above, the term "activatable antibodies" is used herein to refer to activatable antibodies in both their uncleaved (native) and cleaved states. It will be apparent to those skilled in the art that, according to some embodiments, cleaved activatable antibodies lack MM due to cleavage of the CM by a protease, resulting in the release of at least MM (e.g., when the MM is not covalently attached to the activatable antibody (e.g., a disulfite bond between cysteine ​​residues)).

[0298] Activatable or switchable means that the activatable antibody exhibits a first level of target binding (first conformation) under an inhibited, masked, or uncleaved state, and a second level of target binding (i.e., second conformation) under an uninhibited, unmasked, and / or cleaved state, where the second level of target binding is greater than the first level of binding. Generally, target access to the AB of an activatable antibody is higher in the presence of a cleaving agent capable of cleaving the CM than in the absence of such a cleaving agent. Thus, when the activatable antibody is under an uncleaved state, the AB can be inhibited from target binding and masked from target binding (i.e., the first conformation is such that the AB cannot bind the target), and under a cleaved state, the AB is not inhibited or masked from target binding.

[0299] The CM and AB of the activatable antibody are selected so that the AB represents a binding moiety for a predetermined target and the CM represents a substrate for an MMP that is co-localized with the target at a treatment or diagnostic site in a subject. The activatable antibodies disclosed herein are particularly useful, for example, when an MMP capable of cleaving a site in the CM is present at relatively higher levels in target-containing tissue at a treatment or diagnostic site than in tissue at a non-treatment site (e.g., than in healthy tissue).

[0300] According to some embodiments, the activatable antibody provides reduced toxicity and / or adverse side effects resulting from binding of the first AB at a non-therapeutic site when the AB is unmasked or otherwise inhibited from binding its target.

[0301] Generally, an activatable antibody can be designed by selecting for a first AB of interest and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides masking of the AB or reduced binding of the AB to its target. Architectural design criteria should be considered to provide this functional characteristic.

[0302] Activatable antibodies are provided that exhibit a desired dynamic range of switchable phenotypes for target binding in a inhibited conformation relative to an uninhibited conformation. Dynamic range generally refers to the ratio of the maximum detectable level of a parameter under (a) a first set of conditions to the minimum detectable level of that parameter under (b) a second set of conditions. For example, in the case of an activatable antibody, dynamic range refers to the ratio of the maximum detectable level of target protein binding to the activatable antibody in the presence of an MMP capable of cleaving the CM of the activatable antibody to the minimum detectable level of target protein binding to the activatable antibody in the absence of a protease. The dynamic range of an activatable antibody can be calculated as the ratio of the equilibrium dissociation constant of the activatable antibody cleaving agent (e.g., an enzyme) treatment to the equilibrium dissociation constant of the activatable antibody cleaving agent treatment. The greater the dynamic range of the activatable antibody, the better the switchable phenotype of the activatable antibody. Activatable antibodies with a relatively high dynamic range value (e.g., higher than 1) exhibit a more desirable switching phenotype, such that target protein binding by the activatable antibody occurs to a greater extent (e.g., occurs primarily) in the presence of a cleaving agent (e.g., an enzyme) capable of cleaving the CM of the activatable antibody than in the absence of the cleaving agent.

[0303] Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulas for at least a portion of an activatable antibody are provided below. It is specifically contemplated that the MM and CM, in order from the N- to C-terminus of AB, can be reversed within an activatable antibody. It is also specifically contemplated that the CM and MM can overlap in amino acid sequence, e.g., such that a CM is contained within a MM.

[0304] For example, at least a portion of the activatable antibody can be represented, in order from the amino (N) terminal region to the carboxyl (C) terminal region, by the following formula:

[0305] [ka]

[0306] where MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof. Although MM and CM are shown as distinct moieties in the above formula, it is contemplated that, according to all exemplary embodiments (including formulas) disclosed herein, the amino acid sequences of MM and CM can overlap, such that the CM is fully or partially contained within the MM. Additionally, the above formula provides for additional amino acid sequences that can be located N-terminal or C-terminal to the activatable antibody element.

[0307] According to certain embodiments, the MM is not a natural binding partner of AB. According to some embodiments, the MM does not include or contain homology to any natural binding partner of AB. According to other embodiments, the MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0308] In many embodiments, it may be desirable to insert one or more linkers, e.g., flexible linkers, into an activatable antibody construct to provide flexibility at one or more MM-CM junctions, CM-AB junctions, or both. For example, the AB, MM, and / or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, especially Gly) to provide the desired flexibility. The switchable phenotype of such an activatable antibody construct may benefit from the introduction of one or more amino acids to provide a flexible linker. Furthermore, as described below, when an activatable antibody is provided as a conformationally constrained construct, a flexible linker may be operatively introduced to promote the formation and maintenance of a cyclic structure in the uncleaved activatable antibody.

[0309] For example, according to certain embodiments, the activatable antibody comprises one of the following formulas (wherein the formulas represent, from N- to C-terminal or C- to N-terminal, the amino acid sequence):

[0310] [ka]

[0311] wherein MM, CM, and AB are as defined above; and L1 and L2 are each independently, and optionally, the same or different, flexible linkers, which may be present or absent and contain at least one flexible amino acid (e.g., Gly). Additionally, the above formula provides additional amino acid sequences that may be located N-terminally or C-terminally to the activatable antibody element. Examples include, but are not limited to, targeting moieties (e.g., ligands for receptors on cells present in target tissues) and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulins (i.e., IgG) or serum albumins (e.g., human serum albumin (HAS))).

[0312] CM is approximately 0.001-1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250 or 1500 x 10 4 M -1 S -1 is specifically cleaved by at least one MMP at a rate of

[0313] For specific cleavage by an enzyme, contact between the enzyme and the CM is required. When an activatable antibody, including an AB coupled to a MM and a CM, is in the presence of a target and sufficient enzymatic activity, the CM can be cleaved. Sufficient enzymatic activity refers to the ability of the enzyme to contact the CM and effect cleavage. It is readily envisioned that the enzyme may be near the CM but unable to cleave it due to protein modifications by other cellular factors or enzymes.

[0314] Linkers suitable for use in the compositions described herein are generally those that provide flexibility for the modified AB or activatable antibody to facilitate inhibition of binding of at least the first AB to a target. Suitable linkers are readily selected and can be of any suitable different length, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0315] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 1), and (GGGS)n (SEQ ID NO: 2), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore act as neutral tethers between components. Glycine has significantly more access to phi-psi space than alanine, even though it is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, the following sequences: Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 4), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 5), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 7), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 8), and the like. Those skilled in the art will recognize that the design of an activatable antibody can include a linker that is fully or partially flexible, and consequently, the linker can include a flexible linker and one or more moieties that confer a less flexible structure to provide the desired multispecific activatable antibody structure.

