Substrates and other cleavable moieties for matriptase and u-plasminogen activator and methods of use thereof

Abstract

To provide methods of identifying new substrates for proteases and methods of using these substrates in a variety of therapeutic, diagnostic and prophylactic indications.SOLUTION: The invention relates generally to: polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator (uPA); activatable antibodies and other large molecules that include the cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator; and methods of making these polypeptides that include the cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator, and methods of use in a variety of therapeutic, diagnostic and prophylactic indications.SELECTED DRAWING: Figure 4A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 934,619, filed January 31, 2014, and U.S. Provisional Patent Application No. 61 / 971,009, filed March 27, 2014, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to polypeptides containing a cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator (uPA), to activatable antibodies and other large molecules containing a cleavable moiety that is a substrate for at least one protease selected from matriptase and uPA, and to methods of making and using these polypeptides containing a cleavable moiety that is a substrate for at least one protease selected from matriptase and uPA in a variety of therapeutic, diagnostic, and prophylactic applications. [Background technology]

[0003] Proteases are enzymes that cleave peptide bonds between amino acid residues. Some proteases are known to break specific peptide bonds based on the presence of specific amino acid sequences in proteins. Proteases occur naturally in all living organisms and are involved in a variety of physiological reactions, from simple degradation to highly regulated pathways. However, many pathological conditions are associated with deregulated protease expression and / or activity. Thus, inappropriate proteolysis may play a major role in the development and progression of cancer, as well as cardiovascular, inflammatory, neurodegenerative, eukaryotic, bacterial, viral, and parasitic diseases.

[0004] Thus, there is a need to identify new substrates for proteases and to use these substrates in a variety of therapeutic, diagnostic, and prophylactic indications. Summary of the Invention

[0005] The present disclosure provides amino acid sequences containing a cleavable moiety (CM) that is a substrate for at least one protease selected from matriptase (also referred to herein as MT-SP1, matriptase-1, and similar terms meaning matriptase) and u-plasminogen activator (also referred to herein as uPA, urokinase, urokinase-type plasminogen activator, and similar terms meaning uPA). These CMs are useful in a variety of therapeutic, diagnostic, and prophylactic indications.

[0006] In some embodiments, the CM is linked or otherwise attached to the antibody. For example, the CM is used to link one or more agents to an antibody or antigen-binding fragment thereof that binds to a predetermined target, such that upon exposure to proteases, i.e., matriptase and / or uPA, the CM is cleaved and the agents are released from the antibody or antigen-binding fragment thereof. Exemplary targets include, but are not limited to, those listed in Table 1. Exemplary antibodies or antigen-binding fragments thereof include, but are not limited to, those listed in Table 2. In some embodiments, the uncleaved antibody has the following structural arrangement from N- to C-terminus: agent-CM-(antibody or antigen-binding fragment) or (antibody or antigen-binding fragment)-CM-agent. In some embodiments, the antibody comprises a linking peptide between the antibody or antigen-binding fragment and the CM. In some embodiments, the antibody or antigen-binding fragment comprises a linking peptide between the CM and the conjugated agent.

[0007] In some embodiments, the antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), wherein the antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: agent-LP1-CM-LP2-(antibody or antigen-binding fragment) or (antibody or antigen-binding fragment)-LP2-CM-LP1-agent. In some embodiments, the two connecting peptides need not be identical to each other.

[0008] In some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 385) and (GGGS) n (SEQ ID NO: 386), where n is at least one integer.

[0009] 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: 387), GGSGG (SEQ ID NO: 388), GSGSG (SEQ ID NO: 389), GSGGG (SEQ ID NO: 390), GGGSG (SEQ ID NO: 391), and GSSSG (SEQ ID NO: 392).

[0010] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 393), GSSGGSGGSGG (SEQ ID NO: 394), GSSGGSGGSGGS (SEQ ID NO: 395), GSSGGSGGSGGSGGGS (SEQ ID NO: 396), GSSGGSGGSG (SEQ ID NO: 397), or GSSGGSGGSGS (SEQ ID NO: 398).

[0011] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 399), GSSGT (SEQ ID NO: 400), or GSSG (SEQ ID NO: 401).

[0012] In some embodiments, the antibody or antigen-binding fragment thereof has an equilibrium dissociation constant for binding to the target of about 100 nM or less.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds a target. In some embodiments, the antibody or immunologically active fragment thereof that binds the target is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, or a single-domain light chain antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds the target is a murine, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0014] In some embodiments, the protease, ie, matriptase and / or uPA, co-localizes with the target in the tissue, and when the antibody is exposed to the protease, the protease cleaves the CM within the antibody.

[0015] In some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0016] In some embodiments, the CM is a substrate for at least matriptase. In some embodiments, the CM is a substrate for at least uPA. In some embodiments, the CM is a substrate for at least matriptase and uPA.

[0017] In some embodiments, CM is a substrate for matriptase and / or uPA and is resistant to cleavage by at least the other protease. In some embodiments, CM is a substrate for matriptase and / or uPA and is resistant to cleavage by at least plasmin. In some embodiments, CM is a substrate for matriptase and / or uPA and is resistant to cleavage by at least tissue plasminogen activator (tPA).

[0018] In some embodiments, the CM is a substrate for matriptase and / or uPA and comprises a motif sequence recognized by matriptase and / or uPA, with the proviso that for any motif sequence of the present disclosure: (i) the CM does not include any of the following amino acid sequences: TGRGPSWV (SEQ ID NO: 402), SARGPSRW (SEQ ID NO: 403), or TARGPSFK (SEQ ID NO: 404); and the CM does not include a consensus amino acid sequence based on these amino acid sequences, such as, for example, TARGPSW (SEQ ID NO: 405); (ii) the CM does not comprise any of the following amino acid sequences: LSGRSDNH (SEQ ID NO: 406), GGWHTGRN (SEQ ID NO: 407), HTGRSGAL (SEQ ID NO: 408), or PLTGRSGG (SEQ ID NO: 409); and the CM does not comprise a consensus amino acid sequence based on these amino acid sequences, such as, for example, LTGRSGA (SEQ ID NO: 410); and / or (iii) The CM does not include any of the following amino acid sequences: AARGPAIH (SEQ ID NO: 411), RGPAFNPM (SEQ ID NO: 412), SSRGPAYL (SEQ ID NO: 413), or RGPATPIM (SEQ ID NO: 414); and the CM does not include a consensus amino acid sequence based on these amino acid sequences, such as, for example, RGPA (SEQ ID NO: 415).

[0019] In some embodiments, the motif sequence is a substrate for at least matriptase and comprises a core CM consensus sequence set forth in Tables 8A-8J below. In some embodiments, the motif sequence comprises a subgenus, i.e., a subset, of the core CM consensus sequences set forth in Tables 8A-8J below.

[0020] [Table 1]

[0021] [Table 2]

[0022] [Table 3]

[0023] [Table 4]

[0024] [Table 5]

[0025] [Table 6]

[0026] [Table 7]

[0027] [Table 8]

[0028] [Table 9]

[0029] [Table 10]

[0030] In some embodiments, the motif sequence is a substrate for at least matriptase and comprises an extended consensus sequence based on one of the core CM consensus sequences shown in Tables 8A-8J. In some embodiments, the extended consensus sequence is a consensus sequence shown in Tables 9A-9J-3 below.

[0031] Table 11

[0032] Table 12

[0033] Table 13

[0034] Table 14

[0035] Table 15

[0036] Table 16

[0037] Table 17

[0038] Table 18

[0039] Table 19

[0040] Table 20

[0041] Table 21

[0042] Table 22

[0043] Table 23

[0044] Table 24

[0045] Table 25

[0046] Table 26

[0047] Table 27

[0048] Table 28

[0049] Table 29

[0050] Table 30

[0051] Table 31

[0052] [Table 32]

[0053] [Table 33]

[0054] [Table 34]

[0055] [Table 35]

[0056] [Table 36]

[0057] In some embodiments, the CM comprises a core CM consensus 1 sequence comprising the amino acid sequence AAPRS (SEQ ID NO: 163). In some embodiments, the CM comprises an extended core CM consensus 1 sequence comprising the amino acid sequence AAPRSF (SEQ ID NO: 164).

[0058] In some embodiments, the CM comprises a core CM consensus 2 sequence comprising the amino acid sequence SRRVP (SEQ ID NO: 165). In some embodiments, the CM comprises an extended core CM consensus 2 sequence comprising an amino acid sequence selected from the group consisting of QSRRVP (SEQ ID NO: 166), QTRRVP (SEQ ID NO: 167), SRRVPL (SEQ ID NO: 168), SRRVPV (SEQ ID NO: 169), QSRRVPL (SEQ ID NO: 170), QSRRVPV (SEQ ID NO: 171), QTRRVPL (SEQ ID NO: 172), and QTRRVPV (SEQ ID NO: 173).

[0059] In some embodiments, the CM comprises the amino acid sequence QSRRVP (SEQ ID NO: 166). In some embodiments, the CM comprises the amino acid sequence QTRRVP (SEQ ID NO: 167). In some embodiments, the CM comprises the amino acid sequence SRRVPL (SEQ ID NO: 168). In some embodiments, the CM comprises the amino acid sequence SRRVPV (SEQ ID NO: 169). In some embodiments, the CM comprises the amino acid sequence QSRRVPL (SEQ ID NO: 170). In some embodiments, the CM comprises the amino acid sequence QSRRVPV (SEQ ID NO: 171). In some embodiments, the CM comprises the amino acid sequence QTRRVPL (SEQ ID NO: 172). In some embodiments, the CM comprises the amino acid sequence QTRRVPV (SEQ ID NO: 173).

[0060] In some embodiments, the CM comprises a core CM consensus 3 sequence comprising the amino acid sequence PPLGR (SEQ ID NO: 174). In some embodiments, the CM comprises an extended core CM consensus 3 sequence comprising an amino acid sequence selected from the group consisting of GPPLGR (SEQ ID NO: 175), SPPLGR (SEQ ID NO: 176), CGPPLGR (SEQ ID NO: 177), CSPPLGR (SEQ ID NO: 178), GGPPLGR (SEQ ID NO: 179), GSPPLGR (SEQ ID NO: 180), SGPPLGR (SEQ ID NO: 181), SSPPLGR (SEQ ID NO: 182), GCGPPLGR (SEQ ID NO: 183), GCSPPLGR (SEQ ID NO: 184), GGGPPLGR (SEQ ID NO: 185), GGSPPLGR (SEQ ID NO: 186), GSGPPLGR (SEQ ID NO: 187), GSSPPLGR (SEQ ID NO: 188), SCGPPLGR (SEQ ID NO: 189), SCSPPLGR (SEQ ID NO: 190), SGGPPLGR (SEQ ID NO: 191), SGSPPLGR (SEQ ID NO: 192), SSGPPLGR (SEQ ID NO: 193), and SSSPPLGR (SEQ ID NO: 194).

[0061] In some embodiments, the CM comprises the amino acid sequence GPPLGR (SEQ ID NO: 175). In some embodiments, the CM comprises the amino acid sequence SPPLGR (SEQ ID NO: 176). In some embodiments, the CM comprises the amino acid sequence CGPPLGR (SEQ ID NO: 177). In some embodiments, the CM comprises the amino acid sequence CSPPLGR (SEQ ID NO: 178). In some embodiments, the CM comprises the amino acid sequence GGPPLGR (SEQ ID NO: 179). In some embodiments, the CM comprises the amino acid sequence GSPPLGR (SEQ ID NO: 180). In some embodiments, the CM comprises the amino acid sequence SGPPLGR (SEQ ID NO: 181). In some embodiments, the CM comprises the amino acid sequence SSPPLGR (SEQ ID NO: 182). In some embodiments, the CM comprises the amino acid sequence GCGPPLGR (SEQ ID NO: 183). In some embodiments, the CM comprises the amino acid sequence GCSPPLGR (SEQ ID NO: 184). In some embodiments, the CM comprises the amino acid sequence GGGPPLGR (SEQ ID NO: 185). In some embodiments, the CM comprises the amino acid sequence GGSPPLGR (SEQ ID NO: 186). In some embodiments, the CM comprises the amino acid sequence GSGPPLGR (SEQ ID NO: 187). In some embodiments, the CM comprises the amino acid sequence GSSPPLGR (SEQ ID NO: 188). In some embodiments, the CM comprises the amino acid sequence SCGPPLGR (SEQ ID NO: 189). In some embodiments, the CM comprises the amino acid sequence SCSPPLGR (SEQ ID NO: 190). In some embodiments, the CM comprises the amino acid sequence SGGPPLGR (SEQ ID NO: 191). In some embodiments, the CM comprises the amino acid sequence SGSPPLGR (SEQ ID NO: 192). In some embodiments, the CM comprises the amino acid sequence SSGPPLGR (SEQ ID NO: 193). In some embodiments, the CM comprises the amino acid sequence SSSPPLGR (SEQ ID NO: 194).

[0062] In some embodiments, the CM comprises a core CM consensus 4 sequence comprising the amino acid sequence LRSGW (SEQ ID NO: 195). In some embodiments, the CM comprises an extended core CM consensus 4 sequence comprising an amino acid sequence selected from the group consisting of MLRSGW (SEQ ID NO: 196), MLRSGWR (SEQ ID NO: 197), MLRSGWRG (SEQ ID NO: 198), MLRSGWRL (SEQ ID NO: 199), and MLRSGWRS (SEQ ID NO: 200).

[0063] In some embodiments, the CM comprises the amino acid sequence MLRSGW, (SEQ ID NO: 196). In some embodiments, the CM comprises the amino acid sequence MLRSGWR (SEQ ID NO: 197). In some embodiments, the CM comprises the amino acid sequence MLRSGWRG (SEQ ID NO: 198). In some embodiments, the CM comprises the amino acid sequence MLRSGWRL (SEQ ID NO: 199). In some embodiments, the CM comprises the amino acid sequence MLRSGWRS (SEQ ID NO: 200).

[0064] In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence VSRSA (SEQ ID NO: 201). In some embodiments, the CM comprises an extended core CM consensus 5 sequence comprising an amino acid sequence selected from the group consisting of IVSRSA (SEQ ID NO: 202), YIVSRSA (SEQ ID NO: 203), and QYIVSRSA (SEQ ID NO: 204).

[0065] In some embodiments, the CM comprises the amino acid sequence IVSRSA (SEQ ID NO: 202). In some embodiments, the CM comprises the amino acid sequence YIVSRSA (SEQ ID NO: 203). In some embodiments, the CM comprises the amino acid sequence QYIVSRSA (SEQ ID NO: 204).

[0066] In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence ALRAP (SEQ ID NO: 205). In some embodiments, the CM comprises an extended core CM consensus 6 sequence comprising the amino acid sequence RALRAP (SEQ ID NO: 206).

[0067] In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence PAGRR (SEQ ID NO: 207). In some embodiments, the CM comprises an extended core CM consensus 7 sequence comprising an amino acid sequence selected from the group consisting of PAGRRS (SEQ ID NO: 208), PAGRRSL (SEQ ID NO: 209), VPAGRRS (SEQ ID NO: 210), and VPAGRRSL (SEQ ID NO: 211).

[0068] In some embodiments, the CM comprises the amino acid sequence PAGRRS (SEQ ID NO: 208). In some embodiments, the CM comprises the amino acid sequence PAGRRSL (SEQ ID NO: 209). In some embodiments, the CM comprises the amino acid sequence VPAGRRS (SEQ ID NO: 210). In some embodiments, the CM comprises the amino acid sequence VPAGRRSL (SEQ ID NO: 211).

[0069] In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence GRSML (SEQ ID NO: 212). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising GRSMLL (SEQ ID NO: 213), GRSMLM (SEQ ID NO: 214), GRSMLLG (SEQ ID NO: 215), GRSMLLP (SEQ ID NO: 216), GRSMLLS (SEQ ID NO: 217), GRSMLMG (SEQ ID NO: 218), GRSMLMP (SEQ ID NO: 219), GRSMLMS (SEQ ID NO: 220), GRSMLLGG (SEQ ID NO: 221), GRSMLLPG (SEQ ID NO: 222), GRSMLLSG (SEQ ID NO: 223), GRSMLMGG (SEQ ID NO: 224), GRSMLMPG (SEQ ID NO: 225), GRSMLMSG (SEQ ID NO: 226). ), GRSMLLGP (SEQ ID NO: 227), GRSMLLPP (SEQ ID NO: 228), GRSMLLSP (SEQ ID NO: 229), GRSMLMGP (SEQ ID NO: 230), GRSMLMPP (SEQ ID NO: 231), GRSMLMSP (SEQ ID NO: 232), GRSMLLGS (SEQ ID NO: 233), GRSMLLPS (SEQ ID NO: 234), GRSMLLSS (SEQ ID NO: 235), GRSMLMGS (SEQ ID NO: 236), GRSMLMPS (SEQ ID NO: 237), and GRSMLMSS (SEQ ID NO: 238).

[0070] In some embodiments, the CM comprises the amino acid sequence GRSMLL (SEQ ID NO:213). In some embodiments, the CM comprises the amino acid sequence GRSMLM (SEQ ID NO:214). In some embodiments, the CM comprises the amino acid sequence GRSMLLG (SEQ ID NO:215). In some embodiments, the CM comprises the amino acid sequence GRSMLLP (SEQ ID NO:216). In some embodiments, the CM comprises the amino acid sequence GRSMLLS (SEQ ID NO:217). In some embodiments, the CM comprises the amino acid sequence GRSMLMG (SEQ ID NO:218). In some embodiments, the CM comprises the amino acid sequence GRSMLMP (SEQ ID NO:219). In some embodiments, the CM comprises the amino acid sequence GRSMLMS (SEQ ID NO:220). In some embodiments, the CM comprises the amino acid sequence GRSMLLGG (SEQ ID NO:221). In some embodiments, the CM comprises the amino acid sequence GRSMLLPG (SEQ ID NO:222). In some embodiments, the CM comprises the amino acid sequence GRSMLLSG (SEQ ID NO:223). In some embodiments, the CM comprises the amino acid sequence GRSMLMGG (SEQ ID NO:224). In some embodiments, the CM comprises the amino acid sequence GRSMLMPG (SEQ ID NO:225). In some embodiments, the CM comprises the amino acid sequence GRSMLMSG (SEQ ID NO: 226). In some embodiments, the CM comprises the amino acid sequence GRSMLLGP (SEQ ID NO: 227). In some embodiments, the CM comprises the amino acid sequence GRSMLLPP (SEQ ID NO: 228). In some embodiments, the CM comprises the amino acid sequence GRSMLLSP (SEQ ID NO: 229). In some embodiments, the CM comprises the amino acid sequence GRSMLMGP (SEQ ID NO: 230). In some embodiments, the CM comprises the amino acid sequence GRSMLMPP (SEQ ID NO: 231). In some embodiments, the CM comprises the amino acid sequence GRSMLMSP (SEQ ID NO: 232). In some embodiments, the CM comprises the amino acid sequence GRSMLLGS (SEQ ID NO: 233). In some embodiments, the CM comprises the amino acid sequence GRSMLLPS (SEQ ID NO: 234).In some embodiments, the CM comprises the amino acid sequence GRSMLLSS (SEQ ID NO: 235). In some embodiments, the CM comprises the amino acid sequence GRSMLMGS (SEQ ID NO: 236). In some embodiments, the CM comprises the amino acid sequence GRSMLMPS (SEQ ID NO: 237). In some embodiments, the CM comprises the amino acid sequence GRSMLMSS (SEQ ID NO: 238).

[0071] In some embodiments, the CM comprises a core CM consensus 9 sequence comprising the amino acid sequence LARAG (SEQ ID NO: 239). In some embodiments, the CM comprises an extended core CM consensus 9 sequence comprising an amino acid sequence selected from the group consisting of LARAGI (SEQ ID NO: 240), LARAGL (SEQ ID NO: 241), PLARAGI (SEQ ID NO: 242), PLARAGL (SEQ ID NO: 243), RPLARAGI (SEQ ID NO: 244), and RPLARAGL (SEQ ID NO: 245).

[0072] In some embodiments, the CM comprises the amino acid sequence LARAGI (SEQ ID NO: 240). In some embodiments, the CM comprises the amino acid sequence LARAGL (SEQ ID NO: 241). In some embodiments, the CM comprises the amino acid sequence PLARAGI (SEQ ID NO: 242). In some embodiments, the CM comprises the amino acid sequence PLARAGL (SEQ ID NO: 243). In some embodiments, the CM comprises the amino acid sequence RPLARAGI (SEQ ID NO: 244). In some embodiments, the CM comprises the amino acid sequence RPLARAGL (SEQ ID NO: 245).

[0073] In some embodiments, the CM comprises a core CM consensus 10 sequence comprising the amino acid sequence ESRRW (SEQ ID NO: 246). In some embodiments, the CM comprises an extended core CM consensus 10 sequence comprising an amino acid sequence selected from the group consisting of ESRRWM (SEQ ID NO: 247), ESRRWMP (SEQ ID NO: 248), and PESRRWMP (SEQ ID NO: 249).

[0074] In some embodiments, the CM comprises the amino acid sequence ESRRWM (SEQ ID NO: 247). In some embodiments, the CM comprises the amino acid sequence ESRRWMP (SEQ ID NO: 248). In some embodiments, the CM comprises the amino acid sequence PESRRWMP (SEQ ID NO: 249).

[0075] In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of ILPRSPAF (SEQ ID NO: 250), VAGRSMRP (SEQ ID NO: 251), VVPEGRRS (SEQ ID NO: 252), QGRAITFI (SEQ ID NO: 253), VLSKQMSF (SEQ ID NO: 254), LKGRSYYY (SEQ ID NO: 255), KRMPVQFL (SEQ ID NO: 256), PQHRIVSF (SEQ ID NO: 257), YKKFVGSL (SEQ ID NO: 258), HMMQYARH (SEQ ID NO: 259), IPFSWSRF (SEQ ID NO: 260), LSQARWRK (SEQ ID NO: 261), DISHWRRS (SEQ ID NO: 262), RKTVQHWW (SEQ ID NO: 263), RFYRNQFF (SEQ ID NO: 264), RSLVFAPI (SEQ ID NO: 265), RSPSRLKC (SEQ ID NO: 266), and RKMPNITV (SEQ ID NO: 267).

[0076] In some embodiments, the CM comprises the amino acid sequence ILPRSPAF (SEQ ID NO: 250). In some embodiments, the CM comprises the amino acid sequence VAGRSMRP (SEQ ID NO: 251). In some embodiments, the CM comprises the amino acid sequence VVPEGRRS (SEQ ID NO: 252). In some embodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ ID NO: 253). In some embodiments, the CM comprises the amino acid sequence VLSKQMSF (SEQ ID NO: 254). In some embodiments, the CM comprises the amino acid sequence LKGRSYYY (SEQ ID NO: 255). In some embodiments, the CM comprises the amino acid sequence KRMPVQFL (SEQ ID NO: 256). In some embodiments, the CM comprises the amino acid sequence PQHRIVSF (SEQ ID NO: 257). In some embodiments, the CM comprises the amino acid sequence YKKFVGSL (SEQ ID NO: 258). In some embodiments, the CM comprises the amino acid sequence HMMQYARH (SEQ ID NO: 259). In some embodiments, the CM comprises the amino acid sequence IPFSWSRF (SEQ ID NO: 260). In some embodiments, the CM comprises the amino acid sequence LSQARWRK (SEQ ID NO: 261). In some embodiments, the CM comprises the amino acid sequence DISHWRRS (SEQ ID NO: 262). In some embodiments, the CM comprises the amino acid sequence RKTVQHWW (SEQ ID NO: 263). In some embodiments, the CM comprises the amino acid sequence RFYRNQFF (SEQ ID NO: 264). In some embodiments, the CM comprises the amino acid sequence RSLVFAPI (SEQ ID NO: 265). In some embodiments, the CM comprises the amino acid sequence RSPSRLKC (SEQ ID NO: 266). In some embodiments, the CM comprises the amino acid sequence RKMPNITV (SEQ ID NO: 267).

