US 20160289324A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0289324 A1 M00re et al. (43) Pub. Date: Oct. 6, 2016

(54) MATRIX METALLOPROTEASE-CLEAVABLE Publication Classification AND SERINE -CLEAVABLE SUBSTRATES AND METHODS OF USE (51) Int. Cl. C07K 6/28 (2006.01) THEREOF C07K 7/08 (2006.01) A61R 49/00 (2006.01) (71) Applicant: Cytomix Therapeutics, Inc., South San C07K 6/40 (2006.01) Francisco, CA (US) A6II 47/48 (2006.01) C07K I4/00 (2006.01) (72) Inventors: Stephen James Moore, Danville, CA A638/05 (2006.01) (US); Margaret Thy Lulu Nguyen, San (52) U.S. Cl. Francisco, CA (US); Daniel Robert CPC ...... C07K 16/28 (2013.01); C07K 14/00 (2013.01); C07K 7/08 (2013.01); A61K 38/05 Hostetter, Palo Alto, CA (US): Olga (2013.01); C07K 16/2863 (2013.01); C07K Vasiljeva, Cupertino, CA (US); Jason I6/40 (2013.01); A61K 47/48415 (2013.01); Gary Sagert, San Mateo, CA (US); A61K 49/0058 (2013.01); A61K 47/48584 Jonathan Alexander Terrett, (2013.01); C07K 2319/74 (2013.01); C07K Cupertino, CA (US); James William 2319/50 (2013.01); C07K 2317/51 (2013.01); West, Bend, OR (US) C07K 2317/515 (2013.01); C07K 2317/92 (2013.01); A6 IK 2039/505 (2013.01) (57) ABSTRACT (21) Appl. No.: 15/002,131 The invention relates generally to polypeptides that include at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least (22) Filed: Jan. 20, 2016 one serine protease (SP), to activatable antibodies and other larger molecules that include these polypeptides that include at least a CM1 that is a substrate for at least one MMP Related U.S. Application Data protease and at least a CM2 that is a substrate for at least one SP protease, and to methods of making and using these (60) Provisional application No. 62/105,490, filed on Jan. polypeptides that include at least a CM1 that is a substrate 20, 2015, provisional application No. 62/258,015, for at least one MMP protease and at least a CM2 that is a filed on Nov. 20, 2015, provisional application No. substrate for at least one SP protease in a variety of thera 62/278,713, filed on Jan. 14, 2016. peutic, diagnostic and prophylactic indications. Patent Application Publication Oct. 6, 2016 Sheet 1 of 12 US 2016/0289.324 A1

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MATRIX METALLOPROTEASE-CLEAVABLE follows: CM1-CM2 or CM2-CM1. The term “CM1-CM2 AND SERINE PROTEASE-CLEAVABLE Substrates' also encompasses Substrates where at least a SUBSTRATES AND METHODS OF USE portion of the CM1 sequence overlaps with at least a portion THEREOF of the CM2 sequence. 0007. The CM1-CM2 substrates described herein are RELATED APPLICATIONS useful in a variety of therapeutic, diagnostic and prophylac 0001. This application claims the benefit of U.S. Provi tic indications. For example, these CM1-CM2 substrates are sional Application No. 62/105,490, filed Jan. 20, 2015, U.S. useful in activatable antibodies that include antibodies or Provisional Application No. 62/258,015, filed Nov. 20, antigen-binding fragments thereof (AB) that include at least 2015, and U.S. Provisional Application No. 62/278,713, one masking moiety (MM) linked to at least one antigen- or filed Jan. 14, 2016, the contents of each of which are epitope-binding domain of the AB Such that coupling of the incorporated herein by reference in their entirety. MM reduces the ability of the AB to bind its target. 0008. In some embodiments, the activatable antibody includes at least a first CM (CM1) and a second CM (CM2). INCORPORATION OF SEQUENCE LISTING In some embodiments, at least a portion of the CM1 sub 0002. The contents of the text file named strate sequence overlaps with at least a portion of the CM2 “CYTMO37001 US ST25, which was created on Jan. 15, sequence. In some embodiments, the CM1 substrate 2016 and is 456 KB in size, are hereby incorporated by sequence and the CM2 Substrate sequence share at least one reference in their entirety. amino acid residue in common. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence FIELD OF THE INVENTION share at least two amino acid residues in common. In some 0003. The invention relates generally to polypeptides that embodiments, the CM1 substrate sequence and the CM2 include at least a first cleavable moiety (CM1) that is a Substrate sequence share at least three amino acid residues in substrate for at least one matrix metalloprotease (MMP) and common. In some embodiments, the CM1 substrate at least a second cleavable moiety (CM2) that is a substrate sequence and the CM2 Substrate sequence share three or for at least one serine protease (SP), to activatable antibodies more amino acid residues in common. and other larger molecules that include these polypeptides 0009. In some embodiments, CM1 and CM2 are separate that include at least a CM1 that is a substrate for at least one polypeptides that are operably linked together. MMP protease and a CM2 that is a substrate for at least one 0010. In some embodiments, CM1 and CM2 are separate SP protease, and to methods of making and using these polypeptides that are directly linked together, i.e., the N-ter polypeptides that include at least a CM1 that is a substrate minus of one substrate is linked directly to the C-terminus of for at least one MMP protease and a CM2 that is a substrate the other substrate polypeptide. In some embodiments, the for at least one SP protease in a variety of therapeutic, N-terminus of the CM1 is linked directly to the C-terminus diagnostic and prophylactic indications. of the CM2. In some embodiments, the N-terminus of the CM2 is linked directly to the C-terminus of the CM1. BACKGROUND OF THE INVENTION 0011. In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together via at least 0004 are that degrade proteins by one linking moiety. cleaving the peptide bonds between amino acid residues. 0012. In some embodiments, the first cleavable moiety Proteases occur naturally in all organisms and are involved CM1 and the second cleavable moiety CM2 in the activat in a variety of physiological reactions from simple degra able antibody in the uncleaved state have the structural dation to highly regulated pathways. Some proteases are arrangement from N-terminus to C-terminus as follows: known to break specific peptide bonds based on the presence MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2 of a particular amino acid sequence within a protein. CM1-AB, or AB-CM1-CM2-MM. 0005 Accordingly, there exists a need to identify new 0013. In some embodiments, the activatable antibody Substrates for proteases and to use these Substrates in a includes a linking peptide (LP) between CM1 and CM2. In variety of therapeutic, diagnostic and prophylactic indica Some embodiments, the activatable antibody includes a tions. linking peptide (LP") between the masking moiety (MM) and CM1. In some embodiments, the activatable antibody SUMMARY OF THE INVENTION includes a linking peptide (LP") between CM2 and AB. In 0006. The disclosure provides amino acid sequences that Some embodiments, the activatable antibody includes a include at least a first cleavable moiety (CM1) that is a linking peptide (LP") between the MM and CM1 and a substrate for at least one matrix metalloprotease (MMP) and linking peptide (LP") between CM2 and AB. In some at least a second cleavable moiety (CM2) that is a substrate embodiments, the activatable antibody includes a linking for at least one serine protease (SP). These amino acid peptide between the MM and CM1 (LP") and a linking sequences are collectively referred to herein as “CM1-CM2 peptide between CM1 and CM2 (LP). In some embodi Substrates.” This term is not intended to convey any require ments, the activatable antibody includes a linking peptide ment regarding the orientation or other structural arrange (LP") between CM1 and CM2 and a linking peptide (LP") ment of the first cleavable moiety (CM1) that is a substrate between CM2 and AB. In some embodiments, the activat for at least one matrix metalloprotease (MMP) and at least able antibody includes a linking peptide (LP") between the a second cleavable moiety (CM2) that is a substrate for at MM and CM1, a linking peptide (LP) between CM1 and least one serine protease (SP). Thus, the term “CM1-CM2 CM2, and a linking peptide (LP") between CM2 and AB. substrates’ encompasses CM1-CM2 substrates having the 0014. In some embodiments, the activatable antibody structural arrangement from N-terminus to C-terminus as includes a linking peptide (LP) between CM1 and CM2. In US 2016/0289.324 A1 Oct. 6, 2016

