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

Abstract

The present invention generally relates to polypeptides 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), to activatable antibodies and other larger molecules of these polypeptides comprising at least CM1 that is a substrate for at least one MM protease and at least CM2 that is a substrate for at least one SP protease, and to methods of making and using these polypeptides comprising at least CM1 that is a substrate for at least one MM protease and at least CM2 that is a substrate for at least one SP protease in various therapeutic, diagnostic and prophylactic indications.

Description

[0001] This application is a divisional application of an invention patent application with an application date of January 20, 2016, application number 201680016951.5, and invention name "Matrix metalloproteinase-cleavable and serine protease-cleavable substrates and methods of use thereof".

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 105,490, filed January 20, 2015, U.S. Provisional Application No. 62 / 258,015, filed November 20, 2015, and U.S. Provisional Application No. 62 / 278,713, filed January 14, 2016, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Incorporation of Sequence Listing

[0004] The contents of a text file named "CYTM037001WO_ST25" created on January 15, 2016 and 456 KB in size are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention generally relates to polypeptides 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), including activatable antibodies and other larger molecules of these polypeptides comprising at least CM1 that is a substrate for at least one MM protease and CM2 that is a substrate for at least one SP protease, and to methods of making and using these polypeptides comprising at least CM1 that is a substrate for at least one MM protease and CM2 that is a substrate for at least one SP protease in various therapeutic, diagnostic and prophylactic indications. Background Art

[0006] Proteases are enzymes that degrade proteins by cleaving the peptide bonds between the amino acid residues. Proteases are naturally present in all organisms and are involved in a variety of physiological responses ranging from simple degradation to highly regulated pathways. Some proteases are known to destroy specific peptide bonds based on the presence of specific amino acid sequences within proteins.

[0007] Therefore, there is a need to identify new substrates for proteases and to use these substrates in various therapeutic, diagnostic and preventive indications. Summary of the invention

[0008] The present invention provides amino acid sequences comprising at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloproteinase (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". The term is not intended to convey any requirements regarding the orientation or other structural arrangement of the first cleavable portion (CM1) that is a substrate for at least one matrix metalloproteinase (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 CM1-CM2 substrates having the following N-terminal to C-terminal structural arrangement: 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.

[0009] The CM1-CM2 substrates described herein can be used in a variety of therapeutic, diagnostic and preventive indications. For example, these CM1-CM2 substrates can be used in activatable antibodies, which include antibodies or antigen-binding fragments thereof (AB), which include at least one masking moiety (MM) connected to at least one antigen or epitope binding domain of the AB, such that the coupling of the MM reduces the ability of the AB to bind to its target.

[0010] In some embodiments, the 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 at least a portion of the CM2 sequence. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least one common amino acid residue. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least two common amino acid residues. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least three common amino acid residues. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share three or more common amino acid residues.

[0011] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together.

[0012] In some embodiments, CM1 and CM2 are separate polypeptides that are directly linked together, i.e., the N-terminus of one substrate 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.

[0013] In some embodiments, CM1 and CM2 are separate polypeptides operably linked together via at least one linking moiety.

[0014] In some embodiments, the first cleavable moiety CM1 and the second cleavable moiety CM2 in 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.

[0015] In some embodiments, the activatable antibody includes a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between a masking moiety (MM) and CM1. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM2 and AB. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between MM and CM1 and a connecting peptide (LP''') between CM2 and AB. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between MM and CM1 and a connecting peptide (LP'') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM1 and CM2 and a connecting peptide (LP''') between CM2 and AB. In some embodiments, the activatable antibody includes 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.

[0016] In some embodiments, the activatable antibody includes a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between AB and CM1. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM2 and a masking moiety (MM). In some embodiments, the activatable antibody includes a connecting peptide (LP'') between AB and CM1 and a connecting peptide (LP''') between CM2 and MM. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between AB and CM1 and a connecting peptide (LP'') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM1 and CM2 and a connecting peptide (LP''') between CM2 and MM. In some embodiments, the activatable antibody includes 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.

[0017] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0018] 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.

[0019] In some embodiments, CM1 is a substrate of MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11 and / or MMP19. In some embodiments, CM1 is a substrate of MMP2. In some embodiments, CM1 is a substrate of MMP9. In some embodiments, CM1 is a substrate of MMP14. In some embodiments, CM1 is a substrate of two or more MMPs. In some embodiments, CM1 is a substrate of at least MMP9 and MMP14. In some embodiments, CM1 is a substrate of at least MMP2 and MMP9. In some embodiments, CM1 is a substrate of at least MMP2 and MMP14. In some embodiments, CM1 is a substrate of three or more MMPs. In some embodiments, CM1 is a substrate of at least MMP2, MMP9 and MMP14. In some embodiments, CM1 comprises two or more substrates of 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.

[0020] In some embodiments, CM1 is a substrate for MMP and includes 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); LPGGLSPW (SEQ ID NO:361); MGLFSEAG (SEQ ID NO:362); SPLPLRVP (SEQ ID NO:363); RMHLRSLG (SEQ ID NO:364). 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).

[0021] 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 GPSHLVLT (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).

[0022] 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 known as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1), and combinations thereof. By way of non-limiting example, examples of other SPs that cleave CM2 described herein include activated protein C; cathepsin A; cathepsin G; chymotrypsin; coagulation factor proteases, such as, for example, FVIIa, FIXa, FXa, FXIa, FXIIa; elastase; granzyme B; Guanidinobenzoatase; HtrA1; human neutrophil elastase; lactoferrin; Marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine protease (TTSP), such as, for example, DESC1, DPP-4, FAP, Hepsin, Matriptase-2, TMPRSS2, TMPRSS3, and / or TMPRSS4.

[0023] For example, a suitable CM2 is cleaved by at least one serine protease and includes 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); LSGRSGNH (SEQ ID NO: 412); TSTSGRSANPRG (SEQ ID NO: 413); TSGRSANP (SEQ ID NO: 414); SGRSANPRG (SEQ ID NO: 415); 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).

[0024] 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).

[0025] In some embodiments, the CM1-CM2 substrates include 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); LSGRSDNHGGAVGLLAPP (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); 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).;

[0026] 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 ISSGLLSGRSDIH (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).

[0027] In some embodiments, the activatable antibody in the 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.

[0028] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the antibody in the uncleaved state has the following structural arrangement from N-terminus 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, LP1 and LP2 are each peptides of about 1-20 amino acids in length. In some embodiments, the two connecting peptides do not need to be identical to each other.

[0029] In some embodiments, the activatable antibody includes a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between a masking moiety (MM) and CM1. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM2 and AB. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between MM and CM1 and a connecting peptide (LP''') between CM2 and AB. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between MM and CM1 and a connecting peptide (LP'') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM1 and CM2 and a connecting peptide (LP''') between CM2 and AB. In some embodiments, the activatable antibody includes 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.

[0030] In some embodiments, the activatable antibody includes a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between AB and CM1. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM2 and a masking moiety (MM). In some embodiments, the activatable antibody includes a connecting peptide (LP'') between AB and CM1 and a connecting peptide (LP''') between CM2 and MM. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between AB and CM1 and a connecting peptide (LP'') between CM1 and CM2. In some embodiments, the activatable antibody includes a connecting peptide (LP'') between CM1 and CM2 and a connecting peptide (LP''') between CM2 and MM. In some embodiments, the activatable antibody includes 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.

[0031] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0032] 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), wherein n is an integer of at least 1.

[0033] 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).

[0034] 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).

[0035] 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).

[0036] In some embodiments, the AB has a dissociation constant for binding to a target of about 100 nM or less.

[0037] In some embodiments, the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target. In some embodiments, AB is a full-length antibody. In some embodiments, AB is an immunologically active fragment. In some embodiments, AB is an antigen-binding fragment. In some embodiments, AB is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab') 2 Fragments, scFv, scAb, dAb, single domain heavy chain antibodies or single domain light chain antibodies. In some embodiments, such AB is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.

[0038] In some embodiments, the MMP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease cleaves CM1 in the antibody. In some embodiments, the SP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the SP protease cleaves the CM2 substrate in the antibody. In some embodiments, the MMP protease and / or the SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease and / or the SP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and the SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, at least one of the MMP protease and the SP protease cleaves the CM1-CM2 substrate in the antibody.

[0039] In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate are each independently a polypeptide of up to 15 amino acids in length.

[0040] In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from 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 the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 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 the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 90% identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence having no more than 90% or greater identity to any human polypeptide sequence naturally cleaved by the same MMP protease.

[0041] 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 substantially different from 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 substantially different from 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 substantially different from 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 no more than 90% or more 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 no more than 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 having no more than 90% or greater identity to any human polypeptide sequence naturally cleaved by the same SP protease.

[0042] 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 contains 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 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 its target.

[0043] 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.

[0044] In some embodiments, CM1-CM2 is included in an activatable antibody having a light chain amino acid sequence selected from SEQ ID NO: 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.

[0045] In some embodiments, CM1-CM2 is included in an activatable antibody having a light chain amino acid sequence selected from SEQ ID NO: 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.

[0046] In some embodiments, the activatable antibody contains 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 contains 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.

[0047] In some embodiments, CM1-CM2 is included in an activatable antibody having a light chain amino acid sequence selected from SEQ ID NO: 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.

[0048] In some embodiments, CM1-CM2 is included in an activatable antibody having a light chain amino acid sequence selected from SEQ ID NO: 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.

[0049] In some embodiments, CM1-CM2 is included in an activatable antibody having a light chain amino acid sequence selected from SEQ ID NO: 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.

[0050] In some embodiments, the CM1-CM2 substrate is also a substrate for at least one additional protease.

[0051] In some embodiments, the at least one additional protease is a different MMP protease than the MMP protease that cleaves CM1. In some embodiments, the at least one additional protease is a 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.

[0052] In some embodiments, at least one additional protease is a SP protease different from the SP protease that cleaves CM2. In some embodiments, the at least one additional SP protease is selected from uPA; matriptase; activated protein C; cathepsin A; cathepsin G; chymotrypsin; coagulation factor proteases, such as, for example, FVIIa, FIXa, FXa, FXIa, FXIIa; elastase; granzyme B; Guanidinobenzoatase; HtrA1; human neutrophil elastase; lactoferrin; Marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine protease (TTSP), such as, for example, DESC1, DPP-4, FAP, Hepsin, Matriptase-2, TMPRSS2, TMPRSS3 and / or TMPRSS4.

[0053] In some embodiments, the at least one additional protease is selected from those shown in Table 6.

[0054] Table 6: Exemplary proteases and / or enzymes

[0055]

[0056] The present invention also provides an antibody comprising at least a first CM1 and a second CM2 and conjugated to a reagent. In some embodiments, the first CM1 and the second CM2 are each a polypeptide of no more than 15 amino acids in length. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from N-terminus to C-terminus in its uncut state: MM-CM1-CM2-AB-reagent, reagent-AB-CM2-CM1-MM, MM-CM2-CM1-AB-reagent or reagent-AB-CM1-CM2-MM. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from N-terminus to C-terminus in its uncut state: reagent-MM-CM1-CM2-AB, AB-CM2-CM1-MM-reagent, reagent-MM-CM2-CM1-AB or AB-CM1-CM2-MM-reagent. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from N-terminus to C-terminus in its uncleaved state: reagent-MM-CM1-CM2-AB-reagent, reagent-AB-CM2-CM1-MM-reagent, reagent-MM-CM2-CM1-AB-reagent, or reagent-AB-CM1-CM2-MM-reagent.

[0057] 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 the uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-CM2-LP2-AB-reagent, reagent-AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB-reagent, or reagent-AB-LP2-CM1-CM2-LP1-MM. In some embodiments, LP1 and LP2 are each peptides of about 1-20 amino acids in length. In some embodiments, the two connecting peptides do not need to be identical to each other.

[0058] 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 the uncleaved state has the following structural arrangement from N-terminus to C-terminus: reagent-MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM-reagent, reagent-MM1-LP1-CM2-CM1-LP2-AB or AB-LP2-CM1-CM2-LP1-MM-reagent. In some embodiments, LP1 and LP2 are each peptides of about 1-20 amino acids in length. In some embodiments, the two connecting peptides do not need to be identical to each other.

[0059] 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 the uncleaved state has the following structural arrangement from N-terminus to C-terminus: reagent-MM1-LP1-CM1-CM2-LP2-AB-reagent, reagent-AB-LP2-CM2-CM1-LP1-MM-reagent, reagent-MM1-LP1-CM2-CM1-LP2-AB-reagent, or reagent-AB-LP2-CM1-CM2-LP1-MM-reagent. In some embodiments, LP1 and LP2 are each peptides of about 1-20 amino acids in length. In some embodiments, the two connecting peptides do not need to be identical to each other.

[0060] In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between the masking moiety (MM) and CM1. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between CM2 and AB. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between MM and CM1 and a connecting peptide (LP''') between CM2 and AB. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between MM and CM1 and a connecting peptide (LP'') between CM1 and CM2. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between CM1 and CM2 and a connecting peptide (LP''') between CM2 and AB. In some embodiments, the conjugated activatable antibody includes 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.

[0061] In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between AB and CM1. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between CM2 and a masking moiety (MM). In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between AB and CM1 and a connecting peptide (LP''') between CM2 and MM. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between AB and CM1 and a connecting peptide (LP'') between CM1 and CM2. In some embodiments, the conjugated activatable antibody includes a connecting peptide (LP'') between CM1 and CM2 and a connecting peptide (LP''') between CM2 and MM. In some embodiments, the conjugated activatable antibody includes 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.

[0062] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0063] 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), wherein n is an integer of at least 1.

[0064] 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).

[0065] 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).

[0066] 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).

[0067] 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 given target, such that when exposed 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 shown in Table 1. Exemplary ABs include, but are not limited to, the antibodies shown in Table 2.

[0068] In some embodiments, the AB has a dissociation constant for binding to a target of about 100 nM or less.

[0069] In some embodiments, the antibody includes an antibody or an antigen-binding fragment thereof that specifically binds to a target. In some embodiments, the antibody or immunologically active fragment thereof that binds to a target is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab') 2Fragments, scFv, scab, dAb, single domain heavy chain antibodies or single domain light chain antibodies. In some embodiments, such target-binding antibodies or immunologically active fragments thereof are mouse, other rodent, chimeric, humanized or fully human monoclonal antibodies.

[0070] In some embodiments, the MM binds to the AB with a dissociation constant that is no greater than the dissociation constant of the AB to the target.

[0071] In some embodiments, in the cleaved state, the MM does not interfere with or compete with the AB for binding to the target.

[0072] In some embodiments, the MM is a polypeptide of about 2-40 amino acids in length. For example, the MM is a polypeptide of up to about 40 amino acids in length.

[0073] In some embodiments, the MM polypeptide sequence is different from the polypeptide sequence of any natural binding partner of AB. In some embodiments, the MM polypeptide sequence has no more than 50% identity with any natural binding partner of AB. In some embodiments, the MM polypeptide sequence has no more than 40%, 30%, 25%, 20%, 15% or 10% identity with any natural binding partner of AB.

[0074] In some embodiments, the reagent conjugated to AB or AB of an activatable antibody is a therapeutic agent. In some embodiments, the reagent is an antitumor agent. In some embodiments, the reagent is a toxin or a fragment thereof. As used herein, a fragment of a toxin is a fragment that retains toxic activity. In some embodiments, the reagent is conjugated to AB via a cleavable linker. In some embodiments, the reagent is conjugated to AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the reagent is conjugated to AB via a non-cleavable linker. In some embodiments, the reagent is a microtubule inhibitor. In some embodiments, the reagent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the reagent is selected from the reagents listed in Table 3. In some embodiments, the reagent is dolastatin. In some embodiments, the reagent is auristatin or a derivative thereof. In some embodiments, the reagent is auristatin E or a derivative thereof. In some embodiments, the reagent is monomethyl auristatin E (MMAE). In some embodiments, the reagent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

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

[0076] In some embodiments, the antibody and / or activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0077] In some embodiments, the conjugated antibody and / or the conjugated activatable antibody includes a detectable label. In some embodiments, the detectable label includes 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 radioactive isotope. In some embodiments, the radioactive isotope is indium or technetium. In some embodiments, the contrast agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or beta-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 europium derivatives. In some embodiments, the luminescent label comprises an N-methylacrydium derivative. In some embodiments, the label comprises AlexaFluor ® Labels, 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.

[0078] In some embodiments, the antibody and / or AB of the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to include one or more disulfide bonds.

[0079] In some embodiments, antibodies and / or conjugated antibodies are monospecific. In some embodiments, antibodies and / or conjugated antibodies are multispecific, referred to herein as multispecific antibodies and / or conjugated multispecific antibodies. In some embodiments, multispecific antibodies and / or conjugated multispecific antibodies are bispecific or trifunctional. In some embodiments, antibodies and / or conjugated antibodies are formulated as part of a former bispecific T-cell engager (former BITE) molecule. In some embodiments, antibodies and / or conjugated antibodies are formulated as part of a former chimeric antigen receptor (former CAR) modified T-cell or other engineered receptor or other immune effector cell, such as a part of a CAR modified NK cell. In some embodiments, activatable antibodies and / or conjugated activatable antibodies are formulated as part of a former chimeric antigen receptor (CAR) modified T-cell. In some embodiments, activatable antibodies and / or conjugated activatable antibodies are formulated as part of a former chimeric antigen receptor (CAR) modified NK cell.

[0080] In some embodiments, the activatable antibody and / or the conjugated activatable antibody is monospecific. In some embodiments, the activatable antibody and / or the conjugated activatable antibody is multispecific, referred to herein as a multispecific activatable antibody and / or a conjugated multispecific activatable antibody. As used herein, unless otherwise specified, terms such as "activatable antibody" and all grammatical variants thereof are intended to encompass, but not limited to, embodiments in which the activatable antibody is a multispecific activatable antibody of the present invention. As used herein, unless otherwise specified, terms such as "conjugated activatable antibody" and all grammatical variants thereof are intended to encompass, but not limited to, embodiments in which the conjugated activatable antibody is a multispecific activatable antibody of the present invention. In some embodiments, the multispecific activatable antibody and / or the conjugated multispecific activatable antibody is bispecific or trifunctional. In some embodiments, the activatable antibody and / or the conjugated activatable antibody is formulated as a part of the former bispecific T-cell engager (former BITE) molecule. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pre-chimeric antigen receptor (pre-CAR) modified T-cell or other engineered receptor.

[0081] 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 combination with one or more additional agents or combinations of additional agents. Suitable additional agents include current drugs and / or surgical therapies for the intended application, such as, for example, cancer. For example, activatable antibodies, conjugated activatable antibodies, multispecific activatable antibodies, and / or conjugated multispecific activatable antibodies can be used in combination with additional chemotherapeutic agents or anti-tumor agents.