[0077] In some embodiments, the CM comprises a motif sequence that is a substrate for at least uPA and / or matriptase and comprises a core CM consensus sequence set forth in Tables 10A-10J below. In some embodiments, the motif sequence comprises a subgenus, i.e., a subgroup, of the core CM consensus sequences set forth in Tables 10A-10D below.

[0078] [Table 37]

[0079] [Table 38]

[0080] [Table 39]

[0081] [Table 40]

[0082] In some embodiments, the motif sequence is a substrate for at least uPA and / or matriptase and comprises an extended consensus sequence based on one of the core CM consensus sequences shown in Tables 10A-10D. In some embodiments, the extended consensus sequence is a consensus sequence shown in Tables 11A-11D below.

[0083] [Table 41]

[0084] [Table 42]

[0085] [Table 43]

[0086] [Table 44]

[0087] In some embodiments, the CM comprises a core CM consensus 11 sequence comprising the amino acid sequence LSGRSANH (SEQ ID NO: 307) or LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CM comprises an extended core CM consensus 11 sequence comprising the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309), DRLSGRSDNHKK (SEQ ID NO: 310), or NTLSGRSGNHGS (SEQ ID NO: 311).

[0088] In some embodiments, the CM comprises the amino acid sequence LSGRSANH (SEQ ID NO: 307). In some embodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CM comprises the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309). In some embodiments, the CM comprises the amino acid sequence DRLSGRSDNHKK (SEQ ID NO: 310). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 311).

[0089] In some embodiments, the CM comprises the amino acid sequence LSGRSANH (SEQ ID NO: 307). In some embodiments, the CM comprises the amino acid sequence LNGRSDNH (SEQ ID NO: 313). In some embodiments, the CM comprises the amino acid sequence LTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM comprises a core CM consensus 12 sequence comprising the amino acid sequences LSGRSANH (SEQ ID NO: 307), LNGRSDNH (SEQ ID NO: 313), and LTGRSDRH (SEQ ID NO: 314).In some embodiments, the CM is selected from the group consisting of DRLSGRSANHKK (SEQ ID NO: 309), DRLSGRSDNHKK (SEQ ID NO: 310), GPLNGRSDNHKA (SEQ ID NO: 320), GPLNGRSDNHKK (SEQ ID NO: 321), GPLNGRSDNHKR (SEQ ID NO: 322), GPLNGRSDNHQA (SEQ ID NO: 323), GPLNGRSDNHQK (SEQ ID NO: 324), GPLNGRSDNHQR (SEQ ID NO: 325), GPLNGRSDNHRA (SEQ ID NO: 326), GPLNGRSDNHRK ( SEQ ID NO: 327), GPLNGRSDNHRR (SEQ ID NO: 328), RPLNGRSDNHKA (SEQ ID NO: 329), RPLNGRSDNHKK (SEQ ID NO: 330), RPLNGRSDNHKR (SEQ ID NO: 331), RPLNGRSDNHQA (SEQ ID NO: 332), RPLNGRSDNHQK (SEQ ID NO: 333), RPLNGRSDNHQR (SEQ ID NO: 334), RPLNGRSDNHRA (SEQ ID NO: 335), RPLNGRSDNHRK (SEQ ID NO: 336), RPLNGRSDNHRR (SEQ ID NO: 337), G PLSGRSDNHKA (SEQ ID NO: 338), GPLSGRSDNHKK (SEQ ID NO: 339), GPLSGRSDNHKR (SEQ ID NO: 340), GPLSGRSDNHQA (SEQ ID NO: 341), GPLSGRSDNHQK (SEQ ID NO: 342), GPLSGRSDNHQR (SEQ ID NO: 343), GPLSGRSDNHRA (SEQ ID NO: 344), GPLSGRSDNHRK (SEQ ID NO: 345), GPLSGRSDNHRR (SEQ ID NO: 346), RPLSGRSDNHKA (SEQ ID NO: 347), RPLSGRSDNH KK (SEQ ID NO: 348), RPLSGRSDNHKR (SEQ ID NO: 349), RPLSGRSDNHQA (SEQ ID NO: 350), RPLSGRSDNHQK (SEQ ID NO: 351), RPLSGRSDNHQR (SEQ ID NO: 352), RPLSGRSDNHRA (SEQ ID NO: 353), RPLSGRSDNHRK (SEQ ID NO: 354), RPLSGRSDNHRR (SEQ ID NO: 355), and KGLTGRSDRHQA (SEQ ID NO: 356).

[0090] In some embodiments, the CM comprises the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309). In some embodiments, the CM comprises the amino acid sequence DRLSGRSDNHKK (SEQ ID NO: 310). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHKA (SEQ ID NO: 320). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHKK (SEQ ID NO: 321). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHKR (SEQ ID NO: 322). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHQA (SEQ ID NO: 323). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHQK (SEQ ID NO: 324). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHQR (SEQ ID NO: 325). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHRA (SEQ ID NO: 326). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHRK (SEQ ID NO: 327). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHRR (SEQ ID NO: 328). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHKA (SEQ ID NO: 329). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHKK (SEQ ID NO: 330). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHKR (SEQ ID NO: 331). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHQA (SEQ ID NO: 332). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHQK (SEQ ID NO: 333). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHQR (SEQ ID NO: 334). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHRA (SEQ ID NO: 335). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHRK (SEQ ID NO: 336). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHRR (SEQ ID NO: 337).In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHKA (SEQ ID NO: 338). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHKK (SEQ ID NO: 339). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHKR (SEQ ID NO: 340). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHQA (SEQ ID NO: 341). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHQK (SEQ ID NO: 342). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHQR (SEQ ID NO: 343). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHRA (SEQ ID NO: 344). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHRK (SEQ ID NO: 345). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHRR (SEQ ID NO: 346). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHKA (SEQ ID NO: 347). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHKK (SEQ ID NO: 348). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHKR (SEQ ID NO: 349). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHQA (SEQ ID NO: 350). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHQK (SEQ ID NO: 351). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHQR (SEQ ID NO: 352). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHRA (SEQ ID NO: 353). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHRK (SEQ ID NO: 354). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHRR (SEQ ID NO: 355). In some embodiments, the CM comprises the amino acid sequence KGLTGRSDRHQA (SEQ ID NO: 356).

[0091] In some embodiments, the CM comprises a core CM consensus 13 sequence comprising the amino acid sequence RIGRSDNH (SEQ ID NO: 357) or RLGRSDNN (SEQ ID NO: 358). In some embodiments, the CM comprises an extended core CM consensus 13 sequence comprising the amino acid sequence NHRIGRSDNHRR (SEQ ID NO: 359) or TLRLGRSDNNKN (SEQ ID NO: 360).

[0092] In some embodiments, the CM comprises the amino acid sequence RIGRSDNH (SEQ ID NO: 357). In some embodiments, the CM comprises the amino acid sequence RLGRSDNN (SEQ ID NO: 358). In some embodiments, the CM comprises the amino acid sequence NHRIGRSDNHRR (SEQ ID NO: 359). In some embodiments, the CM comprises the amino acid sequence TLRLGRSDNNKN (SEQ ID NO: 360).

[0093] In some embodiments, the CM comprises a core CM consensus 14 sequence comprising an amino acid sequence selected from the group consisting of TSGRSANP (SEQ ID NO: 361), TSGRSGNP (SEQ ID NO: 362), LSGRSANP (SEQ ID NO: 363), and LSGRSGNP (SEQ ID NO: 364). In some embodiments, the CM comprises an extended core CM consensus 14 sequence comprising an amino acid sequence selected from the group consisting of TSTSGRSAMPRG (SEQ ID NO: 365), TSTSGRSGNPRG (SEQ ID NO: 366), TSLSGRSANPRG (SEQ ID NO: 367), and TSLSGRSGNPRG (SEQ ID NO: 368).

[0094] In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 361). In some embodiments, the CM comprises the amino acid sequence TSGRSGNP (SEQ ID NO: 362). In some embodiments, the CM comprises the amino acid sequence LSGRSANP (SEQ ID NO: 363). In some embodiments, the CM comprises the amino acid sequence LSGRSGNP (SEQ ID NO: 364). In some embodiments, the CM comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 365). In some embodiments, the CM comprises the amino acid sequence TSTSGRSGNPRG (SEQ ID NO: 366). In some embodiments, the CM comprises the amino acid sequence TSLSGRSANPRG (SEQ ID NO: 367). In some embodiments, the CM comprises the amino acid sequence TSLSGRSGNPRG (SEQ ID NO: 368).

[0095] In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of LSGRSENH (SEQ ID NO: 369), SIARSDNL (SEQ ID NO: 370), LSGRSVTQ (SEQ ID NO: 371), LSGRSGNH (SEQ ID NO: 308), LTGRSDRH (SEQ ID NO: 314), LYGRSENN (SEQ ID NO: 374), RLGRSDNN (SEQ ID NO: 375), TSGRSANP (SEQ ID NO: 376), NTLSGRSENHSG (SEQ ID NO: 377), PPSIARSDNLAN (SEQ ID NO: 378), TGLSGRSVTQTS (SEQ ID NO: 379), NTLSGRSGNHGS (SEQ ID NO: 311), KGLTGRSDRHQA (SEQ ID NO: 381), KNLYGRSENNGN (SEQ ID NO: 382), TLRLGRSDNNKN (SEQ ID NO: 383), and TSTSGRSANPRG (SEQ ID NO: 384).

[0096] In some embodiments, the CM comprises the amino acid sequence LSGRSENH (SEQ ID NO: 369). In some embodiments, the CM comprises the amino acid sequence SIARSDNL (SEQ ID NO: 370). In some embodiments, the CM comprises the amino acid sequence LSGRSVTQ (SEQ ID NO: 371). In some embodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CM comprises the amino acid sequence LTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM comprises the amino acid sequence LYGRSENN (SEQ ID NO: 374). In some embodiments, the CM comprises the amino acid sequence RLGRSDNN (SEQ ID NO: 375). In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 376). In some embodiments, the CM comprises the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 377). In some embodiments, the CM comprises the amino acid sequence PPSIARSDNLAN (SEQ ID NO: 378). In some embodiments, the CM comprises the amino acid sequence TGLSGRSVTQTS (SEQ ID NO: 379). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 311). In some embodiments, the CM comprises the amino acid sequence KGLTGRSDRHQA (SEQ ID NO: 381). In some embodiments, the CM comprises the amino acid sequence KNLYGRSENNGN (SEQ ID NO: 382). In some embodiments, the CM comprises the amino acid sequence TLRLGRSDNNKN (SEQ ID NO: 383). In some embodiments, the CM comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 384).

[0097] In some embodiments, the CM is a substrate for at least two proteases, in some embodiments, at least one protease is matriptase or uPA, and at least one protease is selected from the group consisting of those shown in Table 7.

[0098] [Table 45]

[0099] In some embodiments, the antibody is attached to at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each polypeptides less than 15 amino acids in length. In some embodiments, the first CM and the second CM in the antibody in its uncleaved state have the following structural arrangement from N-terminus to C-terminus: agent-CM1-CM2-(antibody or antigen-binding fragment), (antibody or antigen-binding fragment thereof)-CM2-CM1-agent, agent-CM2-CM1-(antibody or antigen-binding fragment thereof), or (antibody or antigen-binding fragment thereof)-CM1-CM2-agent. In some embodiments, the antibody comprises a linking peptide between the agent and CM1. In some embodiments, the antibody comprises a linking peptide between CM1 and CM2. In some embodiments, the antibody comprises a linking peptide between CM2 and the antibody or antigen-binding fragment thereof. In some embodiments, the antibody comprises a linking peptide between the agent and CM1 and a linking peptide between CM2 and the antibody or antigen-binding fragment thereof. In some embodiments, the antibody comprises a linking peptide between the agent and CM1 and a linking peptide between CM2 and the antibody or antigen-binding fragment thereof. In some embodiments, the antibody comprises a linking peptide between the agent and CM1 and a linking peptide between CM1 and CM2. In some embodiments, the antibody comprises a connecting peptide between CM1 and CM2, and a connecting peptide between CM2 and the antibody or antigen-binding fragment thereof, hi some embodiments, the antibody comprises a connecting peptide between the agent and CM1, a connecting peptide between CM1 and CM2, and a connecting peptide between CM2 and the antibody or antigen-binding fragment thereof.

[0100] In some embodiments, the antibody comprises at least one first CM comprising a substrate for at least one protease selected from matriptase and uPA, and a second CM comprising a substrate sequence. Exemplary substrates for the second CM (CM2) include, but are not limited to, substrates cleavable by one or more of the following enzymes or proteases listed in Table 7:

[0101] In some embodiments, CM2 is selected for use with a particular protease, ie, CM2 is a substrate for at least one protease selected from the group consisting of matrix metalloproteinases (MMPs), neutrophil elastase, uPA (also called u-plasminogen activator), legumin, matriptase, thrombin, cysteine ​​proteases such as cathepsins, ADAM17, BMP-1, HtrA1, and TMPRSSs such as TMPRSS3 or TMPRSS4.

[0102] In some embodiments, CM2 is a substrate for neutrophil elastase. In some embodiments, CM2 is a substrate for uPA. In some embodiments, CM2 is a substrate for legumin. In some embodiments, CM2 is a substrate for matriptase. In some embodiments, CM2 is a substrate for thrombin. In some embodiments, CM2 is a substrate for cysteine ​​proteases. In some embodiments, CM2 is a substrate for cathepsins. In some embodiments, CM2 is a substrate for ADAM17. In some embodiments, CM2 is a substrate for BMP-1. In some embodiments, CM2 is a substrate for HtrA1. In some embodiments, CM2 is a substrate for TMPRSS. In some embodiments, CM2 is a substrate for TMPRSS3. In some embodiments, CM2 is a substrate for TMPRSS4.

[0103] For example, a suitable CM2 may be cleaved by at least one protease and contain the sequences TGRGPSWV (SEQ ID NO: 402); SARGPSRW (SEQ ID NO: 403); TARGPSFK (SEQ ID NO: 404); TARGPSW (SEQ ID NO: 405); LSGRSDNH (SEQ ID NO: 406); GGWHTGRN (SEQ ID NO: 407); HTGRSGAL (SEQ ID NO: 408); PLTGRSGG (SEQ ID NO: 409); AARGPAIH (SEQ ID NO: 411); RGPAFNPM (SEQ ID NO: 412); SSRGPAYL (SEQ ID NO: 413); Sequence number 413); RGPATPIM (sequence number 414); RGPA (sequence number 415); GGQPSGMWGW (sequence number 416); FPRPLGITGL (sequence number 417); VHMPLGFLGP (sequence number 418); SPLTGRSG (sequence number 419); SAGFSLPA (sequence number 126); LAPLGLQRR (sequence number 420); SGGPLGVR (sequence number 421); PLGL (sequence number 422); GPRSFGL (sequence number 423) and / or GPRSFG (sequence number 424).

[0104] In some embodiments, CM2 comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 402). In some embodiments, CM2 comprises the amino acid sequence SARGPSRW (SEQ ID NO: 403). In some embodiments, CM2 comprises the amino acid sequence TARGPSFK (SEQ ID NO: 404). In some embodiments, CM2 comprises the amino acid sequence TARGPSW (SEQ ID NO: 405). In some embodiments, CM2 comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 406). In some embodiments, CM2 comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 407). In some embodiments, CM2 comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 408). In some embodiments, CM2 comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 409). In some embodiments, CM2 comprises the amino acid sequence AARGPAIH (SEQ ID NO: 411). In some embodiments, CM2 comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 412). In some embodiments, CM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 413). In some embodiments, CM2 comprises the amino acid sequence RGPATPIM (SEQ ID NO: 414). In some embodiments, CM2 comprises the amino acid sequence RGPA (SEQ ID NO: 415). In some embodiments, CM2 comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO: 416). In some embodiments, CM2 comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 417). In some embodiments, CM2 comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 418). In some embodiments, CM2 comprises the amino acid sequence SPLTGRSG (SEQ ID NO: 419). In some embodiments, CM2 comprises the amino acid sequence LAPLGLQRR (SEQ ID NO: 420). In some embodiments, CM2 comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 421). In some embodiments, CM2 comprises the amino acid sequence PLGL (SEQ ID NO: 422). In some embodiments, CM2 comprises the amino acid sequence GPRSFGL (SEQ ID NO: 423). In some embodiments, CM2 comprises the amino acid sequence GPRSFG (SEQ ID NO: 424).

[0105] In some embodiments, CM2 is a substrate for at least one MMP. In some embodiments, CM2 is a substrate for at least one MMP listed in Table 7. In some embodiments, CM2 is a substrate for MMP9. In some embodiments, CM2 is a substrate for MMP14. In some embodiments, CM2 is a substrate for two or more MMPs. In some embodiments, CM2 is a substrate for at least MMP9 or MMP14. In some embodiments, CM2 is a substrate for two or more MMPs. In some embodiments, CM2 is a substrate for at least MMP9 and MMP14.

[0106] In some embodiments, CM2 is a substrate for MMPs and includes the sequences ISSGLLSS (SEQ ID NO: 425); QNQALRMA (SEQ ID NO: 426); AQNLLGMV (SEQ ID NO: 427); STFPFGMF (SEQ ID NO: 428); PVGYTSSL (SEQ ID NO: 429); DWLYWPGI (SEQ ID NO: 430); MIAPVAYR (SEQ ID NO: 431); RPSPMWAY (SEQ ID NO: 432); WATPRPMR (SEQ ID NO: 433); FRLLDWQW (SEQ ID NO: 434); LKAAPRWA (SEQ ID NO: 435); GPSHLLVLT (SEQ ID NO: 436); LPGGLSPW (SEQ ID NO: 437); MGLFSEAG (SEQ ID NO: 438); SPLPLRVP (SEQ ID NO: 439); RMHLRSLG (SEQ ID NO: 440); LAAPLGLL (SEQ ID NO: 441); AVGLLAPP (SEQ ID NO: 442); LLAPSHRA (SEQ ID NO: 443); PAGLWLDP (SEQ ID NO: 444) and / or ISSGLSS (SEQ ID NO: 445).

[0107] In some embodiments, the first cleaving agent and the second cleaving agent are the same protease, and the first CM and the second CM are different substrates for the enzyme. In some embodiments, the first cleaving agent and the second cleaving agent are different proteases. In some embodiments, the first cleaving agent and the second cleaving agent are co-localized in the target tissue. In some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

[0108] In some embodiments, the CM comprises the non-prime side of the protease cleavage site; i.e., the CM comprises at least the P1 and P2 amino acids, and in some embodiments, the P1, P2, and P3 amino acids, and in some embodiments, the P1, P2, P3, and P4 amino acids. In some embodiments, the CM comprises the non-prime side and the prime side of the protease cleavage site. In some embodiments, the CM comprises the non-prime side but lacks at least a portion of the prime side of the protease cleavage site. In some embodiments, the CM comprises the non-prime side but lacks the prime side of the protease cleavage site. Such CMs can be linked directly or through a linker to antibodies or other molecules described herein in this disclosure, such as, but not limited to, detection moieties.

[0109] In some embodiments, the agent conjugated to the antibody or antigen-binding fragment thereof is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or fragment thereof. As used herein, a fragment of a toxin is a fragment that retains toxic activity. In some embodiments, the agent is attached to AB via a cleavable linker. In some embodiments, the agent is attached to AB via a linker comprising at least one cleavable substrate sequence described herein. In some embodiments, the agent is attached 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 intercalator, or other DNA damaging agent. In some embodiments, the agent is an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0110] In some embodiments, the agent is an anti-inflammatory agent.

[0111] In some embodiments, the antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0112] In some embodiments, the conjugated antibody and / or conjugated activatable antibody comprises a detectable label. In some embodiments, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent comprises a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the imaging agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label comprises an N-methylacridinium derivative. In some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0113] In some embodiments, antibodies naturally contain one or more disulfide bonds. In some embodiments, antibodies or antigen-binding fragments thereof can be engineered to contain one or more disulfide bonds.

[0114] In some embodiments, the antibodies and / or conjugated antibodies are monospecific. In some embodiments, the antibodies and / or conjugated antibodies are multispecific, for example, by way of non-limiting example, bispecific or trispecific. In some embodiments, the antibodies and / or conjugated antibodies are formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. In some embodiments, the antibodies and / or conjugated antibodies are formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor.

[0115] In some embodiments, the activatable antibody and / or composite activatable antibody is monospecific. In some embodiments, the activatable antibody and / or composite activatable antibody is multispecific, also referred to herein as a multispecific activatable antibody and / or a multispecific composite activatable antibody. As used herein, the term "activatable antibody" and all grammatical variations thereof, unless otherwise noted, are intended to encompass the multispecific activatable antibody of the present disclosure, without being limited to embodiments in which the activatable antibody is a multispecific activatable antibody of the present disclosure. As used herein, the term "composite activatable antibody" and all grammatical variations thereof, unless otherwise noted, are intended to encompass the multispecific composite activatable antibody of the present disclosure, without being limited to embodiments in which the composite activatable antibody is a multispecific composite activatable antibody of the present disclosure. In some embodiments, the multispecific activatable antibody and / or the multispecific composite activatable antibody is bispecific or trispecific.

[0116] In some embodiments, the conjugated antibodies described herein are conjugated or used in combination with one or more additional agents. Suitable additional agents include, for example, current pharmaceutical and / or surgical therapies for a targeted indication, such as cancer. For example, the conjugated antibodies are used in combination with an additional chemotherapeutic or anti-cancer agent.

[0117] Matriptase and / or uPA substrates of the present disclosure are also useful for activatable antibodies. The activatable antibodies described herein in their activated state bind a predetermined target and include: (i) an antibody or antigen-binding fragment thereof (AB) that specifically binds to the target; (ii) a masking moiety (MM) that inhibits binding of the AB to the target when the activatable antibody is present in an uncleaved state; and (iii) a cleavable moiety (CM) coupled to the AB, where the CM is a polypeptide that functions as a substrate for at least one protease selected from matriptase and / or uPA.

[0118] In some embodiments, the activatable antibody in its uncleaved state has the following structural arrangement from N- to C-terminus: MM-CM-AB or AB-CM-MM.

[0119] In some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM.

[0120] In some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB.

[0121] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and in its uncleaved state, the activatable antibody has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.

[0122] In some embodiments, the two connecting peptides need not be identical to each other.

[0123] In some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 385) and (GGGS) n(SEQ ID NO: 386), where n is at least one integer.

[0124] 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: 387), GGSGG (SEQ ID NO: 388), GSGSG (SEQ ID NO: 389), GSGGG (SEQ ID NO: 390), GGGSG (SEQ ID NO: 391), and GSSSG (SEQ ID NO: 392).

