Matrix metalloprotease-cleavable and serine protease-cleavable substrates and methods of use thereof

Abstract

To identify new substrates for proteases and to use these substrates for a variety of therapeutic, diagnostic and prophylactic applications.SOLUTION: The invention relates generally to: polypeptides that include at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP); activatable antibodies and other large molecules that include these polypeptides that include at least one CM1 that is a substrate for at least one MMP protease and at least one CM2 that is a substrate for at least one SP protease; and methods of making these polypeptides that include at least one CM1 that is a substrate for at least one MMP protease and at least one CM2 that is a substrate for at least one SP protease, and methods of using them in a variety of therapeutic, diagnostic and prophylactic applications.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 105,490, filed January 20, 2015, U.S. Provisional Patent Application No. 62 / 258,015, filed November 20, 2015, and U.S. Provisional Patent Application No. 62 / 278,713, filed January 14, 2016, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Incorporation of sequence listings The contents of the text file named "CYTM037001WO_ST25" (created on January 15, 2016, and 456 KB in size) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION The present invention broadly relates to polypeptides comprising at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP), activatable antibodies and other large molecules comprising these polypeptides that comprise at least CM1 that is a substrate for at least one MMP protease and CM2 that is a substrate for at least one SP protease, and methods for making and using such polypeptides in various therapeutic, diagnostic, and prophylactic applications. [Background technology]

[0004] Proteases are enzymes that degrade 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 degradation 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.

[0005] Thus, there is a need to identify novel substrates for proteases and to use those substrates for a variety of therapeutic, diagnostic and prophylactic applications. Summary of the Invention

[0006] The present disclosure provides amino acid sequences comprising at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP). These amino acid sequences are collectively referred to herein as "CM1-CM2 substrates." This term is not intended to convey any requirement regarding the orientation or other structural arrangement of the first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and the at least second cleavable portion (CM2) that is a substrate for at least one serine protease (SP). Thus, the term "CM1-CM2 substrate" encompasses substrates CM1-CM2 having the following structural arrangement from N-terminus to C-terminus: CM1-CM2 or CM2-CM1. The term "CM1-CM2 substrate" also encompasses substrates in which at least a portion of the CM1 sequence overlaps with at least a portion of the CM2 sequence.

[0007] The CM1-CM2 substrates described herein are useful in a variety of therapeutic, diagnostic, and prophylactic applications, for example, for activatable antibodies, including antibodies or antigen-binding fragments thereof (AB), which contain at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the AB, such that coupling of the MM reduces the ability of the AB to bind to its target.

[0008] In some embodiments, an activatable antibody comprises at least a first CM (CM1) and a second CM (CM2). In some embodiments, at least a portion of the CM1 substrate sequence overlaps with at least a portion of the CM2 sequence. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least one amino acid residue in common. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least two amino acid residues in common. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least three amino acid residues in common. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share three or more amino acid residues in common.

[0009] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together.

[0010] In some embodiments, CM1 and CM2 are separate polypeptides that are directly linked, i.e., the N-terminus of one substrate polypeptide is directly linked to the C-terminus of the other substrate polypeptide. In some embodiments, the N-terminus of CM1 is directly linked to the C-terminus of CM2. In some embodiments, the N-terminus of CM2 is directly linked to the C-terminus of CM1.

[0011] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together via at least one linking moiety.

[0012] In some embodiments, the first cleavable moiety CM1 and the second cleavable moiety CM2 of the activatable antibody in the 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.

[0013] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between the masking moiety (MM) and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1 and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP) between CM1 and CM2, and a connecting peptide (LP'") between CM2 and AB.

[0014] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP"') between CM2 and the masking moiety (MM). In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1, a connecting peptide (LP") between CM1 and CM2, and a connecting peptide (LP'") between CM2 and MM.

[0015] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0016] In some embodiments, CM1 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.

[0017] In some embodiments, CM1 is a substrate for MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and / or MMP19. In some embodiments, CM1 is a substrate for MMP2. In some embodiments, CM1 is a substrate for MMP9. In some embodiments, CM1 is a substrate for MMP14. In some embodiments, CM1 is a substrate for two or more MMPs. In some embodiments, CM1 is a substrate for at least MMP9 and MMP14. In some embodiments, CM1 is a substrate for at least MMP2 and MMP9. In some embodiments, CM1 is a substrate for at least MMP2 and MMP14. In some embodiments, CM1 is a substrate for three or more MMPs. In some embodiments, CM1 is a substrate for at least MMP2, MMP9, and MMP14. In some embodiments, CM1 comprises two or more substrates for the same MMP. In some embodiments, CM1 comprises at least two or more MMP2 substrates. In some embodiments, CM1 comprises at least two or more MMP9 substrates. In some embodiments, CM1 comprises at least two or more MMP14 substrates.

[0018] In some embodiments, CM1 is a substrate for MMPs and is a sequence encoding at least the sequences ISSGLLSS (SEQ ID NO: 20); QNQALRMA (SEQ ID NO: 21); AQNLLGMV (SEQ ID NO: 351); STFPFGMF (SEQ ID NO: 352); PVGYTSSL (SEQ ID NO: 353); DWLYWPGI (SEQ ID NO: 354); MIAPVAYR (SEQ ID NO: 355); RPSPMWAY (SEQ ID NO: 356); WATPRPMR (SEQ ID NO: 357); FRLLDWQW (SEQ ID NO: 358); LKAAPRWA (SEQ ID NO: 359); GPSHLVLT (SEQ ID NO: 360); No. 360); LPGGLSPW (SEQ ID NO: 361); MGLFSEAG (SEQ ID NO: 362); SPLPLRVP (SEQ ID NO: 363); RMHLRSLG (SEQ ID NO: 364); LAAPLGLL (SEQ ID NO: 365); AVGLLAPP (SEQ ID NO: 366); LLAPSHRA (SEQ ID NO: 367); PAGLWLDP (SEQ ID NO: 368); ISSGLSS (SEQ ID NO: 369); ISSGL (SEQ ID NO: 480); ISSGLLS (SEQ ID NO: 481); ISSGLL (SEQ ID NO: 482); and / or VHMPLGFLGP (SEQ ID NO: 411).

[0019] In some embodiments, CM1 comprises the amino acid sequence ISSGLLSS (SEQ ID NO:20). In some embodiments, CM1 comprises the amino acid sequence QNQALRMA (SEQ ID NO:21). In some embodiments, CM1 comprises the amino acid sequence AQNLLGMV (SEQ ID NO:351). In some embodiments, CM1 comprises the amino acid sequence STFPFGMF (SEQ ID NO:352). In some embodiments, CM1 comprises the amino acid sequence PVGYTSSL (SEQ ID NO:353). In some embodiments, CM1 comprises the amino acid sequence DWLYWPGI (SEQ ID NO:354). In some embodiments, CM1 comprises the amino acid sequence MIAPVAYR (SEQ ID NO:355). In some embodiments, CM1 comprises the amino acid sequence RPSPMWAY (SEQ ID NO:356). In some embodiments, CM1 comprises the amino acid sequence WATPRPMR (SEQ ID NO:357). In some embodiments, CM1 comprises the amino acid sequence FRLLDWQW (SEQ ID NO:358). In some embodiments, CM1 comprises the amino acid sequence LKAAPRWA (SEQ ID NO:359). In some embodiments, CM1 comprises the amino acid sequence GPSHLLVLT (SEQ ID NO: 360). In some embodiments, CM1 comprises the amino acid sequence LPGGLSPW (SEQ ID NO: 361). In some embodiments, CM1 comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 362). In some embodiments, CM1 comprises the amino acid sequence SPLPLRVP (SEQ ID NO: 363). In some embodiments, CM1 comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 364). In some embodiments, CM1 comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 365). In some embodiments, CM1 comprises the amino acid sequence AVGLLAPP (SEQ ID NO: 366). In some embodiments, CM1 comprises the amino acid sequence LLAPSHRA (SEQ ID NO: 367). In some embodiments, CM1 comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 368). In some embodiments, CM1 comprises the amino acid sequence ISSGLSS (SEQ ID NO: 369).In some embodiments, CM1 comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 411). In some embodiments, CM1 comprises the amino acid sequence ISSGL (SEQ ID NO: 480). In some embodiments, CM1 comprises the amino acid sequence ISSGLLS (SEQ ID NO: 481). In some embodiments, CM1 comprises the amino acid sequence ISSGLL (SEQ ID NO: 482).

[0020] In some embodiments, CM2 is a substrate for at least one serine protease (SP), in some embodiments, the SP is selected from u-type plasminogen activator (uPA, also referred to as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1), and combinations thereof. Other examples of SPs that cleave CM2 described herein include, by way of non-limiting example, activated protein C; cathepsin A; cathepsin G; chymase; coagulation factor proteases, such as FVIIa, FIXa, FXa, FXIa, FXIIa, etc.; elastase; granzyme B; guanidinobenzoatase; HtrA1; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, TMPRSS2, TMPRSS3, and / or TMPRSS4.

[0021] For example, a suitable CM2 is cleaved by at least one serine protease and has the sequences TGRGPSWV (SEQ ID NO: 370); SARGPSRW (SEQ ID NO: 371); TARGPSFK (SEQ ID NO: 372); LSGRSDNH (SEQ ID NO: 18); GGWHTGRN (SEQ ID NO: 373); HTGRSGAL (SEQ ID NO: 374); PLTGRSGG (SEQ ID NO: 375); AARGPAIH (SEQ ID NO: 376); RGPAFNPM (SEQ ID NO: 377); SSRGPAYL (SEQ ID NO: 378); RGPATPIM (SEQ ID NO: 379); RGPA (SEQ ID NO: 380); LSGRSGN H (SEQ ID NO: 412); TSTSGRSAMPRG (SEQ ID NO: 413); TSGRSAMP (SEQ ID NO: 414); SGRSANPRG (SEQ ID NO: 468); VAGRSMRP (SEQ ID NO: 415); LSGRSDDH (SEQ ID NO: 547); LSGRSDIH (SEQ ID NO: 548); LSGRSDQH (SEQ ID NO: 549); LSGRSDTH (SEQ ID NO: 550); LSGRSDYH (SEQ ID NO: 551); LSGRSDNP (SEQ ID NO: 552); LSGRSANP (SEQ ID NO: 553); LSGRSANI (SEQ ID NO: 554); and / or LSGRSDNI (SEQ ID NO: 71).

[0022] In some embodiments, CM2 comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 370). In some embodiments, CM2 comprises the amino acid sequence SARGPSRW (SEQ ID NO: 371). In some embodiments, CM2 comprises the amino acid sequence TARGPSFK (SEQ ID NO: 372). In some embodiments, CM2 comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 18). In some embodiments, CM2 comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 373). In some embodiments, CM2 comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 374). In some embodiments, CM2 comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 375). In some embodiments, CM2 comprises the amino acid sequence AARGPAIH (SEQ ID NO: 376). In some embodiments, CM2 comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 377). In some embodiments, CM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 378). In some embodiments, CM2 comprises the amino acid sequence RGPATPIM (SEQ ID NO: 379). In some embodiments, CM2 comprises the amino acid sequence RGPA (SEQ ID NO: 380). In some embodiments, CM2 comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 412). In some embodiments, CM2 comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 413). In some embodiments, CM2 comprises the amino acid sequence SGRSANPRG (SEQ ID NO: 468). In some embodiments, CM2 comprises the amino acid sequence TSGRSANP (SEQ ID NO: 414). In some embodiments, CM2 comprises the amino acid sequence VAGRSMRP (SEQ ID NO: 415). In some embodiments, CM2 comprises the amino acid sequence LSGRSDDH (SEQ ID NO: 547). In some embodiments, CM2 comprises the amino acid sequence LSGRSDIH (SEQ ID NO: 548). In some embodiments, CM2 comprises the amino acid sequence LSGRSDQH (SEQ ID NO: 549). In some embodiments, CM2 comprises the amino acid sequence LSGRSDTH (SEQ ID NO: 550).In some embodiments, CM2 comprises the amino acid sequence LSGRSDYH (SEQ ID NO:551). In some embodiments, CM2 comprises the amino acid sequence LSGRSDNP (SEQ ID NO:552). In some embodiments, CM2 comprises the amino acid sequence LSGRSANP (SEQ ID NO:553). In some embodiments, CM2 comprises the amino acid sequence LSGRSANI (SEQ ID NO:554). In some embodiments, CM2 comprises the amino acid sequence LSGRSDNI (SEQ ID NO:71).

[0023] In some embodiments, the CM1-CM2 substrate has the sequences ISSGLLSGRSDNH (SEQ ID NO: 1); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 2); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 3); TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 4); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 5); TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 6); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 7); LSGRSDNHGGAVGLLAP P (SEQ ID NO: 8); VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 9); LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 10); LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 11); LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 12); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 13); LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 14); QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 15); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 16); No. 16); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 17); ISSGLLSGRSGNH (SEQ ID NO: 22); ISSGLLSGRSANPRG (SEQ ID NO: 469); AVGLLAPPTSGRSANPRG (SEQ ID NO: 470); AVGLLAPPSGRSANPRG (SEQ ID NO: 471); ISSGLLSGRSDDH (SEQ ID NO: 483); ISSGLLSGRSDIH (SEQ ID NO: 484); ISSGLLSGRSDQH (SEQ ID NO: 485); ISSGLLSGRSDTH (SEQ ID NO: 486); ISSGLLSGRSDYH ( SEQ ID NO:487);ISSGLLSGRSDNP (SEQ ID NO:488);ISSGLLSGRSANP (SEQ ID NO:489);ISSGLLSGRSANI (SEQ ID NO:490);AVGLLAPPGGLSGRSDDH (SEQ ID NO:515);AVGLLAPPGGLSGRSDIH (SEQ ID NO:516);AVGLLAPPGGLSGRSDQH (SEQ ID NO:517);AVGLLAPPGGLSGRSDTH (SEQ ID NO:518);AVGLLAPPGGLSGRSDYH (SEQ ID NO:519);AVGLLAPPGGLSGRSDNP (SEQ ID NO:520);AVGLLAPPGGLSGRSANP (SEQ ID NO: 521); AVGLLAPPGGLSGRSANI (SEQ ID NO: 522); ISSGLLSGRSDNI (SEQ ID NO: 555); and / or AVGLLAPPGGLSGRSDNI (SEQ ID NO: 557).