[0316] According to some embodiments, the activatable antibodies described herein also encompass an agent conjugated to the activatable antibody. According to some embodiments, the conjugated agent is a therapeutic agent, such as an anti-inflammatory agent and / or an anti-tumor agent. According to such embodiments, the agent is conjugated to a carbohydrate moiety of the activatable antibody; for example, according to some embodiments, the carbohydrate moiety is located on the exterior of the antigen-binding region of the antibody or antigen-binding fragment of the activatable antibody. According to some embodiments, the agent is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment of the activatable antibody.

[0317] According to some embodiments, the agent is a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0318] In some embodiments, the agent is a detectable moiety, such as a label or other marker. For example, the agent may be detected by a radiolabeled amino acid, one or more biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected optically or colorimetrically), one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents.

[0319] The present disclosure also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, e.g., a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate). Suitable cytotoxic agents include, for example, dolastatins and their derivatives (e.g., auristatin E, AFP, MMAD, MMAF, MMAE). For example, the cytotoxic agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a derivative thereof. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0320] According to some embodiments, the agent is linked to A-B using a maleimidocaproyl-citrulline linker or a maleimidoPEG-valine-citrulline linker. According to some embodiments, the agent is linked to A-B using a maleimidocaproyl-valine-citrulline linker. According to some embodiments, the agent is linked to A-B using a maleimidoPEG-valine-citrulline linker. According to some embodiments, the agent is linked to A-B using a maleimidoPEG-valine-citrulline linker. According to some embodiments, the agent is monomethyl auristatin D (MMAD) linked to A-B using a maleimidoPEG-valine-citrulline-para-aminobenzoyloxycarbonyl linker, and this linker-payload construct is referred to herein as "vc-MMAD." According to some embodiments, the agent is monomethyl auristatin E (MMAE) linked to A-B using a maleimidoPEG-valine-citrulline-para-aminobenzoyloxycarbonyl linker, and this linker-payload construct is referred to herein as "vc-MMAE." The structures of vc-MMAD and vc-MMAE are shown below:

[0321] [ka]

[0322] [ka]

[0323] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, croton, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re can be mentioned.

[0324] Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). 14C-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionuclides to antibodies (see WO 94 / 11026).

[0325] Table 3 lists some exemplary agents that may be used in the disclosure described herein, but is by no means meant to be an exhaustive list.

[0326] [Table 25]

[0327] [Table 26]

[0328] Those skilled in the art will recognize that a wide variety of possible moieties can be coupled to the resulting antibodies of the present disclosure (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0329] Coupling can be achieved by any chemical reaction that will link two molecules so that the antibody and other moiety retain their respective activities for as long as possible. This binding can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. However, according to some embodiments, covalent bonding is preferred. Covalent bonding can be achieved by direct condensation of existing side chains or by the use of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various types of coupling agents known in the art, but rather is illustrative of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0330] In addition to the compositions and methods provided herein, according to some embodiments, conjugated activatable antibodies can also be modified for site-specific conjugation through modified amino acid sequences inserted into or otherwise included in the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled substitution and / or administration of conjugated agents within the conjugated activatable antibody. For example, activatable antibodies can be engineered to contain cysteine ​​substitutions at positions on the light and heavy chains that provide reactive thiol groups and do not negatively affect protein folding and assembly or alter antigen binding. According to some embodiments, activatable antibodies can be engineered to contain or otherwise introduce one or more non-naturally occurring amino acid residues into the activatable antibody to provide suitable sites for conjugation. According to some embodiments, activatable antibodies can be engineered to contain or otherwise introduce enzymatically activatable peptide sequences into their activatable antibody sequence.

[0331] Suitable linkers are described in the literature (see, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies by oligopeptide linkers. According to some embodiments, suitable linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (21558G)); (iii) SPDP (succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co. catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co. catalog number 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide, Pierce Chem. Co.) conjugated to EDC. Catalog No. 24510): Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

[0332] The linkers described above contain components with different attributes, thus leading to conjugates with different physico-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond and can form conjugates with enhanced stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in low available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. Carbodiimide coupling (e.g., EDC) when used in conjunction with sulfo-NHS is more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0333] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are multiple linkers. The multiple linkers can all be the same, e.g., cleavable or non-cleavable, or the multiple linkers can be different, e.g., at least one cleavable and at least one non-cleavable.

[0334] The present disclosure utilizes several methods for conjugating an agent to an AB, including: (a) attachment to the carbohydrate moiety of the AB, or (b) attachment to a sulfhydryl group of the AB, or (c) attachment to an amino group of the AB, or (d) attachment to a carboxyl group of the AB. According to the present disclosure, the AB can be covalently attached to the agent through an intermediate linker having at least two reactive groups, where one reactive group reacts with the AB and one group reacts with the agent. The linker, which can comprise any compatible organic compound, is selected so that reaction with the AB (or agent) does not adversely affect the AB reactivity and selectivity. Furthermore, attachment of the linker to the agent does not destroy the activity of the agent. Suitable linkers for reaction with an oxidized antibody or oxidized antibody fragment are those linkers containing an amine selected from the group consisting of primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and thiosemifulvazides. Such reactive functional groups may be present as part of the structure of the linker or may be introduced by appropriate chemical modification of a linker that does not contain such groups.

[0335] According to the present disclosure, suitable linkers for attachment to reduced AB include those linkers having specific reactive groups capable of reacting with sulfhydryl groups on reduced antibodies or fragments. Such reactive groups include, but are not limited to, reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercurybenzoic acid groups, and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

[0336] According to the present disclosure, suitable linkers for attachment to unoxidized or unreduced AB are those linkers that have specific functional groups capable of reacting with primary amines present in unmodified lysine residues in AB. Such reactive groups include, but are not limited to, NHS carboxylic acid or carbonate, sulfo-NHS carboxylic acid or carbonate, 4-nitrophenyl carboxylic acid or carbonate, pentafluorophenyl carboxylic acid or carbonate, acylimidazole, isocyanate, and isocyanate.

[0337] According to the present disclosure, suitable linkers for attachment to unoxidized and unreduced AB include those linkers having specific functional groups activated by an appropriate reagent that can react with carboxylic acid groups present in aspartic acid or glutamic acid residues in AB. Suitable activation reagents include EDC with or without an added NHS or sulfo-NHS, and other dehydrating agents utilized for carboxamide formation. In these cases, functional groups present on suitable linkers include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0338] The agent can be attached to the linker before or after the linker is attached to the AB. Depending on the particular application, it may be desirable to first generate an Ab-linker intermediate in which the linker does not have an associated agent. Depending on the particular application, a particular agent can be covalently attached to the linker. In other embodiments, the AB is first attached to the MM, CM, and associated linker, and then attached to the linker for conjugation purposes.