Some embodiments, the activatable antibody includes a CM1 comprises the amino acid sequence AQNLLGMV linking peptide (LP") between the AB and CM1. In some (SEQ ID NO: 351). In some embodiments, the CM1 com embodiments, the activatable antibody includes a linking prises the amino acid sequence STFPFGMF (SEQ ID NO: peptide (LP") between CM2 and the masking moiety (MM). 352). In some embodiments, the CM1 comprises the amino In some embodiments, the activatable antibody includes a acid sequence PVGYTSSL (SEQ ID NO: 353). In some linking peptide (LP") between the AB and CM1 and a embodiments, the CM1 comprises the amino acid sequence linking peptide (LP") between CM2 and MM. In some DWLYWPGI (SEQID NO:354). In some embodiments, the embodiments, the activatable antibody includes a linking CM1 comprises the amino acid sequence MIAPVAYR (SEQ peptide between the AB and CM1 (LP") and a linking ID NO: 355). In some embodiments, the CM1 comprises the peptide between CM1 and CM2 (LP"). In some embodi amino acid sequence RPSPMWAY (SEQ ID NO: 356). In ments, the activatable antibody includes a linking peptide Some embodiments, the CM1 comprises the amino acid (LP") between CM1 and CM2 and a linking peptide (LP") sequence WATPRPMR (SEQID NO:357). In some embodi between CM2 and MM. In some embodiments, the activat ments, the CM1 comprises the amino acid sequence FRLL able antibody includes a linking peptide (LP") between the DWQW (SEQ ID NO:358). In some embodiments, the AB and CM1, a linking peptide (LP") between CM1 and CM1 comprises the amino acid sequence LKAAPRWA CM2, and a linking peptide (LP") between CM2 and MM. (SEQ ID NO: 359). In some embodiments, the CM1 com 0.015. In some embodiments, LP' is G.G. In some embodi prises the amino acid sequence GPSHLVLT (SEQ ID NO: ments, LP is GGSGGS (SEQ ID NO:350). 360). In some embodiments, the CM1 comprises the amino 0016. In some embodiments, CM1 is a substrate for at acid sequence LPGGLSPW (SEQ ID NO: 361). In some least one matrix metalloprotease (MMP). Examples of embodiments, the CM1 comprises the amino acid sequence MMPs include MMP1; MMP2: MMP3; MMP7; MMP8; MGLFSEAG (SEQID NO:362). In some embodiments, the MMP9; MMP10; MMP11; MMP12: MMP13; MMP14: CM1 comprises the amino acid sequence SPLPLRVP (SEQ MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; ID NO: 363). In some embodiments, the CM1 comprises the MMP24; MMP26; and MMP27. amino acid sequence RMHLRSLG (SEQ ID NO: 364). In 0017. In some embodiments, CM1 is a substrate for Some embodiments, the CM1 comprises the amino acid MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, sequence LAAPLGLL (SEQID NO:365). In some embodi MMP17, MMP11, and/or MMP 19. In some embodiments. ments, the CM1 comprises the amino acid sequence AVGL CM1 is a substrate for MMP2. In some embodiments, CM1 LAPP (SEQ ID NO:366). In some embodiments, the CM1 is a substrate for MMP9. In some embodiments, CM1 is a comprises the amino acid sequence LLAPSHRA (SEQ ID substrate for MMP14. In some embodiments, CM1 is a NO: 367). In some embodiments, the CM1 comprises the substrate for two or more MMPs. In some embodiments, amino acid sequence PAGLWLDP (SEQ ID NO: 368). In CM1 is a Substrate for at least MMP9 and MMP14. In some Some embodiments, the CM1 comprises the amino acid embodiments, CM1 is a substrate for at least MMP2 and sequence ISSGLSS (SEQ ID NO:369). In some embodi MMP9. In some embodiments, CM1 is a substrate for at ments, CM1 comprises the amino acid sequence VHMPL least MMP2 and MMP14. In some embodiments, CM1 is a GFLGP (SEQ ID NO: 411). In some embodiments, CM1 substrate for three or more MMPs. In some embodiments, comprises the amino acid sequence ISSGL (SEQ ID NO: CM1 is a substrate for at least MMP2, MMP9, and MMP14. 