[0082] In some embodiments, the activatable antibody is a multispecific activatable antibody. The multispecific activatable antibody provided herein is such a multispecific antibody that recognizes two or more different antigens or epitopes and includes at least one masking portion (MM) that is connected to at least one antigen or epitope binding domain of the multispecific antibody, so that the coupling of the MM reduces the ability of the antigen or epitope binding domain to bind to 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 acts as a substrate for at least one MMP protease and at least one SP protease. The activatable multispecific antibodies provided herein are stable in circulation, activated at the intended treatment and / or diagnosis site, but not activated in normal (ie, healthy) tissues, and when activated, exhibit binding to the target at least comparable to the corresponding unmodified multispecific antibody.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a Jagged target, e.g., Jagged 1 and / or Jagged 2, and contains 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 VHCDR3 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); and a VH CDR3 sequence comprising at least the amino acid sequence DIGGRSAFDY (SEQ ID NO: 400), and combinations thereof.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1), which specifically binds to a Jagged target, for example, Jagged 1 and / or Jagged 2, and contains 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 VLCDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RASQSISSY (SEQ ID NO: 401); a VL CDR2 sequence comprising at least the amino acid sequence AASSLQS (SEQ ID NO: 402); a VL CDR3 sequence comprising at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 403), and combinations thereof.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a Jagged target, e.g., Jagged 1 and / or Jagged 2, and contains 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 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 VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 399); CDR2 sequences; VHCDR3 sequences comprising sequences that are 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.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a Jagged target, e.g., Jagged 1 and / or Jagged 2, and contains 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 VL CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSISSY (SEQ ID NO: 401); a VL CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQ ID NO: 402); CDR2 sequences; and VL CDR3 sequences 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.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a Jagged target, e.g., Jagged 1 and / or Jagged 2, and contains 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 includes at least the amino acid sequence SYAMS (SEQ ID NO: 398); the VH CDR2 sequence includes at least the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 399); the VH CDR3 sequence includes at least the amino acid sequence DIGGRSAFDY (SEQ ID NO: 400); the VL CDR1 sequence includes at least the amino acid sequence RASQSISSY (SEQ ID NO: 401); the VL CDR2 sequence includes at least the amino acid sequence AASSLQS (SEQ ID NO: 402); NO:402); and the VL CDR3 sequence comprises at least the amino acid sequence QQTVVAPPL (SEQ ID NO:403).

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a Jagged target, e.g., Jagged 1 and / or Jagged 2, and contains 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 VH 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 SIDPEGRQTYYADSVKG (SEQ ID NO: 399); CDR3 sequences include sequences that are 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); VL CDR1 sequences include sequences that are 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); VL CDR2 sequences include sequences that are 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 VLCDR3 sequences include sequences that are 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).

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1), which specifically binds to epidermal growth factor receptor (EGFR) and contains a combination of a VH CDR1 sequence, a VHCDR2 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 including at least the amino acid sequence NYGVH (SEQ ID NO: 404); a VH CDR2 sequence including at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 405); a VH CDR3 sequence including at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 406); and combinations thereof.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1), which specifically binds to EGFR and contains 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 VL CDR1 sequence comprising at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 407); a VL CDR2 sequence comprising at least the amino acid sequence KYASESIS (SEQID NO: 408); and a VLCDR3 sequence comprising at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 409), and combinations thereof.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains 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 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 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 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 ALTYYDYEFAY (SEQ ID NO: 406). NO:406) VH CDR3 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity; and combinations thereof.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains 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 VL CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 407); a VL 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); and a VL 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 QQNNNWPTT (SEQ ID NO: NO:409) VL CDR3 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and combinations thereof.

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1), which specifically binds to EGFR and contains 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 includes at least the amino acid sequence NYGVH (SEQ ID NO: 404); the VH CDR2 sequence includes at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 405); the VH CDR3 sequence includes at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 406); the VL CDR1 sequence includes at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 407); the VL CDR2 sequence includes at least the amino acid sequence KYASESIS (SEQ ID NO: 408); and the VL The CDR3 sequence includes at least the amino acid sequence QQNNNWPTT (SEQ ID NO:409).

[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 invention, include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains 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 VH 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 VIWSGGNTDYNTPFTS (SEQ ID NO: 405); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 406). the VL CDR2 sequences include sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of KYASESIS (SEQ ID NO:408); and the VL CDR3 sequences include sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of QQNNNWPTT (SEQ ID NO:409).

[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 invention, contain a CM1-CM2 substrate comprising an amino acid sequence selected from SEQ ID NO: 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 invention, contain a CM1-CM2 substrate comprising an amino acid sequence selected from 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.

[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 invention, include at least a heavy chain amino acid sequence of SEQ ID NO: 67 and a light chain amino acid sequence selected from SEQ ID NO: 420, 422, 424, 426, 428, 430, 432, 434, 436, 439, 477, 479, 507-514, 539-546, 561 and 562.

[0097] 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 invention, contain a CM1-CM2 substrate comprising an amino acid sequence selected from SEQ ID NO: 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 invention, contain a CM1-CM2 substrate comprising an amino acid sequence selected from 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.

[0098] 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 invention, include at least a heavy chain amino acid sequence of SEQ ID NO: 108 and a light chain amino acid sequence selected from SEQ ID NO: 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 472, 474, 499-506, 531-538, 559 and 560.

[0099] 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 invention, include 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 the amino acid sequence of SEQ ID NO: 420, 422, 424, 426, 428, 430, 432, 434, 436, 439, 477, 479, 507-514, 539-546, 561, and 562.

[0100] 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 invention, include 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 the amino acid sequence of SEQ ID NO: 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 472, 474, 499-506, 531-538, 559 and 560.

[0101] In some embodiments, the activatable antibody further comprises 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 intercalating agent, 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 including at least one CM1-CM2 substrate sequence. In some embodiments, the agent is selected from the agents listed in Table 3. In some embodiments, the agent is dolastatin. 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 monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansine alkaloid or a maytansine alkaloid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0102] In some embodiments, the agent is an anti-inflammatory agent.

[0103] In some embodiments, the activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0104] In some embodiments, the conjugated antibody includes a detectable label. In some embodiments, the detectable label includes 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 radioactive isotope. In some embodiments, the radioactive isotope is indium or technetium. In some embodiments, the contrast agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or beta-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 europium derivatives. In some embodiments, the luminescent label comprises an N-methylacrydium derivative. In some embodiments, the label comprises Alexa Fluor ® Tags, such as AlexFluor ® 680 or Alexa Fluor ® 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0105] In some embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is directly connected to the MM of the activatable antibody. In some embodiments, the spacer is directly connected to the MM of the activatable antibody in a structural arrangement of the N-terminus to the C-terminus of the spacer-MM-CM1-CM2 substrate-AB. An example of a spacer directly connected to the N-terminus of the MM of the activatable antibody is an amino acid sequence selected from the following: QGQSGQ (SEQID 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 includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 410). In some embodiments, the spacer includes at least the amino acid sequence GQSGQ (SEQ ID NO: 416). In some embodiments, the spacer includes at least the amino acid sequence QSGQ (SEQ ID NO: 417). In some embodiments, the spacer comprises at least the amino acid sequence SGQ (SEQ ID NO: 418). In some embodiments, the spacer comprises at least the amino acid sequence GQ. In some embodiments, the spacer comprises at least the amino acid sequence Q.

[0106] In some embodiments, the AB of the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to include one or more disulfide bonds.

[0107] In some embodiments, the serum half-life of the activatable antibody is longer than the serum half-life of the corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 15 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 11 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 9 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 7 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 6 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 5 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 4 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 3 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 2 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 24 hours. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 20 hours. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 18 hours. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 16 hours. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 14 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 6 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 4 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 3 hours.

[0108] The present invention also provides compositions and methods including activatable antibodies, which include antibodies or antibody fragments (AB) that specifically bind to a given target, wherein AB is coupled to a masking portion (MM) that reduces the ability of AB1 to bind to its target. In some embodiments, the activatable antibody also includes a CM1-CM2 substrate that is a substrate for at least one MMP and at least one SP. The compositions and methods provided herein enable one or more reagents to be connected to one or more cysteine ​​residues in 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 enable one or more reagents to be connected to one or more cysteine ​​residues in AB without reducing or otherwise interfering with one or more disulfide bonds within MM. The compositions and methods provided herein produce activatable antibodies that are conjugated to one or more reagents, such as any of a variety of therapeutic agents, diagnostic agents and / or prophylactic agents, for example, in some embodiments, no reagent is conjugated to the MM of the activatable antibody. The compositions and methods provided herein produce conjugated activatable antibodies, wherein the MM retains the ability to effectively and effectively mask the AB of the activatable antibody in the uncut state. The compositions and methods provided herein produce a conjugated activatable antibody, wherein the activatable antibody is still activated, ie, cleaves, in the presence of a MMP that can cleave a CM1-CM2 substrate.

[0109] The activatable antibody has at least one conjugation point for the agent, but in the methods and compositions provided herein, less than all possible conjugation points can be used for conjugation with the agent. In some embodiments, one or more conjugation points are sulfur atoms involved in disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain sulfide bonds, but not sulfur atoms involved in intrachain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. Such residues may be naturally present in the antibody structure, or may be incorporated into the antibody by site-directed mutagenesis, chemical conversion, or mis-incorporation of non-natural amino acids.

[0110] Also provided is a method for preparing a conjugate of an activatable antibody having one or more interchain disulfide bonds in AB and one or more intrachain disulfide bonds in MM, and providing a drug that reacts with free thiols. The method generally includes partially reducing the interchain disulfide bonds in the activatable antibody with a reducing agent (such as, for example, TCEP); and conjugating the drug that reacts with the free thiol groups to the partially reduced activatable antibody. As used herein, the term partially reduced refers to a situation in which the activatable antibody is contacted with a reducing agent and less than all disulfide bonds, such as less than all possible conjugation 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 less than 5% of all possible conjugation sites are reduced.

[0111] In some embodiments, a method for reducing and conjugating a reagent, such as a drug, to an activatable antibody, resulting in a selective method for placing the reagent, is provided. The method generally includes partially reducing the activatable antibody with a reducing agent so that any conjugation site in the masking portion or other non-AB portion of the activatable antibody is not reduced, and the reagent is conjugated to the interchain thiol in the AB. The conjugation site is selected to allow the desired placement of the reagent to allow conjugation to occur at the desired site. The reducing agent is, for example, TCEP. The reduction reaction conditions are identified by identifying the conditions for producing a conjugated activatable antibody in which the MM retains the ability to effectively and effectively mask the AB of the activatable antibody in the uncut state, such as the ratio of the reducing agent to the activatable antibody, the incubation time, the temperature during the incubation, the pH of the reduction reaction solution, etc. The ratio of the reducing agent to the activatable antibody will vary depending on the activatable antibody. In some embodiments, the ratio of reducing agent to activatable antibody will range from about 20: 1 to 1 : 1, about 10: 1 to 1 : 1, about 9: 1 to 1 : 1, about 8: 1 to 1 : 1, about 7: 1 to 1 : 1, about 6: 1 to 1 : 1, about 5: 1 to 1 : 1, about 4: 1 to 1 : 1, about 3: 1 to 1 : 1, about 2: 1 to 1 : 1, about 20: 1 to 1 : 1.5, about 10: 1 to 1 : 1.5, about 9: 1 to 1 : 1.5, about 8: 1 to 1 : 1.5, about 7: 1 to 1 : 1.5, about 6: 1 to 1 : 1.5, about 5: 1 to 1 : 1.5, about 4: 1 to 1 : 1.5, about 3: 1 to 1 : 1.5, about 2: 1 to 1 : 1.5, about 1.5: 1 to 1 : 1.5, or about 1 : 1 to 1 : 1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0112] In some embodiments, a method is provided for reducing the interchain disulfide bonds in the AB of an activatable antibody and conjugating an agent (e.g., a thiol-containing agent, such as a drug) to the resulting interchain thiol to selectively position the agent on the AB. The method generally comprises partially reducing the AB with a reducing agent to form at least two interchain thiols without forming all possible interchain thiols in the activatable antibody; and conjugating the agent to the interchain thiol of the partially reduced AB. For example, the AB of the activatable antibody is partially reduced at about 37° C. for about 1 hour at a desired ratio of reducing agent: activatable antibody. In some embodiments, the ratio of reducing agent to activatable antibody will range from about 20: 1 to 1 : 1, about 10: 1 to 1 : 1, about 9: 1 to 1 : 1, about 8: 1 to 1 : 1, about 7: 1 to 1 : 1, about 6: 1 to 1 : 1, about 5: 1 to 1 : 1, about 4: 1 to 1 : 1, about 3: 1 to 1 : 1, about 2: 1 to 1 : 1, about 20: 1 to 1 : 1.5, about 10: 1 to 1 : 1.5, about 9: 1 to 1 : 1.5, about 8: 1 to 1 : 1.5, about 7: 1 to 1 : 1.5, about 6: 1 to 1 : 1.5, about 5: 1 to 1 : 1.5, about 4: 1 to 1 : 1.5, about 3: 1 to 1 : 1.5, about 2: 1 to 1 : 1.5, about 1.5: 1 to 1 : 1.5, or about 1 : 1 to 1 : 1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0113] 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 using, for example, column chromatography, dialysis, or diafiltration. In some embodiments, the reduced antibody is not purified after partial reduction and prior to conjugation.

[0114] The present invention also provides a partially reduced activatable antibody, wherein at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without interfering with any intrachain disulfide bond in the activatable antibody, wherein the activatable antibody includes an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking portion (MM) that inhibits the binding of the AB of the activatable antibody in an uncut state to the target, and a CM1-CM2 substrate coupled to the AB, wherein the CM1-CM2 substrate is a polypeptide that acts as a substrate for at least one MMP and one SP. In some embodiments, the MM is coupled to the AB via a CM1-CM2 substrate. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disturbed by a reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM in the activatable antibody are not disturbed by a reducing agent. In some embodiments, the activatable antibody in an uncut state has the following N-terminal to C-terminal structural arrangement: MM-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0115] The present invention also provides partially reduced activatable antibodies, including but not limited to the multispecific activatable antibodies of the present invention, wherein at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without interfering with or otherwise impairing the activity and / or efficacy of the activatable antibody, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking portion (MM) that inhibits the binding of the AB of the activatable antibody in an uncut state to the target, and a CM1-CM2 substrate coupled to the AB, and the CM1-CM2 substrate is a polypeptide that acts as a substrate for a protease. By way of non-limiting example, the activity and / or efficacy of the activatable antibody is the masking activity, activation, and / or binding activity of the activated activatable antibody. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not interfered with by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM within the activatable antibody are not interfered with by the reducing agent. In some embodiments, the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2substrate-AB or AB-CM1-CM2substrate-MM. In some embodiments, the reducing agent is TCEP.

[0116] The present invention 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 portion (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, and a CM1-CM2 substrate coupled to the AB, and wherein the CM1-CM2 substrate is a polypeptide that acts as a substrate for at least one MMP protease and at least one SP protease.

[0117] In some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any of several methods for attaching the agent to AB: (a) to the carbohydrate portion of AB, or (b) to the sulfhydryl group of AB, or (c) to the amino group of AB, or (d) to the carboxylate group of AB.

[0118] In some embodiments, the MMAD payload is conjugated to AB via a linker. In some embodiments, the MMAD payload is conjugated to cysteine ​​in AB via a linker. In some embodiments, the MMAD payload is conjugated to lysine in AB via a linker. In some embodiments, the MMAD payload is conjugated to another residue in AB (such as those disclosed herein) via a linker. 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 linkers shown in Tables 5 and 6. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidoPEG-valine-citrulline-p-aminobenzyloxycarbonyl linker. In some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation techniques disclosed herein.

[0119] The present invention also provides polypeptides and other larger molecules including one or more of the CM1-CM2 substrate sequences provided herein. As non-limiting examples, the CM1-CM2 substrate sequences provided herein can be used in prodrug compositions and methods of use thereof. These CM1-CM2 substrate sequences provided herein can also be used in probes and other detection agents and methods of use thereof. For example, the CM1-CM2 substrate sequences provided herein can be used in conjunction with fluorescence and other quenchers to produce detection agents, such as imaging agents and / or other diagnostic agents. Those of ordinary skill in the art will appreciate that the CM1-CM2 substrate sequences provided herein can be used in any composition and / or method of the substrate that can be cut by at least one MMP and at least one SP in the art.

[0120] The present invention also provides isolated nucleic acid molecules encoding antibodies and / or activatable antibodies as described herein, and vectors comprising these isolated nucleic acid sequences. The present invention provides methods for producing antibodies and / or activatable antibodies by culturing cells under conditions leading to expression of antibodies and / or activatable antibodies, wherein the cells comprise such vectors.

[0121] The present invention provides a method for making a conjugated antibody of the present invention that binds a given target by: (a) culturing a cell comprising a nucleic acid construct encoding the antibody under conditions resulting in expression of the antibody, (i) wherein the antibody comprises a CM1-CM2 substrate, and (ii) wherein the CM1-CM2 substrate is a polypeptide that functions as a substrate for matrix metalloproteinases and serine proteases; (b) recovering the antibody; and (c) conjugating the recovered antibody to one or more additional agents.

[0122] The present invention also provides a method for producing an activatable antibody of the present invention that binds to a given target in an activated state by: (a) culturing a cell comprising a nucleic acid construct encoding the activatable antibody under conditions that result in 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 to a target, (i) wherein the CM1-CM2 substrate is a polypeptide that functions as a substrate for MMPs and SPs; and (ii) wherein the CM1-CM2 substrate is located in the activatable antibody such that in the uncleaved state, the MM interferes with the specific binding of the AB to the target, and in the cleaved state, the MM does not interfere with or compete with the specific binding of the AB to the target; and (b) recovering the activatable antibody.

[0123] The present invention also provides a method for producing a conjugated activatable antibody of the present invention that binds to a given target in an activated state by: (a) culturing a cell comprising a nucleic acid construct encoding the activatable antibody under conditions that result in 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 to a target, (i) wherein the CM1-CM2 substrate is a polypeptide that functions as a substrate for MMPs and SPs; and (ii) wherein the CM1-CM2 substrate is located in the activatable antibody such that in the uncleaved state, the MM interferes with the specific binding of the AB to the target, and in the 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) conjugating the recovered antibody to one or more additional agents.

[0124] The present invention provides methods for preventing, delaying progression of, treating, alleviating symptoms of, or otherwise ameliorating a target-associated disease in a subject by administering a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody described herein to a subject in need thereof.

[0125] The present invention provides a method for preventing inflammation and / or inflammatory conditions, delaying the progression of inflammation and / or inflammatory conditions, treating inflammation and / or inflammatory conditions, alleviating the symptoms of inflammation and / or inflammatory conditions, or otherwise improving inflammation and / or inflammatory conditions in a subject by administering a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody as described herein to a subject in need. The present invention also provides a method for preventing cancer, delaying the progression of cancer, treating cancer, alleviating the symptoms of cancer, or otherwise improving cancer in a subject by administering a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody as described herein to a subject in need. The present invention also provides a method for preventing autoimmune diseases, delaying the progression of autoimmune diseases, treating autoimmune diseases, alleviating the symptoms of autoimmune diseases, or otherwise improving autoimmune diseases in a subject by administering a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody as described herein to a subject in need.

[0126] The conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies used in any embodiment of these methods and uses 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 metastasis). The terms subject and patient are used interchangeably herein.

[0127] In some embodiments, the subject is a mammal, such as a human, a non-human primate, a companion animal (e.g., a cat, a dog, a horse), a livestock, a farm animal, or a zoo animal. In some embodiments, the subject is a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal under veterinary care.