[0339] Branched Linkers: According to certain embodiments, branched linkers are used that have multiple sites for attachment of agents. For multi-site linkers, a single covalent bond to AB results in an AB-linker intermediate that can attach agents at multiple sites. The sites can be aldehydes or sulfhydryl groups, or any chemical site to which an agent can be attached.

[0340] According to some embodiments, higher specific activity (or higher ratio of agent to AB) can be achieved by attaching a single-site linker at multiple sites on AB. These multiple sites can be introduced into AB by either of two methods. First, one method can generate multiple aldehyde and / or sulfhydryl groups on the same AB. Second, one method can attach a "branched linker" with multiple functional sites to the aldehydes or sulfhydryls of AB for subsequent attachment to a linker. The functional sites of the branched linker or multi-site linker can be aldehyde or sulfhydryl groups, or any chemical site to which a linker can be attached. Even higher specific activity can be obtained by combining these two approaches, i.e., attaching a multi-site linker at several sites on AB.

[0341] Cleavable linker: A peptide linker that is susceptible to cleavage by an enzyme of the complement system, such as, but not limited to, urokinase, tissue plasminogen activator, trypsin, plasmin, or another enzyme with proteolytic activity, can be used in one embodiment of the present disclosure. According to one method of the present disclosure, an agent is attached via a linker that is susceptible to cleavage by complement. The antibody is selected from a species that can activate complement. The antibody-agent conjugate thus activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, an agent is attached via a linker that is susceptible to cleavage by an enzyme with proteolytic activity, such as urokinase, tissue plasminogen activator, plasmin, or trypsin. These cleavable linkers are useful in conjugated activatable antibodies that contain exotoxins, such as, by way of non-limiting example, any of the exotoxins listed in Table 3.

[0342] Non-limiting examples of cleavable linker sequences are provided in Table 4.

[0343] [Table 27]

[0344] Additionally, the agent can be conjugated to the AB via a disulfide bond (e.g., a disulfide bond on a cysteine ​​residue). Many tumors naturally release high levels of glutathione (a reducing agent), which reduces the disulfide bond and causes subsequent release of the agent at the site of delivery. According to certain embodiments, the reducing agent that modifies the CM will also modify the linker of the conjugated activatable antibody.

[0345] Spacers and Cleavable Elements: According to yet another embodiment, the linker should be configured in such a way as to optimize the spacing between the agent and the AB of the activatable antibody. This can be achieved by using linkers of the following general structure:

[0346] [ka]

[0347] where W is either -N-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer from 0 to 20.

[0348] According to some embodiments, the linker can comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the core of the AB so that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain branched linkers described above can act as spacer elements.

[0349] Throughout this discussion, it should be understood that the attachment of a linker to an agent (or of a spacer element to a cleavable element, or of a cleavable element to an agent) does not require a particular mode of attachment or reaction: any reaction that provides a product of suitable stability and biological compatibility is acceptable.

[0350] Selection of serum complement and linker: According to one method of the present disclosure, if release of an agent is desired, an antibody of a type capable of activating complement, an AB, is used. The resulting conjugate retains both the ability to conjugate antigen and activate the cascade. Thus, according to this embodiment of the present disclosure, the agent is linked to one end of a cleavable linker or cleavable element, and the other end of the linker group is attached to a specific site on the AB. For example, if the agent has a hydroxy or amino group, it can be attached to the carboxy terminus of a peptide, amino acid, or other appropriately selected linker via an ester or amide bond, respectively. For example, such agents can be attached to the linker peptide via a carbodiimide reaction. If the agent contains functional groups that prevent binding to the linker, those interfering functional groups are blocked prior to conjugation and deblocked once the product conjugate or intermediate is prepared. The opposite or amino terminus of the linker is then used, either directly or after further modification, for conjugation to an AB capable of activating complement.

[0351] The linker (or spacer element of the linker) can be of any desired length, and one end is covalently attached to a specific site on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker.

[0352] Thus, when the conjugates bind to an antigen in the presence of complement, the amide or ester bond connecting the agent to the linker is cleaved, resulting in release of the agent in its active form. These conjugates, when administered to a subject, achieve delivery or release of the agent at the target site and are particularly effective for in vivo delivery of drugs, antibiotics, antimetabolites, antiproliferative agents, and the like, as presented (but not limited to) in Table 3.

[0353] Linkers for release without complement activation: According to yet another application of targeted delivery, release of an agent without complement activation is desirable because activation of the complement cascade ultimately lyses target cells. Therefore, this approach is useful when delivery and release of an agent must be achieved without killing the target cells. This is the case when delivery of cellular mediators, such as hormones, enzymes, corticosteroids, neurotransmitters, genes, or enzymes to target cells is desired. These conjugates can be prepared by linking the agent to an AB that cannot activate complement via a linker that is mildly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complement forms quickly, but cleavage of the agent occurs slowly, thereby resulting in release of the compound at the target site.

[0354] Biochemical Crosslinkers: According to other embodiments, activatable antibodies can be conjugated to one or more therapeutic agents using specific biochemical crosslinkers. Crosslinking reagents form molecular bridges that link together functional groups of two different molecules. To link two different proteins in a stepwise fashion, hetero-bifunctional crosslinkers can be used, which eliminates undesired homopolymer formation.

[0355] Peptidyl linkers cleavable by lysosomal proteases, such as Val-Cit, Val-Ala, or other dipeptides, are also useful. Additionally, acid-labile linkers cleavable in the low pH environment of the lysosome, such as bis-sialyl ether, can be used. Other suitable linkers include cathepsin-labile substrates, particularly those that function optimally at acidic pH.

[0356] Exemplary hetero-bifunctional cross-linkers are referenced in Table 5.

[0357] [Table 28]

[0358] Non-cleavable linker or direct bond: According to yet another embodiment of the present disclosure, the conjugate can be designed so that the agent is delivered to the target but not released. This can be achieved by attaching the agent to the AB directly or via a non-cleavable linker.

[0359] These non-cleavable linkers can include amino acids, peptides, D-amino acids, or other organic compounds that can be subsequently modified to include functional groups that can be utilized for attachment to AB by the methods described herein. A general formula for such organic linkers can be:

[0360] [ka]

[0361] where W is either -N-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer from 0 to 20.

[0362] Non-cleavable conjugates: According to some embodiments, the compound may be conjugated to an AB that is incapable of activating complement. When an AB that is incapable of activating complement is used, this conjugation may be achieved using a linker that is susceptible to cleavage by activated complement, or using a linker that is not susceptible to cleavage by activated complement.