[0125] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 393), GSSGGSGGSGG (SEQ ID NO: 394), GSSGGSGGSGGS (SEQ ID NO: 395), GSSGGSGGSGGSGGGS (SEQ ID NO: 396), GSSGGSGGSG (SEQ ID NO: 397), or GSSGGSGGSGS (SEQ ID NO: 398).

[0126] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 399), GSSGT (SEQ ID NO: 400), or GSSG (SEQ ID NO: 401).

[0127] In some embodiments, the AB has an equilibrium dissociation constant for binding to the target of about 100 nM or less.

[0128] In some embodiments, the activatable antibody comprises an antibody or antigen-binding fragment thereof that specifically binds a target. In some embodiments, the antibody or immunologically active fragment thereof that binds the target is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, or a single-domain light chain antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds the target is a murine, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0129] In some embodiments, the MM has an equilibrium dissociation constant for binding to the AB that is greater than the equilibrium dissociation constant of the AB to the target.

[0130] In some embodiments, the MM has an equilibrium dissociation constant for binding to the AB that is less than or equal to the equilibrium dissociation constant of the AB to the target.

[0131] In some embodiments, the MM, when in its cleaved state, does not interfere with or compete with the AB for binding to the target.

[0132] In some embodiments, the MM is a polypeptide between about 2 and 40 amino acids in length, for example, the MM is a polypeptide of 40 or fewer amino acids in length.

[0133] In some embodiments, the MM polypeptide sequence differs from the polypeptide sequence of any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is less than 50% identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is less than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of AB.

[0134] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d At least twice as high.

[0135] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d At least three times higher than

[0136] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d At least five times higher than

[0137] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d is at least 10 times higher than

[0138] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d is at least 20 times higher than

[0139] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d is at least 40 times higher than

[0140] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d At least 100 times higher than

[0141] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d At least 1000 times higher than

[0142] In some embodiments, coupling of the MM reduces the ability of the AB to bind the target, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to the MM towards the target. d At least 10,000 times higher than

[0143] In some embodiments, the protease, ie, matriptase and / or uPA, co-localizes with the target in the tissue, and when the activating antibody is exposed to the protease, the protease cleaves the CM within the activating antibody.

[0144] In some embodiments, in the presence of a target, the MM reduces the ability of the AB to bind a target by at least 90% when the CM is uncleaved compared to when the CM is cleaved, as assayed in vitro using a target displacement assay, such as the assays described in PCT Publication Nos. WO2009 / 025846 and WO2010 / 081173.

[0145] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, resulting in an equilibrium dissociation constant that is at least two-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0146] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, resulting in an equilibrium dissociation constant that is at least 5-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0147] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, resulting in an equilibrium dissociation constant that is at least 10-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0148] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, resulting in an equilibrium dissociation constant that is at least 20-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0149] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, confirming the occurrence of an equilibrium dissociation constant that is at least 40-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions, the AB binds the target.

[0150] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, resulting in an equilibrium dissociation constant that is at least 50-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0151] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, resulting in an equilibrium dissociation constant that is at least 100-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0152] In some embodiments, the CM is located on the activatable antibody such that, under uncleaved conditions, binding of the activatable antibody to the target is reduced, resulting in an equilibrium dissociation constant that is at least 200-fold higher than the equilibrium dissociation constant of the unmodified AB binding to the target, whereas under cleaved conditions (i.e., when the activatable antibody is in the cleaved state), the AB binds the target.

[0153] In some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0154] In some embodiments, the CM is a substrate for at least matriptase. In some embodiments, the CM is a substrate for at least uPA. In some embodiments, the CM is a substrate for at least matriptase and uPA.

[0155] In some embodiments, CM is a substrate for matriptase and / or uPA and is resistant to cleavage by at least the other protease. In some embodiments, CM is a substrate for matriptase and / or uPA and is resistant to cleavage by at least plasmin. In some embodiments, CM is a substrate for matriptase and / or uPA and is resistant to cleavage by at least tissue plasminogen activator (tPA).

[0156] In some embodiments, the CM is a substrate for matriptase and / or uPA and comprises a motif sequence recognized by matriptase and / or uPA, with the proviso that for any motif sequence of the present disclosure: (i) the CM does not include any of the following amino acid sequences: TGRGPSWV (SEQ ID NO: 402), SARGPSRW (SEQ ID NO: 403), or TARGPSFK (SEQ ID NO: 404); and the CM does not include a consensus amino acid sequence based on these amino acid sequences, such as, for example, TARGPSW (SEQ ID NO: 405); (ii) the CM does not comprise any of the following amino acid sequences: LSGRSDNH (SEQ ID NO: 406), GGWHTGRN (SEQ ID NO: 407), HTGRSGAL (SEQ ID NO: 408), or PLTGRSGG (SEQ ID NO: 409); and the CM does not comprise a consensus amino acid sequence based on these amino acid sequences, such as, for example, LTGRSGA (SEQ ID NO: 410); and / or (iii) The CM does not include any of the following amino acid sequences: AARGPAIH (SEQ ID NO: 411), RGPAFNPM (SEQ ID NO: 412), SSRGPAYL (SEQ ID NO: 413), or RGPATPIM (SEQ ID NO: 414); and the CM does not include a consensus amino acid sequence based on these amino acid sequences, such as, for example, RGPA (SEQ ID NO: 415).

[0157] In some embodiments, the motif sequence comprises a core CM consensus sequence shown in Tables 8A-8J. In some embodiments, the motif sequence comprises a subgenus, i.e., a subgroup, of the core CM consensus sequence shown in Tables 8A-8J.

[0158] In some embodiments, the motif sequence comprises an extended consensus sequence based on one of the core CM consensus sequences shown in Tables 8A-8J. In some embodiments, the extended consensus sequence is a consensus sequence shown in Tables 9A-9J-3.

[0159] In some embodiments, the CM comprises a core CM consensus 1 sequence comprising the amino acid sequence AAPRS (SEQ ID NO: 163). In some embodiments, the CM comprises an extended core CM consensus 1 sequence comprising the amino acid sequence AAPRSF (SEQ ID NO: 164).

[0160] In some embodiments, the CM comprises a core CM consensus 2 sequence comprising the amino acid sequence SRRVP (SEQ ID NO: 165). In some embodiments, the CM comprises an extended core CM consensus 2 sequence comprising an amino acid sequence selected from the group consisting of QSRRVP (SEQ ID NO: 166), QTRRVP (SEQ ID NO: 167), SRRVPL (SEQ ID NO: 168), SRRVPV (SEQ ID NO: 169), QSRRVPL (SEQ ID NO: 170), QSRRVPV (SEQ ID NO: 171), QTRRVPL (SEQ ID NO: 172), and QTRRVPV (SEQ ID NO: 173).

[0161] In some embodiments, the CM comprises the amino acid sequence QSRRVP (SEQ ID NO: 166). In some embodiments, the CM comprises the amino acid sequence QTRRVP (SEQ ID NO: 167). In some embodiments, the CM comprises the amino acid sequence SRRVPL (SEQ ID NO: 168). In some embodiments, the CM comprises the amino acid sequence SRRVPV (SEQ ID NO: 169). In some embodiments, the CM comprises the amino acid sequence QSRRVPL (SEQ ID NO: 170). In some embodiments, the CM comprises the amino acid sequence QSRRVPV (SEQ ID NO: 171). In some embodiments, the CM comprises the amino acid sequence QTRRVPL (SEQ ID NO: 172). In some embodiments, the CM comprises the amino acid sequence QTRRVPV (SEQ ID NO: 173).

[0162] In some embodiments, the CM comprises a core CM consensus 3 sequence comprising the amino acid sequence PPLGR (SEQ ID NO: 174). In some embodiments, the CM comprises an extended core CM consensus 3 sequence comprising an amino acid sequence selected from the group consisting of GPPLGR (SEQ ID NO: 175), SPPLGR (SEQ ID NO: 176), CGPPLGR (SEQ ID NO: 177), CSPPLGR (SEQ ID NO: 178), GGPPLGR (SEQ ID NO: 179), GSPPLGR (SEQ ID NO: 180), SGPPLGR (SEQ ID NO: 181), SSPPLGR (SEQ ID NO: 182), GCGPPLGR (SEQ ID NO: 183), GCSPPLGR (SEQ ID NO: 184), GGGPPLGR (SEQ ID NO: 185), GGSPPLGR (SEQ ID NO: 186), GSGPPLGR (SEQ ID NO: 187), GSSPPLGR (SEQ ID NO: 188), SCGPPLGR (SEQ ID NO: 189), SCSPPLGR (SEQ ID NO: 190), SGGPPLGR (SEQ ID NO: 191), SGSPPLGR (SEQ ID NO: 192), SSGPPLGR (SEQ ID NO: 193), and SSSPPLGR (SEQ ID NO: 194).

[0163] In some embodiments, the CM comprises the amino acid sequence GPPLGR (SEQ ID NO: 175). In some embodiments, the CM comprises the amino acid sequence SPPLGR (SEQ ID NO: 176). In some embodiments, the CM comprises the amino acid sequence CGPPLGR (SEQ ID NO: 177). In some embodiments, the CM comprises the amino acid sequence CSPPLGR (SEQ ID NO: 178). In some embodiments, the CM comprises the amino acid sequence GGPPLGR (SEQ ID NO: 179). In some embodiments, the CM comprises the amino acid sequence GSPPLGR (SEQ ID NO: 180). In some embodiments, the CM comprises the amino acid sequence SGPPLGR (SEQ ID NO: 181). In some embodiments, the CM comprises the amino acid sequence SSPPLGR (SEQ ID NO: 182). In some embodiments, the CM comprises the amino acid sequence GCGPPLGR (SEQ ID NO: 183). In some embodiments, the CM comprises the amino acid sequence GCSPPLGR (SEQ ID NO: 184). In some embodiments, the CM comprises the amino acid sequence GGGPPLGR (SEQ ID NO: 185). In some embodiments, the CM comprises the amino acid sequence GGSPPLGR (SEQ ID NO: 186). In some embodiments, the CM comprises the amino acid sequence GSGPPLGR (SEQ ID NO: 187). In some embodiments, the CM comprises the amino acid sequence GSSPPLGR (SEQ ID NO: 188). In some embodiments, the CM comprises the amino acid sequence SCGPPLGR (SEQ ID NO: 189). In some embodiments, the CM comprises the amino acid sequence SCSPPLGR (SEQ ID NO: 190). In some embodiments, the CM comprises the amino acid sequence SGGPPLGR (SEQ ID NO: 191). In some embodiments, the CM comprises the amino acid sequence SGSPPLGR (SEQ ID NO: 192). In some embodiments, the CM comprises the amino acid sequence SSGPPLGR (SEQ ID NO: 193). In some embodiments, the CM comprises the amino acid sequence SSSPPLGR (SEQ ID NO: 194).

[0164] In some embodiments, the CM comprises a core CM consensus 4 sequence comprising the amino acid sequence LRSGW (SEQ ID NO: 195). In some embodiments, the CM comprises an extended core CM consensus 4 sequence comprising an amino acid sequence selected from the group consisting of MLRSGW (SEQ ID NO: 196), MLRSGWR (SEQ ID NO: 197), MLRSGWRG (SEQ ID NO: 198), MLRSGWRL (SEQ ID NO: 199), and MLRSGWRS (SEQ ID NO: 200).

[0165] In some embodiments, the CM comprises the amino acid sequence MLRSGW, (SEQ ID NO: 196). In some embodiments, the CM comprises the amino acid sequence MLRSGWR (SEQ ID NO: 197). In some embodiments, the CM comprises the amino acid sequence MLRSGWRG (SEQ ID NO: 198). In some embodiments, the CM comprises the amino acid sequence MLRSGWRL (SEQ ID NO: 199). In some embodiments, the CM comprises the amino acid sequence MLRSGWRS (SEQ ID NO: 200).

[0166] In some embodiments, the CM comprises a core CM consensus 5 sequence comprising the amino acid sequence VSRSA (SEQ ID NO: 201). In some embodiments, the CM comprises an extended core CM consensus 5 sequence comprising an amino acid sequence selected from the group consisting of IVSRSA (SEQ ID NO: 202), YIVSRSA (SEQ ID NO: 203), and QYIVSRSA (SEQ ID NO: 204).

[0167] In some embodiments, the CM comprises the amino acid sequence IVSRSA (SEQ ID NO: 202). In some embodiments, the CM comprises the amino acid sequence YIVSRSA (SEQ ID NO: 203). In some embodiments, the CM comprises the amino acid sequence QYIVSRSA (SEQ ID NO: 204).

[0168] In some embodiments, the CM comprises a core CM consensus 6 sequence comprising the amino acid sequence ALRAP (SEQ ID NO: 205). In some embodiments, the CM comprises an extended core CM consensus 6 sequence comprising the amino acid sequence RALRAP (SEQ ID NO: 206).

[0169] In some embodiments, the CM comprises a core CM consensus 7 sequence comprising the amino acid sequence PAGRR (SEQ ID NO: 207). In some embodiments, the CM comprises an extended core CM consensus 7 sequence comprising an amino acid sequence selected from the group consisting of PAGRRS (SEQ ID NO: 208), PAGRRSL (SEQ ID NO: 209), VPAGRRS (SEQ ID NO: 210), and VPAGRRSL (SEQ ID NO: 211).

[0170] In some embodiments, the CM comprises the amino acid sequence PAGRRS (SEQ ID NO: 208). In some embodiments, the CM comprises the amino acid sequence PAGRRSL (SEQ ID NO: 209). In some embodiments, the CM comprises the amino acid sequence VPAGRRS (SEQ ID NO: 210). In some embodiments, the CM comprises the amino acid sequence VPAGRRSL (SEQ ID NO: 211).

[0171] In some embodiments, the CM comprises a core CM consensus 8 sequence comprising the amino acid sequence GRSML (SEQ ID NO: 212). In some embodiments, the CM comprises a core CM consensus 8 sequence comprising GRSMLL (SEQ ID NO: 213), GRSMLM (SEQ ID NO: 214), GRSMLLG (SEQ ID NO: 215), GRSMLLP (SEQ ID NO: 216), GRSMLLS (SEQ ID NO: 217), GRSMLMG (SEQ ID NO: 218), GRSMLMP (SEQ ID NO: 219), GRSMLMS (SEQ ID NO: 220), GRSMLLGG (SEQ ID NO: 221), GRSMLLPG (SEQ ID NO: 222), GRSMLLSG (SEQ ID NO: 223), GRSMLMGG (SEQ ID NO: 224), GRSMLMPG (SEQ ID NO: 225), GRSMLMSG (SEQ ID NO: 226). ), GRSMLLGP (SEQ ID NO: 227), GRSMLLPP (SEQ ID NO: 228), GRSMLLSP (SEQ ID NO: 229), GRSMLMGP (SEQ ID NO: 230), GRSMLMPP (SEQ ID NO: 231), GRSMLMSP (SEQ ID NO: 232), GRSMLLGS (SEQ ID NO: 233), GRSMLLPS (SEQ ID NO: 234), GRSMLLSS (SEQ ID NO: 235), GRSMLMGS (SEQ ID NO: 236), GRSMLMPS (SEQ ID NO: 237), and GRSMLMSS (SEQ ID NO: 238).

[0172] In some embodiments, the CM comprises the amino acid sequence GRSMLL (SEQ ID NO:213). In some embodiments, the CM comprises the amino acid sequence GRSMLM (SEQ ID NO:214). In some embodiments, the CM comprises the amino acid sequence GRSMLLG (SEQ ID NO:215). In some embodiments, the CM comprises the amino acid sequence GRSMLLP (SEQ ID NO:216). In some embodiments, the CM comprises the amino acid sequence GRSMLLS (SEQ ID NO:217). In some embodiments, the CM comprises the amino acid sequence GRSMLMG (SEQ ID NO:218). In some embodiments, the CM comprises the amino acid sequence GRSMLMP (SEQ ID NO:219). In some embodiments, the CM comprises the amino acid sequence GRSMLMS (SEQ ID NO:220). In some embodiments, the CM comprises the amino acid sequence GRSMLLGG (SEQ ID NO:221). In some embodiments, the CM comprises the amino acid sequence GRSMLLPG (SEQ ID NO:222). In some embodiments, the CM comprises the amino acid sequence GRSMLLSG (SEQ ID NO:223). In some embodiments, the CM comprises the amino acid sequence GRSMLMGG (SEQ ID NO:224). In some embodiments, the CM comprises the amino acid sequence GRSMLMPG (SEQ ID NO:225). In some embodiments, the CM comprises the amino acid sequence GRSMLMSG (SEQ ID NO: 226). In some embodiments, the CM comprises the amino acid sequence GRSMLLGP (SEQ ID NO: 227). In some embodiments, the CM comprises the amino acid sequence GRSMLLPP (SEQ ID NO: 228). In some embodiments, the CM comprises the amino acid sequence GRSMLLSP (SEQ ID NO: 229). In some embodiments, the CM comprises the amino acid sequence GRSMLMGP (SEQ ID NO: 230). In some embodiments, the CM comprises the amino acid sequence GRSMLMPP (SEQ ID NO: 231). In some embodiments, the CM comprises the amino acid sequence GRSMLMSP (SEQ ID NO: 232). In some embodiments, the CM comprises the amino acid sequence GRSMLLGS (SEQ ID NO: 233). In some embodiments, the CM comprises the amino acid sequence GRSMLLPS (SEQ ID NO: 234).In some embodiments, the CM comprises the amino acid sequence GRSMLLSS (SEQ ID NO: 235). In some embodiments, the CM comprises the amino acid sequence GRSMLMGS (SEQ ID NO: 236). In some embodiments, the CM comprises the amino acid sequence GRSMLMPS (SEQ ID NO: 237). In some embodiments, the CM comprises the amino acid sequence GRSMLMSS (SEQ ID NO: 238).

[0173] In some embodiments, the CM comprises a core CM consensus 9 sequence comprising the amino acid sequence LARAG (SEQ ID NO: 239). In some embodiments, the CM comprises an extended core CM consensus 9 sequence comprising an amino acid sequence selected from the group consisting of LARAGI (SEQ ID NO: 240), LARAGL (SEQ ID NO: 241), PLARAGI (SEQ ID NO: 242), PLARAGL (SEQ ID NO: 243), RPLARAGI (SEQ ID NO: 244), and RPLARAGL (SEQ ID NO: 245).

[0174] In some embodiments, the CM comprises the amino acid sequence LARAGI (SEQ ID NO: 240). In some embodiments, the CM comprises the amino acid sequence LARAGL (SEQ ID NO: 241). In some embodiments, the CM comprises the amino acid sequence PLARAGI (SEQ ID NO: 242). In some embodiments, the CM comprises the amino acid sequence PLARAGL (SEQ ID NO: 243). In some embodiments, the CM comprises the amino acid sequence RPLARAGI (SEQ ID NO: 244). In some embodiments, the CM comprises the amino acid sequence RPLARAGL (SEQ ID NO: 245).

[0175] In some embodiments, the CM comprises a core CM consensus 10 sequence comprising the amino acid sequence ESRRW (SEQ ID NO: 246). In some embodiments, the CM comprises an extended core CM consensus 10 sequence comprising an amino acid sequence selected from the group consisting of ESRRWM (SEQ ID NO: 247), ESRRWMP (SEQ ID NO: 248), and PESRRWMP (SEQ ID NO: 249).

[0176] In some embodiments, the CM comprises the amino acid sequence ESRRWM (SEQ ID NO: 247). In some embodiments, the CM comprises the amino acid sequence ESRRWMP (SEQ ID NO: 248). In some embodiments, the CM comprises the amino acid sequence PESRRWMP (SEQ ID NO: 249).

[0177] In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of ILPRSPAF (SEQ ID NO: 250), VAGRSMRP (SEQ ID NO: 251), VVPEGRRS (SEQ ID NO: 252), QGRAITFI (SEQ ID NO: 253), VLSKQMSF (SEQ ID NO: 254), LKGRSYYY (SEQ ID NO: 255), KRMPVQFL (SEQ ID NO: 256), PQHRIVSF (SEQ ID NO: 257), YKKFVGSL (SEQ ID NO: 258), HMMQYARH (SEQ ID NO: 259), IPFSWSRF (SEQ ID NO: 260), LSQARWRK (SEQ ID NO: 261), DISHWRRS (SEQ ID NO: 262), RKTVQHWW (SEQ ID NO: 263), RFYRNQFF (SEQ ID NO: 264), RSLVFAPI (SEQ ID NO: 265), RSPSRLKC (SEQ ID NO: 266), and RKMPNITV (SEQ ID NO: 267).

[0178] In some embodiments, the CM comprises the amino acid sequence ILPRSPAF (SEQ ID NO: 250). In some embodiments, the CM comprises the amino acid sequence VAGRSMRP (SEQ ID NO: 251). In some embodiments, the CM comprises the amino acid sequence VVPEGRRS (SEQ ID NO: 252). In some embodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ ID NO: 253). In some embodiments, the CM comprises the amino acid sequence VLSKQMSF (SEQ ID NO: 254). In some embodiments, the CM comprises the amino acid sequence LKGRSYYY (SEQ ID NO: 255). In some embodiments, the CM comprises the amino acid sequence KRMPVQFL (SEQ ID NO: 256). In some embodiments, the CM comprises the amino acid sequence PQHRIVSF (SEQ ID NO: 257). In some embodiments, the CM comprises the amino acid sequence YKKFVGSL (SEQ ID NO: 258). In some embodiments, the CM comprises the amino acid sequence HMMQYARH (SEQ ID NO: 259). In some embodiments, the CM comprises the amino acid sequence IPFSWSRF (SEQ ID NO: 260). In some embodiments, the CM comprises the amino acid sequence LSQARWRK (SEQ ID NO: 261). In some embodiments, the CM comprises the amino acid sequence DISHWRRS (SEQ ID NO: 262). In some embodiments, the CM comprises the amino acid sequence RKTVQHWW (SEQ ID NO: 263). In some embodiments, the CM comprises the amino acid sequence RFYRNQFF (SEQ ID NO: 264). In some embodiments, the CM comprises the amino acid sequence RSLVFAPI (SEQ ID NO: 265). In some embodiments, the CM comprises the amino acid sequence RSPSRLKC (SEQ ID NO: 266). In some embodiments, the CM comprises the amino acid sequence RKMPNITV (SEQ ID NO: 267).

[0179] In some embodiments, the motif sequence comprises a core CM consensus sequence shown in Tables 10A-10D. In some embodiments, the motif sequence comprises a subgenus, i.e., a subgroup, of the core CM consensus sequence shown in Tables 10A-10D.

[0180] In some embodiments, the motif sequence comprises an extended consensus sequence based on one of the core CM consensus sequences shown in Tables 10A-10D. In some embodiments, the extended consensus sequence is a consensus sequence shown in Tables 11A-11D.

[0181] In some embodiments, the CM comprises a core CM consensus 11 sequence comprising the amino acid sequence LSGRSANH (SEQ ID NO: 307) or LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CM comprises an extended core CM consensus 11 sequence comprising the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309), DRLSGRSDNHKK (SEQ ID NO: 310), or NTLSGRSGNHGS (SEQ ID NO: 311).