[0128] The conjugated antibody, activatable antibody and / or conjugated activatable antibody and its therapeutic preparation are applied to a subject suffering from or susceptible to abnormal target expression and / or activity-related diseases or conditions.Use any of various methods known in the art to identify a subject suffering from or susceptible to abnormal target expression and / or activity-related diseases or conditions.For example, use any of various clinical and / or laboratory tests (such as physical examination and blood, urine and / or fecal analysis) to identify a subject suffering from cancer or other tumor conditions to assess health status.For example, use any of various clinical and / or laboratory tests (such as physical examination and / or body fluid analysis, such as blood, urine and / or fecal analysis) to identify a subject suffering from inflammation and / or inflammatory conditions to assess health status.

[0129] If any of the various laboratory or clinical goals are achieved, the conjugated antibody, activatable antibody and / or conjugated activatable antibody is applied to a patient suffering from a disease or condition associated with abnormal target expression and / or activity and is considered successful. For example, if one or more of the symptoms associated with the disease or condition are alleviated, reduced, suppressed or do not progress to a further (i.e., worse) state, the conjugated antibody, activatable antibody and / or conjugated activatable antibody is applied to a patient suffering from a disease or condition associated with abnormal target expression and / or activity and is considered successful. If the disease or condition enters remission or does not progress to a further (i.e., worse) state, the conjugated antibody, activatable antibody and / or conjugated activatable antibody is applied to a patient suffering from a disease or condition associated with abnormal target expression and / or activity and is considered successful.

[0130] In some embodiments, the antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies described herein are used in combination with one or more additional agents or combinations of additional agents. Suitable additional agents include current drugs and / or surgical therapies for the intended application, such as, for example, cancer. For example, antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies can be used in combination with additional chemotherapeutic agents or anti-tumor agents.

[0131] In some embodiments, the additional agent is a chemotherapeutic agent, such as a chemotherapeutic agent selected from docetaxel, paclitaxel, abraxane (ie, albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0132] In some embodiments, the additional agent is a checkpoint inhibitor, a kinase inhibitor, an agent targeting an inhibitor in the tumor microenvironment, and / or a T-cell or NK agonist. In some embodiments, the additional agent is radiotherapy alone or in combination with other additional agents such as chemotherapeutic agents or anti-tumor agents. In some embodiments, the additional agent is a vaccine, a tumor virus, and / or a DC-activator, such as, by way of non-limiting example, a toll-like receptor (TLR) agonist and / or α-CD40. In some embodiments, the additional agent is a tumor-targeting antibody designed to kill tumors via ADCC or via direct conjugation with a toxin (e.g., an antibody drug conjugate (ADC)).

[0133] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from CTLA-4, LAG-3, PD-1, PD-1, TIGIT, TIM-3, B7H4, BTLA and Vista. In some embodiments, the kinase inhibitor is selected from 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 IDO inhibitors, α-CSF1R inhibitors, α-CCR4 inhibitors, TGF-β, bone marrow-derived suppressor cells or T regulatory cells. In some embodiments, the agonist is selected from Ox40, GITR, CD137, ICOS, CD27 and HVEM.

[0134] 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 bone marrow-derived suppressor cell. In some embodiments, the inhibitor is a T regulatory cell.

[0135] 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.

[0136] In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody are administered in combination with one or more additional agents during and / or after treatment, such as, for example, chemotherapeutic agents, anti-inflammatory agents and / or immunosuppressants. In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agents are formulated in a single therapeutic composition, and the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agents are administered simultaneously. Alternatively, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agents are separated from each other, for example, each is formulated in a separate therapeutic composition, and the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agents are administered simultaneously, or the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agents are administered at different times during the treatment regimen. For example, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody is administered before the additional agent is administered, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody is administered after the additional agent is administered, or the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent are administered in an alternating manner. As described herein, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent are administered in a single dose or multiple doses.

[0137] In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent are administered simultaneously. For example, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent are administered sequentially, or the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent are administered at different times during the treatment regimen.

[0138] In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody is administered in combination with one or more additional agents during and / or after treatment, such as (by way of 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 chemotherapy, 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 immunomodulator, such as lenolidominde or IL-2. In some embodiments, the additional agent is a radiation agent. In some embodiments, the additional agent is an agent that is considered to be a 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.

[0139] 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 to 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 to a target different from the target of the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody.

[0140] In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent are administered simultaneously. For example, the conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody and additional agent are administered sequentially, or the antibody and / or conjugated antibody and additional agent are administered at different times during the treatment regimen. For example, the antibody and / or conjugated antibody is administered before the additional agent is administered, the antibody and / or conjugated antibody is administered after the additional agent is administered, or the antibody and / or conjugated antibody and additional agent are administered in an alternating manner. As described herein, the antibody and / or conjugated antibody and additional agent are administered in a single dose or multiple doses.

[0141] The invention also provides methods and kits for using the conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies in various diagnostic and / or prophylactic indications.

[0142] The pharmaceutical composition according to the invention may comprise an antibody, a conjugated antibody, an activatable antibody and / or a conjugated activatable antibody of the invention and a carrier. These pharmaceutical compositions may be included in a kit such as, for example, a diagnostic kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0143] Figure 1A and 1B is a series of graphs depicting the results of human Jagged 1 binding ELISA assays demonstrating binding of anti-Jagged antibodies and masked and activated activatable antibodies. Activatable antibodies containing (A) 2001 and (B) 1001 / LP' / 0001 substrates were activated by uPA (serine protease), MMP14 (MMP), and a combination of uPA and MMP14. The activated activatable antibodies showed equivalent binding to the anti-Jagged antibodies.

[0144] Figure 2 is a graph depicting human IgG serum concentrations of anti-Jagged antibody and 1001 / LP' / 0001 and 2001 activatable antibodies after a single 5 mg / kg intravenous dose. Anti-Jagged antibody is rapidly cleared due to target-mediated clearance. In contrast, anti-Jagged activatable antibody remains sequestered in the circulation.

[0145] Figure 3is a graph depicting HCC1806 tumor volume (group mean ± SEM n = 8) plotted against time after initial dose. Groups were dosed on day 1 and day 8 of the study. Both the anti-Jagged-SPDB-DM4 antibody and anti-Jagged 2001 activatable antibody-SPDB-DM4 groups showed tumor regression, while the isotype-SPDB-DM4 group showed no tumor growth inhibition.

[0146] Figure 4 is a graph depicting the percentage of initial body weight (group mean n = 8) plotted against time after initial dose. Groups were dosed on day 1 and day 8 of the study. Animals in the anti-Jagged-SPDB-DM4 ADC treated group showed significant weight loss, while animals treated with PBS, isotype-SPDB-DM4 ADC, and anti-Jagged 2001 activatable antibody-SPDB-DM4 showed no significant weight loss.

[0147] Figure 5 is a graph depicting in vivo activation of EGFR activatable antibody measured in plasma within 8 days following a dose of 12.5 mg / kg of such EGFR activatable antibody in H292 xenograft tumor-bearing mice.

[0148] Figure 6 is a graph depicting the in vivo efficacy of EGFR activating antibodies in H292 xenograft tumor-bearing mice.

[0149] Figure 7 is a graph depicting in situ assessment of EGFR activatable antibody activation in the H292 xenograft tumor microenvironment.

[0150] Fig. 8A , 8B , 8C, 8D, 8E and 8F are depictions of the use of control intravenous immunoglobulin (IVIG, Fig. 8A ), anti-EGFR antibody cetuximab ( Figure 8B ), an anti-EGFR activatable antibody referred to herein as an anti-EGFR 2001 activatable antibody (which includes a heavy chain sequence of SEQ ID NO: 108 and a light chain sequence of SEQ ID NO: 449) ( Figure 8C ); an anti-EGFR activatable antibody referred to herein as an anti-EGFR 2003 activatable antibody (which includes a heavy chain sequence of SEQ ID NO: 108 and a light chain sequence of SEQ ID NO: 472) ( Fig.8D ); or an anti-EGFR activatable antibody referred to herein as an anti-EGFR 2005 activatable antibody (which includes a heavy chain sequence of SEQ ID NO: 108 and a light chain sequence of SEQ ID NO: 474) ( Fig. 8E) is a series of graphs showing the tumor volume of H292 xenograft tumors in nu / nu mice at various time points after administration. Figure 8C and 8D For the data shown in , one animal was lost from each group due to weight loss. Figure 8F is plotted and compared in a single graph Figures 8A-8E Figure 2 shows the data presented in Figure 2.

[0151] Fig. 9 The present invention depicts a method of treating a rat with isotype control intravenous immunoglobulin (IVIG), a 20 mg / kg dose of an anti-Jagged antibody referred to herein as 4D11 (which includes a heavy chain sequence of SEQ ID NO:67 and a light chain sequence of SEQ ID NO:162), a 10 mg / kg dose of the 4D11 antibody, a 5 mg / kg dose of the 4D11 antibody, an anti-Jagged activatable antibody referred to herein as anti-Jagged 2001 activatable antibody (which includes a heavy chain sequence of SEQ ID NO:67 and a light chain sequence of SEQ ID NO:420), an anti-Jagged activatable antibody referred to herein as anti-Jagged 1004 / LP' / 0001 activatable antibody (which includes a heavy chain sequence of SEQ ID NO:67 and a light chain sequence of SEQ ID NO:432), an anti-Jagged 2003 activatable antibody (which includes a heavy chain sequence of SEQ ID NO:67 and a light chain sequence of SEQ ID NO:477), and an anti-Jagged A graph of the toxicity measured by body weight (BW) loss following administration of an anti-Jagged activatable antibody of the 2005 activatable antibody comprising a heavy chain sequence of SEQ ID NO: 67 and a light chain sequence of SEQ ID NO: 477. The results are shown as a percentage (%) of relative body weight (BW) change at various time points during the study.

[0152] Fig.10The figure depicts a 20 mg / kg dose of an anti-Jagged antibody referred to herein as 4D11 (which includes 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 the 4D11 antibody, a 5 mg / kg dose of the 4D11 antibody, an anti-Jagged activatable antibody referred to herein as anti-Jagged 2001 activatable antibody ("2001") (which includes the heavy chain sequence of SEQ ID NO:67 and the light chain sequence of SEQ ID NO:420), an anti-Jagged activatable antibody referred to herein as anti-Jagged 1004 / LP' / 0001 activatable antibody (which includes 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 referred to herein as anti-Jagged 1004 / LP' / 0003 activatable antibody (which includes the heavy chain sequence of SEQ ID NO:67 and the light chain sequence of SEQ ID NO:434). Figure 3 shows the toxicity measured by body weight (BW) loss after administration of 1% lipopolysaccharide (PD-1) (light chain sequence of IDNO: 424). The results are shown as the relative body weight (BW) change percentage (%) at various time points during the study.

[0153] Fig.11A , 11B , 11C, 11D, 11E, 11F, 11G and 11H and Fig.12 is a series of graphs depicting the efficacy of various substrates of the invention when incorporated into an activatable anti-EGFR antibody of the invention. Tumor volume (TV mm 3 ) to evaluate the efficacy of the substrate.

[0154] Fig.13 is a graph depicting toxicity measured as body weight (BW) loss in DBA / 1 mice following administration of control intravenous immunoglobulin (IVIG), anti-Jagged antibodies of the invention, and activatable anti-Jagged antibodies of the invention including various substrates of the invention.

[0155] Fig.14 Graphs depicting 3D reconstructions of FLIT-μCT imaging data of cetuximab and two EGFR activatable antibodies containing tandem substrates in H292 (open arrows) and FaDu (solid arrows) co-implanted xenograft tumor models. Specific implementation plan

[0156] The present invention provides amino acid sequences comprising at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). These CM1-CM2 substrates can be used in a variety of therapeutic, diagnostic and preventive indications. For example, these CM1-CM2 substrates can be used in activatable antibodies, which include antibodies or antigen-binding fragments thereof (AB), which include at least one masking moiety (MM) connected to at least one antigen or epitope binding domain of AB, such that the coupling of the MM reduces the ability of AB to bind to its target.

[0157] The Examples provided herein demonstrate that these CM1-CM2 substrates exhibit a number of desirable cleavage properties when exposed to at least one MMP protease and / or at least one SP protease under specific conditions.

[0158] The present invention also provides antibodies comprising one or more of these CM1-CM2 substrates. For example, when the antibody is conjugated to one or more additional reagents to produce a conjugated antibody, these CM1-CM2 substrates are useful. These CM1-CM2 substrates can also be used in activatable antibodies and / or activatable antibody conjugates.

[0159] Conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies include antibodies or antigen-binding fragments thereof (AB) that specifically bind to a target. Exemplary categories of targets of AB include, but are not necessarily 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, conjugated antibodies and / or activatable antibodies have ABs that bind to extracellular targets (typically extracellular protein targets). In some embodiments, conjugated antibodies and / or activatable antibodies are designed for cellular uptake and are switchable within cells.

[0160] As a non-limiting example, AB is a binding partner for any of the targets listed in Table 1.

[0161] Table 1: Exemplary targets

[0162]

[0163]

[0164] As non-limiting examples, AB is or is derived from an antibody listed in Table 2.

[0165] Table 2: Exemplary Ab sources

[0166] Antibody Trade Name (Antibody Name) Target Avastin™ (bevacizumab) VEGF Lucentis™ (ranibizumab) VEGF Erbitux™ (cetuximab) EGFR Vectibix™ (panitumumab) EGFR Remicade™ (infliximab) TNFα Humira™ (adalimumab) TNFα Tysabri™ (natalizumab) Integrin α4 Simulect™ (basiliximab) IL2R Soliris™ (eculizumab) Complement C5 Raptiva™ (ivacizumab) CD11a Bexxar™ (tositumomab) CD20 Zevalin™ (ibritumomab tiuxetan) CD20 Rituxan™ (rituximab) CD20 Ocrelizumab CD20 Arzerra™ (ofatumumab) CD20 Gazyva™ (obinutuzumab) CD20 Zenapax™ (daclizumab) CD25 Adcetris™ (brentuximab vedotin) CD30 Myelotarg™ (gemtuzumab) CD33 Mylotarg™ (gemtuzumab ozogamicin) CD33 Campath™ (alemtuzumab) CD52 ReoPro™ (abciximab) Glycoprotein receptor IIb / IIIa Xolair™ (omalizumab) IgE Herceptin™ (trastuzumab) Her2 Kadcyla™ (trastuzumab emtansine) Her2 Synagis™ (palivizumab) RSV F protein (Ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (Pertuzumab) Her2-neu (ertumaxomab) CD3 / Her2-neu Orencia™ (abatacept) CTLA-4 (tanezumab) NGF (Baviximab) Phosphatidylserine (zalutumumab) EGFR (mapatumumab) EGFR (matuzumab) EGFR (Nimotuzumab) EGFR ICR62 EGFR mAb 528 EGFR CH806 EGFR MDX-447 EGFR / CD64 (edrecolomab) EpCAM RAV12 RAAG12 huJ591 PSMA Enbrel™ (etanercept) TNF-R Amevive™ (alefacept) 1-92-LFA-3 Antril™, Kineret™ (anakinra) IL-1Ra GC1008 TGFβ Notch, e.g., Notch 1 Jagged 1 or Jagged 2 (Adelimumab) EpCAM (figitumumab) IGF1R (tocilizumab) IL-6 receptor Stelara™ (ustekinumab) IL-12 / IL-23 Prolia™ (denosumab) RANKL

[0167] Exemplary conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies of the invention include, for example, antibodies that bind to interleukin 6 receptor (IL-6R) and include a heavy chain and a light chain, which is or is derived from an antibody referred to herein as an "Av1" antibody, which binds to interleukin-6 receptor (IL-6R). The amino acid sequences of the Av1 heavy chain and the Av1 light chain are shown below in SEQ ID NO: 100 and SEQ ID NO: 101, respectively.

[0168] Av1 antibody heavy chain amino acid sequence:

[0169]

[0170] Av1 antibody light chain amino acid sequence:

[0171] .

[0172] Exemplary activatable antibodies and / or conjugated activatable antibodies of the present invention include, for example, antibodies that bind to interleukin 6 receptor (IL-6R) and include a heavy chain and a light chain, the antibody being or derived from an Av1 antibody and a masking portion. Exemplary activatable antibodies and / or conjugated activatable antibodies of the present invention include an amino acid sequence attached to the N-terminus of the Av1 light chain. These N-terminal amino acid sequences include, for example, YGSCSWNYVHIFMDC (SEQ ID NO: 102); QGDFDIPFPAHWVPIT (SEQID NO: 103); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 104); QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 105); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 106); or QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 107). It will also be appreciated that such an amino acid sequence may be attached to the N-terminus of the AV1 heavy chain or to the C-terminus of the AV1 heavy or light chain.

[0173] Exemplary activatable antibodies of the present invention include, for example, antibodies that bind to epidermal growth factor receptor (EGFR) and include heavy and light chains, the antibodies being and / or derived from antibodies selected from the following: antibodies referred to herein as "c225v5" antibodies (also referred to herein as C225v5 antibodies), antibodies referred to herein as "c225v4" antibodies (also referred to herein as C225v4 antibodies), and antibodies referred to herein as "c225v6" antibodies (also referred to herein as C225v6 antibodies), each of which binds to EGFR. C225v5 antibodies, c225v4 antibodies, and c225v6 antibodies share the same light chain sequence, referred to herein as "c225 light chains." The amino acid sequences of c225v5 heavy chains, c225v4 antibodies, c225v6 antibodies, and c225 light chains are shown below.

[0174] C225v5 antibody heavy chain amino acid sequence:

[0175]

[0176] C225v4 antibody heavy chain amino acid sequence:

[0177]

[0178] C225v6 antibody heavy chain amino acid sequence:

[0179]

[0180] C225 antibody light chain amino acid sequence:

[0181] .

[0182] Exemplary conjugated antibodies and / or activatable antibodies of the invention include, e.g., antibodies that bind to a Jagged target, e.g., Jagged-1, Jagged-2, and / or both Jagged-1 and Jagged-2, and include a combination of a variable heavy chain region and a variable light chain region that are or are derived from the variable heavy chain and variable light chain sequences set forth below.