[0024] In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDNH (SEQ ID NO: 1). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 2). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 3). In some embodiments, the CM1-CM2 substrate comprises the sequence TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 4). In some embodiments, the CM1-CM2 substrate comprises the sequence VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 5). In some embodiments, the CM1-CM2 substrate comprises the sequence TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 6). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 7). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 8). In some embodiments, the CM1-CM2 substrate comprises the sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 9). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 10). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 11). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 12). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 13). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 14). In some embodiments, the CM1-CM2 substrate comprises the sequence QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 15). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 16).In some embodiments, the CM1-CM2 substrate comprises the sequence QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 17). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSGNH (SEQ ID NO: 22). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSANPRG (SEQ ID NO: 469). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPTSGRSANPRG (SEQ ID NO: 470). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPSGRSANPRG (SEQ ID NO: 471). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDDH (SEQ ID NO: 483). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLLSGRSDIH (SEQ ID NO: 484). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDQH (SEQ ID NO: 485). In some embodiments, the CM1-CM2 substrate comprises the sequence In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDTH (SEQ ID NO: 486). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDYH (SEQ ID NO: 487). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDNP (SEQ ID NO: 488). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSANP (SEQ ID NO: 489). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSANI (SEQ ID NO: 490). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDDH (SEQ ID NO: 515). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDIH (SEQ ID NO: 516). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDQH (SEQ ID NO: 517). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDTH (SEQ ID NO: 518).In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDYH (SEQ ID NO: 519). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDNP (SEQ ID NO: 520). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSANP (SEQ ID NO: 521). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSANI (SEQ ID NO: 522). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDNI (SEQ ID NO: 555). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 557).

[0025] In some embodiments, the activatable antibody in its uncleaved state has 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.

[0026] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the antibody in its uncleaved state has the following structural arrangement from N- to C-terminus: MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM. In some embodiments, each of 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.

[0027] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between the masking moiety (MM) and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1 and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP) between CM1 and CM2, and a connecting peptide (LP'") between CM2 and AB.

[0028] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP'") between CM2 and the masking moiety (MM). In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1, a connecting peptide (LP") between CM1 and CM2, and a connecting peptide (LP'") between CM2 and MM.

[0029] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0030] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 381) and (GGGS)n (SEQ ID NO: 382), where n is at least one integer.

[0031] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 383), GGSGG (SEQ ID NO: 384), GSGSG (SEQ ID NO: 385), GSGGG (SEQ ID NO: 386), GGGSG (SEQ ID NO: 387), or GSSSG (SEQ ID NO: 388).

[0032] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 389), GSSGGSGGSGG (SEQ ID NO: 390), GSSGGSGGSGGS (SEQ ID NO: 391), GSSGGSGGSGGSGGGS (SEQ ID NO: 392), GSSGGSGGSG (SEQ ID NO: 393), or GSSGGSGGSGS (SEQ ID NO: 394).

[0033] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 395), GSSGT (SEQ ID NO: 396), or GSSG (SEQ ID NO: 397).

[0034] In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to the target.

[0035] In some embodiments, the activatable antibody comprises an antibody or antigen-binding fragment (AB) thereof that specifically binds to a target. In some embodiments, the AB is a full-length antibody. In some embodiments, the AB is an immunologically active fragment. In some embodiments, the AB is an antigen-binding fragment. In some embodiments, the AB is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, or a single-domain light chain antibody. In some embodiments, the AB is a murine, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0036] In some embodiments, the MMP protease co-localizes with the target in a tissue, and the MMP protease cleaves the CM1 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the SP protease co-localizes with the target in a tissue, and the SP protease cleaves the CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and / or the SP protease co-localizes with the target in a tissue, and the MMP protease and / or the SP protease cleaves the CM1-CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and the SP protease co-localize with the target in a tissue, and at least one of the MMP protease and the SP protease cleaves the CM1-CM2 substrate in the antibody when the antibody is exposed to the protease.

[0037] In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide up to 15 amino acids in length.

[0038] In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate 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. In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate 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.

[0039] In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and comprises a polypeptide sequence that is not substantially identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and comprises a polypeptide sequence that is not substantially identical to any mammalian polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and comprises a polypeptide sequence that is not substantially identical to any human polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and comprises a polypeptide sequence that is at most 90% identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and comprises a polypeptide sequence that is at most 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP 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 SP protease.

[0040] In some embodiments, the CM1-CM2 substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557. In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, an antibody or antigen-binding fragment thereof (AB) that binds to a target, and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to the target.

[0041] In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an anti-Jagged antibody comprising the amino acid sequence of an anti-Jagged antibody disclosed herein. In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 164 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 163.

[0042] In some embodiments, CM1-CM2 are comprised in an activatable antibody having a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 420, 422, 424, 426, 428, 430, 432, 434, 436, 439, 477, 479, 507-514, 539-546, 561, and 562, and a heavy chain amino acid sequence of SEQ ID NO: 67.

[0043] In some embodiments, CM1-CM2 are comprised in an activatable antibody having a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 420, 422, 424, 426, 428, 430, 432, 434, 436, 439, 477, 479, 507-514, 539-546, 561, and 562, and a heavy chain amino acid sequence of SEQ ID NO: 163.

[0044] In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an anti-EGFR antibody comprising the amino acid sequence of an anti-EGFR antibody disclosed herein. In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 111 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110.

[0045] In some embodiments, CM1-CM2 are comprised in an activatable antibody having a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 472, 474, 499-506, 531-538, 559, and 560, and a heavy chain amino acid sequence of SEQ ID NO: 108.

[0046] In some embodiments, CM1-CM2 are comprised in an activatable antibody having a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 472, 474, 499-506, 531-538, 559, and 560, and a heavy chain amino acid sequence of SEQ ID NO: 109.

[0047] In some embodiments, CM1-CM2 are comprised in an activatable antibody having a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 472, 474, 499-506, 531-538, 559, and 560, and a heavy chain amino acid sequence of SEQ ID NO: 110.

[0048] In some embodiments, the CM1-CM2 substrate is also a substrate for at least one additional protease.

[0049] In some embodiments, the at least one additional protease is an MMP protease that is different from the MMP protease that cleaves CM1. In some embodiments, the at least one additional protease is an MMP protease selected from the group consisting of MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.

[0050] In some embodiments, the at least one additional protease is a different SP protease from the SP protease that cleaves CM2. In some embodiments, the at least one additional SP protease is selected from the group consisting of uPA, matriptase, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor proteases such as FVIIa, FIXa, FXa, FXIa, and FXIIa, elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, and type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, TMPRSS2, TMPRSS3, and TMPRSS4.

[0051] In some embodiments, the at least one additional protease is selected from the group consisting of those shown in Table 6. [Table 1]

[0052] The present disclosure also provides an antibody comprising at least a first CM1 and a second CM2 and conjugated to an agent. In some embodiments, the first CM1 and the second CM2 are each polypeptides of 15 amino acids or less in length. In some embodiments, the activatable antibody is a conjugated activatable antibody and, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB-agent, agent-AB-CM2-CM1-MM, MM-CM2-CM1-AB-agent, or agent-AB-CM1-CM2-MM. In some embodiments, the activatable antibody is a conjugated activatable antibody and, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: agent-MM-CM1-CM2-AB, AB-CM2-CM1-MM-agent, agent-MM-CM2-CM1-AB, or AB-CM1-CM2-MM-agent. In some embodiments, the activatable antibody is a conjugated activatable antibody and, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: agent-MM-CM1-CM2-AB-agent, agent-AB-CM2-CM1-MM-agent, agent-MM-CM2-CM1-AB-agent, or agent-AB-CM1-CM2-MM-agent.

[0053] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first connecting peptide (LP1), and a second connecting peptide (LP2), and the antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-CM2-LP2-AB-agent, agent-AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB-agent, or agent-AB-LP2-CM1-CM2-LP1-MM. In some embodiments, each of 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.

[0054] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first connecting peptide (LP1), and a second connecting peptide (LP2), and the antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: agent-MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM-agent, agent-MM1-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM-agent. In some embodiments, each of 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.

[0055] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first connecting peptide (LP1), and a second connecting peptide (LP2), and the antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: agent-MM1-LP1-CM1-CM2-LP2-AB-agent, agent-AB-LP2-CM2-CM1-LP1-MM-agent, agent-MM1-LP1-CM2-CM1-LP2-AB-agent, or agent-AB-LP2-CM1-CM2-LP1-MM-agent. In some embodiments, each of 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.

[0056] In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between the masking moiety (MM) and CM1. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP'") between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between MM and CM1 and ...) between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP') between CM2 and AB, and a connecting peptide (LP'") between CM2 and AB.

[0057] In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between AB and CM1. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP"') between CM2 and the masking moiety (MM). In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP") between AB and CM1, a connecting peptide (LP'), and a connecting peptide (LP'") between CM2 and MM.

[0058] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0059] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 381) and (GGGS)n (SEQ ID NO: 382), where n is at least one integer.

[0060] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 383), GGSGG (SEQ ID NO: 384), GSGSG (SEQ ID NO: 385), GSGGG (SEQ ID NO: 386), GGGSG (SEQ ID NO: 387), or GSSSG (SEQ ID NO: 388).

[0061] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 389), GSSGGSGGSGG (SEQ ID NO: 390), GSSGGSGGSGGS (SEQ ID NO: 391), GSSGGSGGSGGSGGGS (SEQ ID NO: 392), GSSGGSGGSG (SEQ ID NO: 393), or GSSGGSGGSGS (SEQ ID NO: 394).

[0062] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 395), GSSGT (SEQ ID NO: 396), or GSSG (SEQ ID NO: 397).

[0063] In some embodiments, the CM1-CM2 substrate is linked or otherwise attached to an antibody. For example, CM1-CM2 is used to link one or more agents to an antibody or antigen-binding fragment thereof (AB) that binds to a predetermined target, such that upon exposure to MMPs and / or SPs, CM1-CM2 is cleaved and the agent is released from the AB. Exemplary targets include, but are not limited to, those listed in Table 1. Exemplary ABs include, but are not limited to, those listed in Table 2.

[0064] In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to the target.

[0065] In some embodiments, antibodies include antibodies or antigen-binding fragments thereof that specifically bind to a target. In some embodiments, the target-binding antibody or immunologically active fragment thereof is a monoclonal antibody, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single-domain heavy chain antibody, or single-domain light chain antibody. In some embodiments, such target-binding antibodies or immunologically active fragments thereof are murine, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0066] In some embodiments, the MM has a dissociation constant for binding to the AB that is less than or equal to the dissociation constant of the AB for the target.

[0067] In some embodiments, the MM does not interfere with or compete with the AB for binding to the target in the cleaved state.

[0068] In some embodiments, the MM is a polypeptide between about 2 and 40 amino acids in length, for example, the MM is a polypeptide up to about 40 amino acids in length.

[0069] In some embodiments, the MM polypeptide sequence differs from the sequence of any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is 50% or less identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of AB.

[0070] In some embodiments, the agent conjugated to the AB or to the AB of the activatable antibody 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 the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to the 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 a 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 monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin 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. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0071] In some embodiments, the agent is an anti-inflammatory agent.

[0072] In some embodiments, the antibody and / or activatable antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0073] In some embodiments, the conjugated antibody and / or the conjugated activatable antibody comprises a detectable label. In 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. In some embodiments, the imaging agent comprises a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the imaging agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In 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. In some embodiments, the luminescent label comprises an N-methylacridium derivative. In some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0074] In some embodiments, the antibody and / or AB of the activatable antibody naturally contain one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0075] In some embodiments, the antibody and / or conjugated antibody is monospecific. In some embodiments, the antibody and / or conjugated antibody is multispecific, also referred to herein as a multispecific antibody and / or conjugated multispecific antibody. In some embodiments, the multispecific antibody and / or conjugated multispecific antibody is bispecific or trispecific. In some embodiments, the antibody and / or conjugated antibody is formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. In some embodiments, the antibody and / or conjugated antibody is formulated as part of a pro-chimeric antigen receptor (pro-CAR)-modified T cell or other engineered receptor or other immune effector cell, such as a CAR-modified NK cell. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (CAR)-modified T cell. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (CAR)-modified NK cell.

[0076] In some embodiments, the activatable antibody and / or conjugated activatable antibody is monospecific. In 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 conjugated multispecific activatable antibody. As used herein, the term "activatable antibody" and all grammatical variations thereof, unless otherwise specified, are intended to encompass, but are 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, are intended to encompass, but are not limited to, embodiments in which the conjugated activatable antibody is a conjugated multispecific activatable antibody of the present disclosure. In some embodiments, the multispecific activatable antibody and / or conjugated multispecific activatable antibody is bispecific or trispecific. In some embodiments, the activatable antibody and / or conjugated activatable antibody are formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. In some embodiments, the activatable antibody and / or conjugated activatable antibody are formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor.

[0077] In some embodiments, the antibodies, antibody conjugates, 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.

[0078] In 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. In some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a CM1-CM2 substrate that functions as a substrate for at least one MMP protease and at least one SP 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.

[0079] In 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: 398); a VH CDR2 sequence comprising at least the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 399); a VH CDR3 sequence comprising the amino acid sequence DIGGRSAFDY (SEQ ID NO: 400); and combinations thereof.

[0080] In 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: 401); a VH CDR2 sequence comprising at least the amino acid sequence AASSLQS (SEQ ID NO: 402); a VL CDR3 sequence comprising the amino acid sequence QQTVVAPPL (SEQ ID NO: 403); and combinations thereof.

[0081] In 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 CDR 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: 398). a CDR1 sequence; a VH CDR2 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 SIDPEGRQTYYADSVKG (SEQ ID NO: 399); 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: 400); and combinations thereof.

[0082] In 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 CDR 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: 401). a CDR1 sequence; a VH CDR2 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 AASSLQS (SEQ ID NO: 402); 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: 403); and combinations thereof.

[0083] In 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: 398); the VH CDR2 sequence at least comprises the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 399); the VH CDR3 sequence comprises the amino acid sequence DIGGRSAFDY (SEQ ID NO: 400); the VL CDR1 sequence at least comprises the amino acid sequence RASQSISSY (SEQ ID NO: 401); The CDR2 sequence comprises at least the amino acid sequence AASSLQS (SEQ ID NO: 402); and the VL CDR3 sequence comprises the amino acid sequence QQTVVAPPL (SEQ ID NO: 403).

[0084] In 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: 398); The CD2 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 399); 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 DIGGRSAFDY (SEQ ID NO: 400); 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 RASQSISSY (SEQ ID NO: 401); The 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: 402); 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: 403).