[0363] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0364] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be coupled to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-exchange reaction.

[0365] definition Unless otherwise specified, scientific and technical terms used in connection with this disclosure will have the meanings commonly understood by those of ordinary skill in the art. The terms "a" or "an" entity mean one or more of that entity. For example, a compound refers to one or more compounds. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Furthermore, unless otherwise required by context, singular terms will include the plural, and plural terms will include the singular. Generally, the nomenclature used in connection with and in connection with the cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization techniques described herein is that which is well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, or as commonly accomplished in the nuclear art, or as described herein. The foregoing techniques and procedures are generally performed according to conventional techniques well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0366] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0367] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with, or with a lower affinity (K) with, other polypeptides. d >10 -6 ) means to bind. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibodies, single chain, Fab and F(ab')2 fragments, scFv, and an Fab expression library.

[0368] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Generally, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chains present in the molecule. Specific classes have subclasses, e.g., IgG1, IgG2, and others. Furthermore, in humans, the light chains can be kappa or lambda chains.

[0369] The terms "monoclonal antibody" (mAb) or "monoclonal antibody composition," as used herein, refer to a population of antibody molecules containing only one species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0370] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions" or FRs. Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to be bound, and each of the three hypervariable regions of the H and L chains is referred to as the "complementarity-determining regions" or "CDRs." The amino acid assignment to each domain follows the definitions in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0371] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific conformational and charge characteristics. For example, antibodies are raised against N- or C-terminal peptides of polypeptides. An antibody is said to specifically bind an antigen when the dissociation constant is ≦1 μM, e.g., in some embodiments, ≦100 nM, and in some embodiments, ≦10 nM.

[0372] As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of noncovalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed by the dissociation constant (Kd) of the interaction, where a smaller Kd represents a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio Koff / Kon allows for the cancellation of all parameters not related to affinity and gives the dissociation constant K d(See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) An antibody of the present disclosure is said to specifically bind to EGFR if the equilibrium binding constant (Kd), as measured by an assay known to one of skill in the art, e.g., a radioligand binding assay or similar assay, is ≦1 μM, e.g., in some embodiments, ≦100 nM, in some embodiments, ≦10 nM, and in some embodiments, ≦100 pM to about 1 pM.

[0373] The term "isolated polynucleotide," as used herein, means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an "isolated polynucleotide" is one that: (1) is not associated with all or part of a polynucleotide with which the "isolated polynucleotide" is found in nature; (2) is operably linked to a polynucleotide with which it is not linked in nature; or (3) is not found in nature as part of a larger sequence. Polynucleotides of the present disclosure include nucleic acid molecules encoding the heavy chain immunoglobulin molecules set forth herein and nucleic acid molecules encoding the light chain immunoglobulin molecules set forth herein.

[0374] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; by its origin or source of derivation, an "isolated protein" is (1) not associated with a protein found in nature, (2) free from other proteins from the same source, e.g., murine proteins, (3) expressed by cells from a different species, or (4) not naturally occurring.

[0375] The term "polypeptide" is used herein as a genetic term to refer to a naturally occurring protein fragment or analog of a polypeptide sequence. Naturally occurring protein fragments and analogs are therefore species of the polypeptide genus. Polypeptides of the present disclosure include heavy chain immunoglobulin molecules as set forth herein, and light chain immunoglobulin molecules as set forth herein, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules together with light chain immunoglobulin molecules, e.g., kappa light chain immunoglobulins, and vice versa, and fragments and analogs thereof.

[0376] As used herein, the term "naturally occurring," as applied to an object, refers to the fact that the object can be found in nature. For example, it can refer to a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in the laboratory, or that otherwise occurs in nature.

[0377] The term "operably linked," as used herein, means that the positions of the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0378] The term "control sequence," as used herein, refers to polynucleotide sequences necessary to affect the expression and processing of linked coding sequences. The nature of such control sequences varies depending on the host organism in prokaryotes; in eukaryotes, such control sequences generally include a promoter, ribosomal binding site, and transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "polynucleotide," as used herein, refers to a sequence of nucleotides, ribonucleotides or deoxynucleotides, of at least 10 bases in length, or modified forms of either type of nucleotide. The term encompasses single- and double-stranded forms of DNA.

[0379] The term polynucleotide, as used herein, encompasses naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides generally containing 200 or fewer bases in length. In some embodiments, oligonucleotides are 10-60 bases in length, and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length. Oligonucleotides are typically single-stranded for probes, although oligonucleotides can be double-stranded for use in constructing gene variants. Oligonucleotides of the present disclosure can be either sense or antisense oligonucleotides.

[0380] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkages" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoronmidate, and the like. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anticancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). Oligonucleotides can contain a label for detection, if desired.

[0381] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers of the 20 conventional amino acids, unnatural amino acids, such as α-substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids (e.g., D-amino acids), may also be suitable components for the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, δ-N-methylarginine, and other similar amino acids, and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0382] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5'-end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The 5' to 3' addition direction of the nascent RNA transcript is referred to as the transcription direction sequence with the DNA strand having the same sequence as the RNA, and the 5' to 5' end direction of the RNA transcript is referred to as the "upstream sequences," i.e., the region of sequences on the DNA strand having the same sequence as the RNA, and the 3' to 3' end direction of the RNA transcript is referred to as the "downstream sequences."

[0383] The term "substantial identity," as applied to polypeptides, means that two peptide sequences, when optimally aligned with the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity; for example, according to some embodiments, at least 90% sequence identity, according to some embodiments, at least 95% sequence identity, and according to some embodiments, at least 99% sequence identity.

[0384] According to some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0385] As discussed herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are intended to be encompassed by the present disclosure, provided that the variation in amino acid sequence is maintained at least 75%, e.g., according to some embodiments, at least 80%, 90%, 95%, and according to some embodiments, 99%. Conservative amino acid substitutions are particularly contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic-hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. It is reasonable to predict that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the backbone. Whether an amino acid change results in a functional peptide can be easily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by one of skill in the art. Suitable amino- and carboxy-termini of fragments or analogs reside near boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequences to public or proprietary sequence databases. According to some embodiments, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Methods for identifying proteins that fold into known three-dimensional structures are known (Bowie et al. Science 253:164 (1991)). Thus, the foregoing examples demonstrate that one of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present disclosure.