[0183] Variable light chain amino acid sequence Lc4

[0184]

[0185] Variable heavy chain amino acid sequence Hc4

[0186]

[0187] Variable light chain amino acid sequence Lc5

[0188]

[0189] Variable heavy chain amino acid sequence Hc5

[0190]

[0191] Variable light chain amino acid sequence Lc7

[0192]

[0193] Variable heavy chain amino acid sequence Hc7

[0194]

[0195] Variable light chain amino acid sequence Lc8

[0196]

[0197] Variable heavy chain amino acid sequence Hc8

[0198]

[0199] Variable light chain amino acid sequence Lc13

[0200]

[0201] Variable heavy chain amino acid sequence Hc13

[0202]

[0203]

[0204] Variable light chain amino acid sequence Lc16

[0205]

[0206] Variable heavy chain amino acid sequence Hc16

[0207]

[0208] Variable light chain amino acid sequence Lc19

[0209]

[0210] Variable heavy chain amino acid sequence Hc19

[0211]

[0212] Variable light chain amino acid sequence Lc21

[0213]

[0214] Variable heavy chain amino acid sequence Hc21

[0215]

[0216] Variable light chain amino acid sequence Lc24

[0217]

[0218] Variable heavy chain amino acid sequence Hc24

[0219]

[0220] Variable light chain amino acid sequence Lc26

[0221]

[0222] Variable heavy chain amino acid sequence Hc26

[0223]

[0224] Variable light chain amino acid sequence Lc27

[0225]

[0226] Variable heavy chain amino acid sequence Hc27

[0227]

[0228] Variable light chain amino acid sequence Lc28

[0229]

[0230] Variable heavy chain amino acid sequence Hc28

[0231]

[0232] Variable light chain amino acid sequence Lc30

[0233]

[0234] Variable heavy chain amino acid sequence Hc30

[0235]

[0236] Variable light chain amino acid sequence Lc31

[0237]

[0238] Variable heavy chain amino acid sequence Hc31

[0239]

[0240] Variable light chain amino acid sequence Lc32

[0241]

[0242] Variable heavy chain amino acid sequence Hc32

[0243]

[0244]

[0245] Variable light chain amino acid sequence Lc37

[0246]

[0247] Variable heavy chain amino acid sequence Hc37

[0248]

[0249] Variable light chain amino acid sequence Lc39

[0250]

[0251] Variable heavy chain amino acid sequence Hc39

[0252]

[0253] Variable light chain amino acid sequence Lc40

[0254]

[0255] Heavy chain amino acid sequence Hc40

[0256]

[0257] Variable light chain amino acid sequence Lc47

[0258]

[0259] Variable heavy chain amino acid sequence Hc47

[0260]

[0261] Variable 4B2 light chain

[0262]

[0263] Variable 4B2 heavy chain

[0264]

[0265] Variable 4D11 light chain

[0266]

[0267] Variable 4D11 heavy chain

[0268]

[0269] Variable 4E7 light chain

[0270]

[0271] Variable 4E7 heavy chain

[0272]

[0273] Variable 4E11 light chain

[0274]

[0275] Variable 4E11 heavy chain

[0276]

[0277] Variable 6B7 light chain

[0278]

[0279] Variable 6B7 heavy chain

[0280]

[0281] Variable 6F8 light chain

[0282]

[0283] Variable 6F8 heavy chain

[0284] .

[0285] Exemplary conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies of the invention include, for example, antibodies that bind to a Jagged target, such as Jagged-1, Jagged-2 and / or both Jagged-1 and Jagged-2, and include a combination of heavy chain regions and light chain regions that are or are derived from the heavy chain and light chain sequences shown below.

[0286] 4D11 light chain sequence:

[0287]

[0288] 4D11 heavy chain sequence:

[0289]

[0290] 4D11v2 heavy chain sequence

[0291]

[0292] 4D11v2 light chain sequence

[0293] .

[0294] The activatable antibodies and activatable antibody compositions provided herein contain at least an antibody or antibody fragment thereof (collectively referred to as AB throughout the present invention) that specifically binds to a target (eg, a human target), wherein the AB is modified with a masking moiety (MM).

[0295] 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 to EGFR includes an MM comprising the sequence CISPRG (SEQ ID NO: 165). By way of non-limiting example, an MM may include sequences such as 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 way of non-limiting example, GSHCLIPINMGAPSC (SEQ ID NO: 172); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 173); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 174); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 175); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 176); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 177); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 178); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 179); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 180); CNHVYFGTCGCISPRGCPG (SEQ ID NO:181); CNHFTLTTCGCISPRGCPG (SEQ ID NO:182); CHHFTLTTCGCISPRGCPG (SEQ ID NO:183); YNPCATPMCCISPRGCPG (SEQ ID NO:184); CNHHYFYTCGCISPRGCG (SEQ ID NO:185); NO:186); CNHVYFGTCGCISPRGCG (SEQ ID NO:187); CHHVYWGHCGCISPRGCG (SEQ ID NO:188);CPHFTTTSCGCISPRGCG (SEQ ID NO:189);CNHFTLTTCGCISPRGCG (SEQ ID NO:190);CHHFTLTTCGCISPRGCG (SEQ ID NO:191);CPYYTLSYCGCISPRGCG (SEQ ID NO:192);CPHVSFGSCGCISPRGCG (SEQ ID NO:191) NO:193)?ADHVFWGSYGCISPRGCG (SEQ ID NO:194);YNPCATPMCCISPRGCG (SEQ ID NO:195)CHHVYWGHCGCISPRGCG (SEQ ID NO:195). 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 / CISPRGCNAVSGLGS (SEQ ID NO:200).

[0296] Suitable masking moieties for use with antibodies that bind to a Jagged target, e.g., Jagged 1 and / or Jagged 2, include, by way of non-limiting example, masking moieties that include sequences such as 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); YCLQLPHYMQAMCGR (SEQ ID NO: 207); CFLYSCTDVSYWNNT (SEQ ID NO: 208); PWCMQRQDYLRCPQP (SEQ ID NO: 209); 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); (SEQ ID NO:215); CNLWVSGGDCRGLQG (SEQ ID NO:216); CNLWLHGGDCRGWQG (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); ID NO:227); CFLYSCTDVAYWNSA (SEQ ID NO:228);<h2 style=";text-align:left;direction:ltr">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) NO:247);GKSEFCNIWLNGGDCRGWIG (SEQ ID NO:248);GTPGGCNIWANGGDCRGWEG (SEQ ID NO:249);GASQYCNLWINGGDCRGWRG (SEQ ID NO:250);GCNIWLVGGDCRPWVEGG (SEQ ID NO:251);GCNIWAVGGDCRPFVDGG (SEQ ID NO:252);GCNIWLNGGDCRAWVDTG (SEQ ID NO:253);GCNIWIVGGDCRPFINDG (SEQ ID NO:254);GCNIWLNGGDCRPVVFGG (SEQ ID NO:255);GCNIWLSGGDCRMFMNEG (SEQ ID NO:256);GCNIWVNGGDCRSFVYSG (SEQ ID NO:257); GCNIWLNGGDCRGWEASG (SEQ ID NO:258); GCNIWAHGGDCRGFIEPG (SEQ ID NO:259); GCNIWLNGGDCRTFVASG (SEQ ID NO:260); GCNIWAHGGDCRGFIEPG (SEQ ID NO:261); GFLENCNIWLNGGDCRTG (SEQ ID NO:262); GIYENCNIWLNGGDCRMG (SEQ ID NO:263); and / or GIPDNCNIWINGGDCRYG (SEQ ID NO:264).;

[0297] Suitable masking moieties for use with antibodies that bind to an interleukin 6 target, such as the interleukin 6 receptor (IL-6R), include, by way of non-limiting example, masking moieties that include 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); ID NO:274); YGSCNWNYVHIFLDC (SEQ ID NO:275); YTSCNWNYVHIFMDC (SEQ ID NO:276); (SEQ ID NO:280); SGSCKWDYVHIFLDC (SEQ ID NO:281); SRSCIWNYAHIHLDC (SEQ ID NO:282); SMSCYWQYERIFLDC (SEQ ID NO:283); YRSCNWNYVSIFLDC (SEQ ID NO:284); SGSCKWDYVHIFLDC (SEQ ID NO:282); NO:285); YKSCHWDYVHIFLDC (SEQ ID NO:286); YGSCTWNYVHIFMEC (SEQ ID NO:287); FSSCNWNYVHIFLDC (SEQ ID NO:288); WRSCNWNYAHIFLDC (SEQ ID NO:289); (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).;

[0298] When AB is modified with MM and in the presence of target, specific binding of AB to its target is reduced or inhibited compared to specific binding of AB not modified with MM or specific binding of parent AB to the target.

[0299] K of AB modified with MM to target d Compared with the K of AB without MM modification or parental AB to target dat least 5, 10, 25, 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 times or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000 , 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times. In contrast, the binding affinity of the AB modified with MM to the 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 times or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000 times or more, than the binding affinity of the AB not modified with MM or the parent AB to the target. ,000, 10-10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000 or 100,000-10,000,000 times.

[0300] The dissociation constant (K) of MM to AB d ) is usually greater than the K of AB for the target d MM vs. AB's K d The K of AB to target can be compared dAB has a binding affinity that is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times higher than the binding affinity of AB to the target. In contrast, the binding affinity of MM to AB is generally lower than the binding affinity of AB to the target. The binding affinity of MM to AB may be 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 AB to the target.

[0301] When AB is modified with MM and in the presence of target, specific binding of AB to its target is reduced or inhibited compared to specific binding of AB not modified with MM or specific binding of parent AB to the target. The ability of the AB to bind to a target when modified with MM can 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 longer when measured in vivo or in an in vitro assay, when compared to the binding of an AB not modified with MM to the target or the binding of a parental AB to the target.

[0302] MM inhibits binding of AB to target. MM binds to the antigen binding domain of AB and inhibits binding of AB to target. MM can sterically inhibit binding of AB to target. MM can allosterically inhibit binding of AB to its target. In these embodiments, when AB is modified with MM or coupled to MM and in the presence of target, there is no binding of AB to target or substantially no binding of AB to target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% when measured in vivo or in an in vitro assay, compared to binding of AB not modified with MM, parent AB, or AB not coupled to MM to target. , 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% binding of AB to target 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 longer.

[0303] When AB is coupled to or modified by MM, MM "masks" or reduces or otherwise inhibits the specific binding of AB to the target. When AB is coupled to or modified by MM, such coupling or modification can achieve a structural change that reduces or inhibits the ability of AB to specifically bind to its target.

[0304] AB coupled to or modified with MM can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region):

[0305] (MM)-(AB)

[0306] (AB)-(MM)

[0307] (MM)-L-(AB)

[0308] (AB)-L-(MM)

[0309] wherein MM is a masking moiety, AB is an antibody or antibody fragment thereof, and L is a linker. In many embodiments, it may be desirable to insert one or more linkers, for example, flexible linkers, into the composition to provide flexibility.

[0310] In certain embodiments, MM is not a natural binding partner of AB. In some embodiments, MM does not contain or substantially contains no homology with any natural binding partner of AB. In some embodiments, MM has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% similarity with any natural binding partner of AB. In some embodiments, MM has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% identity with any natural binding partner of AB. In some embodiments, MM has no more than 25% identity with any natural binding partner of AB. In some embodiments, MM has no more than 50% identity with any natural binding partner of AB. In some embodiments, MM has no more than 20% identity with any natural binding partner of AB. In some embodiments, the MM has no more than 10% identity with any natural binding partner of the AB.

[0311] In some embodiments, the activatable antibody comprises an AB modified by a MM and further comprises at least one cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (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 to the target of the AB. The activatable antibody generally comprises an antibody or antibody fragment (AB) modified by or coupled to a masking moiety (MM) and a CM1-CM2 substrate.

[0312] The elements of the activatable antibody are arranged so that the MM and the CM1-CM2 substrate are positioned so that in the cleaved (or relatively active) state and in the presence of the target, the AB binds to the target, while in the uncleaved (or relatively inactive) state, in the presence of the target, the specific binding of the AB to its target is reduced or inhibited. The specific binding of the AB to its target can be reduced because the ability of the AB to specifically bind to its target is inhibited or masked by the MM.

[0313] K of AB modified with MM and CM1-CM2 substrates towards the target d Compared with the K of AB or parental AB not modified with MM and CM1-CM2 substrates for target dat 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 times or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1, 000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-10,000,000, 1000-10,000,000, 10,000-10,000,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times. In contrast, the binding affinity of the AB modified with MM and CM1-CM2 substrates to the 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 times or more, or 5-10, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 10-100, 100-100, 100-100, 100-100, 100-100, 100-100, 1000-100 0, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,0 00, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1 ,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000, 000, 10,000-10,000,000, 100,000-1,000,000, 100,000-10,000,000 times.

[0314] When AB is modified with MM and CM1-CM2 substrates and in the presence of the target, but not in the presence of modifying agents (e.g., MMPs and SPs), the specific binding of AB to its target is reduced or inhibited compared to the specific binding of AB not modified with MM and CM1-CM2 substrates or the parent AB to the target. The ability of the AB to bind to a target when modified with MM and CM1-CM2 substrates can 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 longer, when measured in vivo or in an in vitro assay, when compared to the binding of a parent AB to its target or the binding of an AB not modified with MM and CM1-CM2 substrates to its target.

[0315] As used herein, the term cleavage state refers to the state of the activatable antibody after the CM1-CM2 substrate is modified (i.e., cleaved) by at least one matrix metalloproteinase and / or at least one serine protease. As used herein, the term uncut state or completely uncut refers to the state of the activatable antibody in the absence of CM1-CM2 substrates being cleaved by MMP and / or SP. As discussed above, the term "activatable antibody" is used herein to refer to an activatable antibody in its uncut (native) state and in its cleaved state. The activatable antibody in its cleaved state is also referred to herein as an activated antibody and / or an activated activatable antibody. It is apparent to a person of ordinary skill that, in some embodiments, the cleaved activatable antibody may lack MM due to the cleavage of the CM1-CM2 substrate by a protease, resulting in the release of at least MM.

[0316] Activatable or convertible means that the activatable antibody exhibits a first binding level to the target when in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second binding level to the target in an uninhibited, unmasked and / or cleaved state (i.e., a second conformation), wherein the second target binding level is greater than the first binding level. Typically, the access of the target to the AB of the activatable antibody is greater in the presence of a cleavage agent capable of cleaving the CM1-CM2 substrate than in the absence of such a cleavage agent. Thus, when the activatable antibody is in an uncleaved state, the AB is inhibited from target binding and can be masked from target binding (i.e., the first conformation renders the AB incapable of binding the target), and in the cleaved state, the AB is not inhibited or masked from target binding.

[0317] The CM1-CM2 substrate and AB of the activatable antibody are selected so that the AB represents the binding portion of a given target, and the CM1-CM2 substrate represents the substrate of the MMP and SP, wherein the MMP and / or SP are co-localized with the target at the treatment site or diagnostic site in the subject. The activatable antibodies disclosed herein are particularly useful in situations where, for example, the MMP and SP, each of which is capable of cleaving a site in the CM1-CM2 substrate, are present at relatively higher levels in tissue containing the target at the treatment site or diagnostic site than in tissues at non-treatment sites (e.g., healthy tissues).

[0318] In some embodiments, the activatable antibody provides reduced toxicity and / or adverse side effects that might otherwise result from binding of the AB at non-therapeutic sites if the AB were not masked or otherwise inhibited from binding to the target.

[0319] In general, an activatable antibody can be designed by selecting an AB of interest and constructing the remainder of the activatable antibody such that when conformationally constrained, the MM provides masking of the AB or reduced binding of the AB to its target. Structural design criteria can be considered to provide this functional feature.

[0320] Provided is an activatable antibody that exhibits a convertible phenotype of a desired dynamic range for target binding in an inhibitory conformation compared to a non-inhibitory conformation. Dynamic range generally refers to the ratio of the maximum detection level of a parameter under (a) a first set of conditions to the minimum detection value of the parameter under (b) a second set of conditions. For example, in the context of an activatable antibody, the dynamic range refers to the ratio of the maximum detection level of a target protein bound to an activatable antibody (a) in the presence of MMPs and SPs that can cut the CM1-CM2 substrates of the activatable antibody to the minimum detection level of a target protein bound to an activatable antibody (b) in the absence of a protease. The dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of an activatable antibody cleavage agent (e.g., enzyme) treatment to the dissociation constant of an activatable antibody cleavage agent treatment. The larger the dynamic range of an activatable antibody, the better the convertible phenotype of the activatable antibody. Activatable antibodies having relatively higher dynamic range values ​​(e.g., greater than 1) exhibit a more desirable switching phenotype, such that target protein binding of the activatable antibody occurs to a greater extent (e.g., predominantly) in the presence of a cleavage agent (e.g., an enzyme) capable of cleaving the CM1-CM2 substrate of the activatable antibody than in the absence of the cleavage agent.

[0321] Activatable antibodies can be provided in various structural configurations. Exemplary formulas of activatable antibodies are provided below. It is particularly contemplated that the N- to C-terminal order of AB, MM, and CM1-CM2 substrates can be reversed within the activatable antibody. It is also particularly contemplated that the amino acid sequences of CM and MM can overlap, for example, such that the CM1-CM2 substrate is at least partially contained within the MM.

[0322] For example, the activatable antibody can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region):

[0323] (MM)-(CM1-CM2 substrate)-(AB)

[0324] (AB)-(CM1-CM2 substrate)-(MM)

[0325] Wherein MM is a masking moiety, CM1-CM2 substrate is a cleavable moiety, and AB is an antibody or fragment thereof. As indicated above, the term "CM1-CM2 substrate" is not intended to convey any requirements regarding the orientation or other structural arrangement of a first cleavable moiety (CM1) as a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) as a substrate for at least one serine protease (SP). Thus, the term "CM1-CM2 substrate" encompasses CM1-CM2 substrates having the following N-terminal to C-terminal structural arrangement: 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 MM and CM1-CM2 substrates are represented as different components in the above formula, in all exemplary embodiments (including formulas) disclosed herein, it is contemplated that the amino acid sequences of MM and CM1-CM2 substrates may overlap, for example, such that the CM1-CM2 substrate is fully or partially contained within the MM. Furthermore, the above formula provides additional amino acid sequences that can be located at the N-terminus or the C-terminus of the activatable antibody element.

[0326] In certain embodiments, MM is not a natural binding partner of AB. In some embodiments, MM does not contain or substantially contains no homology with any natural binding partner of AB. In some embodiments, MM has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% similarity with any natural binding partner of AB. In some embodiments, MM has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% identity with any natural binding partner of AB. In some embodiments, MM has no more than 50% identity with any natural binding partner of AB. In some embodiments, MM has no more than 25% identity with any natural binding partner of AB. In some embodiments, MM has no more than 20% identity with any natural binding partner of AB. In some embodiments, the MM has no more than 10% identity with any natural binding partner of the AB.

[0327] In many embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, into the activatable antibody construct to provide flexibility at one or more of the MM-CM1-CM2 substrate connection CM1-CM2 substrate-AB connection or both. For example, AB, MM and / or CM1-CM2 substrates may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, especially Gly) to provide the desired flexibility. Therefore, the convertible phenotype of such an activatable antibody construct may benefit from the introduction of one or more amino acids to provide a flexible linker. In addition, as described below, in the case of providing an activatable antibody as a conformationally restricted construct, a flexible linker may be operably inserted to promote the formation and maintenance of the ring structure of the uncut activatable antibody.

[0328] For example, in certain embodiments, the activatable antibody comprises one of the following formulae (wherein the following formulae represent amino acid sequences in either the N- to C-terminal direction or the C- to N-terminal direction):

[0329] (MM)-L1-(CM1-CM2 substrate)-(AB)

[0330] (MM)-(CM1-CM2 substrate)-L2-(AB)

[0331] (MM)-L1-(CM1-CM2 substrate)-L2-(AB)

[0332] Wherein MM, CM1-CM2 substrates and AB are as defined above; Wherein L1 and L2 are each independently and optionally present or absent, and are identical or different flexible linkers, which include at least 1 flexible amino acid (e.g., Gly). In addition, the above formula provides additional amino acid sequences, which may be located at the N-terminus or C-terminus of the activatable antibody element. Examples include, but are not limited to, targeting moieties (e.g., ligands of receptors of cells present in target tissues) and serum half-life extension moieties (e.g., polypeptides that bind serum proteins such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HAS)).

[0333] The CM1-CM2 substrate is activated by at least one MMP at a rate of about 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 The rate of specific cleavage is about 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 Rate-specific cleavage.