[0085] In 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: 404); a VH CDR2 sequence comprising at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 405); a VH CDR3 sequence comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 406); and combinations thereof.

[0086] In 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: 407); a VH CDR2 sequence comprising at least the amino acid sequence KYASESIS (SEQ ID NO: 408); a VH CDR3 sequence comprising the amino acid sequence QQNNNWPTT (SEQ ID NO: 409); and combinations thereof.

[0087] In 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 CDR 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: 404). a CDR1 sequence; a VH CDR2 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 VIWSGGNTDYNTPFTS (SEQ ID NO: 405); 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: 406); and combinations thereof.

[0088] In 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 sequence that 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 RASQSIGTNIH (SEQ ID NO: 407). a CDR1 sequence; a VH CDR2 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 KYASESIS (SEQ ID NO: 408); 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: 409); and combinations thereof.

[0089] In 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: 404); the VH CDR2 sequence at least comprises the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 405); the VH CDR3 sequence comprises the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 406); the VL CDR1 sequence at least comprises the amino acid sequence RASQSIGTNIH (SEQ ID NO: 407); The CDR2 sequence comprises at least the amino acid sequence KYASESIS (SEQ ID NO: 408); and the VL CDR3 sequence comprises the amino acid sequence QQNNNWPTT (SEQ ID NO: 409).

[0090] In 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: 404); The CD2 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 405); 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: 406); 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: 407); The 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 KYASESIS (SEQ ID NO: 408); 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: 409).

[0091] In 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 a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an anti-Jagged antibody comprising the amino acid sequence of an anti-Jagged antibody disclosed herein. In 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 a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 162 or SEQ ID NO: 164 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 163.

[0092] In 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 the heavy chain amino acid sequence of SEQ ID NO: 67 and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 420, 422, 424, 426, 428, 430, 432, 434, 436, 439, 477, 479, 507-514, 539-546, 561, and 562.

[0093] In 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 a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an anti-EGFR antibody comprising the amino acid sequence of an anti-EGFR antibody disclosed herein. In 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 a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and an antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 111 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110.

[0094] In 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 the heavy chain amino acid sequence of SEQ ID NO: 108 and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 472, 474, 499-506, 531-538, 559, and 560.

[0095] In 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 the amino acid sequence of SEQ ID NO: 67, and 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: 420, 422, 424, 426, 428, 430, 432, 434, 436, 439, 477, 479, 507-514, 539-546, 561, and 562.

[0096] In 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 the amino acid sequence of SEQ ID NO: 108, and 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: 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 472, 474, 499-506, 531-538, 559, and 560.

[0097] 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 CM1-CM2 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 monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin 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. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0098] In some embodiments, the agent is an anti-inflammatory agent.

[0099] In some embodiments, the activatable antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0100] In some embodiments, the conjugated antibody comprises a detectable label. In 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. In some embodiments, the imaging agent comprises a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the imaging agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In 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. In some embodiments, the luminescent label comprises an N-methylacridium derivative. In some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0101] In some embodiments, the activatable antibody also comprises a signal peptide. In some embodiments, the signal peptide is attached to the activatable antibody via a spacer. In some embodiments, the spacer is attached to the activatable antibody in the absence of an immediately following peptide. In some embodiments, the spacer is linked directly to the MM of the activatable antibody. In some embodiments, the spacer is linked directly to the MM of the activatable antibody in the N- to C-terminal structural arrangement of spacer-MM-CM1-CM2substrate-AB. Examples of spacers linked directly to the N-terminus of the MM of an activatable antibody are amino acid sequences selected from the group consisting of QGQSGQ (SEQ ID NO: 410), GQSGQ (SEQ ID NO: 416), QSGQ (SEQ ID NO: 417), SGQ (SEQ ID NO: 418), GQ, and Q. In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 410). In some embodiments, the spacer comprises at least the amino acid sequence GQSGQ (SEQ ID NO: 416). In some embodiments, the spacer comprises at least the amino acid sequence QSGQ (SEQ ID NO: 417). In some embodiments, the spacer includes at least the amino acid sequence SGQ (SEQ ID NO: 418). In some embodiments, the spacer includes at least the amino acid sequence GQ. In some embodiments, the spacer includes at least the amino acid sequence Q.

[0102] 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.

[0103] In 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. In some embodiments, the serum half-life of an activatable antibody is similar to the half-life of its corresponding antibody. In some embodiments, the serum half-life of an activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism.In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism.

[0104] The present disclosure also provides compositions and methods comprising activatable antibodies, including antibodies or antibody fragments (AB) that specifically bind a predetermined target, wherein the AB is coupled to a masking moiety (MM) that reduces the ability of the AB to bind its target. In some embodiments, the activatable antibody further comprises a CM1-CM2 substrate that is a substrate for at least one MMP and at least one SP. 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. In 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 that are conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic, and / or prophylactic agents; for example, in some embodiments, none of the agents are conjugated to any of the MMs of the activatable antibody. The compositions and methods provided herein generate conjugated activatable antibodies that retain the ability to effectively and efficiently mask the AB of the activatable antibody when the MMP is not cleaved. The compositions and methods provided herein generate conjugated activatable antibodies that are still activated, i.e., cleaved, in the presence of an MMP that is capable of cleaving the CM1-CM2 substrate.

[0105] Although an activatable antibody has at least one attachment point for an agent, in the methods and compositions provided herein, all available attachment points are available for attachment to an agent. In some embodiments, one or more attachment points are sulfur atoms involved in disulfide bonds. In some embodiments, one or more attachment points are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more attachment points are sulfur atoms involved in interchain sulfide bonds, but not intrachain disulfide bonds. In 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.

[0106] 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 attaching 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 available binding sites, are reduced. In 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 available binding sites are reduced.

[0107] In some embodiments, a method is provided for reducing an agent, e.g., a drug, and binding it to an activatable antibody, resulting in selection under the displacement of the agent. The method involves reducing the activatable antibody with a reducing agent, thereby not reducing any binding sites on either the masking portion or other non-AB portions of the activatable antibody, and then binding the agent to interchain thiols on one or more ABs of the activatable antibody. The binding site(s) are selected to allow for the desired displacement of the agent, allowing binding 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 bound activatable antibody in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody under uncleaved conditions. The ratio of reducing agent to activatable antibody will vary depending on the activatable antibody. In 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, about 1.5:1-1:1.5 or about In some embodiments, the ratio will be in the range of 1:1 to 1:1.5. In some embodiments, the ratio will be in the range of about 5:1 to 1.5:1. In some embodiments, the ratio will be in the range of about 4:1 to 1 ....4:1. In some embodiments, the ratio will be in the range of about 8:1 to about 1:1. In some embodiments, the ratio will be in the range of about 2.5:1 to 1:1.

[0108] In some embodiments, methods are provided for reducing interchain disulfide bonds in the ABs of an activatable antibody and attaching an agent, e.g., a thiol-containing agent, e.g., a drug, to the resulting interchain thiols to selectively locate the agent(s) 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 attaching 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. In 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, about 1.5:1-1:1.5 or about In some embodiments, the ratio will be in the range of 1:1 to 1:1.5. In some embodiments, the ratio will be in the range of about 5:1 to 1.5:1. In some embodiments, the ratio will be in the range of about 4:1 to 1 ....4:1. In some embodiments, the ratio will be in the range of about 8:1 to about 1:1. In some embodiments, the ratio will be in the range of about 2.5:1 to 1:1.

[0109] The thiol-containing reagent can be, for example, cysteine ​​or N-acetylcysteine. The reducing agent can be, for example, TCEP. In 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.

[0110] 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 with 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 CM1-CM2 substrate (a polypeptide that functions as a substrate for at least one MMP and one SP) coupled to the AB. In some embodiments, the MM is coupled to the AB via the CM1-CM2 substrate. In some embodiments, one or more intrachain disulfide bond(s) of the activatable antibody are not disrupted by the reducing agent. In some embodiments, one or more intrachain disulfide bond(s) of the 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-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0111] 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 interfering with or otherwise impairing the activity and / or potency 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 CM1-CM2 substrate (a polypeptide that functions as a substrate for a protease) coupled to the AB. The activity and / or potency of the activatable antibody is, by non-limiting example, masking activity, activation of the activatable antibody, and / or binding activity of the activated activatable antibody. In some embodiments, one or more intrachain disulfide bonds(s) of the activatable antibody are not disrupted by a reducing agent. In some embodiments, one or more intrachain disulfide bonds(s) 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-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0112] 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 CM1-CM2 substrate coupled to the AB, the CM1-CM2 substrate being a polypeptide that functions as a substrate for at least one MMP protease and at least one SP protease.

[0113] In some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any of several methods for attaching an agent to the AB, such as: (a) attachment to a 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 carboxylate group of the AB.

[0114] In some embodiments, the MMAD payload is attached to AB via a linker. In some embodiments, the MMAD payload is attached to a cysteine ​​via a linker. In some embodiments, the MMAD payload is attached to a lysine in AB via a linker. In some embodiments, the MMAD is attached to another residue of 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. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, hi some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation techniques disclosed herein.

[0115] The present disclosure also provides polypeptides and other large molecules comprising one or more CM1-CM2 substrate sequences provided herein. By way of non-limiting example, the CM1-CM2 substrate sequences provided herein are useful in prodrug compositions and methods of use thereof. These CM1-CM2 substrate sequences provided herein are also useful in probes and other detection agents and methods of use thereof. For example, the CM1-CM2 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 CM1-CM2 substrate sequences provided herein are useful in any composition and / or method in the art that employs a substrate cleavable by at least one MMP and at least one SP.

[0116] 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.

[0117] The present disclosure provides methods 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 CM1-CM2 substrate, and (ii) the CM1-CM2 substrate is a polypeptide that functions as a substrate for matrix metalloproteases and serine proteases; (b) recovering the antibody; and (c) conjugating the recovered antibody to one or more additional agents.

[0118] 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 CM1-CM2 substrate, and an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (i) the CM1-CM2 substrate is a polypeptide that functions as a substrate for MMPs and SPs; and (ii) the CM1-CM2 substrate 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.

[0119] The present disclosure also provides a method for producing a conjugated activatable antibody of the present disclosure that binds a predetermined target under an activated state by: (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions conducive to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a CM1-CM2 substrate, and an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (i) the CM1-CM2 substrate is a polypeptide that functions as a substrate for MMPs and SPs; and (ii) the CM1-CM2 substrate is positioned on the activatable antibody such that, under an uncleaved state, the MM interferes with the specific binding of the AB to the target, and under a cleaved state, the MM does not interfere with or compete with the specific binding of the AB to the target; (b) recovering the activatable antibody; and (c) binding one or more additional agents to the recovered antibody.

[0120] The present disclosure provides methods of preventing, delaying the progression of, treating, alleviating symptoms of, or otherwise ameliorating a target-related disease in a subject by administering to the subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody and / or conjugated activatable antibody described herein.

[0121] The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating the symptoms of, or otherwise ameliorating inflammation and / or inflammatory diseases 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. The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating the symptoms of, or otherwise ameliorating cancer 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. The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating the symptoms of, or otherwise ameliorating autoimmune diseases 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.

[0122] 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 the 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.

[0123] 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.

[0124] The conjugated antibody, activatable antibody, and / or conjugated activatable antibody, 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.

[0125] 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.

[0126] In some embodiments, the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies described herein are used in conjunction with or in combination with one or more additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended application, such as cancer. For example, the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be used in conjunction with an additional chemotherapeutic or anti-cancer agent.

[0127] In some embodiments, the additional agent(s) is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, Abraxane (i.e., albumin-bound paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0128] In some embodiments, the additional agent(s) are checkpoint inhibitors, kinase inhibitors, agents targeting inhibitors in the tumor microenvironment, and / or T cell or NK agonists. In some embodiments, the additional agent(s) are radiation therapy alone or in combination with another additional agent(s), such as a chemotherapeutic agent or an anti-neoplastic agent. In some embodiments, the additional agent(s) are vaccines, oncoviruses, and / or CD-activating agents, such as, but not limited to, Toll-like receptor (TLR) agonists and / or α-CD40. In some embodiments, the additional agent(s) are tumor-targeting antibodies designed to kill tumors via ADCC or via direct conjugation to a toxin (e.g., antibody-drug conjugates (ADCs)).

[0129] In some embodiments, the checkpoint inhibitor is selected from the group consisting of CTLA-4, LAG-3, PD-1, PD-1, TIGIT, TIM-3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an α-CSF1R inhibitor, an α-CCR4 inhibitor, TGF-beta, myeloid-derived suppressor cells, or T-regulatory cells. In some embodiments, the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM.

[0130] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is TGF-β. In some embodiments, the inhibitor is a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T-regulatory cell.

[0131] In some embodiments, the agonist is Ox40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.

[0132] In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody is administered in combination with one or more additional agents, e.g., chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressive agents, during and / or after treatment. In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent(s) are formulated into a single therapeutic composition, and the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent(s) are administered simultaneously. Alternatively, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent(s) are separate from one another, e.g., each is formulated into a separate therapeutic composition, and the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent(s) are administered simultaneously, or the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent(s) are administered at different times during the therapeutic regimen. For example, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody may be administered prior to administration of the additional agent, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody may be administered subsequent to administration of the additional agent, or the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent may be administered in alternation. As described herein, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent may be administered in a single dose, or multiple doses.

[0133] In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and additional agent(s) are administered simultaneously. For example, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and additional agent(s) can be formulated into a single composition or administered as multiple separate compositions. In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and additional agent(s) are administered sequentially, or the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and additional agent(s) are administered at different times during a treatment regimen.

[0134] 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.

[0135] In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody. In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody directed against the same target as the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody. In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody directed against a target different from that of the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody.

[0136] In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent(s) are administered simultaneously. For example, the conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent(s) can be formulated in a single composition or administered as multiple separate compositions. In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent(s) are administered sequentially, or the antibody and / or conjugated antibody and additional agent(s) 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 following the administration of the additional agent, or the antibody and / or conjugated antibody and additional agent are administered alternately. As described herein, the antibody and / or conjugated antibody and additional agent(s) can be administered in a single dose or in multiple doses.

[0137] 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.