[0182] In some embodiments, the CM comprises the amino acid sequence LSGRSANH (SEQ ID NO: 307). In some embodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CM comprises the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309). In some embodiments, the CM comprises the amino acid sequence DRLSGRSDNHKK (SEQ ID NO: 310). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 311).

[0183] In some embodiments, the CM comprises the amino acid sequence LSGRSANH (SEQ ID NO: 307). In some embodiments, the CM comprises the amino acid sequence LNGRSDNH (SEQ ID NO: 313). In some embodiments, the CM comprises the amino acid sequence LTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM comprises a core CM consensus 12 sequence comprising the amino acid sequences LSGRSANH (SEQ ID NO: 307), LNGRSDNH (SEQ ID NO: 313), and LTGRSDRH (SEQ ID NO: 314).In some embodiments, the CM is selected from the group consisting of DRLSGRSANHKK (SEQ ID NO: 309), DRLSGRSDNHKK (SEQ ID NO: 310), GPLNGRSDNHKA (SEQ ID NO: 320), GPLNGRSDNHKK (SEQ ID NO: 321), GPLNGRSDNHKR (SEQ ID NO: 322), GPLNGRSDNHQA (SEQ ID NO: 323), GPLNGRSDNHQK (SEQ ID NO: 324), GPLNGRSDNHQR (SEQ ID NO: 325), GPLNGRSDNHRA (SEQ ID NO: 326), GPLNGRSDNHRK ( SEQ ID NO: 327), GPLNGRSDNHRR (SEQ ID NO: 328), RPLNGRSDNHKA (SEQ ID NO: 329), RPLNGRSDNHKK (SEQ ID NO: 330), RPLNGRSDNHKR (SEQ ID NO: 331), RPLNGRSDNHQA (SEQ ID NO: 332), RPLNGRSDNHQK (SEQ ID NO: 333), RPLNGRSDNHQR (SEQ ID NO: 334), RPLNGRSDNHRA (SEQ ID NO: 335), RPLNGRSDNHRK (SEQ ID NO: 336), RPLNGRSDNHRR (SEQ ID NO: 337), G PLSGRSDNHKA (SEQ ID NO: 338), GPLSGRSDNHKK (SEQ ID NO: 339), GPLSGRSDNHKR (SEQ ID NO: 340), GPLSGRSDNHQA (SEQ ID NO: 341), GPLSGRSDNHQK (SEQ ID NO: 342), GPLSGRSDNHQR (SEQ ID NO: 343), GPLSGRSDNHRA (SEQ ID NO: 344), GPLSGRSDNHRK (SEQ ID NO: 345), GPLSGRSDNHRR (SEQ ID NO: 346), RPLSGRSDNHKA (SEQ ID NO: 347), RPLSGRSDNH KK (SEQ ID NO: 348), RPLSGRSDNHKR (SEQ ID NO: 349), RPLSGRSDNHQA (SEQ ID NO: 350), RPLSGRSDNHQK (SEQ ID NO: 351), RPLSGRSDNHQR (SEQ ID NO: 352), RPLSGRSDNHRA (SEQ ID NO: 353), RPLSGRSDNHRK (SEQ ID NO: 354), RPLSGRSDNHRR (SEQ ID NO: 355), and KGLTGRSDRHQA (SEQ ID NO: 356).

[0184] In some embodiments, the CM comprises the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309). In some embodiments, the CM comprises the amino acid sequence DRLSGRSDNHKK (SEQ ID NO: 310). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHKA (SEQ ID NO: 320). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHKK (SEQ ID NO: 321). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHKR (SEQ ID NO: 322). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHQA (SEQ ID NO: 323). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHQK (SEQ ID NO: 324). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHQR (SEQ ID NO: 325). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHRA (SEQ ID NO: 326). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHRK (SEQ ID NO: 327). In some embodiments, the CM comprises the amino acid sequence GPLNGRSDNHRR (SEQ ID NO: 328). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHKA (SEQ ID NO: 329). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHKK (SEQ ID NO: 330). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHKR (SEQ ID NO: 331). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHQA (SEQ ID NO: 332). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHQK (SEQ ID NO: 333). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHQR (SEQ ID NO: 334). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHRA (SEQ ID NO: 335). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHRK (SEQ ID NO: 336). In some embodiments, the CM comprises the amino acid sequence RPLNGRSDNHRR (SEQ ID NO: 337).In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHKA (SEQ ID NO: 338). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHKK (SEQ ID NO: 339). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHKR (SEQ ID NO: 340). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHQA (SEQ ID NO: 341). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHQK (SEQ ID NO: 342). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHQR (SEQ ID NO: 343). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHRA (SEQ ID NO: 344). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHRK (SEQ ID NO: 345). In some embodiments, the CM comprises the amino acid sequence GPLSGRSDNHRR (SEQ ID NO: 346). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHKA (SEQ ID NO: 347). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHKK (SEQ ID NO: 348). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHKR (SEQ ID NO: 349). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHQA (SEQ ID NO: 350). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHQK (SEQ ID NO: 351). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHQR (SEQ ID NO: 352). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHRA (SEQ ID NO: 353). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHRK (SEQ ID NO: 354). In some embodiments, the CM comprises the amino acid sequence RPLSGRSDNHRR (SEQ ID NO: 355). In some embodiments, the CM comprises the amino acid sequence KGLTGRSDRHQA (SEQ ID NO: 356).

[0185] In some embodiments, the CM comprises a core CM consensus 13 sequence comprising the amino acid sequence RIGRSDNH (SEQ ID NO: 357) or RLGRSDNN (SEQ ID NO: 358). In some embodiments, the CM comprises an extended core CM consensus 13 sequence comprising the amino acid sequence NHRIGRSDNHRR (SEQ ID NO: 359) or TLRLGRSDNNKN (SEQ ID NO: 360).

[0186] In some embodiments, the CM comprises the amino acid sequence RIGRSDNH (SEQ ID NO: 357). In some embodiments, the CM comprises the amino acid sequence RLGRSDNN (SEQ ID NO: 358). In some embodiments, the CM comprises the amino acid sequence NHRIGRSDNHRR (SEQ ID NO: 359). In some embodiments, the CM comprises the amino acid sequence TLRLGRSDNNKN (SEQ ID NO: 360).

[0187] In some embodiments, the CM comprises a core CM consensus 14 sequence comprising an amino acid sequence selected from the group consisting of TSGRSANP (SEQ ID NO: 361), TSGRSGNP (SEQ ID NO: 362), LSGRSANP (SEQ ID NO: 363), and LSGRSGNP (SEQ ID NO: 364). In some embodiments, the CM comprises an extended core CM consensus 14 sequence comprising an amino acid sequence selected from the group consisting of TSTSGRSAMPRG (SEQ ID NO: 365), TSTSGRSGNPRG (SEQ ID NO: 366), TSLSGRSANPRG (SEQ ID NO: 367), and TSLSGRSGNPRG (SEQ ID NO: 368).

[0188] In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 361). In some embodiments, the CM comprises the amino acid sequence TSGRSGNP (SEQ ID NO: 362). In some embodiments, the CM comprises the amino acid sequence LSGRSANP (SEQ ID NO: 363). In some embodiments, the CM comprises the amino acid sequence LSGRSGNP (SEQ ID NO: 364). In some embodiments, the CM comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 365). In some embodiments, the CM comprises the amino acid sequence TSTSGRSGNPRG (SEQ ID NO: 366). In some embodiments, the CM comprises the amino acid sequence TSLSGRSANPRG (SEQ ID NO: 367). In some embodiments, the CM comprises the amino acid sequence TSLSGRSGNPRG (SEQ ID NO: 368).

[0189] In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of LSGRSENH (SEQ ID NO: 369), SIARSDNL (SEQ ID NO: 370), LSGRSVTQ (SEQ ID NO: 371), LSGRSGNH (SEQ ID NO: 308), LTGRSDRH (SEQ ID NO: 314), LYGRSENN (SEQ ID NO: 374), RLGRSDNN (SEQ ID NO: 375), TSGRSANP (SEQ ID NO: 376), NTLSGRSENHSG (SEQ ID NO: 377), PPSIARSDNLAN (SEQ ID NO: 378), TGLSGRSVTQTS (SEQ ID NO: 379), NTLSGRSGNHGS (SEQ ID NO: 311), KGLTGRSDRHQA (SEQ ID NO: 381), KNLYGRSENNGN (SEQ ID NO: 382), TLRLGRSDNNKN (SEQ ID NO: 383), and TSTSGRSANPRG (SEQ ID NO: 384).

[0190] In some embodiments, the CM comprises the amino acid sequence LSGRSENH (SEQ ID NO: 369). In some embodiments, the CM comprises the amino acid sequence SIARSDNL (SEQ ID NO: 370). In some embodiments, the CM comprises the amino acid sequence LSGRSVTQ (SEQ ID NO: 371). In some embodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CM comprises the amino acid sequence LTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM comprises the amino acid sequence LYGRSENN (SEQ ID NO: 374). In some embodiments, the CM comprises the amino acid sequence RLGRSDNN (SEQ ID NO: 375). In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 376). In some embodiments, the CM comprises the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 377). In some embodiments, the CM comprises the amino acid sequence PPSIARSDNLAN (SEQ ID NO: 378). In some embodiments, the CM comprises the amino acid sequence TGLSGRSVTQTS (SEQ ID NO: 379). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 311). In some embodiments, the CM comprises the amino acid sequence KGLTGRSDRHQA (SEQ ID NO: 381). In some embodiments, the CM comprises the amino acid sequence KNLYGRSENNGN (SEQ ID NO: 382). In some embodiments, the CM comprises the amino acid sequence TLRLGRSDNNKN (SEQ ID NO: 383). In some embodiments, the CM comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 384).

[0191] In some embodiments, the CM is a substrate for at least two proteases, in some embodiments, at least one protease is selected from matriptase and uPA, and at least one protease is selected from the group consisting of those shown in Table 7.

[0192] In some embodiments, an activatable antibody comprises at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each polypeptides less than 15 amino acids in length. In some embodiments, the first CM and the second CM in an activatable antibody have the following structural arrangement from N-terminus to C-terminus in their uncleaved state: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM. In some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1. In some embodiments, an activatable antibody comprises a connecting peptide between CM1 and CM2. In some embodiments, an activatable antibody comprises a connecting peptide between CM2 and AB. In some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1 and a connecting peptide between CM2 and AB. In some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1 and a connecting peptide between CM2 and AB. In some embodiments, an activatable antibody comprises a connecting peptide between MM and CM1 and a connecting peptide between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide between CM1 and CM2 and a connecting peptide between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide between MM and CM1, a connecting peptide between CM1 and CM2, and a connecting peptide between CM2 and AB.

[0193] In some embodiments, CM2 is selected for use with a particular protease, ie, CM2 is a substrate for at least one protease selected from the group consisting of matrix metalloproteinases (MMPs), neutrophil elastase, uPA, legumin, matriptase, thrombin, cysteine ​​proteases such as cathepsins, ADAM17, BMP-1, HtrA1, and TMPRSSs such as TMPRSS3 or TMPRSS4.

[0194] In some embodiments, CM2 is a substrate for neutrophil elastase. In some embodiments, CM2 is a substrate for uPA. In some embodiments, CM2 is a substrate for legumin. In some embodiments, CM2 is a substrate for matriptase. In some embodiments, CM2 is a substrate for thrombin. In some embodiments, CM2 is a substrate for cysteine ​​proteases. In some embodiments, CM2 is a substrate for cathepsins. In some embodiments, CM2 is a substrate for ADAM17. In some embodiments, CM2 is a substrate for BMP-1. In some embodiments, CM2 is a substrate for HtrA1. In some embodiments, CM2 is a substrate for TMPRSS. In some embodiments, CM2 is a substrate for TMPRSS3. In some embodiments, CM2 is a substrate for TMPRSS4.

[0195] For example, a suitable CM2 may be cleaved by at least one protease and contain the sequences TGRGPSWV (SEQ ID NO: 402); SARGPSRW (SEQ ID NO: 403); TARGPSFK (SEQ ID NO: 404); TARGPSW (SEQ ID NO: 405); LSGRSDNH (SEQ ID NO: 406); GGWHTGRN (SEQ ID NO: 407); HTGRSGAL (SEQ ID NO: 408); PLTGRSGG (SEQ ID NO: 409); AARGPAIH (SEQ ID NO: 411); RGPAFNPM (SEQ ID NO: 412); SSRGPAYL (SEQ ID NO: 413); Sequence number 413); RGPATPIM (sequence number 414); RGPA (sequence number 415); GGQPSGMWGW (sequence number 416); FPRPLGITGL (sequence number 417); VHMPLGFLGP (sequence number 418); SPLTGRSG (sequence number 419); SAGFSLPA (sequence number 126); LAPLGLQRR (sequence number 420); SGGPLGVR (sequence number 421); PLGL (sequence number 422); GPRSFGL (sequence number 423) and / or GPRSFG (sequence number 424).

[0196] In some embodiments, CM2 comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 402). In some embodiments, CM2 comprises the amino acid sequence SARGPSRW (SEQ ID NO: 403). In some embodiments, CM2 comprises the amino acid sequence TARGPSFK (SEQ ID NO: 404). In some embodiments, CM2 comprises the amino acid sequence TARGPSW (SEQ ID NO: 405). In some embodiments, CM2 comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 406). In some embodiments, CM2 comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 407). In some embodiments, CM2 comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 408). In some embodiments, CM2 comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 409). In some embodiments, CM2 comprises the amino acid sequence AARGPAIH (SEQ ID NO: 411). In some embodiments, CM2 comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 412). In some embodiments, CM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 413). In some embodiments, CM2 comprises the amino acid sequence RGPATPIM (SEQ ID NO: 414). In some embodiments, CM2 comprises the amino acid sequence RGPA (SEQ ID NO: 415). In some embodiments, CM2 comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO: 416). In some embodiments, CM2 comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 417). In some embodiments, CM2 comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 418). In some embodiments, CM2 comprises the amino acid sequence SPLTGRSG (SEQ ID NO: 419). In some embodiments, CM2 comprises the amino acid sequence LAPLGLQRR (SEQ ID NO: 420). In some embodiments, CM2 comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 421). In some embodiments, CM2 comprises the amino acid sequence PLGL (SEQ ID NO: 422). In some embodiments, CM2 comprises the amino acid sequence GPRSFGL (SEQ ID NO: 423). In some embodiments, CM2 comprises the amino acid sequence GPRSFG (SEQ ID NO: 424).

[0197] In some embodiments, CM2 is a substrate for at least one MMP. In some embodiments, CM2 is a substrate for at least one MMP listed in Table 7. In some embodiments, CM2 is a substrate for MMP9. In some embodiments, CM2 is a substrate for MMP14. In some embodiments, CM2 is a substrate for two or more MMPs. In some embodiments, CM2 is a substrate for at least MMP9 or MMP14. In some embodiments, CM2 is a substrate for two or more MMPs. In some embodiments, CM2 is a substrate for at least MMP9 and MMP14.

[0198] In some embodiments, CM2 is a substrate for MMPs and is selected from the group consisting of the sequences ISSGLLSS (SEQ ID NO: 425); QNQALRMA (SEQ ID NO: 426); AQNLLGMV (SEQ ID NO: 427); STFPFGMF (SEQ ID NO: 428); PVGYTSSL (SEQ ID NO: 429); DWLYWPGI (SEQ ID NO: 430); MIAPVAYR (SEQ ID NO: 431); RPSPMWAY (SEQ ID NO: 432); WATPRPMR (SEQ ID NO: 433); FRLLDWQW (SEQ ID NO: 434); LKAAPRWA (SEQ ID NO: 435); GPSHLLVLT (SEQ ID NO: 436); LPGGLSPW (SEQ ID NO: 437); MGLFSEAG (SEQ ID NO: 438); SPLPLRVP (SEQ ID NO: 439); RMHLRSLG (SEQ ID NO: 440); LAAPLGLL (SEQ ID NO: 441); AVGLLAPP (SEQ ID NO: 442); LLAPSHRA (SEQ ID NO: 443); PAGLWLDP (SEQ ID NO: 444). and / or ISSGLSS (SEQ ID NO: 445).

[0199] In some embodiments, the first cleavage agent and the second cleavage agent are proteases selected from matriptase and uPA, and the first CM and the second CM are different substrates for the enzymes. In some embodiments, the first cleavage agent and the second cleavage agent are different proteases, where at least one protease is selected from matriptase and uPA. In some embodiments, the first cleavage agent and the second cleavage agent are co-localized within the target tissue. In some embodiments, the first CM and the second CM are cleaved by at least one cleavage agent in the target tissue.

[0200] In some embodiments, the activatable antibody is exposed to and cleaved by a protease selected from matriptase and uPA, such that in the activated or cleaved state, the activated antibody comprises a light chain amino acid sequence that includes at least a portion of the LP2 and / or CM sequence after the protease cleaves the CM.

[0201] In some embodiments, the CM comprises the non-prime side of the protease cleavage site; i.e., the CM comprises at least the P1 and P2 amino acids, and in some embodiments, the P1, P2, and P3 amino acids, and in some embodiments, the P1, P2, P3, and P4 amino acids. In some embodiments, the CM comprises the non-prime side and the prime side of the protease cleavage site. In some embodiments, the CM comprises the non-prime side but lacks at least a portion of the prime side of the protease cleavage site. In some embodiments, the CM comprises the non-prime side but lacks the prime side of the protease cleavage site. Such CMs can be linked directly or through a linker to antibodies or other molecules described herein in this disclosure, such as, but not limited to, detection moieties.

[0202] In some embodiments, the activatable antibody also includes an agent conjugated to AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is attached 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, a DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is attached to AB via a linker comprising at least one MMP-cleavable substrate sequence. In some embodiments, the agent is an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0203] In some embodiments, the agent is an anti-inflammatory agent.

[0204] In some embodiments, the activatable antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0205] In some embodiments, the conjugated antibody comprises a detectable label. In some embodiments, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent comprises a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the imaging agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label comprises an N-methylacridinium derivative. In some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0206] In some embodiments, the activatable antibody also comprises a signal peptide. In some embodiments, the signal peptide is attached to the activatable antibody via a spacer. In some embodiments, the spacer is attached to the activatable antibody without the signal peptide. In some embodiments, the spacer is directly linked to the MM of the activatable antibody. In some embodiments, the spacer is directly linked to the MM of the activatable antibody in the N- to C-terminal structural arrangement of spacer-MM-CM-AB. An example of a spacer directly linked to the N-terminus of the MM of an activatable antibody is QGQSGQ (SEQ ID NO: 446). In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 446).

[0207] In some embodiments, the AB of an activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0208] In some embodiments, the serum half-life of an 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 an activatable antibody is comparable to the serum half-life of the corresponding antibody. In some embodiments, the serum half-life of an activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism.In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism.

[0209] In some embodiments, the activatable antibody and / or conjugated activatable antibody is monospecific. In some embodiments, the activatable antibody and / or conjugated activatable antibody is multispecific, such as, by non-limiting example, bispecific or trifunctional. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-bispecific T cell engaging (BITE) molecule. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (CAR) modified T cell or other engineered receptor.

[0210] The present disclosure also provides compositions and methods comprising activatable antibodies, including antibodies or antibody fragments (AB) that specifically bind a predetermined target, wherein the AB is conjugated to a masking moiety (MM) that reduces the ability of the AB to bind its target. In some embodiments, the activatable antibody further comprises a cleavable moiety (CM) that is a substrate for at least one protease selected from matriptase and uPA. The compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues in the AB without impairing the activity (e.g., masking, activation, or binding activity) of the activatable antibody. In some embodiments, the compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues in the AB without reducing or otherwise disrupting one or more disulfide bonds in the MM. The compositions and methods provided herein produce activatable antibodies conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic, and / or prophylactic agents; for example, in some embodiments, none of the agent(s) is conjugated to the MM of the activatable antibody. The compositions and methods provided herein produce conjugated activatable antibodies that retain the ability to effectively and efficiently mask the AB of an activatable antibody when the MM is uncleaved. The compositions and methods provided herein produce conjugated activatable antibodies, where the activatable antibody is still active, i.e., cleaved, in the presence of proteases capable of cleaving the CM, i.e., matriptase and / or uPA.

[0211] Although an activatable antibody has at least one attachment point for an agent, in the methods and compositions provided herein, fewer than all possible attachment points are available for attachment to an agent. In some embodiments, one or more attachment points are sulfur atoms involved in disulfide bonds. In some embodiments, one or more attachment points are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more attachment points are sulfur atoms involved in interchain sulfide bonds, but not intrachain disulfide bonds. In some embodiments, one or more attachment points are sulfur atoms in cysteine ​​or other amino acid residues that contain sulfur atoms. Such residues can occur naturally in antibody structures or can be incorporated into antibodies by site-directed mutagenesis, chemical conversion, or mis-incorporation of non-natural amino acids.

[0212] Also provided are methods for preparing an activatable antibody having one or more interchain disulfide bonds in AB and one or more intrachain disulfide bonds in MM, and a drug reactive with free thiols is provided. The methods generally involve partially reducing the interchain disulfide bonds in the activatable antibody with a reducing agent, e.g., TCEP; and attaching a drug reactive with free thiols to the partially reduced activatable antibody. As used herein, the term "partial reduction" refers to a situation in which an activatable antibody is contacted with a reducing agent and fewer than all disulfide bonds, e.g., fewer than all potential binding sites, are reduced. In some embodiments, less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of all potential binding sites are reduced.

[0213] In some embodiments, methods are provided for reducing and conjugating an agent, e.g., a drug, to an activatable antibody that provides selectivity under the configuration of the agent. The methods generally involve partially reducing the activatable antibody with a reducing agent, thereby unreducing any binding sites in the masking portion or other non-AB portion of the activatable antibody, and then conjugating the agent to the interchain thiol in the AB. The binding site is selected to allow for the desired configuration of the agent, allowing for conjugation at the desired site. An example of a reducing agent is TCEP. Reduction reaction conditions, such as the ratio of reducing agent to activatable antibody, the length of incubation, the temperature during incubation, the pH of the reduction reaction solution, etc., are determined by identifying conditions that produce a composite activatable antibody that retains the ability to effectively and efficiently mask the AB of the activatable antibody when the MM is uncleaved. The ratio of reducing agent to activatable antibody will vary depending on the activatable antibody. In some embodiments, the ratio of reducing agent:activatable antibody will be in the range of 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 1.5:1. 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.

[0214] In some embodiments, methods are provided for reducing the interchain disulfide bond in AB of an activatable antibody and attaching an agent, e.g., a thiol-containing agent, e.g., a drug, to the resulting interchain thiol to selectively locate the agent on AB. The methods generally involve partially reducing AB with a reducing agent to form at least two interchain thiols, but not all possible interchain thiols, on the activatable antibody; and attaching the agent to the interchain thiol of the partially reduced AB. For example, AB of an activatable antibody is partially reduced at a desired ratio of reducing agent:activatable antibody at about 37°C for about 1 hour. In some embodiments, the ratio of reducing agent:activatable antibody will be in the range of 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 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.

[0215] The thiol-containing reagent can be, for example, cysteine ​​or N-acetylcysteine. The reducing agent can be, for example, TCEP. In some embodiments, the reduced activatable antibody can be purified prior to conjugation, for example, using column chromatography, dialysis, or diafiltration. In some embodiments, the reduced antibody is not purified after partial reduction and prior to conjugation.