[0334] For specific cleavage of the enzyme, contact between the enzyme and the CM1-CM2 substrate is made. When an activatable antibody comprising an AB coupled to the MM and the CM1-CM2 substrate is in the presence of a target and sufficient enzyme activity, the CM1-CM2 substrate can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to contact the CM1-CM2 substrate and achieve cleavage. It can be easily imagined that the enzyme can be in the vicinity of the CM1-CM2 substrate, but cannot cleave due to protein modification by other cytokines or enzymes.

[0335] The joint suitable for compositions described herein is generally a joint that provides the flexibility of the modified AB or activatable antibody to promote the inhibition of the combination of AB and target. Such joints are generally referred to as flexible joints. Suitable joints can be easily selected, and can have any suitable different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 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 long.

[0336] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (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 therefore may be able to act as neutral tethers between components. Glycine gains significantly more phi-psi space than even alanine and is much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, 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), etc. One of ordinary skill will recognize that the design of an activatable antibody can include a fully or partially flexible linker, such that a linker can include a flexible linker and one or more portions that impart a less flexible structure to provide a desired activatable antibody structure.

[0337] In some embodiments, the activatable antibodies described herein further include 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 portion of the activatable antibody, for example, in some embodiments, wherein the carbohydrate portion is located outside the antigen binding region of the antibody or antigen binding fragment in the activatable antibody. In some embodiments, the agent is conjugated to a thiol group of the antibody or antigen binding fragment in the activatable antibody.

[0338] 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 radioactive isotope (ie, a radioconjugate).

[0339] In some embodiments, the agent is a detectable moiety, such as, for example, a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotin moieties detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety is connected by a spacer molecule.

[0340] The present invention also relates to immunoconjugates comprising antibodies conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioactive isotopes (i.e., radioconjugates). Suitable cytotoxic agents include, for example, dolastatin and derivatives thereof (e.g., auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD). In some embodiments, the agent is selected from the agents listed in Table 3. In some embodiments, the agent is dolastatin. 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 monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0341] In some embodiments, the reagent is connected to AB using a maleimidocaproyl-valine-citrulline linker or a maleimido PEG-valine-citrulline linker. In some embodiments, the reagent is connected to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, the reagent is connected to AB using a maleimido PEG-valine-citrulline linker. In some embodiments, the reagent is connected to AB using a maleimido PEG-valine-citrulline linker. In some embodiments, the reagent is monomethyl auristatin D (MMAD) connected to AB using a maleimido PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker, and the linker payload construct is referred to herein as "vc-MMAD". In some embodiments, the reagent is monomethyl auristatin E (MMAE) connected to AB using a maleimido PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker, and the linker payload construct is referred to herein as "vc-MMAE". The structures of vc-MMAD and vc-MMAE are shown below:

[0342] vc-MMAD:

[0343]

[0344] vc-MMAE:

[0345] .

[0346] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, schizonepeta toxin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca Americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes. Various radionuclides can be used to prepare radioconjugated antibodies. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0347] Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium salt derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see WO94 / 11026).

[0348] Table 3 lists some exemplary drugs that may be used with the disclosure described herein, but is by no means intended to be an exhaustive list.

[0349] Table 3: Exemplary drugs for conjugation

[0350]

[0351]

[0352]

[0353] One of ordinary skill in the art will recognize that there are many possible moieties that can be coupled to the antibodies of the invention (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).

[0354] Coupling can be carried out by any chemical reaction that combines two molecules, as long as the antibody and the other part keep their respective activities. The bond may include many chemical mechanisms, such as covalent bonding, affinity bonding, embedding, coordination bonding and complexing. However, in some embodiments, the bonding is covalent bonding. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporating external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules, such as antibodies of the present invention, to other molecules. For example, representative coupling agents may include organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine. This list is not intended to exhaust the various types of coupling agents known in the art, but rather an illustration of 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).

[0355] In some embodiments, in addition to the compositions and methods provided herein, the conjugated activatable antibodies can also be modified for site-specific conjugation by inserting or otherwise including modified amino acid sequences in the activatable antibody sequence. These modified amino acid sequences are designed to allow controlled placement and / or administration of conjugated reagents in the conjugated activatable antibodies. For example, the activatable antibody can be engineered to include cysteine ​​substitutions at the positions of the light and heavy chains, which provide reactive sulfhydryls and do not negatively affect protein folding and assembly, nor change antigen binding. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce one or more non-natural amino acid residues in the activatable antibody to provide a suitable site for conjugation. In some embodiments, the activatable antibody can be engineered to include or otherwise enzymatically introduce an activatable peptide sequence in the activatable antibody sequence.

[0356] Suitable linkers are described in the literature (see, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, describing the use of haloacetyl hydrazide derivatives coupled to antibodies via 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-dithiol)-toluene (Pierce Chem. Co., Catalog No. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithiol) propionamido] hexanoate (Pierce Chem. Co., No. 21651G); (iv) Sulfo-LC-SPDP (Sulfosuccinimidyl 6 [3-(2-pyridyldithiol)-propionamide] hexanoate (Pierce Chem. Co. Catalog No. 2165-G); and (v) Sulfo-NHS (N-hydroxysulfo-succinimidyl: Pierce Chem. Co., Catalog No. 24510) conjugated with EDC. Additional linkers include, but are not limited to, SMCC, Sulfo-SMCC, SPDB, or Sulfo-SPDB.

[0357] The above-mentioned linkers contain elements with different properties, thus producing conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS-esters are more difficult to dissolve than sulfo-NOS esters. In addition, the linker SMPT contains a sterically hindered disulfide bond and can form conjugates with increased stability. Disulfide bonds are generally less stable than other bonds because disulfide bonds are cleaved in vitro, resulting in less available conjugates. Specifically, sulfo-NHS can enhance the stability of carbodiimide linkers. When used in combination with sulfo-NHS, carbodiimide conjugates (such as EDC) form esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0358] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are two or more linkers. The two or more linkers are all the same, i.e., cleavable or non-cleavable, or the two or more linkers are different, i.e., at least one is cleavable and at least one is non-cleavable.

[0359] The present invention utilizes several methods to attach the reagent to Ab: (a) to the carbohydrate portion of AB, or (b) to the sulfhydryl group of AB, or (c) to the amino group of AB, or (d) to the carboxylic acid group of AB. According to the present invention, AB can be covalently attached to the reagent through an intermediate linker having at least two reactive groups, one group reacting with AB and one group reacting with the reagent. The linker, which may include any compatible organic compound, may be selected so that the reaction with AB (or the reagent) does not adversely affect the reactivity and selectivity of AB. In addition, attaching the linker to the reagent may not destroy the activity of the reagent. For reactions with oxidized antibodies or oxidized antibody fragments, suitable linkers include those containing amines selected from primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and semicarbazide groups. Such reactive functional groups may be present as part of the linker structure, or may be introduced by suitable chemical modification of a linker that does not contain such groups.

[0360] According to the present invention, suitable linkers for attachment to the reduced AB include those having certain reactive groups capable of reacting with the sulfhydryl groups of the reduced antibody or fragment. 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, maleimide and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

[0361] According to the present invention, suitable linkers for attachment to non-oxidative and non-reducing Abs include those having certain functional groups capable of reacting with primary amino groups present in unmodified lysine residues of the Ab. Such reactive groups include, but are not limited to, NHS carboxylic acids or carbonates, sulfo-NHS carboxylic acids or carbonates, 4-nitrophenyl carboxylic acids or carbonates, pentafluorophenyl carboxylic acids or carbonates, acyl imidazoles, isocyanates, and isothiocyanates.

[0362] According to the present invention, suitable linkers for attachment to non-oxidative and non-reducing Abs include those having certain functional groups capable of reacting with the carboxylic acid groups present in the aspartic acid or glutamic acid residues of the Ab, which have been activated by suitable reagents. Suitable activating reagents include EDC (with or without added NHS or sulfo-NHS) and other dehydrating agents for carboxamide formation. In these examples, the functional groups present in suitable linkers include primary and secondary amines, hydrazine, hydroxylamine and hydrazide.

[0363] The reagents may be connected to the linker before or after the linker is connected to the AB. In certain applications, it may be desirable to first generate an AB-linker intermediate, wherein the linker does not contain the associated reagents. Depending on the specific application, the specific reagent may then be covalently linked to the linker. In some embodiments, the AB is first connected to the MM, CM1-CM2 substrate, and the associated linker, and then connected to the linker for conjugation purposes.

[0364] Branch connector: In a particular embodiment, a branched linker with multiple sites for reagent attachment is utilized. For a multisite linker, a single covalent attachment to the AB will produce an AB-linker intermediate that is capable of binding reagents at multiple sites. The site may be an aldehyde group or a sulfhydryl group or any chemical site to which a reagent may attach.

[0365] In some embodiments, higher specific activity (or higher reagent:AB ratio) can be obtained by attaching single-site linkers at multiple sites of the AB. Such multiple sites can be introduced into the AB by either of two methods. First, multiple aldehyde groups and / or sulfhydryl groups can be generated on the same AB. Second, the aldehyde or sulfhydryl groups of the AB can be attached to a "branched linker" having multiple functional sites for subsequent linker attachment. The functional site of a branched linker or multi-site linker can be an aldehyde group or a sulfhydryl group, or can be any chemical site to which a linker can be attached. Even higher specific activity can be obtained by combining these two methods, i.e., attaching a multi-site linker at several sites of the AB.

[0366] Cleavable linkers: In one embodiment of the invention, a peptide linker that is easily cleaved 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. According to one method of the invention, the agent is connected via a linker that is sensitive to complement cleavage. The antibody is selected from a class that can activate complement. Thus, the antibody-agent conjugate activates the complement cascade and releases the agent at the target site. According to another method of the invention, the agent is connected via a linker that is easily cleaved by an enzyme with proteolytic activity (e.g., urokinase, tissue plasminogen activator, plasmin, or trypsin). These cleavable links can be used for conjugated activatable antibodies, which include exotoxins, such as any of the exotoxins shown in Table 3 by way of non-limiting examples.

[0367] Non-limiting examples of cleavable linker sequences are provided in Table 4.

[0368] Table 4: Exemplary linker sequences for conjugation

[0369]

[0370]

[0371] In addition, the agent can be linked to the AB via a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Because many tumors naturally release high levels of glutathione (reducing agent), this can reduce the disulfide bond and subsequently release the agent at the delivery site. In certain specific embodiments, the reducing agent that modifies the CM1-CM2 substrate will also modify the linker of the conjugated activatable antibody.

[0372] Spacer and cleavable element : In some embodiments, it may be necessary to construct a linker in such a way as to optimize the spacing between the reagent and the AB of the activatable antibody. This can be achieved by using a linker of the following general structure:

[0373] W – (CH 2 )n – Q

[0374] in

[0375] W is --NH--CH 2 --or--CH 2 --;

[0376] Q is an amino acid, a peptide; and

[0377] n is an integer from 0 to 20.

[0378] In some embodiments, the linker may comprise a spacer element and a cleavable element. The spacer element is used to position the cleavable element away from the core of the AB, making the cleavable element more accessible to the enzyme responsible for cleavage. Certain branched linkers described above may be used as spacer elements.

[0379] Throughout this discussion, it is understood that the connection of the linker to the agent (or spacer element to the cleavable element, or cleavable element to the agent) does not require a specific connection or reaction mode. Any reaction that provides a product with suitable stability and biocompatibility is acceptable.

[0380] Serum complement and adaptor selection:According to a method of the present invention, when the release of the reagent is required, an AB of an antibody of the type that can activate complement is used. The resulting conjugate retains the ability to bind antigens and activate the complement cascade at the same time. Therefore, according to this embodiment of the present invention, the reagent is connected to one end of a cleavable joint or a cleavable element, and the other end of the joint group is connected to a specific site on the AB. For example, if the reagent has a hydroxyl or amino group, it can be connected to the carboxyl end of a peptide, an amino acid or other appropriately selected joint via an ester or amide bond, respectively. For example, such a reagent can be connected to a joint peptide via a carbodiimide reaction. If the reagent contains functional groups that interfere with the connection of the joint, these interfering functional groups can be blocked before connection and unblocked after the preparation of the product conjugate or intermediate. The relative or amino end of the joint is then used directly or after further modification to bind to an AB that can activate complement.

[0381] The linker (or spacer element of the linker) can be of any desired length, one end of which can be covalently linked to a specific site of the AB of the activatable antibody. The other end of the linker or spacer element can be connected to an amino acid or peptide linker.

[0382] Therefore, when these conjugates bind to antigens in the presence of complement, the amide or ester bond connecting the agent to the linker will be cleaved, resulting in the release of the agent in its active form. These conjugates, when administered to a subject, will achieve delivery and release of the agent at the target site, and are particularly effective for in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative drugs, etc., such as those provided in Table 3 (but not limited thereto).

[0383] Linkers for release in the absence of complement activation : In another application of targeted delivery, the release of reagents without complement activation is needed, because the activation of the complement cascade will eventually dissolve the target cells. Therefore, the method is useful when the delivery and release of reagents should be realized without killing the target cells. This is the goal when it is necessary to deliver cell mediators such as hormones, enzymes, corticosteroids, neurotransmitters, genes or enzymes to target cells. These conjugates can be prepared by connecting reagents to AB that cannot activate complement via a joint, and the joint is slightly sensitive to the cutting by serum proteases. When the conjugate is applied to an individual, the antigen-antibody complex will form quickly, and the cutting of the reagent will occur slowly, therefore resulting in the release of the compound on the target site.

[0384] Biochemical cross-linker : In some embodiments, an activatable antibody can be conjugated to one or more therapeutic agents using certain biochemical cross-linkers. Cross-linking agents form molecular bridges that tie together the functional groups of two different molecules. In order to connect two different proteins in a stepwise manner, hetero-bifunctional cross-linkers can be used, which exclude unwanted homopolymer formation.

[0385] Peptide-based linkers that are cleavable by lysosomal proteases, such as Val-Cit, Val-Ala or other dipeptides, can also be used. In addition, acid-labile linkers that are cleavable in the low pH environment of the lysosome, such as bis-sialic acid ethers, can be used. Other suitable linkers include cathepsin-labile substrates, particularly those that exhibit optimal function at acidic pH.

[0386] Exemplary hetero-bifunctional cross-linkers are mentioned in Table 5.

[0387] Table 5: Exemplary hetero-bifunctional cross-linkers

[0388]

[0389]

[0390] Non-cleavable linker or direct ligation : In some embodiments of the invention, the conjugate can be designed so that the agent is delivered to the target without being released. This can be achieved by connecting the agent to AB directly or via a non-cleavable linker.

[0391] These non-cleavable linkers may include amino acids, peptides, D-amino acids, or other organic compounds that may be modified to include functional groups that can then be used to link AB by the methods described herein. The general formula of such organic linkers may be

[0392] W – (CH 2 )n – Q

[0393] in

[0394] W is --NH--CH 2 --or--CH 2 --;

[0395] Q is an amino acid, a peptide; and

[0396] n is an integer from 0 to 20.

[0397] Non-cleavable conjugate : In some embodiments, the compound can be linked to an AB that does not activate complement. When an AB that cannot activate complement is used, the link can be achieved using a linker that is sensitive to cleavage by activated complement or using a linker that is insensitive to cleavage by activated complement.

[0398] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and methods described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556.

[0399] Particularly useful liposomes can be produced by reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of limited pore size to obtain liposomes with the desired diameter. The Fab' fragments of the antibodies of the present invention can be conjugated to the liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982).

[0400] definition:

[0401] Unless otherwise defined, the scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The term "one / kind (a)" entity or "an" entity refers to one / kind or more / kinds of the entity. For example, a compound refers to one or more compounds. Therefore, the terms "one / kind (a)", "an", "one / kind or more / kinds" and "at least one / kind" can be used interchangeably. In addition, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. Generally, the nomenclature and techniques used in conjunction with the cell and tissue culture, molecular biology and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those 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 the manufacturer's instructions or commonly practiced in the art or as described herein. The aforementioned techniques and procedures are generally performed according to conventional methods 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 (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). Nomenclature used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those 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.

[0402] As used in accordance with the present invention, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0403] 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. "Specific binding" or "immunoreacts with" or "immunospecific binding" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with or binds with much lower affinity (K) to other polypeptides. d > 10 -6 ). Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, domain antibodies, single chain antibodies, Fab and F(ab') 2 Fragment, scFv and Fab expression libraries.

[0404] The basic antibody structural unit is known to include a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl terminal portion of each chain defines a constant region, which is primarily responsible for effector function. Typically, antibody molecules obtained from humans refer to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chain present in the molecule. Some classes also have subclasses, such as IgG 1 IgG 2 And others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0405] The term "monoclonal antibody" (mAb) or "monoclonal antibody composition" as used herein refers to a population of antibody molecules containing only one molecular species of antibody molecules, which are composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining region (CDR) of the monoclonal antibody is the same in all molecules of the population. MAbs contain an antigen binding site that can immunoreact with a specific epitope of an antigen and are characterized by having a unique binding affinity for the antigen.

[0406] The term "antigen binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly distinct segments within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interspersed between more conservative flanking segments called "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequence that naturally occurs between and adjacent to the hypervariable regions of an immunoglobulin. 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 bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs." The distribution of amino acids among the domains is based on the definitions in 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).

[0407] As used herein, the term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin, scFv or T-cell receptor. The term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin or a T-cell receptor. Epitope determinants are usually composed of chemically active surface clusters (groupings) of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, antibodies can be generated against the N-terminal or C-terminal peptide of a polypeptide. When the dissociation constant is ≤ 1 μM; in some embodiments ≤ 100 nM and in some embodiments ≤ 10nM, it is considered that the antibody specifically binds to the antigen.

[0408] As used herein, the terms "specific binding", "immunological binding" and "immunological binding properties" refer to the type of non-covalent interaction formed between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of the immunological binding interaction can be expressed as the dissociation constant (K) of the interaction. d ), where K d The smaller the value, the greater the affinity. The immunological binding properties of a selected polypeptide can be quantitatively determined using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "binding rate constant" (K on ) and the “dissociation rate constant” (K off ) can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). off / K on The ratio of 2 allows the elimination of all parameters not related to affinity and is equal to the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). d ) is ≤ 1 μM, in some embodiments ≤ 100 nM, in some embodiments ≤ 10 nM, and in some embodiments ≤ 100 pM to about 1 pM, an antibody of the invention is considered to specifically bind to a target as measured by an assay such as a radioligand binding assay or similar assays known to those of skill in the art.

[0409] As used herein, the term "isolated polynucleotide" shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, depending on its origin, (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" naturally occurs, (2) is operably linked to a polynucleotide to which it is not naturally linked, or (3) does not naturally occur as part of a larger sequence. Polynucleotides according to the present invention include nucleic acid molecules encoding heavy chain immunoglobulin molecules as set forth herein, and nucleic acid molecules encoding light chain immunoglobulin molecules as set forth herein.

[0410] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which, depending on its origin or source of derivation, (1) is not associated with naturally occurring proteins, (2) does not contain other proteins from the same source, e.g., does not contain murine proteins, (3) is expressed by cells of a different species, or (4) does not occur in nature.

[0411] The term "polypeptide" used herein as a generic term refers to a natural protein, fragment or analog having a polypeptide sequence. Therefore, natural protein fragments and analogs are subordinate concepts (species) of the polypeptide superordinate concept (genus). The polypeptide according to the present invention includes the heavy chain immunoglobulin molecules shown herein and the light chain immunoglobulin molecules shown herein, as well as antibody molecules formed by a combination (and vice versa) comprising heavy chain immunoglobulin molecules and light chain immunoglobulin molecules such as κ light chain immunoglobulin molecules, as well as fragments and analogs thereof.