[0138] Pharmaceutical compositions according to the present disclosure can include an antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody of the present disclosure and a carrier. These pharmaceutical compositions can be included in a kit, such as, for example, a diagnostic kit. [Brief explanation of the drawings]

[0139] [Figure 1] Figures 1A and 1B are a series of graphs showing the results of a human Jagged1 binding ELISA assay demonstrating binding of anti-Jagged antibodies, and masked and activated activatable antibodies. Both (A) 2001 and (B) 1001 / LP' / 0001 substrate-containing activatable antibodies were activated by uPA (a serine protease), MMP14 (MMP), and uPA in combination with MMP14. The activated activatable antibodies showed binding equivalent to that of anti-Jagged antibodies.

[0140] [Figure 2] Figure 2 is a graph showing human IgG serum concentrations for anti-Jagged antibodies and 1001 / LP' / 0001 and 2001 activatable antibodies following a single 5 mg / kg intravenous dose. Anti-Jagged antibodies are rapidly cleared due to target-mediated clearance. In contrast, anti-Jagged activatable antibodies remain masked in the circulation.

[0141] [Figure 3] Figure 3 is a graph showing spotted HCC1806 tumor volume (group mean ± SEM, n=8) versus time after first dosing. Groups were dosed on days 1 and 8 of the study. Both the anti-Jagged-SPDB-DM4 antibody and anti-Jagged2001 activatable antibody-SPDB-DM4 groups showed tumor regression, whereas the isotype-SPDB-DM4 group showed no tumor growth inhibition.

[0142] [Figure 4]Figure 4 is a graph showing percent of initial body weight (group mean, n=8) spotted versus time after first dosing. Groups were dosed on days 1 and 8 of the study. Animals in the anti-Jagged-SPDB-DM4 ADC-treated group showed significant weight loss, whereas animals treated with PBS, isotype-SPDB-DM4 ADC, and anti-Jagged2001 activatable antibody-SPDB-DM4 did not show significant weight loss.

[0143] [Figure 5] FIG. 5 is a graph showing in vivo activation of EGFR-activatable antibodies as measured in plasma 8 days after administration of 12.5 mg / kg of such EGFR-activatable antibodies in H292 xenograft tumor-bearing mice.

[0144] [Figure 6] FIG. 6 is a graph showing the in vivo efficacy of EGFR activatable antibodies in H292 xenograft tumor-bearing mice.

[0145] [Figure 7] FIG. 7 is a graph showing in situ assessment of EGFR activatable antibody activation in the H292 xenograft tumor microenvironment.

[0146] [Figure 8A-E]Figures 8A, 8B, 8C, 8D, 8E, and 8F are a series of graphs showing tumor volumes of H292 xenograft tumors in nu / nu mice at various time points after administration of control intravenous immunoglobulin (IVIG, Figure 8A), the anti-EGFR antibody cetuximab (Figure 8B), an anti-EGFR activatable antibody referred to herein as anti-EGFR2001 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 108 and the light chain sequence of SEQ ID NO: 449) (Figure 8C); an anti-EGFR activatable antibody referred to herein as anti-EGFR2003 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 108 and the light chain sequence of SEQ ID NO: 472) (Figure 8D); or an anti-EGFR activatable antibody referred to herein as anti-EGFR2005 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 108 and the light chain sequence of SEQ ID NO: 474) (Figure 8E). For the data shown in Figures 8C and 8D, one animal was lost in each group due to weight loss. FIG. 8F is a graph that plots and compares the data presented in FIGS. 8A-8E in one graph. [Figure 8F] Figures 8A, 8B, 8C, 8D, 8E, and 8F are a series of graphs showing tumor volumes of H292 xenograft tumors in nu / nu mice at various time points after administration of control intravenous immunoglobulin (IVIG, Figure 8A), the anti-EGFR antibody cetuximab (Figure 8B), an anti-EGFR activatable antibody referred to herein as anti-EGFR2001 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 108 and the light chain sequence of SEQ ID NO: 449) (Figure 8C); an anti-EGFR activatable antibody referred to herein as anti-EGFR2003 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 108 and the light chain sequence of SEQ ID NO: 472) (Figure 8D); or an anti-EGFR activatable antibody referred to herein as anti-EGFR2005 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 108 and the light chain sequence of SEQ ID NO: 474) (Figure 8E). For the data shown in Figures 8C and 8D, one animal was lost in each group due to weight loss. FIG. 8F is a graph that plots and compares the data presented in FIGS. 8A-8E in one graph.

[0147] [Figure 9]FIG. 9 shows the results of a comparison of isotype control intravenous immunoglobulin (IVIG), a 20 mg / kg dose of anti-Jagged antibody (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 162), herein referred to as 4D11, a 10 mg / kg dose of 4D11 antibody, a 5 mg / kg dose of 4D11 antibody, an anti-Jagged activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 420), herein referred to as anti-Jagged2001 activatable antibody, and an anti-Jagged1004 / LP' / 0001 activatable antibody. 1 is a graph showing toxicity, as measured by loss of body weight (BW), following administration of an anti-Jagged activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 432), herein referred to as anti-Jagged2003 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 477), and an anti-Jagged activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 479), herein referred to as anti-Jagged2005 activatable antibody. Results are presented as percent (%) relative body weight (BW) change at various time points during the study.

[0148] [Figure 10]10 is a graph showing toxicity as measured by loss of body weight (BW) following administration of isotype control intravenous immunoglobulin (IVIG), a 20 mg / kg dose of anti-Jagged antibody designated herein as 4D11 (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 162), a 10 mg / kg dose of 4D11 antibody, a 5 mg / kg dose of 4D11 antibody, an anti-Jagged activatable antibody designated herein as anti-Jagged2001 activatable antibody ("2001") (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 420), an anti-Jagged activatable antibody designated herein as anti-Jagged1004 / LP' / 0001 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 432), and an anti-Jagged activatable antibody designated herein as anti-Jagged1004 / LP' / 0003 activatable antibody (comprising the heavy chain sequence of SEQ ID NO: 67 and the light chain sequence of SEQ ID NO: 424). Results are presented as percent (%) relative body weight (BW) change at various time points during the study.

[0149] [Figures 11A-H] Figures 11A, 11B, 11C, 11D, 11E, 11F, 11G, 11H, and Figure 12 are a series of graphs showing the efficacy of various substrates of the present disclosure incorporated into activatable anti-EGFR antibodies of the present disclosure. The efficacy of the substrates was assessed by measuring tumor volume (TV mm3) at various time points after administration. [Figure 12] Figures 11A, 11B, 11C, 11D, 11E, 11F, 11G, 11H, and Figure 12 are a series of graphs showing the efficacy of various substrates of the present disclosure incorporated into activatable anti-EGFR antibodies of the present disclosure. The efficacy of the substrates was assessed by measuring tumor volume (TV mm3) at various time points after administration.

[0150] [Figure 13]Figure 13 is a graph showing toxicity measured as a function of body weight (BW) loss in DBA / 1 mice following administration of control intravenous immunoglobulin (IVIG), an anti-Jagged antibody of the present disclosure, and an activatable anti-Jagged antibody of the present disclosure with various substrates of the present disclosure.

[0151] [Figure 14] FIG. 14 is a graph showing 3D reconstruction of FLIT-μCT imaging data for cetuximab and two EGFR-activatable antibodies containing tandem substrates in a xenograft tumor model co-implanted with H292 (open arrow) and FaDu (filled arrow). DETAILED DESCRIPTION OF THE INVENTION

[0152] Detailed Description of the Invention The present disclosure provides amino acid sequences comprising at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). These CM1-CM2 substrates are useful for a variety of therapeutic, diagnostic, and prophylactic applications. For example, these CM1-CM2 substrates are useful for activatable antibodies, including antibodies or antigen-binding fragments thereof (AB), that include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the AB, such that coupling of the MM reduces the ability of the AB to bind to a target.

[0153] The examples provided herein demonstrate that these CM1-CM2 substrates exhibit many desirable cleavage characteristics when exposed to at least one MMP protease and / or at least one SP protease under specified conditions.

[0154] The present disclosure also provides antibodies comprising one or more of these CM1-CM2 substrates. For example, these CM1-CM2 substrates are useful when conjugating an antibody to one or more additional agents to generate a conjugated antibody. These CM1-CM2 substrates are also useful in activatable antibodies and / or activatable antibody conjugates.

[0155] The conjugated antibody, activatable antibody, and / or conjugated 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. In some embodiments, the conjugated antibody and / or activatable antibody has an AB that binds an extracellular target, typically an extracellular protein target. In some embodiments, the conjugated antibody and / or activatable antibody is designed for cellular uptake and is switchable intracellularly.

[0156] As a non-limiting example, AB is a binding partner for any of the targets listed in Table 1. [Table 2]

[0157] As a non-limiting example, AB is or is derived from an antibody listed in Table 2. [Table 3]

[0158] Exemplary conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies of the present disclosure include, for example, antibodies that bind the interleukin-6 receptor (IL-6R) and that are, or comprise heavy and light chains derived from, the antibody referred to herein as the "Av1" antibody, which binds the interleukin-6 receptor (IL-6R). The amino acid sequences for the Av1 heavy chain and Av1 light chain are set forth below as SEQ ID NO:100 and SEQ ID NO:101, respectively. Av1 antibody H chain amino acid sequence: [ka] Av1 antibody L chain amino acid sequence: [ka]

[0159] Exemplary activatable antibodies and / or conjugated activatable antibodies of the present disclosure include, for example, antibodies that bind interleukin-6 receptor (IL-6R) and are Av1 antibodies or include heavy and light chains derived from Av1 antibodies and a masking moiety. Exemplary activatable antibodies and / or conjugated activatable antibodies of the present disclosure include amino acid sequences attached to the N-terminus of the AV1 light chain. These N-terminal amino acid sequences include, for example, YGSCSWNYVHIFMDC (SEQ ID NO: 102); QGDFDIPFPAHWVPIT (SEQ ID NO: 103); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 104); QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 105); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 106); or QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 107). 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.

[0160] Exemplary activatable antibodies of the present disclosure include, for example, antibodies that bind epidermal growth factor receptor (EGFR) and are selected from the group consisting of the antibody referred to herein as the "c22v5" antibody (also referred to herein as the C225v5 antibody), the antibody referred to herein as the "c225v4" antibody (also referred to herein as the C225v4 antibody), and the antibody referred to herein as the "c225v6" antibody (also 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. C225v5 antibody H chain amino acid sequence: [ka] C225v4 antibody H chain amino acid sequence: [ka] C225v6 antibody H chain amino acid sequence: [ka] C225 antibody L chain amino acid sequence: [ka]

[0161] 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. Variable L chain amino acid sequence Lc4 [ka] Variable H chain amino acid sequence Hc4 [ka] Variable L chain amino acid sequence Lc5 [ka] Variable H chain amino acid sequence Hc5 [ka] Variable light chain amino acid sequence Lc7 [ka] Variable H chain amino acid sequence Hc7 [ka] Variable L chain amino acid sequence Lc8 [ka] Variable H chain amino acid sequence Hc8 [ka] Variable L chain amino acid sequence Lc13 [ka] Variable H chain amino acid sequence Hc13 [ka] Variable light chain amino acid sequence Lc16 [ka] Variable H chain amino acid sequence Hc16 [ka] Variable L chain amino acid sequence Lc19 [ka] Variable H chain amino acid sequence Hc19 [ka] Variable light chain amino acid sequence Lc21 [ka] Variable H chain amino acid sequence Hc21 [ka] Variable light chain amino acid sequence Lc24 [ka] Variable H chain amino acid sequence Hc24 [ka] Variable light chain amino acid sequence Lc26 [ka] Variable H chain amino acid sequence Hc26 [ka] Variable light chain amino acid sequence Lc27 [ka] Variable H chain amino acid sequence Hc27 [ka] Variable light chain amino acid sequence Lc28 [ka] Variable H chain amino acid sequence Hc28 [ka] Variable L chain amino acid sequence Lc30 [ka] Variable H chain amino acid sequence Hc30 [ka] Variable light chain amino acid sequence Lc31 [ka] Variable H chain amino acid sequence Hc31 [ka] Variable L chain amino acid sequence Lc32 [ka] Variable H chain amino acid sequence Hc32 [ka] Variable light chain amino acid sequence Lc37 [ka] Variable heavy chain amino acid sequence Hc37 [ka] Variable L chain amino acid sequence Lc39 [ka] Variable H chain amino acid sequence Hc39 [ka] Variable L chain amino acid sequence Lc40 [ka] H chain amino acid sequence Hc40 [ka] Variable light chain amino acid sequence Lc47 [ka] Variable heavy chain amino acid sequence Hc47 [ka] Variable 4B2L chain [ka] Variable 4B2H chain [ka] Variable 4D11L chain [ka] Variable 4D11 heavy chain [ka] Variable 4E7L chain [ka] Variable 4E7H chain [ka] Variable 4E11L chain [ka] Variable 4E11H chain [ka] Variable 6B7L chain [ka] Variable 6B7H chain [ka] Variable 6F8L chain [ka] Variable 6F8H chain [ka]

[0162] Exemplary conjugated antibodies, activatable antibodies and / or conjugated 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 a combination of heavy and light chain regions that are or are derived from the heavy and light chain sequences shown below. 4D11 light chain sequence: [ka] 4D11 heavy chain sequence: [ka] 4D11v2 heavy chain sequence [ka] 4D11v2 light chain sequence [ka]

[0163] 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).

[0164] 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: 165). By non-limiting example, a MM can include the sequence, e.g., CISPRGC (SEQ ID NO: 166); CISPRGCG (SEQ ID NO: 167); CISPRGCPDGPYVMY (SEQ ID NO: 168); 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, such as, 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); CPH FTTTSCGCISPRGCPG (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); CPHV SFGSCGCISPRGCG (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).