[0216] 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 disrupting any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that, in its uncleaved state, inhibits binding of the AB in the activatable antibody to the target, and a cleavable moiety (CM) attached to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease selected from matriptase and uPA. In some embodiments, the MM is attached to the AB via the CM. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disrupted by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM in the activatable antibody are not disrupted by the reducing agent. In some embodiments, the activatable antibody in its uncleaved state has the following structural configuration from N- to C-terminus: MM-CM-AB or AB-CM-MM. In some embodiments, the reducing agent is TCEP.

[0217] The present disclosure also provides a composite activatable antibody comprising an activatable antibody linked to a monomethyl auristatin D (MMAD) payload, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that, in its uncleaved state, inhibits binding of the AB in the activatable antibody to the target, and a cleavable moiety (CM) attached to the AB, wherein the CM is a polypeptide that functions as a substrate for at least one MMP protease.

[0218] In some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any of several methods for attaching an agent to the AB: (a) attachment to a carbohydrate moiety of the AB, or (b) attachment to a sulfhydryl group of the AB, or (c) attachment to an amino group of the AB, or (d) attachment to a carboxylate group of the AB.

[0219] In some embodiments, the MMAD payload is attached to AB via a linker. In some embodiments, the MMAD payload is attached to a cysteine ​​of AB via a linker. In some embodiments, the MMAD payload is attached to a lysine of AB via a linker. In some embodiments, the MMAD payload is attached to another residue of AB via a linker, such as those residues disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is selected from the group consisting of linkers shown in Tables 5 and 6. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidoPEG-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, hi some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation techniques disclosed herein.

[0220] The present disclosure also provides polypeptides and other large molecules that include one or more of the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein. By way of non-limiting example, the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are useful in prodrug compositions and methods of use thereof. These matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are useful in probes and other detectable agents and methods of use thereof. For example, the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are used in conjunction with fluorite or other quenchers to generate detectable agents, such as imaging agents and / or other diagnostic agents. Those skilled in the art will appreciate that the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are useful in any composition and / or method in the art that uses substrates cleavable by matriptase and / or uPA.

[0221] In some embodiments, the matriptase and / or uPA substrates of the present disclosure comprise a large molecule, e.g., (i) at least one moiety (M) that is located amino (N)-terminal to the CM, i.e., located within the large molecule closer to the N-terminus of the large molecule than the CM. N (ii) at least one moiety (M) located carboxyl (C)-terminal to the CM, i.e., located within the large molecule closer to the C-terminus of the large molecule than the CM; C (iii) combinations thereof. In some embodiments, the large molecule is used in an isolated polypeptide comprising at least one additional moiety (M) selected from the group consisting of: N and at least one M C Contains:

[0222] By way of non-limiting example, suitable M groups for use in the large molecules of the present disclosure include: Ncomprises at least one of the following: a masking moiety, an antibody, a protein, a therapeutic agent, an anti-neoplastic agent, a toxic agent, a drug, a detectable moiety, a diagnostic agent, an affinity tag, and combinations thereof.

[0223] By way of non-limiting example, suitable M groups for use in the large molecules of the present disclosure include: C comprises at least one of the following: a masking moiety, an antibody, a protein, a therapeutic agent, an anti-neoplastic agent, a toxic agent, a drug, a detectable moiety, a diagnostic agent, an affinity tag, and combinations thereof.

[0224] The present disclosure also provides isolated nucleic acid molecules encoding the CM-containing molecules of the present disclosure, e.g., CM-containing polypeptides, e.g., the CM-containing probes, antibodies, and / or activatable antibodies described herein, as well as vectors comprising these isolated nucleic acid sequences. The present disclosure provides methods for producing CM-containing polypeptides by culturing cells under conditions conducive to expression of the CM-containing polypeptides, wherein the cells comprise such vectors. The present disclosure provides methods for producing antibodies and / or activatable antibodies by culturing cells under conditions conducive to expression of the antibodies and / or activatable antibodies, wherein the cells comprise such vectors.

[0225] The present disclosure provides methods for producing a CM-containing polypeptide of the present disclosure that binds to a predetermined target by (a) culturing cells containing a nucleic acid construct encoding the CM-containing polypeptide under conditions conducive to expression of the polypeptide, (i) wherein the polypeptide comprises a cleavable moiety (CM), and (ii) wherein the CM is a polypeptide that functions as a substrate for at least one protease selected from matriptase and uPA; and (b) recovering the polypeptide. These methods can also include the further step of (c) binding the recovered polypeptide to one or more additional agents.

[0226] The present disclosure provides a method for producing a composite antibody of the present disclosure that binds to a predetermined target by (a) culturing cells containing a nucleic acid construct encoding the antibody under conditions conducive to expression of the antibody, (i) wherein the antibody comprises a cleavable moiety (CM), and (ii) the CM is a polypeptide that functions as a substrate for at least one protease selected from matriptase and uPA; (b) recovering the antibody; and (c) conjugating the recovered antibody to one or more additional agents.

[0227] The present disclosure also provides a method for producing an activatable antibody of the present disclosure, which binds to a predetermined target in an activated state, by (a) culturing cells containing a nucleic acid construct encoding the activatable antibody under conditions conducive to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (i) the CM is a polypeptide comprising an amino acid sequence that functions as a substrate for a protease selected from matriptase and uPA; and (ii) the CM is located on the activatable antibody such that, under uncleaved conditions, the MM interferes with specific binding of the AB to the target, and under cleaved conditions, the MM does not interfere with or compete with specific binding of the AB to the target; and (b) recovering the activatable antibody.

[0228] The present disclosure also provides methods for producing CM-containing non-polypeptide molecules, including, by way of non-limiting example, prodrugs, non-peptide probes, etc. These CM-containing non-polypeptide molecules can be made using a variety of art-recognized techniques, including standard chemical synthesis and / or conjugation methods.

[0229] The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating symptoms of, or otherwise ameliorating a target-related disease in a subject by administering to a subject in need thereof a therapeutically effective amount of a composite antibody, activatable antibody, and / or composite activatable antibody described herein.

[0230] The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating symptoms, or otherwise ameliorating inflammation and / or inflammatory diseases in a subject by administering to a subject in need thereof a therapeutically effective amount of a composite antibody, activatable antibody, and / or composite activatable antibody described herein. The present disclosure also provides methods of preventing, slowing the progression of, treating, alleviating symptoms, or otherwise ameliorating cancer in a subject by administering to a subject in need thereof a therapeutically effective amount of a composite antibody, activatable antibody, and / or composite activatable antibody described herein. The present disclosure also provides methods of preventing, slowing the progression of, treating, alleviating symptoms, or otherwise ameliorating autoimmune diseases in a subject by administering to a subject in need thereof a therapeutically effective amount of a composite antibody, activatable antibody, and / or composite activatable antibody described herein.

[0231] The composite antibodies, activatable antibodies and / or composite activatable antibodies used in any embodiment of these methods and uses may be administered at any stage of the disease. For example, such composite antibodies, activatable antibodies and / or composite activatable antibodies may be administered to a patient suffering from cancer at any stage, from early stage to metastatic. The terms subject and patient are used interchangeably herein.

[0232] In some embodiments, the subject is a mammal, such as a human, a non-human primate, a companion animal (e.g., a cat, dog, horse), a farm animal, a working animal, or a zoo animal. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal under veterinary care.

[0233] The composite antibody, activatable antibody, and / or composite activatable antibody and therapeutic formulations thereof are administered to a subject having or susceptible to a disease or disorder associated with aberrant target expression and / or activity. Subjects having or susceptible to a disease or disorder associated with aberrant target expression and / or activity can be identified using a variety of methods known in the art. For example, subjects with cancer or other neoplastic conditions can be identified using a variety of clinical and / or laboratory tests, such as physical examinations and blood, urine, and / or stool tests, to assess their health status. For example, subjects suffering from inflammation and / or inflammatory disorders can be identified using any of a variety of clinical and / or laboratory tests, such as physical examinations and body fluid analyses, blood, urine, and / or stool tests, to assess their health status.

[0234] Administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody to a patient with a disease or disorder associated with aberrant target expression and / or activity is considered successful if various experimental or clinical objectives are achieved. For example, administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody to a patient with a disease or disorder associated with aberrant target expression and / or activity is considered successful if one or more symptoms associated with the patient or disorder are alleviated, reduced, inhibited, or do not progress to a further worsening state. Administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody to a patient with a disease or disorder associated with aberrant target expression and / or activity is considered successful if the disease or disorder is ameliorated or does not progress to a further worsening state.

[0235] In some embodiments, the composite antibody, activatable antibody, and / or composite activatable antibody is administered in combination with one or more additional agents, such as, for example, an anti-inflammatory agent, an immunosuppressant, and / or a chemotherapeutic agent, during and / or after treatment. In some embodiments, the composite antibody, activatable antibody, and / or composite activatable antibody and the additional agent(s) are administered simultaneously. For example, the composite antibody, activatable antibody, and / or composite activatable antibody and the additional agent(s) may be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the composite antibody, activatable antibody, and / or composite activatable antibody and the additional agent(s) are administered sequentially, or the antibody and / or composite antibody and the additional agent(s) are administered at different times during the treatment regimen. For example, the antibody and / or composite antibody is administered before the administration of the additional agent, the antibody and / or composite antibody is administered subsequent to the administration of the additional agent, or the antibody and / or composite antibody and the additional agent(s) are administered in an alternating manner. As described herein, the antibodies and / or conjugated antibodies and additional agents may be administered in a single dose or in multiple doses.

[0236] In some embodiments, the composite antibody, activatable antibody, and / or composite activatable antibody are administered during and / or after treatment in combination with one or more additional agents, such as, by way of non-limiting example, anti-inflammatory agents, immunosuppressants, chemotherapeutic agents such as alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics, and / or other nucleic acid damaging agents. 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, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a targeted agent such as another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteosome inhibitor such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulatory agent such as lenalidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent that would be considered standard of care by those of skill in the art. In some embodiments, the additional agent is a chemotherapeutic agent known to those of skill in the art.

[0237] In some embodiments, the additional agent is an antibody, another conjugate antibody, another activatable antibody, and / or another conjugate activatable antibody. In some embodiments, the additional agent is an antibody, another conjugate antibody, another activatable antibody, and / or another conjugate activatable antibody directed against the same target as the first conjugate antibody, activatable antibody, and / or conjugate activatable antibody. In some embodiments, the additional agent is an antibody, another conjugate antibody, another activatable antibody, and / or another conjugate activatable antibody directed against a target different from that of the first conjugate antibody, activatable antibody, and / or conjugate activatable antibody.

[0238] In some embodiments, the composite antibody, activatable antibody, and / or composite activatable antibody, and the additional agent(s) are administered simultaneously. For example, the composite antibody, activatable antibody, and / or composite activatable antibody, and the additional agent(s) may be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the composite antibody, activatable antibody, and / or composite activatable antibody, and the additional agent(s) are administered sequentially, or the antibody and / or composite antibody and the additional agent(s) are administered at different times during a treatment regimen. For example, the antibody and / or composite antibody may be administered before the administration of the additional agent, the antibody and / or composite antibody may be administered subsequent to the administration of the additional agent, or the antibody and / or composite antibody and the additional agent(s) are administered in an alternating manner. As described herein, the antibody and / or composite antibody and the additional agent(s) may be administered in a single dose or multiple doses.

[0239] In some embodiments, a CM is linked or otherwise attached to an activatable antibody comprising an antibody or antigen-binding fragment thereof that specifically binds to a predetermined target coupled to a masking moiety (MM), such that coupling of the MM to the AB reduces the ability of the antibody or antigen-binding fragment thereof to bind to the target. In some embodiments, the MM is coupled via a CM. Exemplary targets include, but are not limited to, those shown in Table 1. Exemplary ABs include, but are not limited to, those shown in Table 2. The activatable antibodies provided herein are stable in the circulation, are activated at the intended therapeutic and / or diagnostic site, but are not normally activated, e.g., in healthy tissues or other tissues not targeted for treatment and / or diagnosis, and, when activated, exhibit binding to the target that is at least comparable to that of the corresponding unmodified antibody.

[0240] The present disclosure also provides methods and kits for using the conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies for various diagnostic and / or prophylactic indications.

[0241] In some embodiments, the disclosure provides a method for (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by a cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds a target of interest, wherein the activatable antibody in its uncleaved, non-activated state has the following structural arrangement from N- to C-terminus: MM-CM-AB or AB-CM-MM, wherein (a) MM is a peptide that inhibits binding of AB to the target, and MM does not have the amino acid sequence of a naturally occurring binding partner of AB and is not a modified form of the natural binding partner of AB, and (b) in its uncleaved, non-activated state, MM binds to the target. and (ii) provides methods and kits for detecting the presence or absence of a cleavage agent and a target of interest in a subject or sample by measuring the level of activated activatable antibody in the subject or sample, wherein a detectable level of activated activatable antibody in the subject or sample indicates that the cleavage agent and target are present in the subject or sample, and wherein the absence of detectable levels of activated activatable antibody in the subject or sample indicates that the cleavage agent, the target, or both are absent and / or insufficiently present in the subject or sample.

[0242] In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is located on the AB. In some embodiments, measuring the level of the activatable antibody in a subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody that comprises a detectable label.

[0243] In some embodiments of the methods and kits, the activatable antibody comprises a detectable label. In some embodiments of the methods and kits, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of the methods and kits, the imaging agent comprises a radioisotope. In some embodiments of the methods and kits, the radioisotope is indium or technetium. In some embodiments of the methods and kits, the contrast agent comprises iodine, gadolinium, or iron oxide. In some embodiments of the methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments of the methods and kits, the luminescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments of the methods and kits, the light-emitting label comprises an N-methylacridinium derivative. In some embodiments of the methods and kits, the label includes an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of the methods and kits, the ligand-based label includes biotin, avidin, streptavidin, or one or more haptens.

[0244] In some embodiments of these methods and kits, the subject is a mammal. In some embodiments of these methods, the subject is a human. In some embodiments of these methods and kits, the subject is a non-human mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), livestock, a working animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0245] In some embodiments of these methods and kits, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method.

[0246] The present disclosure also provides methods for detecting the presence or absence of a cleavage agent in a subject or biological sample by (i) contacting the subject or biological sample with a probe comprising a cleavable moiety (CM) and a detectable label that is released or activated upon cleavage of the CM; and (ii) measuring the level of the detectable label in the subject or biological sample. When such release or activation enhances detection of the label (e.g., stimulates a detectable signal), a detectable level of the detectable label in the subject or biological sample indicates the presence of the cleavage agent in the subject or biological sample, and wherein a decrease in the detectable level of the detectable label in the subject or biological sample indicates the absence of the cleavage agent in the subject or biological sample and / or its absence at detectable levels in the subject or biological sample, resulting in undetectable protease cleavage of the CM. If such release or activation reduces detection of the label, then a detectable level of the detectable label in the subject or biological sample indicates that the cleavage agent is not present or is not present in sufficient amounts in the subject or biological sample at a detectable level, such that protease cleavage of the CM cannot be detected in the subject or biological sample, and wherein a reduced detectable level of the detectable label in the subject or biological sample indicates that the cleavage agent is present in the subject or biological sample.

[0247] In some embodiments of the methods and kits, the CM-containing probe comprises a detectable label. In some embodiments of the methods and kits, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of the methods and kits, the imaging agent comprises a radioisotope. In some embodiments of the methods and kits, the radioisotope is indium or technetium. In some embodiments of the methods and kits, the contrast agent comprises iodine, gadolinium, or iron oxide. In some embodiments of the methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments of the methods and kits, the luminescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments of the methods and kits, the light-emitting label comprises an N-methylacridinium derivative. In some embodiments of the methods and kits, the label includes an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of the methods and kits, the ligand-based label includes biotin, avidin, streptavidin, or one or more haptens.

[0248] In some embodiments of these methods, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method.

[0249] In some embodiments of these methods and kits, the methods are used to identify or otherwise subdivide a patient population appropriate for treatment with an activatable antibody of the present disclosure, followed by administration of that activatable antibody and / or composite activatable antibody to a subject in need thereof. For example, a patient who tests positive for both the target and at least one protease selected from matriptase and uPA that cleaves a substrate in the cleavable moiety (CM) of an activatable antibody tested in these methods is identified as an appropriate candidate for treatment with the activatable antibody containing that CM, and the patient is then administered a therapeutically effective amount of the activatable antibody and / or composite activatable antibody being tested. Similarly, a patient who tests negative for either or both of the target and the protease that cleaves a substrate in the CM of an activatable antibody tested using these methods, i.e., matriptase and / or uPA, would be identified as an appropriate candidate for another form of treatment (i.e., not appropriate for treatment with the activatable antibody being tested). In some embodiments, such patients may be tested with other activatable antibodies until a suitable activatable antibody for treatment is identified, hi some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody and / or composite activatable antibody to which the patient tested positive.

[0250] The present disclosure also provides polypeptides and other large molecules comprising one or more of the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein. By way of non-limiting example, the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are useful in prodrug compositions and methods of use thereof. In some embodiments, the polypeptide comprises a CM linked to a drug, such as a small molecule. Examples of drugs are well known in the art. These matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are useful in probes and other detectable agents and methods of use thereof. For example, the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are used in conjunction with fluorescein or other quenchers to generate detectable agents, such as imaging agents and / or other diagnostic agents. Those skilled in the art will appreciate that the matriptase-cleavable substrate sequences provided herein and / or the uPA-cleavable substrate sequences provided herein are useful in any composition and / or method in the art that uses substrates cleavable by matriptase and / or uPA.

[0251] In some embodiments, the matriptase and / or uPA substrates of the present disclosure comprise a large molecule, e.g., (i) at least one moiety (M) that is located amino (N)-terminal to the CM, i.e., located within the large molecule closer to the N-terminus of the large molecule than the CM. N (ii) at least one moiety (M) located carboxyl (C)-terminal to the CM, i.e., located within the large molecule closer to the C-terminus of the large molecule than the CM; C (iii) combinations thereof. In some embodiments, the large molecule is used in an isolated polypeptide comprising at least one additional moiety (M) selected from the group consisting of: N and at least one M C Contains:

[0252] By way of non-limiting example, suitable M groups for use in the large molecules of the present disclosure include: N comprises at least one of the following: a masking moiety, an antibody, a protein, a therapeutic agent, an anti-neoplastic agent, a toxic agent, a drug, a detectable moiety, a diagnostic agent, an affinity tag, and combinations thereof.

[0253] By way of non-limiting example, suitable M groups for use in the large molecules of the present disclosure include: C comprises at least one of the following: a masking moiety, an antibody, a protein, a therapeutic agent, an anti-neoplastic agent, a toxic agent, a drug, a detectable moiety, a diagnostic agent, an affinity tag, and combinations thereof.

[0254] Pharmaceutical compositions according to the present disclosure can include an antibody of the present disclosure and a carrier. These pharmaceutical compositions can be included in a kit, such as, for example, a diagnostic kit. [Brief explanation of the drawings]

[0255]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

[0256] Detailed Description of the Invention The present disclosure provides amino acid sequences containing cleavable moieties (CMs) that are substrates for at least one protease selected from matriptase and u-plasminogen activator (uPA). These CMs are useful in a variety of therapeutic, diagnostic, and prophylactic indications.

[0257] The present disclosure provides antibodies comprising one or more of these matriptase-cleavable substrates and / or uPA-cleavable substrates. For example, these matriptase-cleavable substrates and / or uPA-cleavable substrates are useful when conjugating an antibody to one or more additional agents to produce a composite antibody. These matriptase-cleavable substrates and / or uPA-cleavable substrates are useful in activatable antibody constructs.

[0258] A conjugated antibody comprises an antibody or antigen-binding fragment thereof that specifically binds to a target, and an activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target. Exemplary classes of targets for antibodies or antigen-binding fragments thereof include, but are not limited to, cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), etc. In some embodiments, the conjugated antibody and / or activatable antibody comprises an antibody or antigen-binding fragment thereof that binds to an extracellular target, typically an extracellular protein target. In some embodiments, the conjugated antibody and / or activatable antibody is designed for cellular uptake and is switchable inside the cell.

[0259] As a non-limiting example, the antibody or antigen-binding fragment and / or activatable antibody AB is a binding partner of any of the targets listed in Table 1.

[0260] [Table 46]

[0261] [Table 47]

[0262] As a non-limiting example, the antibody or antigen-binding fragment and / or activatable antibody AB is or is derived from an antibody listed in Table 2.

[0263] [Table 48]

[0264] [Table 49]

[0265] Representative composite and / or activatable antibodies of the present disclosure include, for example, antibodies that bind to the interleukin-6 receptor (IL-6R) and antibodies that contain heavy and light chains derived from, or are designated herein as the "Av1" antibody, which binds to the interleukin-6 receptor (IL-6R). The amino acid sequences of the Av1 heavy chain and Av1 light chain are set forth below in SEQ ID NO:54 and SEQ ID NO:55, respectively. Av1 antibody heavy chain amino acid sequence: QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGQG SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 447) Av1 antibody light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 448)

[0266] Representative composite and / or activatable antibodies of the present disclosure include, for example, antibodies that bind to the interleukin-6 receptor (IL-6R) and comprise heavy and light chains that are or are derived from the Av1 antibody and masking moieties. Representative composite and / or activatable antibodies of the present disclosure comprise an amino acid sequence attached to the N-terminus of the AV1 light chain. These N-terminal amino acid sequences include, for example, YGSCSWNYVHIFMDC (SEQ ID NO: 449); QGDFDIPFPAHWVPIT (SEQ ID NO: 450); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 451); QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 452); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 453); or QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 454). It should also be understood that such amino acid sequences may be attached to the N-terminus of the AV1 heavy chain or the C-terminus of the AV1 heavy or light chain.