[0412] As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by man in the laboratory or otherwise modified is naturally occurring.

[0413] As used herein, the term "operably linked" refers to the positions of the components described being in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0414] As used herein, the term "control sequence" refers to a polynucleotide sequence necessary for expression and processing of the coding sequence to which they are connected. The nature of such control sequences varies depending on the host organism, and in prokaryotes such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences, and in eukaryotes such control sequences generally include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all elements necessary for expression and processing, and may also include other elements that are advantageous for their presence, such as leader sequences and fusion partner sequences. The term "polynucleotide" mentioned herein means nucleotides of at least 10 bases in length, which are modified forms of ribonucleotides or deoxyribonucleotides or nucleotides of either type. The term includes DNA in single-stranded and double-stranded forms.

[0415] The term oligonucleotide mentioned herein includes naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide bonds (linkage). Oligonucleotide is a polynucleotide subset that generally comprises 200 or less bases in length. In some embodiments, oligonucleotide is 10-60 bases long, and in some embodiments 12,13,14,15,16,17,18,19 or 20-40 bases long. Oligonucleotide is typically single-stranded, for example for probes, but oligonucleotide can also be double-stranded, for example for constructing gene mutants. Oligonucleotide of the present invention is sense or antisense oligonucleotide.

[0416] The term "naturally occurring nucleotides" as referred to herein include deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as referred to herein include nucleotides with modified or substituted sugar groups, etc. The term "oligonucleotide linkage" as referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoronmidate, etc. 。 See, e.g., 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. Anti Cancer 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. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). If desired, the oligonucleotide can include a label for detection.

[0417] As used herein, the 20 conventional amino acids and their abbreviations are used conventionally. 。See Immunology - A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland7Mass. (1991)). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), non-natural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide symbols used herein, according to standard usage and convention, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction.

[0418] 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' joining direction of a nascent RNA transcript is referred to as the transcription direction, and the sequence region on the DNA strand having the same sequence as the RNA and at the 5' end of the 5' end of the RNA transcript is referred to as the "upstream sequence", and the sequence region on the DNA strand having the same sequence as the RNA and at the 3' end of the 3' end of the RNA transcript is referred to as the "downstream sequence".

[0419] The term "substantial identity" when applied to polypeptides means that two peptide sequences, when optimally aligned such as by the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity, 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.

[0420] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0421] As discussed herein, minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated to be included in the present invention, provided that the changes in the amino acid sequence remain at least 75%, in some embodiments at least 80%, 90%, 95%, and in some embodiments 99%. Specifically, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur in a family of amino acids that are related on their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid, glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, 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) serine and threonine, which are aliphatic hydroxyl families; (ii) asparagine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan and tyrosine, which are aromatic families. For example, it is reasonable to expect that a single replacement of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar replacement of amino acids with structurally related amino acids will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve amino acids within framework sites. Whether an amino acid change produces a functional peptide can be easily determined by measuring the specific activity of a polypeptide derivative. The assay is described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be easily prepared by those of ordinary skill in the art. The amino-terminal and carboxyl-terminal of suitable fragments or analogs appear near the border of the functional domain. The structure and function domain can be identified by comparing nucleotide and / or amino acid sequence data with a disclosed or proprietary sequence database. In some embodiments, computer comparison methods are used to identify sequence motifs or predicted protein conformation domains that appear in other proteins with known structures and / or functions. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Bowie et al. Science 253:164 (1991). Therefore, the aforementioned examples confirm that those skilled in the art can identify sequence motifs and structural conformations that can be used to define structure and function domains according to the present invention.

[0422] 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 may include various mutant proteins having sequences that are not naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally occurring sequence, such as in portions of the polypeptide outside of the domains that form intermolecular contacts. Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the replacement amino acid should not tend to interrupt helices present in the parent sequence, or disrupt other types of secondary structures characteristic of the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH 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).

[0423] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or one or more internal deletions, but wherein the remaining amino acid sequence is identical to the corresponding positions of the naturally occurring sequence inferred from, for example, a full-length cDNA sequence. A fragment is typically at least 5, 6, 8 or 10 amino acids long, in some embodiments at least 14 amino acids long, in some embodiments at least 20 amino acids long, typically at least 50 amino acids long, and in some embodiments at least 70 amino acids long. As used herein, the term "analog" refers to a polypeptide consisting of a segment of at least 25 amino acids, which has substantial identity with a portion of the inferred amino acid sequence and specifically binds to a target under suitable binding conditions. Typically, a polypeptide analog comprises conservative amino acid substitutions (or additions or deletions) relative to a naturally occurring sequence. Analogs are typically at least 20 amino acids long, in some embodiments at least 50 amino acids long or longer, and typically can be as long as a full-length naturally occurring polypeptide.

[0424] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from biological material.

[0425] 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 attachment to a polypeptide of a biotinyl moiety detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity detectable by optical or calorimetric methods). In some cases, the label or marker may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups, predetermined polypeptide epitopes recognized by second reporter molecules (e.g., leucine zipper pair sequences, binding sites of second antibodies, metal binding domains, epitope tags). In some embodiments, the labels are connected by spacer arms of various lengths to reduce potential steric hindrance. As used herein, the term "drug or pharmaceutical" refers to a compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient.

[0426] Other chemical terms are used herein according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S. ed., McGraw-Hill, San Francisco (1985)).

[0427] As used herein, "substantially pure" means that the subject species is the predominant species present (i.e., it is more abundant on a molar basis than any other individual species in the composition), and in some embodiments, a substantially purified fraction is a composition in which the subject species comprises at least about 50% (on a molar basis) of all macromolecular species present.

[0428] Typically, a substantially pure composition will contain more than about 80% of all macromolecular species present in the composition, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the subject species is purified to substantial homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0429] The term patient includes both human and veterinary subjects.

[0430] The activatable antibodies of the present invention specifically bind to a given target, such as a human target protein. Also included in the present invention are activatable antibodies that bind to the same epitope as the activatable antibodies described herein.

[0431] Those skilled in the art will recognize that it is possible to determine whether the former has the same specificity as the latter by determining whether a monoclonal antibody (e.g., mouse monoclonal or humanized antibody) prevents the monoclonal antibody used in the methods described herein from binding to the target without excessive experimentation. If the tested monoclonal antibody competes with the monoclonal antibody of the present invention, as shown by the reduction in the binding of the monoclonal antibody of the present invention, the two monoclonal antibodies bind to the same or closely related epitopes. The method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody of the present invention is to pre-incubate the monoclonal antibody of the present invention with the target, then add the monoclonal antibody to be tested, to determine whether the tested monoclonal antibody is suppressed in terms of its ability to bind to the target. If the tested monoclonal antibody is suppressed, it is likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention.

[0432] Multispecific activatable antibodies

[0433] The present invention also provides multi-specific activatable antibodies. The multi-specific activatable antibodies provided herein are such multi-specific antibodies that recognize two or more different antigens or epitopes and include at least one masking portion (MM), which is connected to at least one antigen or epitope binding domain of the multi-specific antibody, so that the coupling of the MM reduces the ability of the antigen or epitope binding domain to bind to its target. In some embodiments, the MM is coupled to the antigen or epitope binding domain of the multi-specific antibody via a CM1-CM2 substrate that acts as a substrate of at least one MMP protease and at least one SP. The activatable multi-specific antibodies provided herein are stable in circulation, activated at the intended treatment and / or diagnosis site, but not activated in normal (ie, healthy) tissues, and when activated, exhibit binding to the target at least comparable to the corresponding unmodified multi-specific antibody.

[0434] In some embodiments, multispecific activatable antibodies are designed to engage immune effector cells, also referred to herein as immune effector cell engagement multispecific activatable antibodies. In some embodiments, multispecific activatable antibodies are designed to engage leukocytes, also referred to herein as leukocyte engagement multispecific activatable antibodies. In some embodiments, multispecific activatable antibodies are designed to engage T-cells, also referred to herein as T-cell engagement multispecific activatable antibodies. In some embodiments, multispecific activatable antibodies engage surface antigens on leukocytes (such as T-cells, natural killer (NK) cells, bone marrow mononuclear cells, macrophages, and / or another immune effector cell). In some embodiments, immune effector cells are leukocytes. In some embodiments, immune effector cells are T-cells. In some embodiments, immune effector cells are NK cells. In some embodiments, immune effector cells are monocytes, such as bone marrow mononuclear cells. In some embodiments, multispecific activatable antibodies are designed to bind to or otherwise interact with more than one target and / or more than one epitope, also referred to herein as multi-antigen targeting activatable antibodies. As used herein, the terms "target" and "antigen" are used interchangeably.

[0435] In some embodiments, the immune effector cell engagement multispecific activatable antibody of the present invention comprises a targeting antibody or an antigen binding fragment thereof and an immune effector cell engagement antibody or an antigen binding portion thereof, wherein at least one of the targeting antibody or its antigen binding fragment and / or the immune effector cell engagement antibody or its antigen binding portion is masked. In some embodiments, the immune effector cell engagement antibody or its antigen binding fragment comprises a first antibody or its antigen binding fragment (AB1) that binds to a first immune effector cell engagement target, wherein AB1 is connected to a masking portion (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the targeting antibody or its antigen binding fragment comprises a second antibody or its fragment, which comprises a second antibody or its antigen binding fragment (AB2) that binds to a second target, wherein AB2 is connected to a masking portion (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the immune effector cell engagement antibody or its antigen binding fragment comprises a first antibody or its antigen binding fragment (AB1) that binds to a first immune effector cell engagement target, wherein AB1 is connected to a masking portion (MM1) so that the coupling of MM1 reduces the ability of AB1 to bind to the first target, and the targeting antibody or its antigen binding fragment comprises a second antibody or its fragment, which comprises a second antibody or its antigen binding fragment (AB2) that binds to a second target, wherein AB2 is connected to a masking portion (MM2) so that the coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the non-immune effector cell engagement antibody is a cancer targeting antibody. In some embodiments, the non-immune cell effector antibody is an IgG. In some embodiments, the non-immune effector cell engagement antibody is a scFv. In some embodiments, the targeting antibody (e.g., a non-immune cell effector antibody) is an IgG and the immune effector cell engagement antibody is a 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 cell is a NK cell. In some embodiments, the immune effector cell is a bone marrow mononuclear cell.

[0436] In some embodiments, the T-cell engaging multispecific activatable antibody of the present invention comprises a targeting antibody or an antigen binding fragment thereof and a T-cell engaging antibody or an antigen binding portion thereof, wherein at least one of the targeting antibody or an antigen binding fragment thereof and / or the T-cell engaging antibody or an antigen binding portion thereof is masked. In some embodiments, the T-cell engaging antibody or an antigen binding fragment thereof comprises a first antibody or an antigen binding fragment thereof (AB1) that binds to a first T-cell engaging target, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the targeting antibody or an antigen binding fragment thereof comprises a second antibody or a fragment thereof, which comprises a second antibody or an antigen binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to 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 AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target, and the targeting antibody or antigen binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target.

[0437] 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 to a first T-cell engaging target, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the cancer targeting antibody or antigen binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a second cancer-associated target. In some embodiments, the T-cell engaging antibodies or antigen binding fragments thereof include a first antibody or antigen binding fragment thereof (AB1) that binds to a first T-cell engaging target, wherein AB1 is linked to a masking moiety (MM1) such that conjugation of MM1 reduces the ability of AB1 to bind to the first target, and the cancer targeting antibodies or antigen binding fragments thereof include a second antibody or fragment thereof, which includes a second antibody or antigen binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that conjugation of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0438] In some embodiments, the T-cell engaging multispecific activatable antibody comprises a cancer-targeting IgG antibody or an antigen-binding fragment thereof and a T-cell engaging scFv, wherein at least one of the cancer-targeting IgG antibody or an antigen-binding fragment thereof and / or the T-cell engaging antibody or or its antigen-binding portion is masked. In some embodiments, the T-cell engaging antibody or its antigen-binding fragment comprises a first antibody or its antigen-binding fragment (AB1) that binds to a first T-cell engaging target, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the cancer-targeting IgG antibody or its antigen-binding fragment comprises a second antibody or its fragment, which comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a second cancer-associated target. In some embodiments, the T-cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen binding fragment thereof (AB1) that binds to a first T-cell engaging target, wherein AB1 is linked to a masking moiety (MM1) such that conjugation of MM1 reduces the ability of AB1 to bind to the first target, and the cancer targeting IgG antibody or antigen binding fragment thereof includes a second antibody or fragment thereof, which includes a second antibody or antigen binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that conjugation of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0439] In some embodiments of immune effector cell-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, such as, 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 the other antigen is typically a stimulatory or inhibitory receptor present on the surface of a T-cell, a natural killer (NK) cell, a bone marrow mononuclear cell, a macrophage, and / or other immune effector cell, 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 a T cell or NK cell; examples of such stimulatory receptors include, but are not limited to CD3, CD27, CD28, CD137 (also known as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments, the antigen is an inhibitory receptor present on the surface of a T cell; examples of such inhibitory receptors include, but are not limited to BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIRs. The antibody domain that confers specificity to a T-cell surface antigen can also be replaced by a ligand or ligand domain that binds to a T cell receptor, a NK cell receptor, a macrophage receptor, and / or other immune effector cell receptors, such as, for example, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9.

[0440] One embodiment of the present invention is a multi-specific activatable antibody that can be activated in a cancer microenvironment, and includes antibodies (e.g., IgG or scFv) for tumor targets and agonist antibodies (e.g., IgG or scFv) for costimulatory receptors expressed on the surface of activated T cells or NK cells, wherein at least one of the cancer target antibodies and / or agonist antibodies is masked. Examples of costimulatory receptors include, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, and OX40. In this embodiment, multi-specific activatable antibodies, once activated by tumor-associated proteases, will effectively crosslink and activate the costimulatory receptors expressed by T cells or NK cells in a tumor-dependent manner, to enhance the activity of T cells in response to any tumor antigen via their endogenous T cell antigens or NK activation receptors. The activation-dependent properties of these T cells or NK cell costimulatory receptors will focus on the activity of multi-specific activatable antibodies for the activation of tumor-specific T cells, without activating all T cells, which are independent of their antigenic specificity. In one embodiment, at least the co-stimulatory 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.

[0441] One embodiment of the present invention is a multi-specific activatable antibody, which can be activated in a disease characterized by excessive stimulation of T cells (such as, but not limited to, an autoimmune disease or an inflammatory disease microenvironment). Such multi-specific activatable antibodies include antibodies (such as IgG or scFv) for surface antigens expressed in tissues targeted by T cells in autoimmune or inflammatory diseases, and antibodies (such as IgG or scFv) for inhibitory receptors expressed on the surface of T cells or NK cells, wherein at least one of the disease tissue target antibodies and / or T cell inhibitory receptor antibodies is masked. Examples of inhibitory receptors include, but are not limited to BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3 and NK-expressed KIRs. Examples of tissue antigens targeted by T cells in autoimmune diseases include, but are not limited to, surface antigens expressed on myelin sheaths or nerve cells in multiple sclerosis or surface antigens expressed on islet cells in type 1 diabetes. In this embodiment, when located in a tissue under autoimmune attack or inflammation, the multispecific activatable antibodies are activated and co-engage T cell or NK cell inhibitory receptors to inhibit the activity of autoreactive T cells that respond to any disease tissue targeted antigen via their endogenous TCR or activating receptors. In one embodiment, at least one or more antibodies are masked to prevent inhibition of T cell responses in non-disease tissues where the target antigen may also be expressed.

[0442] In some embodiments, the T cell engagement multispecific activatable antibody comprises an anti-CD3ε (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 to CD3ε, wherein AB1 is connected to a masking portion (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen binding fragment thereof (AB2), which binds to a second target, wherein AB2 is connected to a masking portion (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind to a second target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target.

[0443] In some embodiments, the T-cell engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer targeting antibody or an antigen binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer targeting antibody or an antigen binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or an antigen binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is connected to a masking portion (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer targeting antibody or an antigen binding fragment thereof comprises a second antibody or a fragment thereof, which comprises a second antibody or an antigen binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is connected to a masking portion (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind to a second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0444] In some embodiments, the T-cell engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeted IgG antibody or an antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeted IgG antibody or an antigen-binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is connected to a masking portion (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer-targeted IgG antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, which comprises a second antibody or an antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is connected to a masking portion (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting antibody IgG or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0445] In some embodiments, the T-cell engaging multispecific activatable antibody comprises an anti-CD3epsilon (CD3ε) scFv derived from OKT3, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or 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 to CD3ε, wherein AB1 is linked to a masking portion (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking portion (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a 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 AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target.

[0446] 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 to CD3ε, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer targeting antibody or antigen binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a 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 to CD3ε, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0447] In some embodiments, the T-cell engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeted IgG antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeted 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 to CD3ε, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer-targeted IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a second cancer-associated target. In some embodiments, the OKT3 scFv or OKT3-derived scFv includes a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting antibody IgG or antigen-binding fragment thereof includes a second antibody or fragment thereof, which includes a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0448] 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 to CTLA-4, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CTLA-4. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a second target. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CTLA-4, wherein AB1 is linked to a masking moiety (MM1) such that conjugation of MM1 reduces the ability of AB1 to bind to CTLA-4, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking moiety (MM2) such that conjugation of MM2 reduces the ability of AB2 to bind to the second target.

[0449] 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 fragment thereof is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CTLA-4, wherein AB1 is linked to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CTLA-4. In some embodiments, the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to a second target. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CTLA-4, wherein AB1 is linked to a masking moiety (MM1) such that conjugation of MM1 reduces the ability of AB1 to bind to CTLA-4, and the targeting antibody IgG or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is linked to a masking moiety (MM2) such that conjugation of MM2 reduces the ability of AB2 to bind to the second target.

[0450] In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody at least includes a first antibody or an antigen-binding fragment thereof that binds to a first target and / or a first epitope and a second antibody or an antigen-binding fragment thereof that binds to a second target and / or a second epitope. In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody binds to two or more different targets. In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody binds to two or more different epitopes on the same target. In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody binds to a combination of two or more different targets and two or more different epitopes on the same target.

[0451] In some embodiments, the multispecific activatable antibody comprising IgG has a masked IgG variable domain. In some embodiments, the multispecific activatable antibody comprising scFv has a masked scFv domain. In some embodiments, the multispecific activatable antibody has both IgG variable domains and scFv domains, wherein at least one of the IgG variable domains is coupled to a masking portion. In some embodiments, the multispecific activatable antibody has both IgG variable domains and scFv domains, wherein at least one of the scFv domains is coupled to a masking portion. In some embodiments, the multispecific activatable antibody has both IgG variable domains and scFv domains, wherein at least one of the IgG variable domains is coupled to a masking portion, and at least one of the scFv domains is coupled to a masking portion. In some embodiments, the multispecific activatable antibody has both IgG variable domains and scFv domains, wherein each of the IgG variable domains and the scFv domains is coupled to its own masking portion. In some embodiments, one antibody domain of a 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 a 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 a multispecific activatable antibody has specificity for one epitope of a target antigen, and another antibody domain has specificity for another epitope of a target antigen.