[0165] Suitable masking moieties for use with antibodies that bind Jagged targets, such as Jagged1 and / or Jagged2, include, by non-limiting example, the sequences: QGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 201); QGQSGQGQQQWCNIWINGGDCRGWNG (SEQ ID NO: 202); PWCMQRQDFLRCPQP (SEQ ID NO: 203); QLGLPAYMCTFECLR (SEQ ID NO: 204); CNLWVSGGDCGGLQG (SEQ ID NO: 205); SCSLWTSGSCLPHSP (SEQ ID NO: 206); YCLQLP HYMQAMCGR (SEQ ID NO: 207); CFLYSCTDVSYWNNT (SEQ ID NO: 208); PWCMQRQDYLRCPQP (SEQ ID NO: 209); CNLWISGGDCRGLAG (SEQ ID NO: 210); CNLWVSGGDCRGVQG (SEQ ID NO: 211); CNLWVSGGDCRGLRG (SEQ ID NO: 212); CNLWISGGDCRGLPG (SEQ ID NO: 213); CNLWVSGGDCRDAPW (SEQ ID NO: 214); CNLWVSGGDCRDLLG (SEQ ID NO: 215); CNLWVSGGDCRGLQG (SEQ ID NO: 216); CNL WLHGGDCRGWQG (SEQ ID NO: 217); CNIWLVGGDCRGWQG (SEQ ID NO: 218); CTTWFCGGDCGVMRG (SEQ ID NO: 219); CNIWGPSVDCGALLG (SEQ ID NO: 220); CNIWVNGGDCRSFEG (SEQ ID NO: 221); YCLNLPRYMQDMCWA (SEQ ID NO: 222); YCLALPHYMQADCAR (SEQ ID NO: 223); CFLYSCGDVSYWGSA (SEQ ID NO: 224); CYLYSCTDSAFWNNR (SEQ ID NO: 225); CYLYSCNDVSYWSNT (SEQ ID NO: 226); CFLYSCTDVSYW (SEQ ID NO: 227); CFLYSCTDVAYWNSA (SEQ ID NO: 228); CFLYSCTDVSYWGDT (SEQ ID NO: 229); CFLYSCTDVSYWGNS (SEQ ID NO: 230); CFLYSCTDVAYWNNT (SEQ ID NO: 231); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 232); CFLYSCTDVAYWSGL (SEQ ID NO: 233); CYLYSCTDGSYWNST (SEQ ID NO: 234); CFLYSCSDVSYWGNI (SEQ ID NO: 235); CFLYSCTDVAYW (SEQ ID NO: 236);CFLYSCTDVSYWGST (SEQ ID NO: 237); CFLYSCTDVAYWGDT (SEQ ID NO: 238); GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 239); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 240); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 241); GCNIWANGGDCRGWIDNIDG (SEQ ID NO: 242); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 243); GCNIWLVGGDCRGWLEEAVG (SEQ ID NO: 244); GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 245); GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 246); GQQQWCNIWINGGDCRGWNG (SEQ ID NO: 247); GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 248); GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 249); GASQYCNLWINGGDCRGWRG (SEQ ID NO: 250); and / or GIPDNCNIWINGGDCRYG (SEQ ID NO: 264).

[0166] Masking moieties for use with antibodies that bind interleukin 6 targets, such as interleukin 6 receptor (IL-6R), can, by non-limiting example, be masked with sequences such as QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 265); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 266); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 267); YRSCNWNYVSIFLDC (SEQ ID NO: 268); PGAFDIPFPAHWVPNT (SEQ ID NO: 269); ESSCVWNYVHIYMDC (SEQ ID NO: 270); YPGCKWNYDRIFLDC (SEQ ID NO: 271); YRTCSWNYVGIFLDC (SEQ ID NO: 272); YGSCSWNYVHIFMDC (SEQ ID NO: 273); YGSCSWNYVHIFLDC (SEQ ID NO: 274); YGSCNWNYVHIFLDC (SEQ ID NO: 275); YTSCNWNYVHIFMDC (SEQ ID NO: 276); YPGCKWNYDRIFLDC (SEQ ID NO: 277); WRSCNWNYAHIFLDC (SEQ ID NO: 278); WSNCHWNYVHIFLDC (SEQ ID NO: 279); DRSCTWNYVRISYDC (SEQ ID NO: 28 0);SGSCKWDYVHIFLDC (SEQ ID NO: 281);SRSCIWNYAHIHLDC (SEQ ID NO: 282);SMSCYWQYERIFLDC (SEQ ID NO: 283);YRSCNWNYVSIFLDC (SEQ ID NO: 284);SGSCKWDYVHIFLDC (SEQ ID NO: 285);YKSCHWDYVHIFLDC (SEQ ID NO: 286);YGSCTWNYVHIFMEC (SEQ ID NO: 287);FSSCNWNYVHIFLDC (SEQ ID NO: 288);WRSCNWNYAHIFLDC (SEQ ID NO: 289);YGSCQWNYVHIFLDC (SEQ ID NO: No. 290); YRSCNWNYVHIFLDC (SEQ ID NO: 291); NMSCHWDYVHIFLDC (SEQ ID NO: 292); FGPCTWNYARISWDC (SEQ ID NO: 293); XXsCXWXYvhIfXdC (SEQ ID NO: 294); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 295); RDTGGQCRWDYVHIFMDC (SEQ ID NO: 296); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 297); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 298); DGGPAGCSWNYVHIFMEC (SEQ ID NO: 299);AVGPAGCWWNYVHIFMEC (SEQ ID NO: 300); CTWNYVHIFMDCGEGEGP (SEQ ID NO: 301); GGVPEGCTWNYAHIFMEC (SEQ ID NO: 302); AEVPAGCWWNYVHIFMEC (SEQ ID NO: 303); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 304); SGASGGCKWNYVHIFMDC (SEQ ID NO: 305); TPGCRWNYVHIFMECEAL (SEQ ID NO: 306); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 307); PGAFDIPFPAHWVPNT (SEQ ID NO: 308); RGACDIPFPAHWIPNT (SEQ ID NO: 309) );QGDFDIPFPAHWVPIT (SEQ ID NO: 310);XGafDIPFPAHWvPnT (SEQ ID NO: 311);RGDGNDSDIPFPAHWVPRT (SEQ ID NO: 312);SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 313);WAGGNDCDIPFPAHWIPNT (SEQ ID NO: 314);WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 315);AGSGNDSDIPFPAHWVPRT (SEQ ID NO: 316);ESRSGYADIPFPAHWVPRT (SEQ ID NO: 317);and / or RECGRCGDIPFPAHWVPRT (SEQ ID NO: 318).

[0167] 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.

[0168] 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; 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 00, 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 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 or more than the binding affinity of an AB or parent AB not modified with a MM to a target. 0, 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 lower.

[0169] The dissociation constant of MM with AB (K d ) is generally the K of AB against the target dHigher than AB. d is the K of AB against the target d The binding affinity of the MM to the AB is generally 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 AB to the target.

[0170] 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 binding of an AB not modified with a MM or the binding of the parent AB to a target, may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% 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.

[0171] 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. In these embodiments, when the AB is modified or coupled to the MM and in the presence of the target, the MM inhibits the binding of AB to its target for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, as measured in an in vivo or in vitro assay, or For 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, there is no, or substantially no, or only 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target compared to binding of the AB unmodified with the MM, the parent AB, or an AB not coupled to the MM.

[0172] 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.

[0173] An AB coupled to or modified with a MM can be represented by the following formula (from the amino (N)-terminal region to the carboxyl (C)-terminal region): (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) 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 include one or more linkers, e.g., flexible linkers, in the composition to provide flexibility.

[0174] In certain embodiments, the MM is not a natural binding partner of AB. In some embodiments, the MM does not include or contain homology to any natural binding partner of AB. In other 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. In 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. In some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0175] In some embodiments, an activatable antibody comprises an AB modified with a masking moiety (MM) and also comprising at least one cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). 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 with or coupled to a masking moiety (MM) and a CM1-CM2 substrate.

[0176] The elements in the activatable antibody are positioned such that the MM and CM1-CM2 substrate 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.

[0177] K of AB modified by MM and CM1-CM2 substrates against the target d is the K of the AB unmodified by the MM and CM1-CM2 substrates, 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 00, 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 CM1-CM2 substrates 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 greater than the binding affinity of the parent AB to a target or AB that is not modified with MM and CM1-CM2 substrates; 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 00, 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 lower.

[0178] When an AB is modified with MM and CM1-CM2 substrates and in the presence of its target, but not in the presence of a modifying agent (e.g., MMP and SP), the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that has not been modified with MM and CM1-CM2 substrates or the specific binding of the parent AB to its target. The ability of the AB to bind a target when modified with MM and CM1-CM2 substrates may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% 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, when compared to the binding of the parent AB or the binding of an AB that is not modified with MM and CM1-CM2 substrates to a target.

[0179] As used herein, the term "cleaved state" refers to the state of an activatable antibody following modification, i.e., cleavage, of the CM1-CM2 substrate by at least one matrix metalloprotease. The term "uncleaved state" or "fully uncleaved state," as used herein, refers to the state of an activatable antibody in the absence of cleavage of the CM1-CM2 substrate by MMPs and / or SPs. As discussed above, the term "activatable antibodies" is used herein to refer to activatable antibodies in both their uncleaved (native) state and their cleaved state. An activatable antibody in its cleaved state is also referred to herein as an activated antibody and / or an activated activatable antibody. It will be apparent to those skilled in the art that in some embodiments, a cleaved activatable antibody lacks MM due to cleavage of the CM1-CM2 substrate by a protease, resulting in the release of at least MM.

[0180] 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., a 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 CM1-CM2 substrate 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.

[0181] The CM1-CM2 substrate and AB of the activatable antibody are selected such that AB represents a binding moiety for a predetermined target, and the CM1-CM2 substrate represents a substrate for an MMP and SP, where the MMP and / or SP co-localize 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 CM1-CM2 substrate 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).

[0182] In 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.

[0183] Generally, an activatable antibody can be designed by selecting for a first AB of interest and then 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.

[0184] Activatable antibodies are provided that exhibit a desired dynamic range of switchable phenotypes for target binding in a inhibited versus 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 MMPs and SPs capable of cleaving the CM1-CM2 substrate of the activatable antibody to the minimum detectable level of target protein binding to the activatable antibody in the absence of proteases. The dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of the activatable antibody cleaving agent (e.g., enzyme) treatment to the 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 relatively high dynamic range values ​​(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 CM1-CM2 substrate of the activatable antibody than in the absence of the cleaving agent.

[0185] 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 CM1-CM2 substrates, 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 the CM1-CM2 substrate is at least partially contained within the MM.

[0186] 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: (MM)-(CM1-CM2 substrate)-(AB) (AB)-(CM1-CM2 substrate)-(MM) where MM is a masking moiety, CM1-CM2 substrate is a cleavable moiety, and AB is an antibody or fragment thereof. As previously noted, the term "CM1-CM2 substrate" is not intended to convey any requirement regarding the orientation or other structural arrangement of the first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and the second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). Thus, the term "CM1-CM2 substrate" encompasses CM1-CM2 substrates having the following structural arrangements from N-terminus to C-terminus: CM1-CM2 or CM2-CM1. The term "CM1-CM2 substrate" also encompasses substrates in which at least a portion of the CM1 sequence overlaps with at least a portion of the CM2 sequence. It should also be noted that although the MM and CM1-CM2 substrates are shown as separate components in the formula above, it is contemplated that, according to all exemplary embodiments (including formulas) disclosed herein, the amino acid sequences of the MM and CM1-CM2 substrates can overlap, such that the CM1-CM2 substrate is fully or partially contained within the MM. Additionally, the formula above provides for additional amino acid sequences that can be located N-terminal or C-terminal to the activatable antibody element.

[0187] In certain embodiments, the MM is not a natural binding partner of AB. In some embodiments, the MM does not include or contain homology to any natural binding partner of AB. In other 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. In 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. In some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0188] In many embodiments, it is desirable to insert one or more linkers, e.g., flexible linkers, into an activatable antibody construct to provide flexibility at one or more MM-CM1-CM2 substrate bonds, CM1-CM2 substrate-AB bonds, or both. For example, the AB, MM, and / or CM1-CM2 substrates 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.

[0189] For example, in 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): (MM)-L1-(CM1-CM2 substrate)-(AB) (MM)-(CM1-CM2 substrate)-L2-(AB) (MM)-L1-(CM1-CM2 substrate)-L2-(AB) wherein MM, CM1-CM2 substrate, and AB are as defined above; L1 and L2 are each independently, and optionally, present or absent, the same or different flexible linkers comprising 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))).

[0190] 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 and is specifically cleaved by at least one MMP at a rate of about 0.001-1500 x 10 4 M -1 S -1or 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 SP at a rate of

[0191] For specific cleavage by an enzyme, contact between the enzyme and the CM1-CM2 substrate is required. When an activatable antibody, including an AB coupled to a MM and a CM1-CM2 substrate, is in the presence of the target and sufficient enzymatic activity, the CM1-CM2 substrate can be cleaved. Sufficient enzymatic activity refers to the ability of the enzyme to contact the CM1-CM2 substrate and effect cleavage. It is readily envisioned that an enzyme may be in proximity to the CM1-CM2 substrate but unable to cleave it due to other cellular factors or protein modifications of the enzyme.

[0192] Linkers suitable for use in the compositions described herein are generally linkers 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.

[0193] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 381), and (GGGS)n (SEQ ID NO: 382), 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: 383), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 384), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 385), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 386), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 387), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 388), 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.

[0194] In some embodiments, the activatable antibodies described herein also encompass an agent conjugated to the activatable antibody. In some embodiments, the conjugated agent is a therapeutic agent, such as an anti-inflammatory agent and / or an anti-tumor agent. In such embodiments, the agent is conjugated to a carbohydrate moiety of the activatable antibody; for example, in 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. In some embodiments, the agent is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment of the activatable antibody.

[0195] In some embodiments, the agent is a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (ie, a radioconjugate).

[0196] In some embodiments, the agent is a detectable moiety, such as a label or other marker. For example, the agent may be detectable 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.

[0197] 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. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0198] In some embodiments, the agent is linked to AB using a maleimidocaproyl-citrulline linker or a maleimidoPEG-valine-citrulline linker. In some embodiments, the agent is linked to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, the agent is linked to AB using a maleimidoPEG-valine-citrulline linker. In some embodiments, the agent is linked to AB using a maleimidoPEG-valine-citrulline linker. In some embodiments, the agent is monomethyl auristatin D (MMAD) linked to AB using a maleimidoPEG-valine-citrulline-para-aminobenzoyloxycarbonyl linker, and this linker-payload construct is referred to herein as "vc-MMAD." In some embodiments, the agent is monomethyl auristatin E (MMAE) linked to AB 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: vc-MMAD: [ka] vc-MMAE: [ka]

[0199] 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 proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, croton, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the 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.

[0200] Conjugates of antibodies and cytotoxic agents are prepared using a variety of 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). C-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionuclides to antibodies (see WO 94 / 11026).

[0201] 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. [Table 4]

[0202] 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).