[0267] Representative activatable antibodies of the disclosure include, for example, antibodies that bind to epidermal growth factor receptor (EGFR), and antibodies comprising heavy and light chains that are or are derived from an antibody selected from the group consisting of the antibody designated herein as "c225v5," the antibody designated herein as "c225v4," and the antibody designated herein as "c225v6," each of which binds to EGFR. The c225v5, c225v4, and c225v6 antibodies share the same light chain sequence, designated herein as the "c225 light chain." The amino acid sequences of the c225v5 heavy chain, c225v4 antibody, c225v6 antibody, and c225 light chain are shown below. C225v5 antibody heavy chain amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 455) C225v4 antibody heavy chain amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 456) C225v6 antibody heavy chain amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 457) C225 antibody light chain amino acid sequence: QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 458)

[0268] Representative activatable antibodies of the disclosure include, for example, antibodies that bind to EGFR and comprise heavy and light chains that are or are derived from the c225v5 antibody, and that comprise a masking moiety, a first connecting peptide, a cleavable moiety, and a second connecting peptide. In some embodiments, the heavy and / or light chains comprise a signal peptide. The heavy and light chain amino acid sequences of c225v5 without the signal peptide are set forth above in SEQ ID NO:455 (heavy chain without signal peptide) and SEQ ID NO:458 (light chain without signal peptide). In some embodiments, the activatable anti-EGFR antibody comprises the amino acid sequence set forth in SEQ ID NO:455, SEQ ID NO:458, and / or a combination of the nucleic acid and amino acid sequences set forth below: C225v5 antibody heavy chain nucleic acid sequence with signal peptide: ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG Underlined: signal peptide C225v5 antibody heavy chain amino acid sequence with signal peptide: MYRMQLLSCIALSLALVTNS QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 685) Underlined: signal peptide 3954-2787-c225 light chain nucleic acid sequence with signal peptide: ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGCAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTACCTCCACCTCCGGCCGTTCCGCGAACCCGCGTGGTGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 686) Underlined part: Signal peptide 3954-2787-c225 light chain amino acid sequence having a signal peptide: MYRMQLLSCIALSLALVTNSQGQSGQCISPRGCPDGPYVMYGSSGGSGGSGGSGTSTSGRSANPRGGSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 687) Underlined: signal peptide 3954-2787-c225 light chain nucleic acid sequence (without signal peptide): CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTACCTCCACCTCCGGCCGTTCCGGAACCCGCGTGGTGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGG GCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGGCGAAAGCATTAGCGGCATTCGAGCCGCTTTAGCGGCAGGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGAT ATTGCGGATTATTGCCAGCAGAACAACAACTGGCCGACCACCCTTTGGCCGGGCACCAAACTGGAACTGAAACGTACGGTGGCCTGCACCATCTGTTCTCCGCCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGGCCTGCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGGTGTCACAGAGCAGACAGCAAGGACACCTACAGCCTCAGCAGCCAGCCAGCCTGAGCCTGAGCCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGGCCTGGAGCTCGCGCGTCACAAAGAGCTTCAACAGGGGGAGAGTGTTAG (sequence number 688) 3954‐2787‐C225 QGQSGQCISPRGCPDGPYVMYGSSGGSGGSGGSGTSTSGRSANPRGGSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 689)

[0269] Representative composite and / or activatable antibodies of the present disclosure include, for example, antibodies that bind to the Jagged targets, Jagged-1, Jagged-2, and / or both Jagged-1 and Jagged-2, as well as antibodies that contain combinations of variable heavy and light chain regions that are or are derived from the variable heavy and light chain sequences set forth below. Variable light chain amino acid sequence Lc4 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 459) Variable heavy chain amino acid sequence Hc4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 460) Variable light chain amino acid sequence Lc5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 461) Variable heavy chain amino acid sequence Hc5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYHGQFDYWGQGTLVTVSS (SEQ ID NO: 462) Variable light chain amino acid sequence Lc7 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 463) Variable heavy chain amino acid sequence Hc7 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 464) Variable light chain amino acid sequence Lc8 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 465) Variable heavy chain amino acid sequence Hc8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHIGRTNPFDYWGQGTLVTVSS (SEQ ID NO: 466) Variable light chain amino acid sequence Lc13 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 467) Variable heavy chain amino acid sequence Hc13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 468) Variable light chain amino acid sequence Lc16 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 469) Variable heavy chain amino acid sequence Hc16 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYYGQFDYWGQGTLVTVSS (SEQ ID NO: 470) Variable light chain amino acid sequence Lc19 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 471) Variable heavy chain amino acid sequence Hc19 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 472) Variable light chain amino acid sequence Lc21 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 473) Variable heavy chain amino acid sequence Hc21 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 474) Variable light chain amino acid sequence Lc24 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 475) Variable heavy chain amino acid sequence Hc24 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 476) Variable light chain amino acid sequence Lc26 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 477) Variable heavy chain amino acid sequence Hc26 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 478) Variable light chain amino acid sequence Lc27 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 479) Variable heavy chain amino acid sequence Hc27 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFYGQFDYWGQGTLVTVSS (SEQ ID NO: 480) Variable light chain amino acid sequence Lc28 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 481) Variable heavy chain amino acid sequence Hc28 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 482) Variable light chain amino acid sequence Lc30 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 483) Variable heavy chain amino acid sequence Hc30 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 484) Variable light chain amino acid sequence Lc31 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 485) Variable heavy chain amino acid sequence Hc31 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 486) Variable light chain amino acid sequence Lc32 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 487) Variable heavy chain amino acid sequence Hc32 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 488) Variable light chain amino acid sequence Lc37 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 489) Variable heavy chain amino acid sequence Hc37 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPHNGQFDYWGQGTLVTVSS (SEQ ID NO: 490) Variable light chain amino acid sequence Lc39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 491) Variable heavy chain amino acid sequence Hc39 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 492) Variable light chain amino acid sequence Lc40 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 493) Heavy chain amino acid sequence Hc40 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 494) Variable light chain amino acid sequence Lc47 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 495) Variable heavy chain amino acid sequence Hc47 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 496) Variable 4B2 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQGTKVEIKR (SEQ ID NO: 497) Variable 4B2 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 498) Variable 4D11 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKR (SEQ ID NO: 499) Variable 4D11 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 500) Variable 4E7 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQGTKVEIKR (SEQ ID NO: 501) Variable 4E7 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 502) Variable 4E11 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQGTKVEIKR (SEQ ID NO: 503) Variable 4E11 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 504) Variable 6B7 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR (SEQ ID NO: 505) Variable 6B7 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 506) Variable 6F8 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR (SEQ ID NO: 507) Variable 6F8 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 508)

[0270] Representative composite and / or activatable antibodies of the present disclosure include, for example, antibodies that bind to the Jagged targets, Jagged-1, Jagged-2, and / or both Jagged-1 and Jagged-2, as well as antibodies that contain combinations of heavy and light chain regions that are or are derived from the heavy and light chain sequences set forth below. 4D11 light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 509) 4D11 heavy chain sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 510) 4D11v2 heavy chain sequence EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 511) 4D11v2 light chain sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 512)

[0271] The activatable antibodies provided herein include at least an antibody or antibody fragment thereof (collectively referred to throughout this disclosure as AB) that specifically binds to a target, e.g., a human target, wherein the AB is modified by a masking moiety (MM).

[0272] In some embodiments, a masking moiety is selected for use with a specific antibody or antibody fragment. For example, a masking moiety suitable for use with an antibody that binds to EGFR includes a MM comprising the sequence CISPRG (SEQ ID NO: 513). By way of non-limiting example, the MM can include sequences such as CISPRGC (SEQ ID NO: 690), CISPRGCG (SEQ ID NO: 514), CISPRGCPDGPYVMY (SEQ ID NO: 515), CISPRGCPDGPYVM (SEQ ID NO: 516), CISPRGCEPGTYVPT (SEQ ID NO: 517), and CISPRGCPGQIWHPP (SEQ ID NO: 518).Other suitable masking moieties include, by way of non-limiting example, GSHCLIPINMGAPSC (SEQ ID NO: 519); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 520); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 521); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 522); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 523); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 524); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 525) Sequence number 525); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 526); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 527); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 528); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 529); CHHFTLTTCGCISPRGCPG (SEQ ID NO: 530); YNPCATPMCCISPRGCPG (SEQ ID NO: 531); CNHHYFYTCGCISPRGCG (SEQ ID NO: 532); CNHHYHYYCGCISPRGCG (SEQ ID NO: 533); C NHVYFGTCGCISPRGCG (SEQ ID NO: 534); CHHVYWGHCGCISPRGCG (SEQ ID NO: 535); CPHFTTTSCGCISPRGCG (SEQ ID NO: 536); CNHFTLTTCGCISPRGCG (SEQ ID NO: 537); CHHFTLTTCGCISPRGCG (SEQ ID NO: 538); CPYYTLSYCGCISPRGCG (SEQ ID NO: 539); CPHVSFGSCGCISPRGCG (SEQ ID NO: 540); ADHVFWGSYGCISPRGCG (SEQ ID NO: 541); YNPCATPMCCISPR GCG (SEQ ID NO: 542); CHHVYWGHCGCISPRGCG (SEQ ID NO: 543); 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: 544); CISPRGCGQPIPSVK ​​(SEQ ID NO: 545); CISPRGCTQPYHVSR (SEQ ID NO: 546); and / or any of the EGFR-specific masks disclosed in PCT Application Publication WO2010 / 081173, such as CISPRGCNAVSGLGS (SEQ ID NO: 547).

[0273] Suitable masking moieties for use with antibodies that bind to Jagged targets (e.g., Jagged1 and / or Jagged2) include, by way of example and not limitation, QGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 691); QGQSGQGQQQWCNIWINGGDCRGWNG (SEQ ID NO: 548); PWCMQRQDFLRCPQP (SEQ ID NO: 549); QLGLPAYMCTFECLR (SEQ ID NO: 550); CNLWVSGGDCGGLQG (SEQ ID NO: 551); SCSLWTSGSCLPHSP (SEQ ID NO: 552); YCL QLPHYMQAMCGR (SEQ ID NO: 553); CFLYSCTDVSYWNNT (SEQ ID NO: 554); PWCMQRQDYLRCPQP (SEQ ID NO: 555); CNLWISGGDCRGLAG (SEQ ID NO: 556); CNLWVSGGDCRGVQG (SEQ ID NO: 557); CNLWVSGGDCRGLRG (SEQ ID NO: 558); CNLWISGGDCRGLPG (SEQ ID NO: 559); CNLWVSGGDCRDAPW (SEQ ID NO: 560); CNLWVSGGDCRDLLG (SEQ ID NO: 561); CNLWVSGGDCRGLQG (SEQ ID NO: 562); C NLWLHGGDCRGWQG (SEQ ID NO: 563); CNIWLVGGDCRGWQG (SEQ ID NO: 564); CTTWFCGGDCGVMRG (SEQ ID NO: 565); CNIWGPSVDCGALLG (SEQ ID NO: 566); CNIWVNGGDCRSFEG (SEQ ID NO: 567); YCLNLPRYMQDMCWA (SEQ ID NO: 568); YCLALPHYMQADCAR (SEQ ID NO: 569); CFLYSCGDVSYWGSA (SEQ ID NO: 570); CYLYSCTDSAFWNNR (SEQ ID NO: 571); CYLYSCNDVSYWSNT (SEQ ID NO: 572) CFLYSCTDVSYW (SEQ ID NO: 573); CFLYSCTDVAYWNSA (SEQ ID NO: 574); CFLYSCTDVSYWGDT (SEQ ID NO: 575); CFLYSCTDVSYWGNS (SEQ ID NO: 576); CFLYSCTDVAYWNNT (SEQ ID NO: 577); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 578); CFLYSCTDVAYWSGL (SEQ ID NO: 579); CYLYSCTDGSYWNST (SEQ ID NO: 580); CFLYSCSDVSYWGNI (SEQ ID NO: 581); CFLYSCTDVAYW (SEQ ID NO: 582);CFLYSCTDVSYWGST (SEQ ID NO: 583); CFLYSCTDVAYWGDT (SEQ ID NO: 584); GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 585); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 586); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 587); GCNIWANGGDCRGWIDNIDG (SEQ ID NO: 588); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 589); GCNIWLVGGDCRGWLEEAVG (SEQ ID NO: 590); GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 591); GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 592); GQQQWCNIWINGGDCRGWNG (SEQ ID NO: 593); GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 594); GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 595); GASQYCNLWINGGDCRGWRG (SEQ ID NO: 596); GCNI Masking moieties include sequences such as WLVGGDCRPWVEGG (SEQ ID NO: 597); GCNIWAVGGDCRPFVDGG (SEQ ID NO: 598); GCNIWLNGGDCRAWVDTG (SEQ ID NO: 599); GCNIWIVGGDCRPFINDG (SEQ ID NO: 600); GCNIWLNGGDCRPVVFGG (SEQ ID NO: 601); GCNIWLSGGDCRMFMNEG (SEQ ID NO: 602); GCNIWVNGGDCRSFVYSG (SEQ ID NO: 603); GCNIWLNGGDCRGWEASG (SEQ ID NO: 604); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 605); GCNIWLNGGDCRTFVASG (SEQ ID NO: 606); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 607); GFLENCNIWLNGGDCRTG (SEQ ID NO: 608); GIYENCNIWLNGGDCRMG (SEQ ID NO: 609); and / or GIPDNCNIWINGGDCRYG (SEQ ID NO: 610).

[0274] Suitable masking moieties for use with antibodies that bind to an interleukin-6 target, e.g., interleukin-6 receptor (IL-6R), include, by way of non-limiting example, QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 611); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 612); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 613); YRSCNWNYVSIFLDC (SEQ ID NO: 614); PGAFDIPFPAHWVPNT (SEQ ID NO: 615); ESSCVWNYVHIYMDC (SEQ ID NO: 6 16);YPGCKWNYDRIFLDC (SEQ ID NO: 617);YRTCSWNYVGIFLDC (SEQ ID NO: 618);YGSCSWNYVHIFMDC (SEQ ID NO: 619);YGSCSWNYVHIFLDC (SEQ ID NO: 620);YGSCNWNYVHIFLDC (SEQ ID NO: 621);YTSCNWNYVHIFMDC (SEQ ID NO: 622);YPGCKWNYDRIFLDC (SEQ ID NO: 623);WRSCNWNYAHIFLDC (SEQ ID NO: 624);WSNCHWNYVHIFLDC (SEQ ID NO: 625);DRSCTWNYVRISYDC (SEQ ID NO: 626); No. 626); SGSCKWDYVHIFLDC (SEQ ID NO: 627); SRSCIWNYAHIHLDC (SEQ ID NO: 628); SMSCYWQYERIFLDC (SEQ ID NO: 629); YRSCNWNYVSIFLDC (SEQ ID NO: 630); SGSCKWDYVHIFLDC (SEQ ID NO: 631); YKSCHWDYVHIFLDC (SEQ ID NO: 632); YGSCTWNYVHIFMEC (SEQ ID NO: 633); FSSCNWNYVHIFLDC (SEQ ID NO: 634); WRSCNWNYAHIFLDC (SEQ ID NO: 635); YGSCQWNYVHIFLDC (SEQ ID NO: 636); Sequence number 636); YRSCNWNYVHIFLDC (SEQ ID NO: 637); NMSCHWDYVHIFLDC (SEQ ID NO: 638); FGPCTWNYARISWDC (SEQ ID NO: 639); XXsCXWXYvhIfXdC (SEQ ID NO: 640); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 641); RDTGGQCRWDYVHIFMDC (SEQ ID NO: 642); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 643); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 644); DGGPAGCSWNYVHIFMEC (SEQ ID NO: 645);AVGPAGCWWNYVHIFMEC (SEQ ID NO: 646); CTWNYVHIFMDCGEGEGP (SEQ ID NO: 647); GGVPEGCTWNYAHIFMEC (SEQ ID NO: 648); AEVPAGCWWNYVHIFMEC (SEQ ID NO: 649); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 650); SGASGGCKWNYVHIFMDC (SEQ ID NO: 651); TPGCRWNYVHIFMECEAL (SEQ ID NO: 652); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 653); PGAFDIPFPAHWVPNT (SEQ ID NO: 654); RGACDIPFPAHWIPNT (SEQ ID NO: 655); QG DFDIPFPAHWVPIT (SEQ ID NO: 656); XGafDIPFPAHWvPnT (SEQ ID NO: 657); RGDGNDSDIPFPAHWVPRT (SEQ ID NO: 658); SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 659); WAGGNDCDIPFPAHWIPNT (SEQ ID NO: 660); WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 661); AGSGNDSDIPFPAHWVPRT (SEQ ID NO: 662); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 663); and / or RECGRCGDIPFPAHWVPRT (SEQ ID NO: 664).

[0275] When an AB is modified with a MM and in the presence of a target, the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to the target.

[0276] K of MM-modified AB against target d is the K of the AB not modified by MM or the parent AB to the target. dor more than 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 more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100, 000, 10-10,000, 000, 10-10,000, 000, 100-1,000, 000, 10-10,000, 000, 100-1,000, 000 00, 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, 10,000-10,000,000, 100,000-10,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of an AB modified with a MM for a target is at least 2, 3, 4, 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 more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100, 00, or more, than the binding affinity of an AB not modified with a MM or the parent AB for the target. 0, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 10,000-10,000,000, 100,000-10,000,000 or 100,000-10,000,000 times lower.

[0277] The dissociation constant of MM with AB (K d ) is generally the K of AB against the target dK of MM against AB is larger than d is the K of AB against the target d The binding affinity of the MM to the AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the binding affinity of the MM to the AB. Conversely, the binding affinity of the MM to the AB is generally lower than the binding affinity of the AB to the target. The binding affinity of the MM to the AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times lower than the binding affinity of the AB to the target.

[0278] When an AB is modified with a MM and in the presence of a target, the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to the target. The ability of the AB to bind a target when modified with a MM, when compared to the binding of an AB not modified with a MM or the binding of the parent AB to a target, may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer, as measured by in vivo or in vitro assays.

[0279] The MM inhibits binding of the AB to a target. The MM binds the antigen-binding domain of the AB and inhibits binding of the AB to a target. The MM can sterically inhibit binding of the AB to a target. The MM can allosterically inhibit binding of the AB to its target. In these embodiments, when the AB is modified and coupled to a MM in the presence of a target, binding of the AB to its target is inhibited for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 240, 300, 360, 480, 600, 720, 84, or 96 hours, as measured in an in vivo or in vitro assay, compared to binding of the unmodified AB, the parent AB, or an AB not coupled to a MM to a target. There is no or substantially no binding of AB to the target, or at most 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of AB to the target for 20, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer.

[0280] When an AB is coupled to or modified by an MM, the MM "masks" or reduces or otherwise inhibits the specific binding of the AB to a target. When an AB is coupled to or modified by an MM, such coupling or modification can result in a conformational change that reduces or inhibits the ability of the AB to specifically bind its target.

[0281] The AB coupled to or modified by the MM can be represented by the following formula, from the amino (N) terminal region to the carboxyl (C) terminal region: (MM)-(AB) (AB)-(MM) (MM)‐L‐(AB) (AB)-L-(MM) where MM is a masking moiety, AB is an antibody or antibody fragment, and L is a linker. In many embodiments, it is desirable to include one or more linkers, e.g., flexible linkers, in the composition to provide flexibility.

[0282] In some embodiments, the MM is not a natural binding partner of AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. In some embodiments, the MM is 50% or less identical to any natural binding partner of AB. In some embodiments, the MM is 20% or less identical to any natural binding partner of AB. In some embodiments, the MM is 10% or less identical to any natural binding partner of AB.

[0283] In some embodiments, an activatable antibody comprises an AB modified by a MM and also comprises one or more cleavable moieties (CM). Such an activatable antibody exhibits activatable / switchable binding of the AB to a target. An activatable antibody generally comprises an antibody or antibody fragment (AB) modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM comprises an amino acid sequence that acts as a substrate for at least one protease selected from matriptase and uPA.

[0284] The activatable antibody components are positioned such that the MM and CM are positioned such that in the cleaved (or relatively active) state and in the presence of a target, the AB binds the target, whereas in the uncleaved (or relatively inactive) state and in the presence of a target, specific binding of the AB to its target is reduced or inhibited. Specific binding of the AB to its target can be reduced due to inhibition or masking of the ability of the AB to specifically bind its target by the MM.

[0285] K of AB modified with MM and CM against target d is the K of the AB unmodified by MM and CM, or the parent AB, against the target. dor more than 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 more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100, 000, 10-10,000, 000, 10-10,000, 000, 100-1,000, 000, 10-10,000, 000, 100-1,000, 000 00, 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, 10,000-10,000,000, 100,000-10,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of an AB modified with MM and CM to a target 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 more, or 5-10, 10-10 ... 00, 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, 10,000-1,000,000, 10,000-10,000,000, 10,000-10,000,000, 100,000-10,000,000 or 100,000-10,000,000 times lower.

[0286] When an AB is modified with a MM and a CM and in the presence of a target but not in the presence of a modifying agent (e.g., at least one protease selected from matriptase and uPA), the specific binding of the AB to the target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM and a CM or the parent AB to the target. The ability of the AB to bind a target when modified with MM and CM compared to the binding of the parent AB or the binding of an AB not modified with MM and CM to its target may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or longer, as measured by in vivo or in vitro assays.

[0287] As used herein, the term cleaved state refers to the state of an activatable antibody following modification of the CM by at least one protease selected from matriptase and uPA. The term uncleaved state, as used herein, refers to the state of an activatable antibody in the absence of cleavage of the CM by a protease selected from matriptase and uPA. As discussed above, the term "activatable antibody" is used herein to refer to an activatable antibody in its uncleaved (native) state and its cleaved state. It will be apparent to one of skill in the art that in some embodiments, a cleaved activatable antibody lacks MM due to cleavage of the CM by a protease, resulting in release of at least MM (e.g., MM is not covalently linked to the activatable antibody (e.g., disulfide bond between cysteine ​​residues)).

[0288] Activatable or switchable means that the activatable antibody exhibits a first level of target binding in an inhibited, masked, or uncleaved state (i.e., a first conformation), and a second level of target binding in an uninhibited, unmasked, and / or cleaved state (i.e., a second conformation), where the second level of target binding is greater than the first level of binding. Generally, the accessibility of the target to the AB of an activatable antibody is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease selected from matriptase and uPA, than in the absence of such a cleaving agent. Thus, when the activatable antibody is present in an uncleaved state, the AB can be inhibited from target binding and masked from target binding (i.e., in the first conformation, such an AB cannot bind the target), and in the cleaved state, the AB is uninhibited from or unmasked from target binding.

[0289] The CM and AB of an activatable antibody are selected such that AB represents a binding moiety for a predetermined target, and the CM represents a substrate for a protease selected from matriptase and uPA. In some embodiments, the protease co-localizes with the target at a therapeutic or diagnostic site in a subject. As used herein, co-localized refers to being at the same site or relatively close by. In some embodiments, the protease cleaves the CM, yielding an activated antibody that binds to the target located near the cleavage site. The activatable antibodies disclosed herein are particularly useful, for example, when a protease capable of cleaving a site within the CM, i.e., a protease selected from matriptase and uPA, is present at a relatively higher level in target-containing tissue at a therapeutic or diagnostic site than in tissue at a non-therapeutic site (e.g., healthy tissue). In some embodiments, the CM of the present disclosure is also cleaved by one or more other proteases. In some embodiments, the other proteases co-localize with the target and may be responsible for cleavage of the CM in vivo.

[0290] In some embodiments, the activatable antibody provides reduced toxicity and / or adverse side effects resulting from binding of the AB at non-therapeutic sites when the AB is unmasked or inhibited from binding the target.

[0291] In general, an activatable antibody can be designed by selecting an AB of interest and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides masking of the AB or reduced binding of the AB to its target. Structural design criteria should be considered to provide this functional feature.

[0292] Activatable antibodies are provided that exhibit a desired dynamic range of switchable phenotypes for target binding in the inhibited conformation relative to the uninhibited conformation. Dynamic range generally refers to the ratio of the maximum detectable level of a parameter under (a) a first set of conditions to the minimum detectable value of the parameter under (b) a second set of conditions. For example, in the case of an activatable antibody, dynamic range refers to the ratio of the maximum detectable level of target protein, i.e., target, that binds to the activatable antibody in the presence of at least one protease selected from matriptase and uPA capable of cleaving the CM of the activatable antibody to the minimum detectable level of target protein that binds to the activatable antibody in the absence of the protease (b). The dynamic range of an activatable antibody can be calculated as the ratio of the equilibrium dissociation constant of treatment with an activatable antibody cleaving agent (e.g., an enzyme) to the equilibrium dissociation constant of treatment with the activatable antibody cleaving agent. The higher the dynamic range of the activatable antibody, the better the switchable phenotype of the activatable antibody. Activatable antibodies with a relatively high dynamic range (e.g., 1 or more) exhibit a more desirable switchable phenotype, and as a result, target protein binding by the activatable antibody occurs to a greater extent (e.g., occurs primarily) in the presence of a cleaving agent (e.g., an enzyme) capable of cleaving the CM of the activatable antibody than in the absence of the cleaving agent.