[0386] Suitable amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence (in portions of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., amino acid substitutions should not tend to disrupt helices present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Principles of Proteins, Structure and Molecules (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0387] The term "polypeptide fragment," as used herein, refers to a polypeptide having an amino- and / or carboxy-terminal deletion and / or one or more internal deletions, but in which the remaining amino acid sequence is identical to the corresponding positions in a naturally occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, e.g., at least 14 amino acids in length, in some embodiments, at least 20 amino acids in length, usually at least 50 amino acids in length, and in some embodiments, 70 amino acids in length. The term "analog," as used herein, refers to a polypeptide comprised of a segment of at least 25 amino acids that has substantial identity to a portion of the deduced amino acid sequence and has specific binding to a target under appropriate binding conditions. Typically, a polypeptide analog contains conservative amino acid substitutions (or additions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, e.g., in some embodiments at least 50 or more amino acids in length, and can often be as long as a full-length naturally occurring polypeptide.

[0388] The term "agent" is used herein to denote a compound, a mixture of compounds, a biopolymer, or an extract made from biomaterials.

[0389] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment to the polypeptide of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected optically or colorimetrically). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug," as used herein, refers to a compound or composition capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0390] Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0391] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and according to some embodiments, a substantially purified fraction is a composition in which the target species accounts for at least about 50% (on a molar basis) of all macromolecular species present.

[0392] Generally, a substantially pure composition will comprise greater than about 80%, and in some embodiments, greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity (contaminating species in the composition cannot be detected by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0393] The term "patient" includes human and veterinary subjects.

[0394] The activatable antibodies of the present disclosure specifically bind a predetermined target, e.g., a human target protein. Activatable antibodies that bind to the same epitope as the activatable antibodies described herein are also conjugated to the present disclosure.

[0395] Those skilled in the art will recognize that it is possible, without undue experimentation, to determine whether a monoclonal antibody (e.g., a murine monoclonal or a humanized antibody) has the same specificity as the monoclonal antibody used in the methods described herein by determining whether the former prevents the latter from binding to its target. If the monoclonal antibody being tested competes with the monoclonal antibody of the present disclosure, as indicated by a decrease in binding by the monoclonal antibody of the present disclosure, then the two monoclonal antibodies bind to the same or closely related epitopes. A method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody of the present disclosure is to preincubate the monoclonal antibody of the present disclosure with the target, and then add the monoclonal antibody being tested and determine whether the monoclonal antibody being tested inhibits its ability to bind the target. If the monoclonal antibody being tested is inhibited, then it almost certainly has the same, or a functionally equivalent, epitope specificity as the monoclonal antibody of the present disclosure.

[0396] Multispecific activatable antibodies The present disclosure also provides multispecific activatable antibodies. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize multiple antigens or epitopes and include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody, such that coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. According to some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for at least one MMP protease. The multispecific activatable antibodies provided herein are stable in the circulation, are activated at intended sites of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit target binding at least comparable to that of the corresponding, unmodified multispecific antibody.

[0397] According to some embodiments, the multispecific activatable antibody is designed to engage immune effector cells, also referred to herein as immune effector-engaging multispecific activatable antibodies. According to some embodiments, the multispecific activatable antibody is designed to engage leukocytes, also referred to herein as leukemia-engaging multispecific activatable antibodies. According to some embodiments, the multispecific activatable antibody is designed to engage T-cells, also referred to herein as T-cell-engaging multispecific activatable antibodies. According to some embodiments, the multispecific activatable antibody engages a surface antigen on a leukocyte, e.g., a T cell, a natural killer (NK) cell, a myelomononuclear cell, a macrophage, and / or another immune effector cell. According to some embodiments, the immune effector cell is a leukocyte. According to some embodiments, the immune effector cell is a T cell. According to some embodiments, the immune effector cell is an NK cell. According to some embodiments, the immune effector cell is a mononuclear cell, e.g., a myelomononuclear cell. According to some embodiments, a multispecific activatable antibody is designed to bind or otherwise react with multiple targets and / or multiple epitopes, also referred to herein as a multi-antigen-targeting activatable antibody. As used herein, the terms "target" and "antigen" are used interchangeably.

[0398] According to some embodiments, the immune effector cell-engaging multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and an immune effector cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the immune effector cell-engaging antibody or antigen-binding portion thereof is masked. According to some embodiments, the immune effector cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the immune effector cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell-engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of said MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target.According to some embodiments, the immune effector cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the non-immune effector cell-engaging antibody is a cancer-targeting antibody. According to some embodiments, the immune effector cell-engaging antibody is an scFv. According to some embodiments, the targeting antibody (e.g., non-immune cell effector antibody) is an IgG and the immune effector cell-engaging antibody is an scFv. According to some embodiments, the immune effector cells are leukocytes. According to some embodiments, the immune effector cells are T cells. According to some embodiments, the immune effector cells are NK cells. According to some embodiments, the immune effector cells are bone marrow mononuclear cells.

[0399] According to some embodiments, the T-cell-engaging multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and a T-cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the T-cell-engaging antibody or antigen-binding portion thereof is masked. According to some embodiments, the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the T-cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and said targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment (AB2) that binds a second target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0400] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises a cancer-targeting antibody or antigen-binding fragment thereof and a T-cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the cancer-targeting antibody or antigen-binding fragment thereof and / or the T-cell-engaging antibody or antigen-binding portion thereof is masked. According to some embodiments, the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind a second cancer-associated target. According to some embodiments, the T-cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and said cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment (AB2) that binds a second cancer-associated target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0401] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof and a T-cell-engaging scFv, wherein at least one of the cancer-targeting IgG antibody or antigen-binding fragment thereof and / or the T-cell-engaging antibody or antigen-binding portion thereof is masked. According to some embodiments, the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind a second cancer-associated target. According to some embodiments, the T-cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and said cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment (AB2) that binds a second cancer-associated target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0402] According to some embodiments of the immune effector-engaging multispecific activatable antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, for example, but not limited to, any of the targets listed in Table 1, such as, but not limited to, EGGR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and another antigen is typically a stimulatory or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. According to some embodiments, the antigen is a stimulatory receptor present on the surface of T cells or NK cells; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. According to some embodiments, the antigen is an inhibitory receptor present on the surface of T cells; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIR. An antibody that confers specificity for a T-cell surface antigen may be substituted with a ligand or ligand domain that binds to an effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9.

[0403] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the cancer microenvironment and includes an antibody, e.g., an IgG or scFv, directed against a tumor target and an agonist antibody, e.g., an IgG or scFv directed against a costimulatory receptor expressed on the surface of activated T cells or NK cells, wherein at least one of the cancer-targeting antibody and / or agonist antibody is masked. Examples of costimulatory receptors include, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, and OX40. According to this embodiment, the multispecific activatable antibody, upon activation by tumor-associated proteases, will effectively crosslink and activate T cell- or NK cell-expressed costimulatory receptors in a tumor-dependent manner to enhance the activity of T cells responding to any tumor antigen via their endogenous T cell antigen or NK-activating receptor. The activation-dependent nature of these T cell or NK cell costimulatory receptors focuses the activity of the activated multispecific activatable antibody on tumor-specific T cells, rather than activating all T cells, regardless of their antigen specificity. According to one embodiment, at least the costimulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of autoreactive T cells that may be present in tissues that also express the antigen recognized by the tumor-targeting antibody in the multispecific activatable antibody, but whose activity is limited by the lack of co-receptor engagement.