[0203] Coupling can be achieved by any chemical reaction that will link two molecules together 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, in 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)).

[0204] In some embodiments, in addition to the compositions and methods provided herein, 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. In 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. In some embodiments, activatable antibodies can be engineered to contain or otherwise introduce enzymatically activatable peptide sequences into their activatable antibody sequences.

[0205] 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. In 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.) coupled to EDC. Catalog No. 24510): Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

[0206] 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 combination with sulfo-NHS is more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0207] 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 are all the same, e.g., cleavable or non-cleavable, or the multiple linkers are different, e.g., at least one is cleavable and at least one is non-cleavable.

[0208] The present disclosure utilizes several methods for attaching 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.

[0209] 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).

[0210] 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.

[0211] 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 attached 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.

[0212] 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, CM1-CM2 substrate, and associated linker, and then attached to the linker for conjugation purposes.

[0213] Branched Linkers: In 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 aldehyde or sulfhydryl groups, or any chemical site to which an agent can be attached.

[0214] In 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 the AB. These multiple sites can be introduced into the 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 the AB for subsequent attachment to a linker. The functional sites of the branched 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., by attaching a multi-site linker at several sites on the AB.

[0215] 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.

[0216] Non-limiting examples of cleavable linker sequences are provided in Table 4. [Table 5]

[0217] Additionally, the agent can be attached to 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. In certain embodiments, the reducing agent that modifies the CM1-CM2 substrate will also modify the linker of the attached activatable antibody.

[0218] Spacers and Cleavable Elements: In 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 a linker of the following general structure: W-(CH2) n -Q {In the formula, W is either -N-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer between 0 and 20}.

[0219] In 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.

[0220] It should be understood throughout this discussion 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.

[0221] 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 bind antigen and activate the cascade. Thus, in 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.

[0222] 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.

[0223] Thus, when these 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.

[0224] 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, while cleavage of the agent occurs slowly, thereby resulting in release of the compound at the target site.

[0225] Biochemical Crosslinkers: In 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.

[0226] 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.

[0227] Exemplary hetero-bifunctional cross-linkers are referenced in Table 5. [Table 6]

[0228] Non-cleavable linker or direct conjugation: In yet other embodiments of the present disclosure, conjugates can be designed so that the agent is delivered to a target but not released. This can be achieved by attaching the agent to the AB directly or via a non-cleavable linker.

[0229] 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: W-(CH2) n -Q {In the formula, W is either -N-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer between 0 and 20}.

[0230] Non-cleavable conjugates: In some embodiments, a compound can be conjugated to an AB that is incapable of activating complement. When an AB that is incapable of activating complement is used, this conjugation can 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0235] 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 binding. 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.

[0236] 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.

[0237] 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.

[0238] 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).

[0239] 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 can be 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.

[0240] As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of non-covalent 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 is determined by the dissociation constant (K d ), where the smaller K d indicates 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 measurement of 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, the "on rate constant" (K on ) and the "off rate constant" (K off Both K and K can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K onThe ratio of α to β allows for the cancellation of all parameters not related to affinity, and the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present disclosure have binding constants (K d ) is said to specifically bind to EGFR when it 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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 mutants. Oligonucleotides of the present disclosure can be either sense or antisense oligonucleotides.

[0248] 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.

[0249] 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.

[0250] 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."

[0251] 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, e.g., in some embodiments, at least 90% sequence identity, in some embodiments, at least 95% sequence identity, and in some embodiments, at least 99% sequence identity.

[0252] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0253] 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., in some embodiments, at least 80%, 90%, 95%, and in 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. In 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.

[0254] 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 mutations 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 domain(s) 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).

[0255] 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., in some embodiments, 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, at least 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.

[0256] The term "agent" is used herein to denote a compound, a mixture of compounds, a biopolymer, or an extract made from biomaterials.

[0257] 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 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, 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.

[0258] 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)).

[0259] 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 in 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.

[0260] 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.

[0261] The term "patient" includes human and veterinary subjects.

[0262] 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.

[0263] 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 reduced 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.

[0264] 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. In some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM1-CM2 substrate) that functions as a substrate for at least one MMP protease and at least one SP. 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.

[0265] In some embodiments, the multispecific activatable antibody is designed to engage immune effector cells, also referred to herein as immune effector-engaging multispecific activatable antibodies. In some embodiments, the multispecific activatable antibody is designed to engage leukocytes, also referred to herein as leukemia-engaging multispecific activatable antibodies. In some embodiments, the multispecific activatable antibody is designed to engage T-cells, also referred to herein as T-cell-engaging multispecific activatable antibodies. In 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. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell is a mononuclear cell, e.g., a myelomononuclear cell. In 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.

[0266] In 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. In 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 the coupling of the MM1 reduces the ability of AB1 to bind the first target. In 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 the coupling of the MM2 reduces the ability of AB2 to bind the second target. In 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.In 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 (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. In some embodiments, the non-immune effector cell-engaging antibody is a cancer-targeting antibody. In some embodiments, the immune effector cell-engaging antibody is an scFv. In 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. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are bone marrow mononuclear cells.

[0267] In 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. In 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. In 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 the coupling of the MM2 reduces the ability of AB2 to bind the second target. In 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.

[0268] In 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. In 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. In 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. In 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.

[0269] In 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. In 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. In 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. In 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 the 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 the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0270] In some embodiments of immune effector-engaging multispecific activatable antibodies, 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, EGFR, 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. In 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. In 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.

[0271] 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. In 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 without activating all T cells, regardless of their antigen specificity. In 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.

[0272] 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. In 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 tissue-targeted antigens in any diseased tissue. In 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.

[0273] In 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. In 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. In 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 MM2 reduces the ability of AB2 to bind the second target. In 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 targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof 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.

[0274] In 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. In 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. In 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. In 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 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.

[0275] In 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. In 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. In 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. In 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.

[0276] In 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. In 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. In 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. In 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, 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.

[0277] In 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. In 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. In 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. In 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 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.

[0278] In 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. In 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. In 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. In 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.

[0279] In 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. In 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. In 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. In 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 the coupling of 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 the coupling of MM2 reduces the ability of AB2 to bind the second target.

[0280] In 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. In 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. In 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. In 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 the coupling of 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 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.

[0281] In 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. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different targets. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different epitopes on the same target. In 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.

[0282] In some embodiments, a multispecific activatable antibody comprising an IgG has a masked IgG variable domain. In some embodiments, a multispecific activatable antibody comprising an scFv has a masked scFv domain. In some embodiments, a 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. In some embodiments, a 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. In some embodiments, a 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. In some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein each of the IgG variable domain and the scFv domain is coupled to its own masking moiety. In 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. In 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. In 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.

[0283] 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. In some embodiments, a masking moiety (MM) linked to the CM1-CM2 substrate is attached to and masks the antigen-binding domain of an IgG. In some embodiments, a masking moiety (MM) linked to the CM1-CM2 substrate is attached to and masks the antigen-binding domain of at least one scFv. In some embodiments, the masking moiety (MM) linked to the CM1-CM2 substrate is linked to and masks the antigen-binding domain of an IgG, and the masking moiety (MM) linked to the CM1-CM2 substrate is linked to and masks the antigen-binding domain of at least one scFv.

[0284] 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-CM1-CM2 substrate-L1-MM)2;(VL-CL)2:(VH-CH1-CH2-CH3-L4-VL * -L3-VH * -L2-CM1-CM2 substrate-L1-MM)2;(MM-L1-CM1-CM2 substrate-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VH * -L3-VL * )2;(MM-L1-CM1-CM2 substrate-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VL* -L3-VH * )2;(VL - CL)2:(MM - L1 - CM1 - CM2 matrix - L2 - VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL - CL)2:(MM - L1 - CM1 - CM2 matrix - L2 - VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(MM - L1 - CM1 - CM2 matrix - L2 - VL - CL)2:(VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(MM - L1 - CM1 - CM2 matrix - L2 - VL - CL)2:(VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(VL - CL - L4 - VH * -L3-VL * -L2-CM1-CM2 matrix - L1-MM)2:(VH-CH1-CH2-CH3)2;(VL - CL - L4 - VL * -L3-VH * -L2-CM1-CM2 matrix - L1-MM)2:(VH-CH1-CH2-CH3)2;(MM - L1 - CM1 - CM2 matrix - L2 - VL * -L3-VH * -L4-VL-CL)2:(VH-CH1-CH2-CH3)2;(MM - L1 - CM1 - CM2 matrix - L2 - VH * -L3-VL * -L4-VL-CL)2:(VH-CH1-CH2-CH3)2;(VL - CL - L4 - VH * -L3-VL * -L2-CM1-CM2 matrix - L1-MM)2:(MM - L1 - CM1 - CM2 matrix - L2 - VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL - CL - L4 - VH * -L3-VL * -L2-CM1-CM2 matrix - L1-MM)2:(MM - L1 - CM1 - CM2 matrix - L2 - VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(VL - CL - L4 - VL* -L3-VH * -L2-CM1-CM2 matrix-L1-MM)2:(MM-L1-CM1-CM2 matrix-L2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL * -L3-VH * -L2-CM1-CM2 matrix-L1-MM)2:(MM-L1-CM1-CM2 matrix-L2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH * -L3-VL * )2:(MM-L1-CM1-CM2 matrix-L2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH * -L3-VL * )2:(MM-L1-CM1-CM2 matrix-L2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL * -L3-VH * )2:(MM-L1-CM1-CM2 matrix-L2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL * -L3-VH * )2:(MM-L1-CM1-CM2 matrix-L2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH * -L3-VL * -L2-CM1-CM2 matrix-L1-MM)2:(VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH * -L3-VL * -L2-CM1-CM2 matrix-L1-MM)2:(VH * -L3-VL *-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL * -L3-VH * -L2-CM1-CM2 substrate-L1-MM)2:(VL * -L3-VH * -L4-VH-CH1-CH2-CH3); or (VL-CL-L4-VL * -L3-VH * -L2-CM1-CM2 substrate-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 CM1-CM2 substrate; L2 is a linker peptide connecting the CM1-CM2 substrate 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 directed against any antigen or epitope.

[0285] In 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 is typically 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. In 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.

[0286] 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.

[0287] In some embodiments, the targeting antibody can exist in the form of an activatable antibody. In some embodiments, the scFv can exist in the form of a pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0288] In some embodiments, the scFv is specific for binding of CD3ε and is an antibody or fragment thereof that binds CD3ε, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, the scFv is specific for binding of CTLA-4 (also referred to herein as CTLA and CTLA4).

[0289] In some embodiments, the anti-CTLA-4 scFv comprises the following amino acid sequence: [ka]

[0290] In 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:347.

[0291] In some embodiments, the anti-CD3ε scFv comprises the following amino acid sequence: [ka]

[0292] In 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:349.

[0293] In some embodiments, the scFv is specific for binding one or more T-cells, one or more NK cells, and / or one or more macrophages, hi 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.

[0294] In some embodiments, the multispecific activatable antibody also includes an agent conjugated to AB. 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 the multispecific activatable antibody via a linker. 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 CM1-CM2 substrate sequence. In some embodiments, the linker is 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 a DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is trastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin 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. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0295] In some embodiments, the multispecific activatable antibody can also comprise a detectable moiety, hi some embodiments, the detectable moiety is a diagnostic agent.

[0296] In some embodiments, the multispecific activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the multispecific activatable antibody can be engineered to contain one or more disulfide bonds.

[0297] 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. In some embodiments, the cells comprise such vectors.

[0298] 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.

[0299] 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 first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that binds a second target or 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. In some embodiments, MM1 is coupled to AB1 via a CM1-CM2 substrate for MMPs and SPs, wherein at least one of the MMPs and SPs 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 the circulation, are activated at the intended site of treatment 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.

[0300] In some embodiments, the multispecific activatable antibody comprises a linking peptide between the MM1 and CM1-CM2 substrates.

[0301] In some embodiments, the multispecific activatable antibody comprises a linking peptide between the CM1-CM2 substrate and AB1.

[0302] In 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-CM2 substrate-LP2-AB1 or AB1-LP2-CM1-CM2 substrate-LP1-MM1. In some embodiments, the two connecting peptides need not be identical to each other.

[0303] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO:381), and (GGGS)n (SEQ ID NO:382), where n is at least one integer. In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:383), GGSGG (SEQ ID NO:384), GSGSG (SEQ ID NO:385), GSGGG (SEQ ID NO:386), GGGSG (SEQ ID NO:387), and GSSSG (SEQ ID NO:388).

[0304] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2.

[0305] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1), a second connecting peptide (LP2), and a connecting peptide (LP') between CM1 and CM2, and at least a portion of the multispecific activatable antibody has the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM1-LP1-CM1-CM2 substrate-LP2-AB1 or AB1-LP2-CM1-CM2 substrate-LP1-MM1. In some embodiments, the connecting peptides need not be identical to each other.

[0306] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0307] In 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. In 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. In some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

[0308] In some embodiments, each AB in the multispecific activatable antibody has a dissociation constant of about 100 nM or less for binding to its corresponding target or epitope.

[0309] In some embodiments, MM1 has a dissociation constant for binding to its corresponding AB that is higher than the dissociation constant for binding of AB to its corresponding target or epitope.

[0310] In some embodiments, MM1 has a dissociation constant for binding to its corresponding AB that is no more than the dissociation constant for binding of the AB to its corresponding target or epitope.

[0311] In 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.

[0312] In some embodiments, MM1 is a polypeptide about 2-40 amino acids in length. In some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 or less amino acids in length.

[0313] In some embodiments, MM1 has a polypeptide sequence that differs from the sequence of the target of its corresponding AB.

[0314] In some embodiments, MM1 has a polypeptide sequence that is 50% or less identical to any natural binding partner of its corresponding AB. In some embodiments, MM1 has a polypeptide sequence that is 25% or less identical to any natural binding partner of its corresponding AB. In some embodiments, MM1 has a polypeptide sequence that is 10% or less identical to any natural binding partner of its corresponding AB.

[0315] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the 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

[0316] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the 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

[0317] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope dAt least 100 times higher than

[0318] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the 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

[0319] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the 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

[0320] In some embodiments, MM1 is an amino acid sequence selected from the MMs shown in the Examples provided herein.

[0321] In 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 an additional cleavable moiety (CM') coupled to AB2, which is either a CM1-CM2 substrate or a polypeptide that functions as a substrate for a second protease. In some embodiments, the CM' is a polypeptide 15 amino acids or less in length. In some embodiments, the CM' is a CM1-CM2 substrate, where each of the CM1 and CM2 of the CM1-CM2 substrate is independently a polypeptide 15 amino acids or less in length.