[0293] Activatable antibodies can be provided in a variety of structural conformations. Exemplary formulas for activatable antibodies are provided below. It is specifically contemplated that the N- to C-terminal order of AB, MM, and CM can be reversed within an activatable antibody. It is also specifically contemplated that CM and MM overlap in amino acid sequence, e.g., resulting in a CM being contained within a MM.

[0294] For example, an activatable antibody can be represented, from amino (N) terminal region to carboxyl (C) terminal region, by the formula: (MM)-(CM)-(AB) (AB)-(CM)-(MM) wherein MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof. Although MM and CM are shown as distinct components in the above formula, it is intended that in all exemplary embodiments (including formulas) disclosed herein, the amino acid sequences of MM and CM overlap, e.g., such that CM is fully or partially contained within MM. Additionally, the above formula provides additional amino acid sequences that can be positioned N-terminally or C-terminally to the activatable antibody element.

[0295] In certain embodiments, the MM is not a natural binding partner of AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0296] In many embodiments, it is desirable to insert one or more linkers, e.g., flexible linkers, into an activatable antibody construct to provide flexibility at one or more MM-CM junctions, CM-AB junctions, or both. For example, the AB, MM, and / or CM do not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, especially Gly) to provide the desired flexibility. The cleavable phenotype of such an activatable antibody construct can benefit from the introduction of one or more amino acids to provide a flexible linker. Furthermore, as described below, when the activatable is provided as a conformationally constrained construct, a flexible linker can be operatively inserted to promote the formation and maintenance of a cyclic structure in the cleaved activatable antibody.

[0297] For example, in one embodiment, the activatable antibody comprises one of the following formulas (wherein the formula represents the amino acid sequence in either the N- to C-terminal direction or the C- to N-terminal direction): (MM)‐L1‐(CM)‐(AB) (MM)-(CM)-L2-(AB) (MM)‐L1‐(CM)‐L2‐(AB) wherein MM, CM, and AB are as defined above; and L1 and L2 are each independently and optionally, the same or different flexible linkers, which may be present or absent and comprise at least one flexible amino acid (e.g., Gly). Additionally, the above formula provides additional amino acid sequences that can be positioned N-terminally or C-terminally to the activatable antibody element. Examples include, but are not limited to, targeting moieties (e.g., ligands for receptors on cells present in target tissues), and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HAS))).

[0298] CM is approximately 0.001 to 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 In some embodiments, the CM is specifically cleaved by the enzyme at a rate of about 100,000 M -1 S -1 In some embodiments, the CM is specifically cleaved at a rate of about 1 x 10E 2 ~1x10E 6 10 4 M -1 S -1 (i.e., about 1×10 2 ~Approx. 1×10 6 M-1 S -1 ) is specifically cleaved at a rate of

[0299] For specific cleavage by an enzyme, contact between the enzyme and the CM is required. When an activatable antibody, including an MM and an AB coupled to the CM, is in the presence of the target and sufficient enzymatic activity, the CM can be cleaved. Sufficient enzymatic activity refers to the ability of the enzyme to contact the CM and effect cleavage. It is easily conceivable that the enzyme may be in close proximity to the CM but be unable to cleave due to other cellular factors or protein modifications of the enzyme.

[0300] Linkers suitable for use in the compositions described herein are generally those that provide flexibility to the modified AB or activatable antibody to facilitate inhibition of AB binding to a target. Such linkers are commonly referred to as flexible linkers. Suitable linkers can be readily selected and can be of any suitable length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids in length, from 2 to 15 amino acids in length, from 3 to 12 amino acids in length, from 4 to 10 amino acids in length, from 5 to 9 amino acids in length, from 6 to 8 amino acids in length, or from 7 to 8 amino acids in length, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0301] A typical flexible linker is a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 385) and (GGGS) n(SEQ ID NO: 386), where n is an integer of at least one, glycine-alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between components. Glycine has significantly more access to phi-psi (phi-psi) space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly (SEQ ID NO: 387), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 388), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 389), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 390), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 391), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 392), and the like. Those skilled in the art will understand that designs of activatable antibodies can include linkers that are fully or partially flexible, and consequently, the linker can include a flexible linker and one or more moieties that provide less flexible conformations to provide the desired activatable antibody structure.

[0302] The conjugated activatable antibodies described herein also 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-neoplastic agent. In such embodiments, for example, in some embodiments, the agent is conjugated to a carbohydrate moiety of the activatable antibody, where the carbohydrate moiety is located outside the antigen-binding region of the antibody or antigen-binding fragment within the activatable antibody. In some embodiments, the agent is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment within the activatable antibody.

[0303] In some embodiments, the agent is a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope (i.e., a radioconjugate). Suitable cytotoxic agents include, for example, any of the cytotoxic agents listed in Table 4.

[0304] In some embodiments, the agent is a detectable moiety, such as, for example, a label or other marker. For example, the agent comprises a radiolabeled amino acid, one or more biotinyl residues detectable by tagged avidin (e.g., streptavidin-containing fluorescent labels or enzymatic activity detectable 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 attached by a spacer molecule.

[0305] The present disclosure also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragment thereof) or a radioactive isotope (i.e., a radioconjugate). Suitable cytotoxic agents include, for example, dolastatin and its derivatives (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 an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0306] In some embodiments, the agent is connected to AB using a maleimidocaproyl-valine-citrulline linker or a maleimidoPEG-valine-citrulline linker. In some embodiments, the agent is connected to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, the agent is connected to AB using a maleimidoPEG-valine-citrulline linker. In some embodiments, the agent is connected to AB using a maleimidoPEG-valine-citrulline linker. In some embodiments, the agent is monomethyl auristatin D (MMAD) connected to AB using a maleimidoPEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, this linker payload construct being referred to herein as "vc-MMAD." In some embodiments, the agent is monomethyl auristatin E (MMAE) connected to AB using a maleimidoPEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, this linker payload construct being referred to herein as "vc-MMAE." The structures of vc-MMAD and vc-MMAE are shown below: vc-MMAD:

[0307] [ka]

[0308] vc-MMAE:

[0309] [ka]

[0310] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.

[0311] Conjugation of antibodies to cytotoxic agents can be achieved using a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl subate), aldehydes (e.g., glutaraldehyde), bis-azido derivatives (e.g., bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, 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 a typical chelating agent for the attachment of radionuclides to antibodies (see WO 94 / 11026).

[0312] Table 3 lists some exemplary pharmaceutical agents that may be used in the disclosure described herein, but is not limited to these.

[0313] [Table 50]

[0314] [Table 51]

[0315] Those skilled in the art will appreciate that a wide variety of possible moieties can be attached to the resulting antibodies of this disclosure (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989); the entire contents of which are incorporated herein by reference).

[0316] In some embodiments, in addition to the compositions and methods provided herein, the composite activatable antibody also Coupling can be achieved by using any chemical reaction that will link the two molecules so that the antibody and the other moiety retain their respective activities for as long as possible. This binding can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. In some embodiments, however, the binding is covalent. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various types of coupling agents known in the art, but rather is exemplary of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0317] In some embodiments, in addition to the compositions and methods provided herein, composite activatable antibodies can also be modified for site-specific conjugation through modified amino acid sequences inserted or otherwise included in the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled placement and / or administration of conjugated agents within the composite activatable antibody. For example, activatable antibodies can be constructed to contain cysteine ​​substitutions at positions on the light and heavy chains that provide reactive thio groups and do not alter antigen binding or negatively affect protein folding and assembly. In some embodiments, activatable antibodies can be constructed to contain or otherwise incorporate one or more non-natural amino acid residues into the activatable antibody to provide suitable sites for conjugation. In some embodiments, activatable antibodies can be constructed to contain or otherwise incorporate enzymatically activatable peptide sequences into the activatable antibody sequence.

[0318] Suitable linkers are described in the literature (see, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives attached to antibodies by oligopeptide linkers. In some embodiments, suitable linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., catalog number 2165G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co., catalog number 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide) coupled to EDC (Pierce Chem. Co., catalog number 24510). Additional linkers include, but are not limited to, SMCC, sulfo-SMqCC, SPDB, or sulfo-SPDB.

[0319] The linkers contain moieties with different attributes, thus leading to conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SHPT contains a sterically hindered disulfide bond and can form conjugates with enhanced stability. Disulfide bonds are generally less stable than other linkages because they are cleaved in vitro, resulting in less available conjugate. Sulfo-NHS can particularly enhance the stability of carbodiimide coupling. When used in conjugation with sulfo-NHS, carbodiimide coupling (such as EDC) forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0320] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are multiple linkers. The multiple linkers are all the same, i.e., cleavable or non-cleavable, or the multiple linkers are different, i.e., at least one cleavable and at least one non-cleavable.

[0321] The present disclosure utilizes several methods for attaching an agent to an AB, including: (a) attachment to the carbohydrate moiety of the AB, or (b) attachment to a sulfhydryl group of the AB, or (c) attachment to an amino group of the AB, or (d) attachment to a carboxylate group of the AB. According to the present disclosure, the AB can be covalently attached to the agent via an intermediate linker having at least two reactive groups (one reactive with the AB and one reactive with the agent). Linkers containing any suitable organic compound are selected so that reaction with the AB (or agent) does not adversely affect the reactivity and selectivity of the AB. Furthermore, attachment of the linker to the agent does not destroy the activity of the agent. Suitable linkers for reaction with an oxidized antibody or oxidized antibody fragment include those containing an amine selected from the group consisting of primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and thiosemicarbazides. Such reactive functional groups can be present as part of the structure of the linker or can be introduced by appropriate chemical modification of a linker that does not contain such groups.

[0322] According to the present disclosure, suitable linkers for attachment to reduced AB include those having certain reactive groups capable of reacting with sulfhydryl groups on reduced antibodies or fragments. Such reactive groups include, but are not limited to, reactive haloalkyl groups (e.g., haloacetyl groups), p-mercurybenzoate groups, and groups capable of Michael-type addition reactions (e.g., maleimide, and groups described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

[0323] According to the present disclosure, suitable linkers for attachment to non-oxidized and non-reduced Abs include those having certain functional groups capable of reacting with primary amino groups present on unmodified lysine residues in the Ab. Such reactive groups include, but are not limited to, NHS carboxylic acid or carbonate esters, pentafluorophenyl carboxylic acid or carbonate esters, acylimidazoles, isocyanates, and isothiocyanates.

[0324] According to the present disclosure, suitable linkers for attachment to unoxidized or unreduced ABs include those having certain functional groups that can react with carboxylic acid groups present in aspartic acid or glutamic acid residues in Abs that have been activated with an appropriate reagent. Suitable activation reagents include EDC with or without an added NHS or sulfo-NHS, and other dehydrating agents used for carboxamide formation. In those cases, functional groups present in suitable linkers include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0325] The agent can be attached to the linker before or after the linker is attached to the AB. For certain applications, it may be desirable to first generate an AB-linker intermediate in which the linker does not have an associated agent. Depending on the particular application, a particular agent can be covalently attached to the linker. In other embodiments, the AB is first attached to the MM, CM, and associated linker, and then attached to the linker for conjugation purposes.

[0326] Branched Linkers: In certain embodiments, branched linkers are used that have multiple sites for attachment of agents. For multi-site linkers, a single covalent bond to AB results in an AB-linker intermediate that can attach agents at many sites. The sites can be aldehyde or sulfhydryl groups, or any chemical site to which an agent can be attached.

[0327] On the other hand, higher specific activity (or a higher ratio of agent to AB) can be achieved by attaching a single-site linker at multiple sites on the AB. These multiple sites can be introduced into the AB by either of two methods. The first method can generate multiple aldehyde and / or sulfhydryl groups on the same AB. The second method can attach a "branched linker" with multiple functional sites for subsequent attachment to a linker to the aldehyde or sulfhydryl groups of the AB. The functional sites in the branched or multi-site linker can be aldehyde or sulfhydryl groups, or any chemical site to which a linker can be attached. Even higher specific activity can be obtained by combining these two approaches, i.e., by attaching a multi-site linker at several sites on the AB.

[0328] Cleavable linkers: Peptide linkers that are susceptible to degradation by enzymes of the complement system, such as, but not limited to, u-plasminogen activator, tissue plasminogen activator, trypsin, plasmin, or another enzyme with proteolytic activity, can be used in one embodiment of the present disclosure. According to one method of the present disclosure, an agent is attached via a linker that is susceptible to cleavage by complement. The antibody is selected from a species that can activate complement. The antibody-agent conjugate thus activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, an agent is attached via a linker that is susceptible to cleavage by an enzyme with proteolytic activity, such as u-plasminogen activator, tissue plasminogen activator, plasmin, or trypsin. These cleavable linkers are useful in conjugated activatable antibodies with exotoxins, such as, by way of non-limiting example, any of the exotoxins listed in Table 3.

[0329] Non-limiting examples of cleavable linker sequences are provided in Table 4.

[0330] [Table 52]

[0331] Additionally, the agent can be attached to the AB via a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Many tumors naturally release high levels of glutathione (a reducing agent), which reduces the disulfide bond and subsequently releases the agent at the site of delivery. In some embodiments, the reducing agent that modifies the CM will also modify the linker of the conjugated activatable antibody.

[0332] Spacers and Cleavable Elements: In some embodiments, it is necessary to construct the linker in such a way as to optimize the spacing between the agent and the AB of the activatable antibody. This can be achieved by using linkers of the following general structure: W‐(CH2) n -Q wherein W is either -NH-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer of 0 to 20.

[0333] In some embodiments, the linker can comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the core of the AB, thereby making the cleavable element more accessible to the enzyme responsible for cleavage. Certain branched linkers described above can serve as spacer elements.

[0334] Throughout this discussion, the attachment of a linker to an agent (or a spacer element to a cleavable element, or a cleavable element to an agent) does not require a particular mode of bond or reaction: any reaction that provides a product of suitable stability and biocompatibility is acceptable.

[0335] Selection of serum complement and linker: According to one method of the present disclosure, if release of an agent is desired, an antibody of a type capable of activating complement, an AB, is used. The resulting conjugate retains both the ability to bind antigen and activate the complement cascade. Thus, in an embodiment of the present disclosure, the agent is linked to one end of a cleavable linker or cleavable element, and the other end of the linker group is attached to a specific site on the AB. For example, if the agent has a hydroxyl or amino group, it can be attached to the carboxy terminus of a peptide, amino acid, or other appropriately selected linker via an ester or amide bond, respectively. For example, such agents can be attached to a linker peptide via a carbodiimide reaction. If the agent contains functional groups that interfere with binding to the linker, those interfering functional groups can be blocked prior to conjugation and then unblocked once the product conjugate or intermediate is prepared. The opposite or amino terminus of the linker is then used, either directly or after further modification, for conjugation to an AB capable of activating complement.

[0336] The linker (or spacer element of the linker) can be of any desired length, and one end can be covalently attached to a specific site on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker.

[0337] Thus, when these conjugates bind to an antigen in the presence of complement, the amide or ester bond connecting the agent to the linker is cleaved, resulting in the release of the agent in its active form. These conjugates, when administered to a subject, achieve delivery and release of the agent at the target site and are particularly effective for the in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative agents, and the like, as provided in Table 3, but not limited to:

[0338] Linkers for release without complement activation: According to yet another application of targeted delivery, release of an agent without complement activation is desired, since activation of the complement cascade ultimately lyses target cells. Therefore, this approach is useful when delivery and release of an agent can be achieved without killing the target cells. This is the goal when delivery of cellular mediators, such as hormones, enzymes, corticosteroids, neurotransmitters, genes, or enzymes to target cells is desired. These conjugates can be prepared by linking the agent to an AB that cannot activate complement via a linker that is mildly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, the antigen-antibody complex forms rapidly, while cleavage of the agent occurs slowly, resulting in release of the compound at the target site.

[0339] Biochemical Crosslinkers: In some embodiments, activatable antibodies can be conjugated to one or more therapeutic agents using certain biochemical crosslinkers. Crosslinking reagents form molecular bridges that link together functional groups of two different molecules. To link two different proteins in a stepwise fashion, heterobifunctional crosslinkers can be used, which eliminates undesired homopolymer formation.

[0340] Peptidyl linkers that can be cleaved by lysosomal proteases, such as Val-Cit, Val-Ala, or other dipeptides, are also useful. Additionally, acid-labile linkers that can be cleaved in the low-pH environment of the lysosome, such as bis-sialyl ether, can be used. Other suitable linkers include cathepsin-labile substrates, particularly those that function optimally at acidic pH.

[0341] Representative heterobifunctional crosslinkers are referenced in Table 5.

[0342] [Table 53]

[0343] Non-cleavable linker or direct conjugation: In some embodiments of the present disclosure, conjugates can be designed to deliver the agent to a target but not release it. This can be achieved by attaching the agent to AB directly or via a non-cleavable linker.

[0344] These non-cleavable linkers can include amino acids, peptides, D-amino acids, or other organic compounds that can be subsequently modified to contain functional groups that can be utilized in attachment to AB by the methods described herein. A general formula for such organic linkers can be: W‐(CH2) n -Q wherein W is either -NH-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer of 0 to 20.

[0345] Non-cleavable conjugates: In some embodiments, a compound can be conjugated to an AB that does not activate complement. When an AB that cannot activate complement is used, this conjugation can be achieved using a linker that is susceptible to cleavage by activated complement or a linker that is not susceptible to cleavage by activated complement.

[0346] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0347] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be coupled to liposomes via a disulfide-exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982).

[0348] Definition: Unless otherwise specified, scientific and technical terms used in connection with this disclosure will have the meanings commonly understood by those of ordinary skill in the art. Terms such as "a" entity or "an" entity refer to one or more of that entity. For example, "a" compound refers to one or more compounds. Thus, terms such as "a," "an," "one or more," and "at least one" can be used interchangeably. Furthermore, unless otherwise required, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are well known to those of ordinary skill in the art and are commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and as described in various general and specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques, are well known to those skilled in the art and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0349] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0350] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides and with a lower affinity (K d >10-6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibodies, single chain, Fab and F(ab')2 fragments, scFvs, and Fab expression libraries.

[0351] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminus of each chain defines a constant region primarily responsible for effector function. Antibody molecules obtained from humans generally belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chain present in the molecule. Particular classes also have subclasses, e.g., IgG1, IgG2, and others. Furthermore, in humans, the light chains can be kappa or lambda chains.

[0352] The terms "monoclonal antibody" (mAb) or "monoclonal antibody composition," as used herein, refer to a population of antibody molecules containing only one species of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a specific epitope of an antigen characterized by a unique binding affinity for that epitope.

[0353] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the V regions of the H and L chains, referred to as "hypervariable regions," are interposed between more conserved adjacent stretches known as "framework regions" or "FR." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the L chain and the three hypervariable regions of the H 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 conformational surface of a bound antigen, and the three hypervariable regions of each H and L chain are referred to as "complementarity-determining regions" or "CDRs." The amino acid assignments for each domain follow the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or the definitions in Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0354] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific conformational and charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. An antibody is said to specifically bind an antigen if the dissociation constant is ≦1 μM; in some embodiments, ≦100 mM, and in some embodiments, ≦10 nM.

[0355] As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ), where the smaller K d represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence both rates. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation (see Nature 361:186-87 (1993)). The ratio Koff / Kon allows for the cancellation of all parameters not related to affinity, and the dissociation constant K d(See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present disclosure have an equilibrium binding constant (K d ) is ≦1 μM, in some embodiments ≦100 nM, in some embodiments ≦10 nM, and in some embodiments ≦100 M to about 1 pM.

[0356] The term "isolated polynucleotide," as used herein, refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an "isolated polynucleotide" is one that: (1) is not associated with all or part of a polynucleotide with which it is found in nature; (2) is operably linked to a polynucleotide with which it is not linked in nature; or (3) is not found in nature as part of a larger sequence. Polynucleotides of the present disclosure include nucleic acid molecules encoding the heavy chain immunoglobulin molecules set forth herein and nucleic acid molecules encoding the light chain immunoglobulin molecules described herein.

[0357] As used herein, the term "isolated protein" refers to a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; according to its origin or source of derivation, an "isolated protein" is (1) not associated with a protein found in nature, (2) free from other proteins from the same source, e.g., free from murine proteins, (3) expressed by cells from a different species, or (4) not found in nature.

[0358] The term "polypeptide" is used herein as a generic term to refer to a naturally occurring protein, fragment, or analog of a polypeptide sequence. Thus, naturally occurring proteins, fragments, and analogs are species of the polypeptide genus. Polypeptides of the present disclosure include antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules as set forth herein, and light chain immunoglobulin molecules as set forth herein, as well as light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, and fragments and analogs thereof.

[0359] As used herein, the term "naturally-occurring," when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide present in an organism (e.g., a virus) has been isolated from a natural source and has not been intentionally modified by man in the laboratory, or is otherwise naturally occurring.

[0360] The term "operably linked," as used herein, refers to the positioning of the described components in a relationship permitting such components to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0361] The term "control sequences," as used herein, refers to polynucleotide sequences necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences varies depending on the host organism in prokaryotes; such control sequences generally include a promoter, ribosomal binding site, and transcription termination sequence in eukaryotes; generally, such control sequences include a promoter and a transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "polynucleotide," as used herein, means a sequence of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term encompasses single- and double-stranded forms of DNA.

[0362] The term "oligonucleotide" as referred to herein encompasses naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising 200 or fewer bases in length. In some embodiments, oligonucleotides are 10-60 bases in length, and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length. Oligonucleotides are typically single-stranded, e.g., for probes, whereas oligonucleotides can be double-stranded for use in constructing gene mutants. Oligonucleotides of the present disclosure may be sense or antisense oligonucleotides.

[0363] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugar groups and the like. The term "oligonucleotide linkage" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilate, phosphoraniladate, phosphoronmidate, and the like. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anticancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). Oligonucleotides can contain a label for detection, if desired.

[0364] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as α-, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, -N,N,N,N-trimethyllysine, -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0365] 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 direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction, and sequences on the DNA strand that have the same sequence as the RNA and that are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences," and sequences on the DNA strand that have the same sequence as the RNA and that are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences."

[0366] The term "substantial identity," as applied to polypeptides, means that two peptide sequences, when properly aligned according to the programs GAP or GESTFIT 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.

[0367] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0368] As discussed herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are intended to be encompassed by the present disclosure if the variation in the amino acid sequence is at least 75%, in some embodiments at least 80%, 90%, 95%, and in some embodiments, 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those substitutions that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic-hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, it is reasonable to expect that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of one amino acid with a structurally related amino acid will have no major effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within the backbone site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by one of skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of nucleotide and / or amino acid sequence data with public or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are well known (Bowie et al. Science 253:164 (1991)). Thus, the foregoing examples demonstrate that one of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present disclosure.