[0404] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the microenvironment of diseases characterized by T cell hyperstimulation, such as, but not limited to, autoimmune or inflammatory diseases. Such multispecific activatable antibodies include antibodies, e.g., IgG or scFv, directed against targets including surface antigens expressed in tissues targeted by T cells in autoimmune inflammatory diseases, and antibodies, e.g., IgG or scFv, directed against inhibitory receptors expressed on the surface of T cells or NK cells, wherein at least one of the disease tissue-targeting antibody and / or T cell receptor antibody is masked. Examples of inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIR. Examples of tissue antigens targeted by T cells in autoimmune diseases include, but are not limited to, surface antigens expressed on myelin or nerve cells in multiple sclerosis, or surface antigens expressed on pancreatic islet cells in type 1 diabetes. According to this embodiment, the multispecific activatable antibodies, when localized to tissues under autoimmune attack or inflammation, become activated and co-engage T cell or NK cell inhibitory receptors via their endogenous TCRs or activating receptors to suppress the activity of autoreactive T cells responding to any diseased tissue-targeted antigen. According to one embodiment, at least one or more antibodies are masked to prevent suppression of desired T cell responses in non-diseased tissues where the target antigen may also be expressed.

[0405] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CD3 epsilon (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, comprising a second antibody or antigen-binding fragment thereof (AB2), wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0406] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0407] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0408] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an OKT3-derived anti-CD3 epsilon (CD3ε) scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the OKT3 scFv or OKT3-derived scFv is masked. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, comprising a second antibody or antigen-binding fragment thereof (AB2), wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second target.

[0409] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0410] According to some embodiments, the T-cell engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0411] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the anti-CTLA-4 scFv is masked. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target.

[0412] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv and a targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CTLA-4 scFv and / or the targeting IgG antibody or antigen-binding portion thereof is masked. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4. According to some embodiments, the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target.

[0413] According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody comprises at least a first antibody or antigen-binding fragment thereof that binds a first target and / or a first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and / or a second epitope. According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different targets. According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different epitopes on the same target. According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds a combination of multiple different targets and multiple different epitopes on the same target.

[0414] According to some embodiments, the multispecific activatable antibody comprising an IgG has a masked IgG variable domain. According to some embodiments, the multispecific activatable antibody comprising an scFv has a masked scFv domain. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein each of the IgG variable domains and the scFv domains is coupled to its own masking moiety. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for another target antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for an epitope of the target antigen and another antibody domain has specificity for another epitope of the target antigen.

[0415] In multispecific activatable antibodies, scFvs can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, the carboxyl terminus of the light chain of an IgG activatable antibody, or the carboxyl terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, the amino terminus of the light chain of an IgG activatable antibody, or the amino terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to the antigen-binding domain of an IgG and masks that domain. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to the antigen-binding domain of at least one scFv and masks that domain. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of an IgG, and a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of at least one scFv.

[0416] The present disclosure provides examples of multispecific activatable antibody structures, including, but not limited to, the following: (VL-CL)2:(VH-CH1-CH2-CH3-L4-VH * -L3-VL * -L2-CM-L1-MM)2; (VL-CL)2:(VH-CH1-CH2-CH3-L4-VL * -L3-VH * -L2-CM-L1-MM)2; (MM-L1-CM-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VH * -L3-VL * )2; (MM-L1-CM-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VL* -L3-VH * )2; (VL-CL)2:(MM-L1-CM-L2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; (VL-CL)2:(MM-L1-CM-L2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL-CL)2:(VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL-CL)2:(VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2:(VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL * -L3-VH * -L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VH * -L3-VL * -L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2: (MM-L1-CM-L2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2: (MM-L1-CM-L2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2: (MM-L1-CM-L2-VL* -L3-VH * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2: (MM-L1-CM-L2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * )2: (MM-L1-CM-L2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * 2: (MM-L1-CM-L2-VH) * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL * -L3-VH * )2: (MM-L1-CM-L2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL * -L3-VH * 2: (MM-L1-CM-L2-VH) * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2: (VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2: (VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2: (VL * -L3-VH *-L4-VH-CH1-CH2-CH3); or (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2: (VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2, where VL and VH represent the light and heavy variable domains of the first specificity contained in IgG; VL * and VH * represents the variable domain of the second specificity contained in the scFv; L1 is a linker peptide connecting the masking moiety (MM) and the cleavable moiety (CM); L2 is a linker peptide connecting the cleavable moiety (CM) and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is a light chain constant domain; and CH1, CH2, and CH3 are heavy chain constant domains. The first and second specificities can be against any antigen or epitope.

[0417] According to some embodiments of the T-cell engaging multispecific activatable antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, such as, but not limited to, any of the targets listed in Table 1, such as EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or The antigen may be, but is not limited to, CD71 (transferrin receptor), and another antigen may typically be a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of T-cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain that provides specificity for a T-cell surface antigen may also be replaced with a ligand or ligand domain that binds to a T-cell receptor, an NK-cell receptor, a macrophage receptor and / or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2 or TNFSF9. According to some embodiments of the multi-antigen targeted activatable antigen, one antigen is selected from the target group listed in Table 1, and another antigen is selected from the target group listed in Table 1.

[0418] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v4, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v6, which is specific for binding to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which is specific for binding to human and mouse Jagged1 and Jagged2. In some embodiments, the targeting antibody is 4D11v2, which is specific for binding to human and mouse Jagged1 and Jagged2.

[0419] In some embodiments, the targeting antibody can be in the form of an activatable antibody. In some embodiments, the scFv can be in the form of a pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0420] According to some embodiments, the scFv is specific for binding CD3ε and is or is derived from an antibody or fragment thereof that binds CD3ε, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. According to some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).

[0421] According to some embodiments, the anti-CTLA-4 scFv comprises the following amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGWQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVTVSSAS (SEQ ID NO: 510)

[0422] According to some embodiments, the anti-CTLA-4 scFv comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:510.

[0423] According to some embodiments, the anti-CD3ε scFv comprises the following amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 511)

[0424] According to some embodiments, the anti-CD3ε scFv comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:511.