[0322] In some embodiments, the MMP protease, SP protease, and / or second protease co-localizes with a second target or epitope in a tissue, and the MMP protease, SP protease, and / or second protease cleaves the CM' in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the MMP protease, SP protease, and / or second protease. In some embodiments, the MMP protease, SP protease, and / or second protease co-localizes with the first target or epitope and the second target or epitope in a tissue. In some embodiments, the MMP protease, SP protease, and / or second protease are the same protease. In some embodiments, the MMP protease, SP protease, and / or second protease are not the same MMP protease and are not the same SP protease. In some embodiments, the CM1-CM2 substrate and CM' are different substrates for the same MMP protease and the same SP protease. In some embodiments, the protease that cleaves CM' is a protease selected from the group consisting of those shown in Table 6.

[0323] In some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has a dissociation constant for binding to its corresponding AB that is higher than the dissociation constant for binding to its corresponding target or epitope of AB.

[0324] In some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has a dissociation constant for binding to its corresponding AB that is equal to or less than the dissociation constant for binding to its corresponding target or epitope of AB.

[0325] In some embodiments, each MM in a 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.

[0326] In some embodiments, each MM in the multispecific activatable antibody is a polypeptide about 2-40 amino acids in length, hi some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 amino acids or less in length.

[0327] In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that differs from the sequence of the target of the corresponding AB.

[0328] In 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. In 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. In 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.

[0329] In 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

[0330] In 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 dAt least 40 times higher than

[0331] In 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

[0332] In 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

[0333] In 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

[0334] In some embodiments, each MM is an amino acid sequence selected from the MMs disclosed herein.

[0335] In some embodiments, the protease that cleaves the CM1-CM2 substrate sequence co-localizes in a tissue with the AB1 target in the multispecific activatable antibody, and an MMP protease and / or an SP protease, i.e., at least one of the MMP protease and the SP protease, cleaves the CM1-CM2 substrate in the multispecific activatable antibody when the multispecific activatable antibody is exposed to that protease.

[0336] In some embodiments, the multispecific activatable antibody comprises multiple CM1-CM2 substrate sequences, and an MMP protease and / or SP protease that cleaves at least one CM1-CM2 substrate sequence 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 MMP protease and / or SP protease cleaves the CM1-CM2 substrate in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0337] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least two-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0338] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least three times higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0339] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least four times higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0340] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least 5-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0341] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least 10-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0342] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least 20-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0343] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least 40-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0344] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least 50-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0345] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least 100-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0346] In some embodiments, the CM1-CM2 substrate 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 a dissociation constant at least 200-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, the AB binds its target.

[0347] The present disclosure also provides compositions and methods including multispecific activatable antibodies comprising at least a first antibody or fragment (AB1) that specifically binds a target, and a second antibody or fragment (AB2), wherein at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that reduces the ability of AB1 to bind its target. In some embodiments, each AB is coupled to a MM that reduces the ability of its corresponding AB to bind its respective target. For example, in a multispecific activatable antibody embodiment, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, and AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target. In some embodiments, the multispecific activatable antibody comprises two or more AB regions; in such embodiments, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target, AB3 is coupled to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody.

[0348] In some embodiments, the multispecific activatable antibody further comprises at least one CM1-CM2 substrate that is a substrate for MMP proteases and SP proteases, where the CM1-CM2 substrate links the MM to the AB. For example, in some embodiments, the multispecific activatable antibody comprises at least a first antibody or antibody fragment (AB1) and a second antibody or antibody fragment (AB2) that specifically bind a target, and at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that reduces the ability of AB1 to bind its target via a first CM1-CM2 substrate. In some multispecific activatable antibody embodiments, AB1 is coupled to MM1 via CM1, and AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target via a second CM1-CM2 substrate. In some embodiments, the multispecific activatable antibody comprises two or more AB regions; according to some of those embodiments, AB1 is coupled to MM1 via a first CM1-CM2 substrate, AB2 is coupled to MM2 via a second CM1-CM2 substrate, and AB3 is coupled via a third CM1-CM2 substrate to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target, and similarly for each AB in the multispecific activatable antibody.

[0349] Activatable antibodies having a non-binding steric moiety or a binding partner for a non-binding steric moiety The present disclosure also provides activatable antibodies comprising a non-binding steric moiety (NB), or a binding partner (BP) for the non-binding steric moiety, where the BP recruits or otherwise attracts the NB to the activatable antibody. The activatable antibodies provided herein include, for example, activatable antibodies comprising a non-binding steric moiety (NB), a CM1-CM2 substrate, and an antibody or antibody fragment (AB) that binds a target; activatable antibodies comprising a binding partner for the non-binding steric moiety (BP), a CM1-CM2 substrate, and AB1; and activatable antibodies comprising a BP to which the NB has been recruited, a CM1-CM2 substrate, and an AB that binds a target. Activatable antibodies in which the NB is covalently bound to the CM1-CM2 substrate and AB of the activatable antibody, or which are associated by interaction with a BP that is covalently bound to the CM1-CM2 substrate and AB of the activatable antibody, are referred to herein as "NB-containing activatable antibodies." Activatable or switchable means that the activatable antibody exhibits a first level of target binding (first conformation) when under inhibited, masked, or uncleaved conditions, and a second level of target binding (i.e., second conformation) when under uninhibited, unmasked, and / or cleaved conditions, where the second level of target binding is greater than the first level of binding. Activatable antibody compositions can exhibit enhanced bioavailability and better biodistribution compared to conventional antibody therapies.

[0350] In some embodiments, the activatable antibodies provide reduced toxicity and / or adverse side effects resulting from binding of non-therapeutic and / or non-diagnostic moieties when the AB is not masked or otherwise inhibited from binding to such moieties.

[0351] In one embodiment, the activatable antibody comprises a non-binding steric portion (NB); a CM1-CM2 substrate; and at least an antibody or antibody fragment (AB) that specifically binds to a target, where NB is a polypeptide that does not specifically bind to AB; the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; the CM1-CM2 substrate is positioned such that, in its uncleaved state, NB interferes with binding of AB to its target, and in its cleaved state, NB does not interfere with binding of AB to its target; and NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. As used herein and throughout, the term polypeptide refers to any polypeptide comprising at least two amino acid residues, e.g., larger polypeptides, full-length proteins, and fragments thereof, and the term polypeptide is not limited to single-chain polypeptides, but can encompass multiple units, e.g., multi-chain polypeptides. The terms peptide and polypeptide are used interchangeably herein when the polypeptide is of short length, e.g., a total of 50 amino acids or less, and the terms polypeptide and protein are used interchangeably herein when the polypeptide is of longer length, e.g., 50 or more amino acids.

[0352] In one embodiment, an activatable antibody comprises a non-binding steric moiety (NB); a CM1-CM2 substrate; and at least an antibody or antibody fragment (AB) that specifically binds to a target, where (i) NB is a polypeptide that does not specifically bind to AB; (ii) the CM1-CM2 substrate is a polypeptide up to 50 amino acids in length that includes a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that, in its uncleaved state, NB interferes with binding of AB to its target, and in its cleaved state, NB does not interfere with binding of AB to its target; and (iv) NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. For example, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate independently has a length of up to 15 amino acids.

[0353] In one embodiment, the activatable antibody comprises a non-binding steric moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) NB is a polypeptide that does not specifically bind to AB; (ii) the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that, in the uncleaved state, NB interferes with binding of AB to its target, and in the cleaved state, NB does not interfere with binding of AB to its target; (iv) NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (v) at least a portion of the multispecific activatable antibody, in the uncleaved state, has the following structural configuration from N- to C-terminus: NB-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-NB.

[0354] In one embodiment, the activatable antibody comprises a non-binding steric moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) NB is a polypeptide that does not specifically bind to AB; (ii) the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that, in the uncleaved state, NB interferes with binding of AB to its target, and in the cleaved state, NB does not interfere with binding of AB to its target; and the NB in ​​the uncleaved activatable antibody reduces the ability of AB to bind its target by at least 50%, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, compared to the ability of cleaved AB to bind its target; and (iv) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. The reduction in the ability of the AB to bind its target is determined, for example, using an assay as described herein or an in vitro target displacement assay, such as the assay described in WO 2009 / 025846 and WO 2010 / 081173.

[0355] In one embodiment, the activatable antibody comprises a binding partner (BP) for a non-binding steric moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB1) that specifically binds to a target, where the BP is a polypeptide that binds to the NB when exposed to the NB; the NB is a polypeptide that does not specifically bind to the AB; the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; the CM1-CM2 substrate is positioned such that, in the presence of the NB, in its uncleaved state, the NB interferes with the binding of AB1 to its target, and in its cleaved state, the NB does not interfere with the binding of AB1 to its target, and the BP does not interfere with the binding of AB to its target; and the NB and BP do not inhibit cleavage of the CM1-CM2 substrate by the enzyme. According to some examples of this embodiment, the BP of the activatable antibody is optionally bound to the NB. In one embodiment, the NB is recruited in vivo by the BP of the activatable antibody.

[0356] According to some examples of any of these activatable antibody embodiments, the activatable antibody is formulated as a composition. According to some of these embodiments, the composition also includes a NB, where the NB is co-formulated with the activatable antibody, including the BP, a CM1-CM2 substrate, and an AB. According to some examples of this embodiment, the BP is selected from the group consisting of an albumin-binding peptide, a fibrinogen-binding peptide, a fibronectin-binding peptide, a hemoglobin-binding peptide, a transferrin-binding peptide, an immunoglobulin domain-binding peptide, and another serum protein-binding peptide.

[0357] According to some examples of any of these activatable antibody embodiments, the NB is a soluble globular protein. According to some examples of any of these activatable antibody embodiments, the NB is a protein that circulates in the bloodstream. According to some examples of any of these activatable antibody embodiments, the NB is selected from the group consisting of albumin, fibrinogen, fibronectin, hemoglobin, transferrin, immunoglobulin domains, and other serum proteins.

[0358] According to some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for a protease. According to some examples of any of these activatable antibody embodiments, the protease co-localizes with its target in a tissue, and the protease cleaves the CM1-CM2 substrate on the activatable antibody when the activatable antibody is exposed to the protease. According to some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide up to 50 amino acids in length. According to some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a substrate (S) up to 15 amino acids in length, e.g., 3 amino acids in length, 4 amino acids in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, or 15 amino acids in length.

[0359] According to some examples of any of these activatable antibody embodiments, the activatable antibody has the following N- to C-terminal structural arrangement in its uncleaved state: NB-CM1-CM2 substrate-AB, AB-CM1-CM2 substrate-NB, BP-CM1-CM2 substrate-AB, or AB-CM1-CM2 substrate-BP. In embodiments in which the activatable antibody comprises a BP and is in the presence of its corresponding NB, the activatable antibody has the following N- to C-terminal structural arrangement in its uncleaved state: NB:BP-CM1-CM2-AB, NB:BP-CM2-CM1-AB, AB-CM1-CM2-BP:NB, or AB-CM2-CM1-BP:NB, where ":" represents an interaction, e.g., binding, between the NB and the BP.

[0360] According to some examples of any of these activatable antibody embodiments, the activatable antibody specifically binds a predetermined target and includes antibodies or antigen-binding fragments thereof that are monoclonal antibodies, domain antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scabs, dAbs, single domain heavy chain antibodies, and single domain light chain antibodies. In some embodiments, such antibodies or immunologically active fragments thereof that bind the target are murine, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0361] According to some examples of any of these activatable antibody embodiments, the activatable antibody comprises a combination of a variable heavy chain region comprising an amino acid sequence provided herein and a variable light chain region comprising an amino acid sequence provided herein. In some embodiments, the activatable antibody comprises a combination of a variable heavy chain region comprising an 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 provided herein and a variable light chain region comprising an 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 provided herein.

[0362] According to some examples of any of these activatable antibody 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 conjugated to AB via a non-cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 3. 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 intercalator, or other DNA damaging agent. 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 monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin 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. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0363] According to some examples of any of these activatable antibody embodiments, the activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0364] According to some examples of any of these activatable antibody embodiments, the activatable antibody also includes a spacer. According to some examples of any of these activatable antibody embodiments, the activatable antibody also includes a signal peptide. In some embodiments, the signal peptide is attached to the activatable antibody via a spacer. According to some examples of any of these activatable antibody embodiments, the spacer is attached directly to the MM of the activatable antibody.

[0365] In 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. In some embodiments, the serum half-life of an activatable antibody is similar to the half-life of its corresponding multi-antibody. In some embodiments, the serum half-life of an activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism.In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism.

[0366] The present disclosure also provides isolated nucleic acid molecules encoding any of these activatable antibodies, and vectors comprising these isolated nucleic acid sequences. The present disclosure provides methods for producing activatable antibodies by culturing cells comprising such nucleic acid sequences under conditions conducive to expression of the activatable antibodies. In some embodiments, the cells comprise such vectors.

[0367] The number of dissociation events of NB-containing activatable antibodies against the target (K d ) is the K of AB against the target when AB is not associated with NB or NB:BP. d The dissociation constant of the NB-containing activatable antibody for the target is higher than the K d For example, the K of an NB-containing activatable antibody against a target d If AB is not related to NB or NB:BP, then the K of AB d , or the K of parent AB relative to the target dor more than at least 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 10, 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 the NB-containing activatable antibody for the target is lower than the binding affinity of the AB when the AB is not associated with the NB or NB:BP, or is lower than the binding affinity of the parent AB for the target. For example, the binding affinity of the NB-containing activatable antibody for a target is at least 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 than the binding affinity of the AB when the AB is not associated with the NB or NB:BP, or the binding affinity of the parent AB for the target, 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 00, 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 lower.

[0368] When the NB-containing activatable antibody is in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB when the AB is not associated with the NB or NB:BP. When the NB-containing activatable antibody is in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the parent AB to its target. The ability of the NB-containing activatable antibody to bind its target, as measured, for example, in vitro and / or in vivo, when compared to the binding of an AB unrelated to the NB or NB:BP, or the binding of the parent AB, to its target, is at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even greater, when measured in an in vivo or in vitro assay, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more. It can be lowered by 100%.