[0452] In multi-specific activatable antibodies, scFv can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, to the carboxyl terminus of the light chain of an IgG activatable antibody, or to the carboxyl terminus of both the heavy chain and the light chain of an IgG activatable antibody. In multi-specific activatable antibodies, scFv can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, to the amino terminus of the light chain of an IgG activatable antibody, or to the amino terminus of both the heavy chain and the light chain of an IgG activatable antibody. In multi-specific activatable antibodies, scFv 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, the masking portion (MM) connected to the CM1-CM2 substrate is connected to the antigen binding domain of IgG and masks it. In some embodiments, the masking portion (MM) connected to the CM1-CM2 substrate is connected to the antigen binding domain of at least one scFv and masks it. In some embodiments, the masking moiety (MM) linked to the CM1-CM2 substrate is linked to and masks the antigen binding domain of 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.

[0453] The present invention provides examples of multispecific activatable antibody structures, which include but are not limited to the following: (VL-CL) 2 :(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM1-CM2Substrate-L1-MM) 2 ; (VL-CL) 2 :(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM1-CM2Substrate-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 substrate-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2 ; (VL-CL) 2 :(MM-L1-CM1-CM2 substrate-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) 2; (MM-L1-CM1-CM2 substrate-L2-VL-CL) 2 :(VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2 ; (MM-L1-CM1-CM2 substrate-L2-VL-CL) 2 :(VH*-L3-VL*-L4-VH-CH1-CH2-CH3) 2 ; (VL-CL-L4-VH*-L3-VL*-L2-CM1-CM2 substrate-L1-MM) 2 :(VH-CH1-CH2-CH3) 2 ; (VL-CL-L4-VL*-L3-VH*-L2-CM1-CM2 substrate-L1-MM) 2 :(VH-CH1-CH2-CH3) 2 ; (MM-L1-CM1-CM2 substrate-L2-VL*-L3-VH*-L4-VL-CL) 2 :(VH-CH1-CH2-CH3) 2 ; (MM-L1-CM1-CM2 substrate-L2-VH*-L3-VL*-L4-VL-CL) 2 :(VH-CH1-CH2-CH3) 2 ; (VL-CL-L4-VH*-L3-VL*-L2-CM1-CM2 substrate-L1-MM) 2 : (MM-L1-CM1-CM2Substrate-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2 ; (VL-CL-L4-VH*-L3-VL*-L2-CM1-CM2 substrate-L1-MM) 2 : (MM-L1-CM1-CM2Substrate-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) 2 ; (VL-CL-L4-VL*-L3-VH*-L2-CM1-CM2 substrate-L1-MM) 2 : (MM-L1-CM1-CM2Substrate-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2 ; (VL-CL-L4-VL*-L3-VH*-L2-CM1-CM2 substrate-L1-MM) 2 :(MM-L1-CM1-CM2 substrate-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) 2 ;(VL-CL-L4-VH*-L3-VL*)2 :(MM-L1-CM1-CM2 substrate-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2 ;(VL-CL-L4-VH*-L3-VL*) 2 :(MM-L1-CM1-CM2 substrate-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3) 2 ;(VL-CL-L4-VL*-L3-VH*) 2 :(MM-L1-CM1-CM2 substrate-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2 ;(VL-CL-L4-VL*-L3-VH*) 2 :(MM-L1-CM1-CM2 substrate-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-L2-CM1-CM2 substrate-L1-MM) 2 : (VL*-L3-VH*-L4-VH-CH1-CH2-CH3) 2 ; (VL-CL-L4-VH*-L3-VL*-L2-CM1-CM2 substrate-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) 2 ; or (VL-CL-L4-VL*-L3-VH*-L2-CM1-CM2 substrate-L1-MM) 2 : (VH*-L3-VL*-L4-VH-CH1-CH2-CH3) 2 , wherein: VL and VH represent the light chain and heavy chain variable domains with the first specificity contained in IgG; VL* and VH* represent the variable domains with the second specificity contained in scFv; L1 is a linker peptide connecting the masking part (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 scFv; L4 is a linker peptide connecting the antibody with the first specificity and the antibody with the second specificity; CL is a light chain constant domain; and CH1, CH2, CH3 are heavy chain constant domains. The first and second specificities can be directed to any antigen or epitope.

[0454] 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, but not limited to, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and the other antigen is typically a stimulatory (also referred to herein as an activating) or inhibitory receptor present on the surface of T-cells, natural killer (NK) cells, myelomonocytes, 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 confers specificity to a T-cell surface antigen may also be replaced by a ligand or ligand domain that binds to a T cell receptor, NK cell receptor, macrophage receptor, and / or other immune effector cell receptor, such as, for example but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9. In some embodiments of the multi-antigen targeting activatable antibody, one antigen is selected from the targets listed in Table 1, and the other antigen is selected from the targets listed in Table 1.

[0455] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which specifically binds to EGFR. In some embodiments, the targeting antibody is C225, which specifically binds to EGFR. In some embodiments, the targeting antibody is C225v4, which specifically binds to EGFR. In some embodiments, the targeting antibody is C225v6, which specifically binds to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which specifically binds to human and mouse Jagged 1 and Jagged 2. In some embodiments, the targeting antibody is 4D11v2, which specifically binds to human and mouse Jagged 1 and Jagged 2.

[0456] In some embodiments, the targeting antibody may be in the form of an activatable antibody. In some embodiments, the scFv may be in the form of a Pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0457] In some embodiments, the scFv is specific for binding to CD3ε and is or is derived from an antibody or fragment thereof that binds to CD3ε, such as CH2527, FN18, H2C, OKT3, 2C11, UCHT1 or V9. In some embodiments, the scFv is specific for binding to CTLA-4 (also referred to herein as CTLA and CTLA4).

[0458] In some embodiments, the anti-CTLA-4 scFv comprises the following amino acid sequence:

[0459] .

[0460] 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.

[0461] In some embodiments, the anti-CD3ε scFv comprises the following amino acid sequence:

[0462] .

[0463] 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.

[0464] In some embodiments, the scFv is specific for binding to one or more T cells, one or more NK cells, and / or one or more macrophages. In some embodiments, the scFv is specific for binding to 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.

[0465] In some embodiments, the multispecific activatable antibody further comprises 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 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 intercalating agent, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is selected from the agents listed in Table 4. In some embodiments, the agent is dolastatin. 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 monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0466] In some embodiments, the multispecific activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0467] In some embodiments, a multispecifically activatable antibody naturally contains one or more disulfide bonds. In some embodiments, a multispecifically activatable antibody can be engineered to include one or more disulfide bonds.

[0468] The present invention also provides isolated nucleic acid molecules encoding the multispecific activatable antibodies described herein, and vectors comprising these isolated nucleic acid sequences. The present invention provides methods for producing multispecific activatable antibodies by culturing cells under conditions that result in expression of activatable antibodies, wherein the cells comprise such nucleic acid molecules. In some embodiments, the cells comprise such vectors.

[0469] The present invention also provides a method for preparing a multispecifically activatable antibody of the present invention by: (a) culturing a cell comprising a nucleic acid construct encoding the multispecifically activatable antibody under conditions that result in expression of the multispecifically activatable antibody, and (b) recovering the multispecifically activatable antibody.

[0470] The present invention also provides multi-specific activatable antibodies and / or multi-specific activatable antibody compositions, which include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a first target or a first epitope and a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target or a second epitope, wherein at least AB1 is coupled to a masking portion (MM1) or otherwise connected, such that the coupling of MM1 reduces the ability of AB1 to bind to its target. In some embodiments, MM1 is coupled to AB1 via a CM1-CM2 substrate of an MMP and SP, wherein at least one of the MMP and SP is co-localized with the target of AB1 at a treatment site or a diagnostic site in a subject. The multi-specific activatable antibodies provided herein are stable in circulation, activated at the intended treatment and / or diagnostic site, but not activated in normal (ie, healthy) tissues, and when activated exhibit binding to the target of AB1 that is at least comparable to that of the corresponding unmodified multi-specific antibody.

[0471] In some embodiments, the multispecifically activatable antibody comprises a linker peptide between MM1 and the CM1-CM2 substrate.

[0472] In some embodiments, the multispecifically activatable antibody comprises a linker peptide between the CM1-CM2 substrate and AB1.

[0473] 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 an 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.

[0474] In some embodiments, at least one of LP1 or LP2 comprises a member selected from (GS) n 、(GGS) n , (GSGGS) n (SEQ ID NO:381) and (GGGS) n(SEQ ID NO: 382), wherein n is an integer of at least 1. 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).

[0475] In some embodiments, the activatable antibody includes a connecting peptide (LP') between CM1 and CM2.

[0476] 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 in the 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 connecting peptides do not need to be identical to each other.

[0477] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 350).

[0478] In some embodiments, the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a first target or a first epitope and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds to a second target or a second epitope. In some embodiments, each AB in the multispecific activatable antibody is independently selected from a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab') 2 Fragments, scFv, scAb, dAb, single domain heavy chain antibodies, and single domain light chain antibodies. In some embodiments, each AB in the multispecific activatable antibody is a rodent (eg, mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

[0479] In some embodiments, each AB in the multispecifically activatable antibody has a dissociation constant for binding to its corresponding target or epitope of about 100 nM or less.

[0480] In some embodiments, the dissociation constant for binding of MM1 to its corresponding AB is greater than the dissociation constant for binding of AB to its corresponding target or epitope.

[0481] In some embodiments, the dissociation constant for binding of MM1 to its corresponding AB is no greater than the dissociation constant for binding of the AB to its corresponding target or epitope.

[0482] In some embodiments, when the multispecifically activatable antibody is in a cleaved state, MM1 does not interfere with or compete with its corresponding AB for binding to a corresponding target or epitope.

[0483] In some embodiments, MM1 is a polypeptide of about 2-40 amino acids in length. In some embodiments, each MM in the multispecific activatable antibody is a polypeptide of no more than 40 amino acids in length.

[0484] In some embodiments, the polypeptide sequence of MM1 is different from the sequence of the target of the corresponding AB.

[0485] In some embodiments, the polypeptide sequence of MM1 has no more than 50% identity with any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of MM1 has no more than 25% identity with any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of MM1 has no more than 10% identity with any natural binding partner of the corresponding AB.

[0486] In some embodiments, the conjugation of MM1 reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to MM1 is d ) is the K of AB for its corresponding target or epitope when not coupled to MM1 d At least 20 times.

[0487] In some embodiments, the conjugation of MM1 reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to MM1 is d ) is the K of AB for its corresponding target or epitope when not coupled to MM1 d At least 40 times.

[0488] In some embodiments, the conjugation of MM1 reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to MM1 is d ) is the K of AB for its corresponding target or epitope when not coupled to MM1 d At least 100 times.

[0489] In some embodiments, the conjugation of MM1 reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to MM1 isd ) is the K of AB for its corresponding target or epitope when not coupled to MM1 d At least 1000 times.

[0490] In some embodiments, the conjugation of MM1 reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to MM1 is d ) is the K of AB for its corresponding target or epitope when not coupled to MM1 d At least 10,000 times.

[0491] In some embodiments, MM1 is an amino acid sequence selected from the MMs disclosed herein.

[0492] In some embodiments, the multispecific activatable antibody comprises at least: a second masking portion (MM2) that inhibits binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state; and an additional cleavable portion (CM') coupled to AB2, wherein CM' is a CM1-CM2 substrate or polypeptide that acts as a substrate for a second protease. In some embodiments, CM' is a polypeptide of no more than 15 amino acids in length. In some embodiments, CM' is a CM1-CM2 substrate, wherein CM1 and CM2 in the CM1-CM2 substrate are each independently no more than 15 amino acids in length.

[0493] In some embodiments, the MMP protease, SP protease and / or the second protease are co-localized with the second target or epitope in the tissue, and wherein the MMP protease, SP protease and / or the second protease cleaves CM' in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the MMP protease, SP protease and / or the second protease. In some embodiments, the MMP protease, SP protease and / or the second protease are co-localized with the first target or epitope and the second target or epitope in the tissue. In some embodiments, the MMP protease, SP protease and / or the second protease are the same MMP protease and the same SP protease. In some embodiments, the MMP protease, SP protease and / or the 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 of the same MMP protease and the same SP protease. In some embodiments, the protease that cleaves CM' is selected from those shown in Table 6.

[0494] In some embodiments, each MM in the multispecifically activatable antibody (eg, MM1 and at least MM2) binds to its corresponding AB with a dissociation constant that is greater than the dissociation constant for binding of the AB to its corresponding target or epitope.

[0495] In some embodiments, the dissociation constant for binding of each MM in the multispecifically activatable antibody to its corresponding AB is no greater than the dissociation constant for binding of the AB to its corresponding target or epitope.

[0496] In some embodiments, when the multispecifically activatable antibody is in a cleaved state, each MM in the multispecifically activatable antibody does not interfere with or compete with its corresponding AB for binding to the corresponding target or epitope.

[0497] In some embodiments, each MM in the multispecific activatable antibody is a polypeptide of about 2-40 amino acids in length. In some embodiments, each MM in the multispecific activatable antibody is a polypeptide of no more than 40 amino acids in length.

[0498] In some embodiments, the polypeptide sequence of each MM in the multispecifically activatable antibody is different from the polypeptide sequence of the target of the corresponding AB.

[0499] In some embodiments, the polypeptide sequence of each MM in the multispecific activatable antibody has no more than 50% identity with any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of each MM in the multispecific activatable antibody has no more than 25% identity with any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of each MM in the multispecific activatable antibody has no more than 10% identity with any natural binding partner of the corresponding AB.

[0500] In some embodiments, the conjugation of each MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to the MM is d ) is the K of AB for its corresponding target or epitope when not coupled to MM d At least 20 times.

[0501] In some embodiments, the conjugation of each MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to the MM is d ) is the K of AB for its corresponding target or epitope when not coupled to MM d At least 40 times.

[0502] In some embodiments, the conjugation of each MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to the MM is d ) is the K of AB for its corresponding target or epitope when not coupled to MM d At least 100 times.

[0503] In some embodiments, the conjugation of each MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to the MM is d ) is the K of AB for its corresponding target or epitope when not coupled to MM d At least 1000 times.

[0504] In some embodiments, the conjugation of each MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB for its corresponding target or epitope when conjugated to the MM is d ) is the K of AB for its corresponding target or epitope when not coupled to MM d At least 10,000 times.

[0505] In some embodiments, each MM is an amino acid sequence selected from the MMs disclosed herein.

[0506] In some embodiments, a protease that cleaves the CM1-CM2 substrate sequence is co-localized with the target of AB1 in the multispecific activatable antibody in a tissue, and the MMP protease and / or the 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 the protease.

[0507] In some embodiments, the multispecific activatable antibody comprises more than one CM1-CM2 substrate sequence, and an MMP protease and / or SP protease that cleaves at least one CM1-CM2 substrate sequence is co-localized with a target of at least one of the AB regions in the multispecific activatable antibody in a tissue, 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.

[0508] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 2-fold greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0509] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 3-fold greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0510] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 4-fold greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0511] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 5-fold greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0512] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 10-fold greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0513] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 20 times greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0514] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 40-fold greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0515] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 50 times greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0516] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 100-fold greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0517] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that in the uncleaved state, binding of the multispecific activatable antibody to a target of one of the AB regions is reduced to a dissociation constant that is at least 200 times greater than the dissociation constant for binding of the unmodified AB to its target, whereas in the cleaved state, the AB binds to its target.

[0518] The present invention also provides compositions and methods comprising multispecific activatable antibodies, the multispecific activatable antibodies comprising at least a first antibody or antibody fragment (AB1) and a second antibody or antibody fragment (AB2) that specifically binds to a target, wherein at least the first AB in the multispecific activatable antibody is coupled to a masking portion (MM1) that reduces the ability of AB1 to bind to its target. In some embodiments, each AB is coupled to an MM that reduces the ability of its corresponding AB to each target. For example, in a bispecific activatable antibody embodiment, AB1 is coupled to a first masking portion (MM1) that reduces the ability of AB1 to bind to its target, and AB2 is coupled to a second masking portion (MM2) that reduces the ability of AB2 to bind to its target. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in such embodiments, for each AB in the multispecific activatable antibody, 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.

[0519] 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, wherein the CM1-CM2 substrate connects the MM to the AB. For example, in some embodiments, the multispecific activatable antibody comprises at least a first antibody or antibody fragment (AB1) that specifically binds to a target and a second antibody or antibody fragment (AB2), wherein at least the first AB in the multispecific activatable antibody is coupled to a masking portion (MM1) that reduces the ability of AB1 to bind to its target via a first CM1-CM2 substrate. In some bispecific activatable antibody embodiments, AB1 is coupled to MM1 via a first CM1-CM2 substrate, and AB2 is coupled to a second masking portion (MM2) that reduces the ability of AB2 to bind to its target via a second CM1-CM2 substrate. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in some of these embodiments, for each AB in the multispecific activatable antibody, 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 to a third masking moiety (MM3) that reduces the ability of AB3 to bind to its target via a third CM1-CM2 substrate, and so on.

[0520] Activatable antibodies with non-binding steric moiety or binding partner of non-binding steric moiety

[0521] The present invention also provides an activatable antibody comprising a non-binding steric portion (NB) or a binding partner (BP) of the non-binding steric portion, wherein the BP recruits or otherwise attracts the NB to the activatable antibody. The activatable antibodies provided herein include, for example, an activatable antibody comprising a non-binding steric portion (NB), a CM1-CM2 substrate, and an antibody or antibody fragment (AB) that binds to a target; an activatable antibody comprising a binding partner (BP) of a non-binding steric portion, a CM1-CM2 substrate, and an AB; and an activatable antibody comprising a BP that has recruited a NB, a CM1-CM2 substrate, and an AB that binds to a target. An activatable antibody in which the NB is covalently linked to the CM1-CM2 substrate and AB of the activatable antibody or is associated by interacting with the BP covalently linked to the CM1-CM2 substrate and AB of the activatable antibody is referred to herein as an "activatable antibody containing a NB". Activatable or convertible means that the activatable antibody exhibits a first level of binding to the target when the activatable antibody is in an inhibited, masked or uncleaved state (ie, a first conformation), and exhibits a second level of binding to the target when the activatable antibody is in an uninhibited, unmasked and / or cleaved state (ie, a second conformation, ie, an activated antibody), wherein the second target binding level is greater than the first target binding level. Activatable antibody compositions can exhibit increased bioavailability and more favorable biodistribution compared to conventional antibody therapeutics.

[0522] In some embodiments, the activatable antibody provides reduced toxicity and / or adverse side effects that might otherwise result from binding at non-therapeutic and / or non-diagnostic sites if the AB is not masked or otherwise inhibited from binding to such sites.

[0523] In one embodiment, the activatable antibody includes a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein 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 so that in the uncut state, the NB interferes with the binding of the AB to the target, and in the cut state, the NB does not interfere with the binding of the AB to the target; and the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. As used herein and throughout, the term polypeptide refers to a polypeptide comprising at least two amino acid residues, including larger polypeptides, full-length proteins, and fragments thereof, and the term polypeptide is not limited to single-chain polypeptides, and may include multi-monomers, e.g., multi-chain, polypeptides. In the case of a polypeptide having a shorter length (e.g., less than 50 amino acids in total), the terms peptide and polypeptide are used interchangeably herein, and in the case of a polypeptide having a longer length (e.g., 50 amino acids or more), the terms polypeptide and protein are used interchangeably herein.