[0425] According to some embodiments, the scFv is specific for binding one or more T-cells, one or more NK cells, and / or one or more macrophages. According to some embodiments, the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.

[0426] According to some embodiments, the multispecific activatable antibody also includes an agent conjugated to AB. According to some embodiments, the agent is an anti-tumor agent. According to some embodiments, the agent is a toxin or a fragment thereof. According to some embodiments, the agent is conjugated to the multispecific activatable antibody via a linker. According to some embodiments, the agent is conjugated to AB via a cleavable linker. According to some embodiments, the agent is conjugated to AB via a linker comprising at least one MMP-cleavable substrate sequence. According to some embodiments, the linker is a non-cleavable linker. According to some embodiments, the agent is a microtubule inhibitor. According to some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent, or a DNA intercalator, or other DNA damaging agent. According to some embodiments, the linker is a cleavable linker. According to some embodiments, the agent is an agent selected from the group listed in Table 4. According to some embodiments, the agent is trastatin. According to some embodiments, the agent is auristatin or a derivative thereof. According to some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0427] According to some embodiments, the multispecific activatable antibody can also comprise a detectable moiety. According to some embodiments, the detectable moiety is a diagnostic agent.

[0428] According to some embodiments, the multispecific activatable antibody naturally contains one or more disulfide bonds. According to some embodiments, the multispecific activatable antibody can be engineered to contain one or more disulfide bonds.

[0429] The present disclosure also provides isolated nucleic acid molecules encoding the multispecific activatable antibodies described herein, and vectors comprising those isolated nucleic acid sequences. The present disclosure provides methods for producing multispecific antibodies by culturing cells comprising such nucleic acid molecules under conditions conducive to expression of the activatable antibodies. According to some embodiments, the cells comprise such vectors.

[0430] The present disclosure also provides methods for producing the multispecific activatable antibodies of the present disclosure by (a) culturing cells containing a nucleic acid construct encoding the multispecific activatable antibody under conditions conducive to expression of the multispecific activatable antibody, and (b) recovering the multispecific activatable antibody.

[0431] The present disclosure also provides multispecific antibody and / or multispecific activatable antibody compositions comprising at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that binds a second target or a second epitope, wherein at least AB1 is coupled to or otherwise bound to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind its target. According to some embodiments, MM1 is coupled to AB1 via a first cleavable moiety (CM1) sequence that comprises a substrate for a protease, e.g., a protease that co-localizes with the target of AB1 at a therapeutic or diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, are activated at the intended site of therapy and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding of AB1 to the target at least comparable to that of the corresponding unmodified multispecific antibody.

[0432] According to some embodiments, the multispecific activatable antibody comprises a linking peptide between MM1 and CM1.

[0433] According to some embodiments, the multispecific activatable antibody comprises a linking peptide between CM1 and AB1.

[0434] According to some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and at least a portion of the multispecific activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. According to some embodiments, the two connecting peptides need not be identical to each other.

[0435] According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO:2), where n is at least one integer. According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), and GSSSG (SEQ ID NO:8).

[0436] According to some embodiments, the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds a second target or a second epitope. According to some embodiments, each AB in the multispecific activatable antibody is independently selected from the group consisting of monoclonal antibodies, domains, antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single-domain heavy chain antibodies, and single-domain light chain antibodies. According to some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

[0437] According to some embodiments, each AB in the multispecific activatable antibody has an equilibrium dissociation constant of about 100 nM or less for binding to its corresponding target or epitope.

[0438] According to some embodiments, MM1 has an equilibrium dissociation constant for binding to its corresponding AB that is higher than the equilibrium dissociation constant for binding of AB to its corresponding target or epitope.

[0439] According to some embodiments, MM1 has an equilibrium dissociation constant for binding to its corresponding AB that is no more than the equilibrium dissociation constant for binding of AB to its corresponding target or epitope.

[0440] According to some embodiments, MM1 does not block or compete with the corresponding AB for binding to its corresponding target or epitope when the multispecific activatable antibody is present in a cleaved state.

[0441] According to some embodiments, MM1 is a polypeptide about 2-40 amino acids in length. According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 amino acids or less in length.

[0442] According to some embodiments, MM1 has a polypeptide sequence that differs from the sequence of the target of its corresponding AB.

[0443] According to some embodiments, MM1 has a polypeptide sequence that is no more than 50% identical to any natural binding partner of its corresponding AB. According to some embodiments, MM1 has a polypeptide sequence that is no more than 25% identical to any natural binding partner of its corresponding AB. According to some embodiments, MM1 has a polypeptide sequence that is no more than 10% identical to any natural binding partner of its corresponding AB.

[0444] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 20 times higher than

[0445] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 40 times higher than

[0446] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitoped At least 100 times higher than

[0447] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 1000 times higher than

[0448] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 10,000 times higher than

[0449] According to some embodiments, MM1 is an amino acid sequence selected from the MMs shown in the Examples provided herein.

[0450] According to some embodiments, the multispecific activatable antibody comprises at least a second masking moiety (MM2), which inhibits binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state, and a second cleavable moiety (CM2), coupled to AB2, which functions as a substrate for a second protease. According to some embodiments, CM2 is a polypeptide 15 amino acids or less in length. According to some embodiments, the second protease is co-localized with a second target or epitope in a tissue, and the second protease cleaves CM2 on the multispecific activatable antibody when the multispecific activatable antibody is exposed to the second protease. According to some embodiments, the first protease and the second protease are co-localized with the first target or epitope and the second target or epitope in a tissue. According to some embodiments, the first protease and the second protease are the same protease. According to some embodiments, CM1 and CM2 are different substrates for the same protease. According to some embodiments, the protease is selected from the group consisting of those shown in Table 7. According to some embodiments, the first protease and the second protease are different proteases. According to some embodiments, the first protease and the second protease are different proteases selected from the group consisting of those shown in Table 7.

[0451] According to some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has an equilibrium dissociation constant for binding to its corresponding AB that is higher than the equilibrium dissociation constant for binding to its corresponding target or epitope of AB.

[0452] According to some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has an equilibrium dissociation constant for binding to its corresponding AB that is less than or equal to the equilibrium dissociation constant for binding to its corresponding target or epitope of AB.

[0453] According to some embodiments, each MM in the multispecific activatable antibody does not interfere with or compete with its corresponding AB for binding to the corresponding target or epitope when the multispecific activatable antibody is in a cleaved state.

[0454] According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide about 2-40 amino acids in length. According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 amino acids or less in length.

[0455] According to some embodiments, each MM in the multispecific activatable antibody has a polypeptide sequence that differs from the sequence of the target of the corresponding AB.

[0456] According to some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 50% identical to any natural binding partner of its corresponding AB. According to some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 25% identical to any natural binding partner of its corresponding AB. According to some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 10% identical to any natural binding partner of its corresponding AB.