[0369] When the NB-containing activatable antibody is in the presence of its target, but not in the presence of a modifying agent (e.g., a protease or other enzyme), specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB when the AB is not associated with the NB or NB:BP. When the NB-containing activatable antibody is in the presence of its target, but not in the presence of a modifying agent (e.g., a protease, other enzyme, reducing agent, or light), specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the parent AB to its target. The ability of the NB-containing activatable antibody to bind its target, as measured, for example, in vitro and / or in vivo, when compared to the binding of an AB unrelated to the NB or NB:BP, or the binding of the parent AB, to its target, is at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even greater, when measured in an in vivo or in vitro assay, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more. It can be lowered by 100%.

[0370] According to some examples of any of these activatable antibody embodiments, the activatable antibody also includes an agent that is conjugated to AB to generate the activatable antibody conjugate. In some embodiments of the activatable antibody conjugate, the agent is a therapeutic agent. In some embodiments, the agent is a diagnostic agent. In some embodiments, the agent is a detectable marker. In some embodiments of the activatable antibody conjugate, the agent is an anti-tumor agent. In some embodiments of the activatable antibody conjugate, the agent is a toxin or a fragment thereof. In some embodiments of the activatable antibody conjugate, the agent is conjugated to AB via a linker. In some embodiments of the activatable antibody conjugate, the linker is a cleavable linker. 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 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 monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin 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. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0371] According to some examples of any of these activatable antibody embodiments, the activatable antibody is a dual-target binding activatable antibody. Such dual-target binding activatable antibodies comprise two Abs capable of binding to the same or different targets. In certain embodiments, the dual-targeting activatable antibody comprises a bispecific antibody or fragment thereof.

[0372] A dual-target-binding activatable antibody is designed to have a CM1-CM2 substrate that can be cleaved by a cleaving agent co-localized in a target tissue with one or both targets that can bind to the AB of the activatable antibody. A dual-target-binding activatable antibody with multiple ABs for the same or different targets can be designed to have multiple CM1-CM2 substrates, where a first CM1-CM2 substrate can be cleaved by a cleaving agent in a first target tissue and a second CM1-CM2 substrate can be cleaved by a cleaving agent in a second target tissue, and one or more targets bind to the AB of the activatable antibody. In one embodiment, the first and second target tissues are spatially separated, for example, at different sites in an organism. In one embodiment, the first and second target tissues are the same tissue but temporally separated, for example, the same tissue at two different time points, for example, where the first time point is when the tissue is an early-stage tumor and the second time point is when the tissue is a later-stage tumor.

[0373] The present disclosure also provides nucleic acid molecules encoding the activatable antibodies described herein. The present disclosure also provides vectors containing those nucleic acids. The activatable antibodies described herein can be produced by culturing cells containing those nucleic acid molecules or vectors under conditions conducive to expression of the activatable antibodies.

[0374] The present disclosure also provides a method for producing an activatable antibody. In one embodiment, the method includes the steps of: (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions conducive to expression of the activatable antibody, wherein the activatable antibody comprises (i) a non-binding steric moiety (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; (3) the CM1-CM2 substrate is positioned such that, in its uncleaved state, the NB interferes with binding of the AB to the target and, in its cleaved state, the NB does not interfere with binding of the AB to the target; and (4) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody.

[0375] In another embodiment, the method includes the steps of (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions conducive to expression of the activatable antibody, wherein the activatable antibody comprises (i) a binding partner (BP) for a non-binding steric moiety (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for the enzyme; (3) the CM1-CM2 substrate is positioned such that, in an uncleaved state and in the presence of the NB, the NB interferes with binding of the AB to the target, and in a cleaved state, the NB does not interfere with binding of the AB to the target, and the BP does not interfere with binding of the AB to the target; and (4) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody. According to some examples of this embodiment, the BP of the activatable antibody is bound to the NB.

[0376] Uses of activatable antibodies and conjugated activatable antibodies It will be understood that administration of therapeutic entities according to the present disclosure will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and the like. Numerous suitable formulations can be found in formularies known to all pharmacists: Blaug, Seymour, Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), especially Chapter 87. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., Lipofectin®), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures are suitable in treatment and therapy in accordance with the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation, and the formulation is physiologically compatible and acceptable to the route of administration.For additional information related to formulations, excipients, and carriers familiar to pharmacists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance," Regul. Toxicol Pharmacol. 32(2):210-8(2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals," Int. J. Pharm. 203(1-2):1-60(2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts," J Pharm Sci. 89(8):967-78(2000), Powell et al. "Compendium of excipients for parenteral formulations," PDA J Pharm Sci Technol. 52:238-311(1998), and references therein.

[0377] Therapeutic formulations of the present disclosure comprising a conjugated antibody, activatable antibody, and / or conjugated activatable antibody are used to prevent, treat, or otherwise ameliorate diseases or disorders associated with aberrant target expression and / or activity. For example, therapeutic formulations of the present disclosure comprising a conjugated antibody, activatable antibody, and / or conjugated activatable antibody are used to treat or otherwise ameliorate inflammation, inflammatory disorders, autoimmune diseases, and / or cancer or other neoplastic conditions. In some embodiments, the cancer is a solid tumor or hematopoietic malignancy in which the target is expressed. In some embodiments, the cancer is a solid tumor in which the target is expressed. In some embodiments, the cancer is a hematopoietic malignancy in which the target is expressed. In some embodiments, the target is expressed on parenchymal tissue (the portion of an organ or tissue that often carries out the function of the organ or tissue, e.g., in cancer). In some embodiments, the target is expressed on a cell, tissue, or organ. In some embodiments, the target is expressed on the stroma (i.e., the connective supporting framework of a cell, tissue, or organ). In some embodiments, the target is expressed on osteoblasts. In some embodiments, the target is expressed on endothelium (vasculature). In some embodiments, the target is expressed on cancer stem cells. In some embodiments, the agent to which the activatable antibody is conjugated is a microtubule inhibitor. In some embodiments, the agent to which the activatable antibody is conjugated is a nucleic acid damaging agent.

[0378] Efficacy of prevention, amelioration, or treatment is determined in association with any known method for diagnosing or treating a disease or disorder associated with target expression and / or activity, e.g., aberrant target expression and / or activity. Prolonging the survival of the subject, or otherwise slowing the progression of a disease or disorder associated with target expression and / or activity, e.g., aberrant target expression and / or activity, in the subject, indicates that the conjugated antibody, activatable antibody, and / or conjugated activatable antibody confers a clinical benefit.

[0379] The conjugated antibody, activatable antibody, and / or conjugated activatable antibody can be combined in the form of a pharmaceutical composition. Principles and considerations involved in the preparation of such compositions, and guidance for the selection of ingredients, are provided, for example, in the following references: Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0380] In some embodiments in which antibody fragments are used, the smallest fragment that specifically binds to the target protein binding domain is selected. For example, peptide molecules that retain the ability to bind the target protein sequence can be designed based on the variable region sequences of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). Formulations can also contain multiple active compounds necessary for the particular indication being treated, e.g., those compounds with complementary activities that, in some embodiments, do not adversely affect each other. In some embodiments, or in addition, the composition can include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0381] The active ingredient may be encapsulated in microcapsules, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsions, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, respectively.

[0382] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0383] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutanate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods.

[0384] In some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody comprise a detectable label. Intact antibodies or fragments thereof (e.g., Fab, scFv, or F(ab)2) are used. The term "labeled," with respect to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody with a fluorescently labeled secondary antibody and end-labeling of a DNA antibody with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to encompass tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, blood and fractions or components of blood, such as serum, plasma, or lymph, are included within the usage of the term "biological sample." That is, the detection methods of the present disclosure can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro te...

Claims

1. 1. An isolated polypeptide comprising a CM1-CM2 substrate comprising a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP), the N-terminus to C-terminus configuration of the CM1-CM2 substrate is CM1-CM2 or CM2-CM1; The isolated polypeptide, wherein the CM1-CM2 substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:483-490 and 515-522, and wherein at least one of the MMPs is MMP14.

2. 1. An isolated polypeptide comprising an antibody or antigen binding fragment thereof (AB) that binds to a target, and a CM1-CM2 substrate comprising a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP), the N-terminus to C-terminus configuration of the CM1-CM2 substrate is CM1-CM2 or CM2-CM1; The isolated polypeptide, wherein the CM1-CM2 substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:483-490 and 515-522, and wherein at least one of the MMPs is MMP14.

3. The isolated polypeptide of claim 2 , wherein the MMP, the SP, or both the MMP and the SP are co-localized in a tissue with the target.

4. The antigen-binding fragment thereof may be a Fab fragment, F(ab') ２ 4. The isolated polypeptide of claim 2 or 3, 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.

5. The AB is linked to the CM1, wherein: the AB is directly linked to the CM1; or The AB is linked to the CM1 via a connecting peptide. An isolated polypeptide according to any one of claims 2 to 4.

6. The AB is linked to the CM2, wherein: the AB is directly linked to CM2; or The AB is linked to the CM2 via a connecting peptide. An isolated polypeptide according to any one of claims 2 to 4.

7. The isolated polypeptide comprises a masking moiety (MM) and wherein: a. the MM has a dissociation constant for binding to the AB that is higher than the dissociation constant of the AB for binding to the target; and / or b. The MM is a polypeptide less than or equal to 40 amino acids in length. An isolated polypeptide according to any one of claims 2 to 6.

8. wherein the MM is linked to the CM1 such that the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB or AB-CM2-CM1-MM; a. the isolated polypeptide comprises a connecting peptide between the MM and the CM1; b. the isolated polypeptide comprises a connecting peptide between the CM2 and the AB; c. the isolated polypeptide comprises a connecting peptide between the CM1 and the CM2; and / or d. the isolated polypeptide comprises a third connecting peptide (LP3) between the CM1 and the CM2.

8. The isolated polypeptide of claim 7.

9. The MM is linked to the CM2 such that the isolated polypeptide comprises the following structural arrangement from N-terminus to C-terminus: MM-CM2-CM1-AB or AB-CM1-CM2-MM, and wherein: a. the isolated polypeptide comprises a connecting peptide between the MM and the CM2; b. the isolated polypeptide comprises a connecting peptide between the CM1 and the AB; c. the isolated polypeptide comprises a connecting peptide between the CM1 and the CM2; and / or d. the isolated polypeptide comprises a third connecting peptide (LP3) between the CM1 and the CM2; 8. The isolated polypeptide of claim 7.

10. The isolated polypeptide comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and wherein the isolated polypeptide has the following N-terminus to C-terminus structural arrangement: MM-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM, and wherein: a. LP1 and LP2 are not identical to each other; b. each of LP1 and LP2 is a peptide between 1 and 20 amino acids in length; and / or c. the isolated polypeptide comprises a third connecting peptide (LP3) between CM1 and CM2 8. The isolated polypeptide of claim 7.

11. 11. The isolated polypeptide of any one of claims 7 to 10, wherein the amino acid sequence of the MM is different from that of the target and is less than 10% identical to the amino acid sequence of the natural binding partner of the AB.

12. 12. The isolated polypeptide of any one of claims 7 to 11, wherein the MM, upon cleavage of the CM, does not interfere with or compete with the AB for binding to the target.

13. the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs:477 and 507-514, and a heavy chain amino acid sequence of SEQ ID NO:67; or The isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 472, 499-506, and 531-538, and a heavy chain amino acid sequence of SEQ ID NO:

108. An isolated polypeptide according to any one of claims 1 to 12.

14. A conjugate comprising an agent and an isolated polypeptide according to any one of claims 1 to 13 linked to said agent.

15. The conjugate of claim 14 , wherein the agent is attached to the AB via a linker.

16. The conjugate of claim 15, wherein the linker is a cleavable or non-cleavable linker.

17. the agent is a toxin or a fragment thereof; the agent is a microtubule inhibitor; the agent is a nucleic acid damaging agent; the agent is selected from the group consisting of dolastatin or a derivative thereof, auristatin or a derivative thereof, maytansinoid or a derivative thereof, duocarmycin or a derivative thereof, and calicheamicin or a derivative thereof; The agent is auristatin E or a derivative thereof; The agent is monomethyl auristatin E (MMAE). the agent is monomethyl auristatin D (MMAD); or the agent is a maytansinoid selected from the group consisting of DM1 and DM4; The conjugate of claim 14.

18. The conjugate of claim 14 , wherein the agent is a detectable moiety.

19. The conjugate of claim 18 , wherein the detectable moiety is a diagnostic agent.

20. A pharmaceutical composition comprising an isolated polypeptide according to any one of claims 1 to 13 or a conjugate according to any one of claims 14 to 19 and a carrier, and optionally an additional agent.

21. 21. The pharmaceutical composition of claim 20, wherein the additional agent is a therapeutic agent.

22. An isolated nucleic acid molecule encoding the isolated polypeptide of any one of claims 1 to 13.

23. 23. A vector comprising the isolated nucleic acid molecule of claim 22.

24. 23. A method for producing an isolated polypeptide by culturing a cell under conditions conducive to expression of the isolated polypeptide of any one of claims 1 to 13, wherein said cell comprises a nucleic acid molecule of claim 22.

25. 1. A method for producing an activatable antibody that binds to a target in an activated state, comprising: (a) culturing a cell comprising a nucleic acid construct encoding the isolated polypeptide of any one of claims 1 to 13; and (b) harvesting the activatable antibody; The method includes:

26. 14. The isolated polypeptide of any one of claims 1 to 13 for use in treating a disorder or disease, alleviating the symptoms of a disorder or disease, or delaying the progression of a disorder or disease.

27. 27. The isolated polypeptide of claim 26, wherein the disorder or disease is cancer.

28. The conjugate of any one of claims 14 to 19 for use in treating a disorder or disease, alleviating the symptoms of a disorder or disease, or delaying the progression of a disorder or disease.

29. 29. The conjugate of claim 28, wherein the disorder or disease is cancer.

30. 22. The pharmaceutical composition of claim 20 or 21 for use in treating a disorder or disease, alleviating the symptoms of a disorder or disease, or delaying the progression of a disorder or disease.

31. 31. The pharmaceutical composition of claim 30, wherein the disorder or disease is cancer.

32. 22. Use of an isolated polypeptide according to any one of claims 1 to 13, a conjugate according to any one of claims 14 to 19, or a pharmaceutical composition according to claim 20 or 21 in the manufacture of a medicament for treating a disorder or disease, alleviating symptoms of a disorder or disease, or delaying the progression of a disorder or disease.

33. 33. The use according to claim 32, wherein the disorder or disease is cancer.

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