[0369] Suitable amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence that forms intermolecular contacts (e.g., portions of the polypeptide outside the domain). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt helices occurring in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0370] The term "polypeptide fragment," as used herein, refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion and / or one or more internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in a naturally occurring sequence deduced from, for example, a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, in some embodiments at least 14 amino acids in length, in some embodiments at least 20 amino acids in length, usually at least 50 amino acids in length, and in some embodiments at least 70 amino acids in length. The term "analog," as used herein, refers to a polypeptide comprised of a segment of at least 25 amino acids that has substantial identity to a portion of the deduced amino acid sequence and has specific binding to a target under appropriate binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) with respect to the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, in some embodiments at least 50 or more amino acids in length, and can often be as long as a full-length naturally occurring polypeptide.

[0371] The term "agent" is used herein to denote a chemical substance, a mixture of chemical substances and biological macromolecules, or an extract made from biological material.

[0372] As used herein, the terms "label" or "labeled" refer to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment to a polypeptide of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). Under certain circumstances, the label or marker can also be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are well known and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., H, C, N, S, Y, Tc, In, I, I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, zeotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug," as used herein, refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0373] Other chemical terms herein are used in accordance with conventional usage in the nuclear art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0374] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, is more abundant than any other individual species in the composition), and in some embodiments, a substantially pure fraction is a composition in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present.

[0375] Generally, a substantially pure composition will contain about 80% or more of all macromolecular species present in the composition, and in some embodiments, about 85%, 90%, 95%, and 99% or more. In some embodiments, the target species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0376] The term patient includes human and animal species.

[0377] The activatable antibodies of the present disclosure bind to a predetermined target, e.g., a human target protein. Activatable antibodies that bind to the same epitope as the activatable antibodies described herein are also included in the present disclosure.

[0378] Those skilled in the art will recognize that it is possible, without undue experimentation, to determine whether a monoclonal antibody (e.g., a murine or humanized monoclonal antibody) has the same specificity as the monoclonal antibody used in the methods described herein by determining whether the former prevents the latter from binding to the target. If the monoclonal antibody being tested competes with the monoclonal antibody of the present disclosure, as indicated by a decrease in binding by the monoclonal antibody of the present disclosure, the two monoclonal antibodies bind to the same or closely related epitopes. Another method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody of the present disclosure is to preincubate the monoclonal antibody of the present disclosure with the target and then add the monoclonal antibody being tested to determine whether it inhibits its ability to bind the target. If the monoclonal antibody being tested is inhibited, then it most likely has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present disclosure. Multispecific activatable antibodies

[0379] The present disclosure also provides multispecific activatable antibodies. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize two or more distinct antigens or epitopes and include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody, where coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. In some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for at least one protease selected from uPA and matriptase. The multispecific activatable antibodies provided herein are stable in the circulation, are activated at intended sites of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit target binding at least comparable to that of the corresponding, unmodified multispecific antibody.

[0380] In some embodiments, the multispecific activatable antibody is designed to recruit immune effector cells, also referred to herein as immune effector cell-recruiting multispecific activatable antibodies. In some embodiments, the multispecific activatable antibody is designed to recruit leukocytes, also referred to herein as leukemia-recruiting multispecific activatable antibodies. In some embodiments, the multispecific activatable antibody is designed to recruit T cells, also referred to herein as T cell-recruiting multispecific activatable antibodies. In some embodiments, the multispecific activatable antibody recruits a surface antigen on a leukocyte, e.g., a T cell, a natural killer (NK) cell, a bone marrow mononuclear cell, a macrophage, and / or another immune effector cell. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell is a mononuclear cell, e.g., a bone marrow mononuclear cell. In some embodiments, a multispecific activatable antibody is designed to bind or otherwise react with multiple targets and / or multiple epitopes, also referred to herein as a multi-antigen-targeting antibody and / or a multi-antigen-targeting, activatable antibody. As used herein, the terms "target" and "antigen" are used interchangeably.

[0381] In some embodiments, the immune effector cell-recruiting multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and an immune effector cell-recruiting antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the immune effector cell-recruiting antibody or antigen-binding portion thereof is masked. In some embodiments, the immune effector cell-recruiting antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell-recruiting target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the immune effector cell-recruiting antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell-recruiting target, where AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, where AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the non-immune effector cell-recruiting antibody is a cancer-targeting antibody. In some embodiments, the immune effector cell-recruiting antibody is an scFv. In some embodiments, the targeting antibody (e.g., non-immune cell effector antibody) is an IgG, and the immune effector cell-recruiting antibody is an scFv. In some embodiments, the immune effector cells are leukocytes. In some embodiments, the immune effector cells are T cells. In some embodiments, the immune effector cells are NK cells.In some embodiments, the immune effector cells are bone marrow mononuclear cells.

[0382] In some embodiments, the T cell-engaging multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and a T cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the T cell-engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the T cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the T cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment (AB2) that binds a second target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0383] In some embodiments, the T cell-engaging multispecific activatable antibody comprises a cancer-targeting antibody or antigen-binding fragment thereof and a T cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the cancer-targeting antibody or antigen-binding fragment thereof and / or the T cell-engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the T cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind a second cancer-associated target. In some embodiments, the T cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell-engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0384] In some embodiments, the T cell-engaging multispecific activatable antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof and a T cell-engaging scFv, wherein at least one of the cancer-targeting IgG antibody or antigen-binding fragment thereof and / or the T cell-engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the T cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind a second cancer-associated target. In some embodiments, the T cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell-engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0385] In some embodiments of immune effector-recruiting multispecific activatable antibodies, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, for example, but not limited to, any of the targets listed in Table 1, such as, but not limited to, EGGR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and another antigen is typically a stimulatory or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of T cells or NK cells; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments, the antigen is an inhibitory receptor present on the surface of T cells; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIR. An antibody that confers specificity for the T cell surface antigen may be substituted with a ligand or ligand domain that binds to an effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9.

[0386] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the cancer microenvironment and includes an antibody, e.g., an IgG or scFv, directed against a tumor target and an agonist antibody, e.g., an IgG or scFv directed against a costimulatory receptor expressed on the surface of activated T cells or NK cells, wherein at least one of the cancer-targeting antibody and / or agonist antibody is masked. Examples of costimulatory receptors include, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, and OX40. In this embodiment, the multispecific activatable antibody, upon activation by tumor-associated proteases, will effectively crosslink and activate T cell- or NK cell-expressed costimulatory receptors in a tumor-dependent manner to enhance the activity of T cells responding to any tumor antigen via their endogenous T cell antigen or NK-activating receptor. The activation-dependent nature of these T cell or NK cell costimulatory receptors focuses the activity of the activated multispecific activatable antibody on tumor-specific T cells without activating all T cells, regardless of their antigen specificity. In one embodiment, at least the costimulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of autoreactive T cells that may be present in tissues that also express the antigen recognized by the tumor-targeting antibody in the multispecific activatable antibody, but whose activity is limited by the lack of co-receptor recruitment.

[0387] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the microenvironment of diseases characterized by T cell hyperstimulation, such as, but not limited to, autoimmune or inflammatory diseases. Such multispecific activatable antibodies include antibodies, e.g., IgG or scFv, directed against targets including surface antigens expressed in tissues targeted by T cells in autoimmune inflammatory diseases, and antibodies, e.g., IgG or scFv, directed against inhibitory receptors expressed on the surface of T cells or NK cells, wherein at least one of the disease tissue-targeting antibody and / or T cell receptor antibody is masked. Examples of inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIR. Examples of tissue antigens targeted by T cells in autoimmune diseases include, but are not limited to, surface antigens expressed on myelin or nerve cells in multiple sclerosis, or surface antigens expressed on pancreatic islet cells in type 1 diabetes. In this embodiment, the multispecific activatable antibodies, when localized to tissues under autoimmune attack or inflammation, become activated and co-recruit T cell or NK cell inhibitory receptors via their endogenous TCRs or activating receptors to suppress the activity of autoreactive T cells responding to any diseased tissue-targeted antigens. In one embodiment, at least one or more antibodies are masked to prevent suppression of T cell responses in non-diseased tissues where the target antigen may also be expressed.

[0388] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an anti-CD3 epsilon (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the targeting antibody or antigen-binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target.

[0389] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0390] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0391] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an OKT3-derived anti-CD3 epsilon (CD3ε) scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the OKT3 scFv or OKT3-derived scFv is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second target.

[0392] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0393] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0394] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the anti-CTLA-4 scFv is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, comprising a second antibody or antigen-binding fragment thereof (AB2), wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target.

[0395] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv and a targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CTLA-4 scFv and / or the targeting IgG antibody or antigen-binding portion thereof is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4. In some embodiments, the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, comprising a second antibody or antigen-binding fragment thereof (AB2), wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target.

[0396] In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody comprises at least a first antibody or antigen-binding fragment thereof that binds a first target and / or a first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and / or a second epitope. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different targets. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different epitopes on the same target. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds a combination of multiple different targets and multiple different epitopes on the same target.

[0397] In some embodiments, a multispecific activatable antibody comprising an IgG has a masked IgG variable domain. In some embodiments, a multispecific activatable antibody comprising an scFv has a masked scFv domain. In some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein each of the IgG variable domain and the scFv domain is coupled to its own masking moiety. In some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T cell surface antigen. In some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for another target antigen. In some embodiments, one antibody domain of the multispecific activatable antibody has specificity for an epitope of the target antigen and another antibody domain has specificity for another epitope of the target antigen.

[0398] In multispecific activatable antibodies, scFvs can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, the carboxyl terminus of the light chain of an IgG activatable antibody, or the carboxyl terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, the amino terminus of the light chain of an IgG activatable antibody, or the amino terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to the antigen-binding domain of an IgG and masks that domain. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to the antigen-binding domain of at least one scFv and masks that domain. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of an IgG, and a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of at least one scFv.

[0399] The present disclosure provides examples of multispecific activatable antibody structures, including, but not limited to, the following: (VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM-L1-MM)2; (VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1-MM)2; (MM-L1-CM-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*)2; (MM-L1-CM-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*)2; (VL-CL)2:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL)2:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL-CL)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL-CL)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2:(VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL*-L3-VH*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VH*-L3-VL*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; or (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2, where VL and VH represent the light and heavy variable domains of a first specificity contained in an IgG; VL* and VH* represent the variable domains of a second specificity contained in an scFv; L1 is a linker peptide connecting the masking moiety (MM) and the cleavable moiety (CM); L2 is a linker peptide connecting the cleavable moiety (CM) and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the first specificity antibody to the second specificity antibody; CL is a light chain constant domain; and CH1, CH2, and CH3 are heavy chain constant domains. The first and second specificities can be directed against any antigen or epitope.

[0400] In some embodiments of the T cell-engaging multispecific activatable antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, such as, but not limited to, any of the targets listed in Table 1, such as EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or The antigen is typically a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain that provides specificity for a T cell surface antigen may also be replaced with a ligand or ligand domain that binds to a T cell receptor, an NK-cell receptor, a macrophage receptor, and / or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9. In some embodiments of the multi-antigen targeted activatable antigen, one antigen is selected from the target group listed in Table 1, and another antigen is selected from the target group listed in Table 1.

[0401] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v4, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v6, which is specific for binding to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which is specific for binding to human and mouse Jagged1 and Jagged2. In some embodiments, the targeting antibody is 4D11v2, which is specific for binding to human and mouse Jagged1 and Jagged2.

[0402] In some embodiments, the targeting antibody can exist in the form of an activatable antibody. In some embodiments, the scFv can exist in the form of a pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0403] In some embodiments, the scFv is specific for binding of CD3ε and is an antibody or fragment thereof that binds CD3ε, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, the scFv is specific for binding of CTLA-4 (also referred to herein as CTLA and CTLA4).

[0404] In some embodiments, the anti-CTLA-4 scFv comprises the following amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVTVSSAS (SEQ ID NO: 692)

[0405] 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:692.

[0406] In some embodiments, the anti-CD3ε scFv comprises the following amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 693)

[0407] 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:693.

[0408] In some embodiments, the scFv is specific for binding one or more T cells, one or more NK-cells, and / or one or more macrophages. In some embodiments, the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.

[0409] In some embodiments, the multispecific activatable antibody also includes an agent conjugated to AB. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the multispecific activatable antibody via a linker. In some embodiments, the agent is conjugated to AB via a cleavable linker. In some embodiments, the agent is conjugated to AB via a linker comprising at least one uPA-cleavable substrate sequence or at least one matriptase-cleavable substrate sequence. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent, or a DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is trastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0410] In some embodiments, the multispecific activatable antibody can also comprise a detectable moiety, hi some embodiments, the detectable moiety is a diagnostic agent.

[0411] In some embodiments, the multispecific activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the multispecific activatable antibody can be engineered to contain one or more disulfide bonds.

[0412] The present disclosure also provides isolated nucleic acid molecules encoding the multispecific activatable antibodies described herein, and vectors comprising those isolated nucleic acid sequences. The present disclosure provides methods for producing multispecific activatable antibodies by culturing cells comprising such nucleic acid molecules under conditions conducive to expression of the activatable antibodies. In some embodiments, the cells comprise such vectors.

[0413] The present disclosure also provides a method of producing a multispecific activatable antibody of the present disclosure by (a) culturing cells containing a nucleic acid construct encoding the multispecific activatable antibody under conditions conducive to expression of the multispecific activatable antibody, and (b) recovering the multispecific activatable antibody.

[0414] The present disclosure also provides multispecific activatable antibody compositions comprising at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that binds a second target or a second epitope, wherein at least AB1 is coupled to or otherwise bound to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind its target. In some embodiments, MM1 is coupled to AB1 via a first cleavable moiety (CM1) sequence that comprises a substrate for a protease, e.g., a protease that co-localizes with the target of AB1 at a therapeutic or diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, are activated at the intended site of therapy and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding of AB1 to its target that is at least comparable to that of its corresponding unmodified multispecific antibody.

[0415] In some embodiments, the multispecific activatable antibody comprises a linking peptide between MM1 and CM1.

[0416] In some embodiments, the multispecific activatable antibody comprises a linking peptide between CM1 and AB1.

[0417] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and at least a portion of the multispecific activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. In some embodiments, the two connecting peptides need not be identical to each other.

[0418] 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:385), and (GGGS)n (SEQ ID NO:386), where n is at least one integer. In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:387), GGSGG (SEQ ID NO:388), GSGSG (SEQ ID NO:389), GSGGG (SEQ ID NO:390), GGGSG (SEQ ID NO:391), and GSSSG (SEQ ID NO:392).

[0419] In some embodiments, the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds a second target or a second epitope. In some embodiments, each AB in the multispecific activatable antibody is independently selected from the group consisting of monoclonal antibodies, domains, antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single-domain heavy chain antibodies, and single-domain light chain antibodies. In some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

[0420] In some embodiments, each AB in the multispecific activatable antibody has an equilibrium dissociation constant of about 100 nM or less for binding to its corresponding target or epitope.

[0421] In some embodiments, MM1 has an equilibrium dissociation constant for binding to its corresponding AB that is higher than the equilibrium dissociation constant for binding of AB to its corresponding target or epitope.

[0422] In some embodiments, MM1 has an equilibrium dissociation constant for binding to its corresponding AB that is no more than the equilibrium dissociation constant for binding of AB to its corresponding target or epitope.

[0423] In some embodiments, MM1 does not block or compete with the corresponding AB for binding to its corresponding target or epitope when the multispecific activatable antibody is present in a cleaved state.

[0424] In some embodiments, MM1 is a polypeptide about 2 to 40 amino acids in length. In some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 or less amino acids in length.

[0425] In some embodiments, MM1 has a polypeptide sequence that differs from the sequence of the target of its corresponding AB.

[0426] In some embodiments, MM1 has a polypeptide sequence that is 50% or less identical to any natural binding partner of its corresponding AB. In some embodiments, MM1 has a polypeptide sequence that is 25% or less identical to any natural binding partner of its corresponding AB. In some embodiments, MM1 has a polypeptide sequence that is 10% or less identical to any natural binding partner of its corresponding AB.

[0427] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 20 times higher than

[0428] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 40 times higher than

[0429] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitoped At least 100 times higher than

[0430] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 1000 times higher than

[0431] In some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 10,000 times higher than

[0432] In some embodiments, MM1 is an amino acid sequence selected from the MMs set forth in the Examples presented herein.

[0433] In some embodiments, the multispecific activatable antibody comprises at least a second masking moiety (MM2), which inhibits binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state, and a second cleavable moiety (CM2), coupled to AB2, which functions as a substrate for a second protease. In some embodiments, CM2 is a polypeptide 15 amino acids or less in length. In some embodiments, the second protease co-localizes with a second target or epitope in a tissue, and the second protease cleaves CM2 on the multispecific activatable antibody when the multispecific activatable antibody is exposed to the second protease. In some embodiments, the first protease and the second protease co-localize with the first target or epitope and the second target or epitope in a tissue. In some embodiments, the first protease and the second protease are the same protease. In some embodiments, CM1 and CM2 are different substrates for the same protease. In some embodiments, the protease is selected from the group consisting of those shown in Table 7. In some embodiments, the first protease and the second protease are different proteases. In some embodiments, the first protease and the second protease are different proteases selected from the group consisting of those shown in Table 7.

[0434] In some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has an equilibrium dissociation constant for binding to its corresponding AB that is higher than the equilibrium dissociation constant for binding to its corresponding target or epitope of AB.

[0435] In some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has an equilibrium dissociation constant for binding to its corresponding AB that is less than or equal to the equilibrium dissociation constant for binding to its corresponding target or epitope of AB.

[0436] In some embodiments, each MM in a multispecific activatable antibody does not interfere with or compete with its corresponding AB for binding to the corresponding target or epitope when the multispecific activatable antibody is in a cleaved state.

[0437] In some embodiments, each MM in the multispecific activatable antibody is a polypeptide about 2-40 amino acids in length, hi some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 amino acids or less in length.

[0438] In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that differs from the sequence of the target of the corresponding AB.

[0439] In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 50% identical to any natural binding partner of its corresponding AB. In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 25% identical to any natural binding partner of its corresponding AB. In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 10% identical to any natural binding partner of its corresponding AB.

[0440] In some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 20 times higher than

[0441] In some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 40 times higher than

[0442] In some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 100 times higher than

[0443] In some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 1000 times higher than

[0444] In some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 10,000 times higher than

[0445] In some embodiments, each MM is an amino acid sequence selected from the MMs set forth in the Examples disclosed herein.

[0446] In some embodiments, at least one of CM1 and / or CM2 is cleaved by at least one protease selected from uPA and matriptase. In some embodiments, at least one of CM1 and / or CM2 comprises an amino acid sequence selected from the group consisting of a core CM consensus sequence shown in Tables 8A-8J, a subgenus of the core CM consensus sequence shown in Tables 8A-8J, or an extended consensus sequence based on one of the core CM consensus sequences shown in Tables 8A-8J. In some embodiments, the extended consensus sequence is a consensus sequence shown in Tables 9A-9J-3, a core CM consensus sequence shown in Tables 10A-10D, a subgenus of the core CM consensus sequence shown in Tables 10A-10D, or a consensus sequence shown in Tables 11A-11D.

[0447] In some embodiments, at least one of CM1 and / or CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 163-267.

[0448] In some embodiments, a protease that cleaves the first cleavable portion (CM1) sequence co-localizes with the target of AB1 in the multispecific activatable antibody in a tissue, and the protease cleaves CM1 in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0449] In some embodiments, the multispecific activatable antibody comprises multiple cleavable subsequences, and a protease that cleaves at least one cleavable subsequence is co-localized in a tissue with a target of at least one region of the AB region in the multispecific activatable antibody, and the protease cleaves the CM in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0450] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least two-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0451] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least three times higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0452] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least four times higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0453] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 5-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0454] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 10-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0455] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 20-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0456] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 40-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0457] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 50-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0458] In some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilib...

Claims

1. 1. An isolated polypeptide comprising a cleavable moiety (CM) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307, 308 and 311, the cleavable moiety is a substrate for at least one protease selected from matriptase protease and u-plasminogen activator (uPA) protease; The isolated polypeptide comprises an antibody or antigen-binding fragment thereof (AB) that binds to a target, and a masking moiety (MM) that inhibits the ability of the AB to bind to the target, wherein the amino acid sequence of the MM differs from the amino acid sequence of the target, and the CM is between the AB and the MM.

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

3. The isolated polypeptide of claim 1 or 2, wherein the AB is directly linked to the CM.

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

5. The MM has the following characteristics: the MM has an equilibrium dissociation constant for binding to the AB that is higher than the equilibrium dissociation constant of the AB for binding to the target; the MM has an equilibrium dissociation constant for binding to the AB that is less than or equal to the equilibrium dissociation constant of the AB for binding to the target; and the MM has an equilibrium dissociation constant for binding to the AB that is less than or equal to the equilibrium dissociation constant of the AB for binding to the target, and the AB has an equilibrium dissociation constant for binding to the target of about 100 nM or less; The isolated polypeptide of any one of claims 1 to 4, having one of the following:

6. The isolated polypeptide of any one of claims 1 to 5, wherein the MM is a polypeptide less than 40 amino acids in length.

7. The isolated polypeptide of any one of claims 1 to 6, wherein the MM is linked to the CM, such that the isolated polypeptide comprises the following structural configuration from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM.

8. The isolated polypeptide of any one of claims 1 to 7, wherein the isolated polypeptide comprises a connecting peptide between the MM and the CM, and / or the isolated polypeptide comprises a connecting peptide between the CM and the AB.

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

10. The isolated polypeptide of claim 9, wherein LP1 and LP2 are not identical to each other.

11. 11. The isolated polypeptide of claim 9 or 10, wherein each of LP1 and LP2 is a peptide of 2 to 20 amino acids in length.

12. 12. The isolated polypeptide of any one of claims 1 to 11, wherein the MM does not interfere with or compete with the AB for binding of the CM to the target.

13. 13. The isolated polypeptide of any one of claims 1 to 12, further comprising a T cell-engaging scFv linked to said AB.

14. The isolated polypeptide of claim 13 , wherein the T cell-engaging scFv comprises a masking moiety.

15. 15. The isolated polypeptide of any one of claims 1 to 14, conjugated to an agent.

16. 16. The isolated polypeptide of claim 15, wherein the agent is a toxin.

17. 17. The isolated polypeptide of claim 15 or 16, wherein the agent is selected from the group consisting of dolastatin, auristatin, maytansinoid, duocarmycin, calicheamicin, auristatin E, monomethylauristatin E (MMAE), monomethylauristatin D (MMAD), DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), and DM4 (N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine).

18. The isolated polypeptide of claim 15 , wherein the agent is a detectable moiety.

19. 20. The isolated polypeptide of claim 18, wherein the detectable moiety is a diagnostic agent.

20. 20. The isolated polypeptide of any one of claims 15 to 19, wherein the agent is attached to the polypeptide via a linker.

21. 21. The isolated polypeptide of claim 20, wherein the linker is a cleavable linker.

22. An isolated nucleic acid molecule encoding the isolated polypeptide of any one of claims 1 to 14.

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

24. 25. A method for producing an isolated polypeptide according to any one of claims 1 to 14, comprising culturing a cell under conditions conducive to expression of said polypeptide, said cell comprising the isolated nucleic acid molecule of claim 22 or the vector of claim 23.

Images