[0457] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 20 times higher than

[0458] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 40 times higher than

[0459] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 100 times higher than

[0460] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 1000 times higher than

[0461] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 10,000 times higher than

[0462] According to some embodiments, each MM is an amino acid sequence selected from the MMs disclosed herein.

[0463] According to some embodiments, at least one of CM1 and / or CM2 is cleaved by at least one MMP protease. At least one of CM1 and / or CM2 comprises an amino acid sequence selected from the group consisting of the sequences ISSGLLSS (SEQ ID NO:14); QNQALRMA (SEQ ID NO:15); AQNLLGMV (SEQ ID NO:16); STFPFGMF (SEQ ID NO:17); PVGYTSSL (SEQ ID NO:18); DWLYWPGI (SEQ ID NO:19); MIAPVAYR (SEQ ID NO:20); RPSPMWAY (SEQ ID NO:21); WATPRPMR (SEQ ID NO:22); FRLLDWQW (SEQ ID NO:23); LKAAPRWA (SEQ ID NO:24); GPSHLVLT (SEQ ID NO:25); LPGGLSPW (SEQ ID NO:26); MGLFSEAG (SEQ ID NO:27); SPLPLRVP (SEQ ID NO:28); RMHLRSLG (SEQ ID NO:29); LAAPLGLL (SEQ ID NO:30); AVGLLAPP (SEQ ID NO:31); LLAPSHRA (SEQ ID NO:32); PAGLWLDP (SEQ ID NO:33); and / or ISSGLSS (SEQ ID NO:159).

[0464] According to some embodiments, at least one of CM1 and / or CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 364-370, 379-393, 402-409, 420-424, 434, 435, 450-452, 457, 470-472, 474 and 483.

[0465] According to some embodiments, at least one of CM1 and / or CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 328, 336-339 and 348-351.

[0466] According to some embodiments, a protease that cleaves the first cleavable moiety (CM1) sequence co-localizes in a tissue with the target of AB1 in the multispecific activatable antibody, and the protease cleaves CM1 in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0467] According to some embodiments, the multispecific activatable antibody comprises multiple cleavable subsequences, and a protease that cleaves at least one cleavable subsequence is co-localized in a tissue with a target of at least one region of the AB region in the multispecific activatable antibody, and the protease cleaves the CM in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0468] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least two-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0469] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least three times higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0470] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least four times higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0471] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 5-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0472] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 10-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0473] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 20-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0474] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 40-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0475] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 50-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0476] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 100-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0477] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 200-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0478] According to some embodiments, each CM in the multispecific activatable antibody is a polypeptide up to 15 amino acids in length.

[0479] According to some embodiments, at least one CM in the multispecific activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-33 and 159, and another CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 26). According to some embodiments, at least one CM in the multispecific activatable antibody comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 26). According to some embodiments, at least one cleavable moiety is selected for use with a particular protease, e.g., a protease known to co-localize with at least one target of the multispecific activatable antibody. For example, suitable cleavable moieties for use in the multispecific activatable antibodies of the present disclosure are cleaved by at least proteases such as urokinase, legumain, and / or matriptase (also referred to herein as MT-SP1 or MTSSP1). According to some embodiments, suitable cleavable moieties comprise at least one of the following sequences: TGRGPSWV (SEQ ID NO:34); SARGPSRW (SEQ ID NO:35); TARGPSFK (SEQ ID NO:36); LSGRSDNH (SEQ ID NO:37); GGWHTGRN (SEQ ID NO:38); HTGRSGAL (SEQ ID NO:39); PLTGRSGG (SEQ ID NO:40); AARGPAIH (SEQ ID NO:41); RGPAFNPM (SEQ ID NO:42); SSRGPAYL (SEQ ID NO:43); RGPATPIM (SEQ ID NO:44); RGPA (SEQ ID NO:45); GGQPSGMWGW (SEQ ID NO:46); FPRPLGITGL (SEQ ID NO:47); VHMPLGFLGP (SEQ ID NO:48); SPLTGRSG (SEQ ID NO:49); SAGFSLPA (SEQ ID NO:126); LAPLGLQRR (SEQ ID NO:50); SGGPLGVR (SEQ ID NO:51); and / or PLGL (SEQ ID NO:52).

[0480] According to...

Claims

1. An isolated polypeptide comprising a cleavable moiety (CM) comprising the amino acid sequence of SEQ ID NO: 15, wherein the cleavable moiety is a substrate for a matrix metalloproteinase (MMP), and the polypeptide comprises an antibody or antigen-binding fragment thereof (AB) that binds to a target.

2. The AB is a Fab fragment, F(ab') 2 2. The isolated polypeptide of claim 1, which is selected from the group consisting of a fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

3. 3. The isolated polypeptide of claim 1 or 2, wherein the CM is a substrate for a matrix metalloproteinase (MMP) that is co-localized in a tissue with the target.

4. The isolated polypeptide of any one of claims 1 to 3, wherein the AB is linked to the CM.

5. The isolated polypeptide of any one of claims 1 to 4, wherein the AB is directly linked to the CM.

6. 5. The isolated polypeptide of claim 1, wherein the AB is linked to the CM via a connecting peptide.

7. The isolated polypeptide of any one of claims 1 to 6, wherein the isolated polypeptide comprises a masking moiety (MM).

8. 8. The isolated polypeptide of claim 7, wherein the MM has an equilibrium dissociation constant for binding to the AB that is higher than the equilibrium dissociation constant of the AB for binding to the target.

9. 9. The isolated polypeptide of claim 7 or 8, wherein the MM is a polypeptide of 40 amino acids or less in length.

10. 10. The isolated polypeptide of any one of claims 7 to 9, wherein the MM is linked to the CM such that the isolated polypeptide in its uncleaved state comprises the following structural configuration from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM.

11. The isolated polypeptide of claim 10, wherein the isolated polypeptide comprises a connecting peptide between the MM and the CM, and a connecting peptide between the CM and the AB.

12. 12. The isolated polypeptide of any one of claims 8 to 11, wherein the isolated polypeptide comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB, or AB-LP2-CM-LP1-MM.

13. 13. The isolated polypeptide of claim 12, wherein the two connecting peptides are not identical to each other.

14. 14. The isolated polypeptide of claim 12 or 13, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

15. 15. The isolated polypeptide of any one of claims 10 to 14, wherein the amino acid sequence of the MM is different from that of the target and is less than 50% identical to the amino acid sequence of the natural binding partner of the AB.

16. 16. The isolated polypeptide of any one of claims 10 to 15, wherein the MM does not interfere with or compete with the AB for target binding in the cleaved state.

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