[0524] In one embodiment, the activatable antibody comprises a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide of up to 50 amino acids in length that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target; and (iv) the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. For example, the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate each independently have a length of up to 15 amino acids.

[0525] In one embodiment, the activatable antibody comprises a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to AB; (ii) the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of AB to the target, and in the cleaved state, the NB does not interfere with the binding of AB to the target; (iv) the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme; and (v) the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: NB-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-NB.

[0526] In one embodiment, the activatable antibody comprises a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target, and wherein the NB in ​​the cleaved activatable antibody reduces the ability of the AB to bind to the 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%, at least 100% compared to the ability of the cleaved AB to bind to the 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 a target is determined, for example, using an assay as described herein or an in vitro target displacement assay, such as the assays described in PCT Publication Nos. WO 2009 / 025846 and WO 2010 / 081173.

[0527] In one embodiment, the activatable antibody comprises a binding partner of a non-binding stereogenic portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein the BP is a polypeptide that binds to it when exposed to the NB; the NB 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 so that in the uncleaved state, in the absence of the NB, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target and the BP does not interfere with the binding of the AB to the target; and the NB and BP do not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. In some embodiments 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.

[0528] In some examples of any of these activatable antibody embodiments, the activatable antibody is formulated into a composition. In some of these embodiments, the composition further comprises a NB, wherein the NB is co-formulated with an activatable antibody comprising a BP, a CM1-CM2 substrate, and an AB. In some embodiments of this embodiment, the BP is selected from 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 other serum protein binding peptides.

[0529] In some examples of any of these activatable antibody embodiments, the NB is a soluble globular protein. In some examples of any of these activatable antibody embodiments, the NB is a protein that circulates in the bloodstream. In some examples of any of these activatable antibody embodiments, the NB is selected from albumin, fibrinogen, fibronectin, hemoglobin, transferrin, immunoglobulin domains, and other serum proteins.

[0530] In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a substrate (S) of a protease. In some examples of any of these activatable antibody embodiments, the protease is co-localized with the target in the tissue, and when the activatable antibody is exposed to the protease, the protease cuts the CM1-CM2 substrate in the activatable antibody. In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide up to 50 amino acids long. In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a length of up to 15 amino acids, for example, 3 amino acids long, 4 amino acids long, 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long or 15 amino acids long.

[0531] In some examples of any of these activatable antibody embodiments, the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: NB-CM1-CM2 substrate-AB, AB-CM1-CM2 substrate-NB, BP-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-BP. In embodiments where the activatable antibody includes a BP and the activatable antibody is in the presence of a corresponding NB, the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: NB:BP-CM1-CM2-AB, NB:BP-CM2-CM1-AB, AB-CM1-CM2-BP:NB or AB-CM2-CM1-BP:NB, wherein ":" represents an interaction, such as binding, between NB and BP.

[0532] In some examples of any of these activatable antibody embodiments, the activatable antibody includes an antibody or antigen-binding fragment thereof that specifically binds to a given target and is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab') 2 Fragments, scFv, scab, dAb, single domain heavy chain antibodies or single domain light chain antibodies. In some embodiments, such target-binding antibodies or immunologically active fragments thereof are mouse, other rodent, chimeric, humanized or fully human monoclonal antibodies.

[0533] In some examples of any one of these activatable antibody embodiments, the activatable antibody includes a variable heavy chain region comprising an amino acid sequence presented herein and a combination of a variable light chain region comprising an amino acid sequence presented herein. In some embodiments, in some embodiments, the activatable antibody includes a variable heavy chain region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence presented herein and a variable light chain region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence presented herein.

[0534] In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises 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 selected from the agents 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 a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the agent is dolastatin. 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 monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansine alkaloid or a maytansine alkaloid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0535] In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0536] In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises a spacer. In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some examples of any of these activatable antibody embodiments, the spacer is directly connected to the MM of the activatable antibody.

[0537] In some embodiments, the serum half-life of the activatable antibody is longer than the serum half-life of the corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 15 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 11 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 9 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 7 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 6 days. In some examples of any of these activatable antibody embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 5 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 4 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 3 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 2 days. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 24 hours. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 20 hours. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 18 hours. In some embodiments, when applied to an organism, the serum half-life of the activatable antibody is at least 16 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 14 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 6 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 4 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 3 hours.

[0538] The present invention also provides isolated nucleic acid molecules encoding any of these activatable antibodies, and vectors comprising these isolated nucleic acid sequences. The present invention provides methods for producing activatable antibodies by culturing cells under conditions leading to expression of activatable antibodies, wherein the cells comprise such nucleic acid sequences. In some embodiments, the cells comprise such vectors.

[0539] The dissociation constant (K) of the activatable antibody containing NB to the target d ) is greater than the K of AB for the target when it is not associated with NB or NB:BP d The dissociation constant (K) of the activatable antibody containing NB to the target d ) is greater than the K of parent AB to target d For example, the K of AB for the target when it is not associated with NB or NB:BP d or the K of the parental AB to the target d In contrast, the K of the activatable antibody containing NB against the target d5, 10, 25, 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 greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000,000, 100-1,000,000, 100-10,000,000, 1,000-10 In contrast, the activatable antibody containing a NB has a binding affinity for the target that is lower than the binding affinity of the AB for the target when it is not associated with a NB or NB:BP or lower than the binding affinity of the parent AB for the target. For example, the activatable antibody containing a NB has a binding affinity for the target that is at least 5, 10, 25, 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 greater, or between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 3, 4, 5, 6, 7, 8, 9, 10, 15 20, 25 30, 25 31 5-10, 10-100, 10-1,000, 10-10,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000,000, 100-1,000,000, 100-10,000,000, 1,000-10 ,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times.

[0540] When an activatable antibody containing a NB is in the presence of a target, specific binding of the AB to the target is reduced or inhibited compared to specific binding of the AB to the target when it is not associated with a NB or NB:BP. When an activatable antibody containing a NB is in the presence of a target, specific binding of the AB to the target is reduced or inhibited compared to specific binding of the parent AB to the target. The ability of the activatable antibody containing a NB to bind to a target is reduced, e.g., by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%, when measured in vivo and / or in vitro, 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 longer, when compared to binding of an AB not associated with a NB or NB:BP to the target, or binding of a parent AB to the target.

[0541] When an activatable antibody containing a NB is in the presence of the target but in the absence of a modifying agent (e.g., a protease or other enzyme), the specific binding of the AB to the target is reduced or inhibited compared to the specific binding of the AB to the target when it is not associated with the NB or NB:BP. When an activatable antibody containing a NB is in the presence of the target but in the absence of a modifying agent (e.g., a protease, other enzyme, a reducing agent, or light), the specific binding of the AB to the target is reduced or inhibited compared to the specific binding of the parent AB to the target. The ability of the activatable antibody containing a NB to bind to a target is reduced, e.g., by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%, when measured in vivo and / or in vitro, 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 longer, when compared to binding of an AB not associated with a NB or NB:BP to the target, or binding of a parent AB to the target.

[0542] In some examples of any of these activatable antibody embodiments, the activatable antibody includes an agent conjugated to AB to produce an 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 a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the agent is selected from the agents listed in Table 3. In some embodiments, the agent is dolastatin. 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 monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0543] In some examples of any of these activatable antibody embodiments, the activatable antibody is a dual target binding activatable antibody. Such a dual target binding activatable antibody contains two Abs that can bind to the same or different targets. In a specific embodiment, the dual targeting activatable antibody contains a bispecific antibody or antibody fragment.

[0544] Dual target binding activatable antibodies are designed to have CM1-CM2 substrates that can be cleaved by a cleavage agent, and the cleavage agent is co-localized with one or both of the targets of the AB that can bind to the activatable antibody in the target tissue. Dual target binding activatable antibodies with more than one AB for the same or different targets can be designed to have more than one CM1-CM2 substrate, wherein the first CM1-CM2 substrate can be cleaved by a cleavage agent in the first target tissue, and wherein the second CM1-CM2 substrate can be cleaved by a cleavage agent in the second target tissue, wherein one or more of the targets bind to the AB of the activatable antibody. In one embodiment, the first and second target tissues are separated in space, such as at different sites in an organism. In one embodiment, the first and second target tissues are the same tissue separated in time, such as the same tissue at two different time points, such as the first time point when the tissue is an early tumor, and the second time point when the tissue is a late tumor.

[0545] The present invention also provides nucleic acid molecules encoding the activatable antibodies described herein. The present invention also provides vectors comprising these nucleic acids. The activatable antibodies described herein are produced by culturing cells under conditions that lead to expression of the activatable antibodies, wherein the cells include these nucleic acid molecules or vectors.

[0546] The present invention also provides a method for preparing an activatable antibody. In one embodiment, the method comprises the following steps: (a) culturing a cell comprising a nucleic acid construct encoding the activatable antibody under conditions that result in expression of the activatable antibody, wherein the activatable antibody comprises (i) a non-binding stereogenic portion (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) of an enzyme; (3) the CM1-CM2 substrate is positioned so that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target; and (4) the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody.

[0547] In some embodiments, the method comprises the following steps: (a) culturing a cell comprising a nucleic acid construct encoding the activatable antibody under conditions that result in expression of the activatable antibody, wherein the activatable antibody comprises (i) a binding partner (BP) of a non-binding stereogenic moiety (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) for an enzyme; (3) the CM1-CM2 substrate is positioned such that in the uncleaved state, in the absence of the NB, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target and the BP does not interfere with the binding of the AB to the target; and (4) the NB and the BP do not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody. In some embodiments of this embodiment, the BP of the activatable antibody is bound to the NB.

[0548] Uses of activatable antibodies and conjugated activatable antibodies

[0549] It should be understood that the administration of the therapeutic entities of the present invention will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. Most suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th Edition, Mack Publishing Company, Easton, PA (1975)), especially Chapter 87 of Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cations or anions) (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions of polyethylene glycol (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. Any of the aforementioned mixtures may be suitable for the treatment and therapy of the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerated with the route of administration. For additional information on formulations, excipients, and carriers well known to pharmaceutical chemists, 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.

[0550] The therapeutic preparations of the present invention including conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies are used to prevent, treat or otherwise improve diseases or conditions associated with abnormal target expression and / or activity. For example, therapeutic preparations of the present invention including conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies are used to treat or improve inflammation, inflammatory conditions, autoimmune diseases and / or cancer or other tumor conditions. In some embodiments, cancer is a solid tumor or hematological malignancy in which the target is expressed. In some embodiments, cancer is a solid tumor in which the target is expressed. In some embodiments, cancer is a hematological malignancy in which the target is expressed. In some embodiments, the target is expressed on parenchyma (for example, in cancer, the part of an organ or tissue that generally implements the function of an organ or tissue). In some embodiments, the target is expressed on cells, tissues or organs. In some embodiments, the target is expressed on a matrix (i.e., a connective support 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 (vascular system). In some embodiments, the target is expressed on cancer stem cells. In some embodiments, the agent conjugated to the activatable antibody is a microtubule inhibitor. In some embodiments, the agent conjugated to the activatable antibody is a nucleic acid damaging agent.

[0551] The effectiveness of prevention, improvement or treatment is determined in relation to any known method for diagnosing or treating a disease or condition associated with target expression and / or activity (such as, for example, abnormal target expression and / or activity). Prolonging the survival of a subject or otherwise delaying the progression of a disease or condition associated with target expression and / or activity (e.g., abnormal target expression and / or activity) in a subject indicates that the conjugated antibody, activatable antibody and / or conjugated activatable antibody confers clinical benefit.

[0552] The conjugated antibody, activatable antibody and / or conjugated activatable antibody can be administered in the form of a pharmaceutical composition. The principles and considerations involved in preparing such compositions and guidance for the selection of components are provided in, for example, Remington: The Science and Practice Of Pharmacy 19th Edition (Alfonso R. Gennaro, et al., ed.) 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.

[0553] In some embodiments where antibody fragments are used, the smallest fragment of the binding domain of the target protein is selected to specifically bind. For example, according to the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The preparation may also contain more than one active compound necessary for the specific indication treated, for example, in some embodiments, those with supplementary activity, which do not adversely affect each other. In some embodiments or in addition, the composition may include an agent that improves its function, such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent or a growth inhibitor. Such molecules are suitably combined in an amount effective for the intended purpose.

[0554] The active ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and p...

Claims

1. An isolated polypeptide comprising a substrate, wherein the substrate comprises 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), wherein the substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 547, 550 and 71.

2. The isolated polypeptide of claim 1, wherein the MMP is MMP2, MMP9 or MMP14.

3. The isolated polypeptide of claim 1 or claim 2, wherein the SP is uPA or matriptase.

4. An isolated polypeptide comprising an antibody or antigen-binding fragment thereof AB, and 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, wherein the substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 547, 550 and 71.

5. The isolated polypeptide of claim 4, wherein the MMP, SP, or both the MMP and SP are co-localized with the target in a tissue.

6. The isolated polypeptide of claim 4, wherein the antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

7. The isolated polypeptide of claim 4, wherein said AB is linked to said CM1.

8. The isolated polypeptide of claim 7, wherein said AB is directly linked to said CM1.

9. The isolated polypeptide of claim 7, wherein the AB is linked to the CM1 via a linker peptide.

10. The isolated polypeptide of claim 4, wherein the AB is linked to CM2.

11. The isolated polypeptide of claim 10, wherein the AB is directly linked to CM2.

12. The isolated polypeptide of claim 10, wherein the AB is linked to the CM1 via a linker peptide.

13. The isolated polypeptide of claim 4, wherein the MMP is MMP2, MMP9 or MMP14.

14. The isolated polypeptide of claim 4, wherein the SP is uPA or matriptase.

15. The isolated polypeptide of claim 4, wherein the isolated polypeptide comprises a masking moiety MM.

16. The isolated polypeptide of claim 15, wherein the MM binds to the AB with a dissociation constant greater than the dissociation constant of the AB binding to the target.

17. The isolated polypeptide of claim 15, wherein the MM is a polypeptide of no more than 40 amino acids in length.

18. The isolated polypeptide of claim 15, wherein the substrate comprises both CM1 and CM2, and the MM is linked to the CM1 such that the isolated polypeptide in the uncleaved state comprises the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB or AB-CM2-CM1-MM.

19. The isolated polypeptide of claim 18, wherein the isolated polypeptide comprises a connecting peptide between the MM and the CM1.

20. The isolated polypeptide of claim 18, wherein the isolated polypeptide comprises a connecting peptide between CM2 and the AB.

21. The isolated polypeptide of claim 15, wherein the substrate comprises both CM1 and CM2, and the MM is linked to CM2 such that the isolated polypeptide in the uncleaved state comprises the following structural arrangement from N-terminus to C-terminus: MM-CM2-CM1-AB or AB-CM1-CM2-MM.

22. The isolated polypeptide of claim 21, wherein the isolated polypeptide comprises a connecting peptide between the MM and CM2.

23. The isolated polypeptide of claim 21, wherein the isolated polypeptide comprises a connecting peptide between CM1 and the AB.

24. The isolated polypeptide of claim 15, wherein the substrate comprises both CM1 and CM2, and the isolated polypeptide comprises a first connecting peptide LP1 and a second connecting peptide LP2, and wherein the isolated polypeptide in an uncleaved state has the following structural arrangement from N-terminus to C-terminus: 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.

25. The isolated polypeptide of claim 24, wherein the two connecting peptides need not be identical to each other.

26. The isolated polypeptide of claim 24, wherein LP1 and LP2 are each peptides 1-20 amino acids in length.

27. The isolated polypeptide of claim 15, wherein the amino acid sequence of the MM is different from the amino acid sequence of the target and has no more than 10% identity with the amino acid sequence of the natural binding partner of the AB.

28. The isolated polypeptide of claim 15, wherein in the cleaved state, the MM does not interfere with or compete with the AB for binding to the target.

29. The isolated polypeptide of claim 15, wherein the MMP is MMP2, MMP9 or MMP14.

30. The isolated polypeptide of claim 15, wherein the SP is uPA or matriptase.

31. A conjugated activatable antibody comprising the isolated polypeptide of claim 15 conjugated to an agent.

32. The conjugated, activatable antibody of claim 31, wherein the agent is conjugated to the AB via a linker.

33. The conjugated, activatable antibody of claim 32, wherein the linker is a cleavable linker.

34. The conjugated, activatable antibody of claim 32, wherein the linker is a non-cleavable linker.

35. The conjugated, activatable antibody of claim 31, wherein the agent is a toxin or a fragment thereof.

36. The conjugated, activatable antibody of claim 31, wherein the agent is a microtubule inhibitor.

37. The conjugated, activatable antibody of claim 31, wherein the agent is a nucleic acid damaging agent.

38. The conjugated, activatable antibody of claim 35, wherein the agent is selected from the group consisting of dolastatin or a derivative thereof, auristatin or a derivative thereof, maytansinoids or a derivative thereof, duocarmycins or a derivative thereof, and calicheamicins or a derivative thereof.

39. The conjugated, activatable antibody of claim 38, wherein the agent is auristatin E or a derivative thereof.

40. The conjugated, activatable antibody of claim 38, wherein the agent is monomethyl auristatin E (MMAE).

41. The conjugated, activatable antibody of claim 38, wherein the agent is monomethyl auristatin D (MMAD).

42. The conjugated, activatable antibody of claim 38, wherein the agent is a maytansinoid selected from the group consisting of DM1 and DM4.

43. The conjugated, activatable antibody of claim 31, wherein the agent is a detectable moiety.

44. The conjugated, activatable antibody of claim 43, wherein the detectable moiety is a diagnostic agent.

45. A pharmaceutical composition comprising the isolated polypeptide as claimed in any one of claims 4 to 30 and a carrier.

46. ​​The pharmaceutical composition of claim 45, comprising an additional agent.

47. The pharmaceutical composition of claim 46, wherein the additional agent is a therapeutic agent.

48. An isolated nucleic acid molecule encoding an isolated polypeptide as described in any one of claims 1-30.

49. A vector comprising the isolated nucleic acid molecule of claim 48.

50. A method of producing an antibody or an activatable antibody by culturing a cell under conditions resulting in the expression of an isolated polypeptide as claimed in any one of claims 1 to 30, wherein the cell comprises a nucleic acid molecule according to claim 48.

51. A method for producing an activatable antibody that binds a target in an activated state, the method comprising: (a) culturing a cell comprising a nucleic acid construct encoding an isolated polypeptide as described in any one of claims 15 to 30; and (b) recovering the activatable antibody.

52. Use of an isolated polypeptide as described in any one of claims 4 to 30, a conjugated activatable antibody as described in any one of claims 31 to 44, or a pharmaceutical composition as described in any one of claims 45 to 47 in the preparation of a medicament for treating, alleviating the symptoms of a disorder or disease, or delaying the progression of a disorder or disease, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide as described in any one of claims 4 to 30, a conjugated activatable antibody as described in any one of claims 31 to 44, or a pharmaceutical composition as described in any one of claims 45 to 47, wherein the disorder or disease is cancer.

53. The use of claim 52, wherein treating, ameliorating symptoms of, or delaying progression of a condition or disease comprises administering an additional agent to the subject.

54. The use of claim 53, wherein the additional agent is a therapeutic agent.

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