480). In some embodiments, CM1 comprises the amino acid In some embodiments, the CM1 comprises two or more sequence ISSGLLS (SEQ ID NO: 481). In some embodi substrates for the same MMP. In some embodiments, the ments, CM1 comprises the amino acid sequence ISSGLL CM1 comprises at least two or more MMP2 substrates. In (SEQ ID NO: 482). some embodiments, the CM1 comprises at least two or more 0020. In some embodiments, CM2 is a substrate for at MMP9 substrates. In some embodiments, the CM1 com least one serine protease (SP). In some embodiments, the SP prises at least two or more MMP14 substrates. is selected from u-type plasminogen activator (uPA, also 0.018. In some embodiments, CM1 is a substrate for an referred to as urokinase), matriptase (also referred to herein MMP and includes at least the sequence ISSGLLSS (SEQ as MT-SP1 or MTSP1), and combinations thereof. Examples ID NO: 20); QNQALRMA (SEQID NO: 21); AQNLLGMV of other SP that cleave a CM2 described herein include, by (SEQ ID NO: 351); STFPFGMF (SEQ ID NO: 352); way of non-limiting example, activated protein C. Cathepsin PVGYTSSL (SEQ ID NO: 353); DWLYWPGI (SEQ ID A. Cathepsin G. Chymase; a coagulation factor protease NO: 354); MIAPVAYR (SEQ ID NO: 355); RPSPMWAY such as, e.g., FVIIa, FIXa, FXa. FXIa, FXIIa; Elastase: (SEQ ID NO: 356); WATPRPMR (SEQ ID NO: 357); Granzyme B; Guanidinobenzoatase: Htra1; Human Neutro FRLLDWQW (SEQ ID NO:358); LKAAPRWA (SEQ ID phil Elastase; Lactoferrin; Marapsin; NS3/4A, PACE4: Plas NO:359); GPSHLVLT (SEQ ID NO: 360); LPGGLSPW min; PSA; thA; Thrombin; Tryptase; a Type II Transmem (SEQ ID NO: 361); MGLFSEAG (SEQ ID NO: 362); brane Serine Protease (TTSP) such as, e.g., DESC1, DPP-4, SPLPLRVP (SEQID NO:363); RMHLRSLG (SEQID NO: FAP. Hepsin, Matriptase-2, TMPRSS2, TMPRSS3, and/or 364); LAAPLGLL (SEQ ID NO: 365); AVGLLAPP (SEQ TMPRSS4. ID NO:366); LLAPSHRA (SEQID NO:367); PAGLWLDP 0021 For example, suitable CM2 are cleaved by at least (SEQ ID NO:368): ISSGLSS (SEQ ID NO:369): ISSGL one serine protease and include the sequence TGRGPSWV (SEQ ID NO:480): ISSGLLS (SEQ ID NO: 481): ISSGLL (SEQ ID NO: 370); SARGPSRW (SEQ ID NO:371); (SEQ ID NO: 482); and/or VHMPLGFLGP (SEQ ID NO: TARGPSFK (SEQ ID NO: 372); LSGRSDNH (SEQ ID 411). NO: 18); GGWHTGRN (SEQ ID NO:373); HTGRSGAL 0019. In some embodiments, the CM1 comprises the (SEQ ID NO: 374); PLTGRSGG (SEQ ID NO: 375); amino acid sequence ISSGLLSS (SEQID NO: 20). In some AARGPAIH (SEQ ID NO:376); RGPAFNPM (SEQ ID embodiments, the CM1 comprises the amino acid sequence NO: 377); SSRGPAYL (SEQ ID NO: 378); RGPATPIM QNQALRMA (SEQID NO: 21). In some embodiments, the (SEQID NO:379); RGPA (SEQID NO:380); LSGRSGNH US 2016/0289.324 A1 Oct. 6, 2016

(SEQ ID NO. 412); TSTSGRSANPRG (SEQ ID NO:413): GFLGP (SEQ ID NO: 10); TSGRSANP (SEQ ID NO. 414): SGRSANPRG (SEQ ID LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 11): NO: 468); VAGRSMRP (SEQ ID NO. 415); LSGRSDDH LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 12): ISS (SEQ ID NO: 547); LSGRSDIH (SEQ ID NO: 548); GLLSSGGSGGSLSGRSGNH (SEQ ID NO: 13); LSGRS LSGRSDQH (SEQ ID NO. 549); LSGRSDTH (SEQ ID DNHGGSGGSQNQALRMA (SEQ ID NO: 14); QNQAL NO: 550); LSGRSDYH (SEQ ID NO. 551); LSGRSDNP RMAGGSGGSLSGRSDNH (SEQ ID NO: 15); (SEQ ID NO. 552); LSGRSANP (SEQ ID NO. 553); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 16); LSGRSANI (SEQ ID NO: 554); and/or LSGRSDNI (SEQ QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 17); ID NO: 71). ISSGLLSGRSGNH (SEQ ID NO: 22): ISSGLLSGRSAN 0022. In some embodiments, CM2 comprises the amino PRG (SEQ ID NO: 469); AVGLLAPPTSGRSANPRG acid sequence TGRGPSWV (SEQ ID NO: 370). In some (SEQ ID NO: 470); AVGLLAPPSGRSANPRG (SEQ ID embodiments, CM2 comprises the amino acid sequence NO: 471): ISSGLLSGRSDDH (SEQ ID NO. 483): ISS SARGPSRW (SEQ ID NO:371). In some embodiments. GLLSGRSDIH (SEQ ID NO: 484); ISSGLLSGRSDQH CM2 comprises the amino acid sequence TARGPSFK (SEQ (SEQ ID NO:485): ISSGLLSGRSDTH (SEQ ID NO:486); ID NO: 372). In some embodiments, CM2 comprises the ISSGLLSGRSDYH (SEQ ID NO. 487): ISSGLLSGRS amino acid sequence LSGRSDNH (SEQ ID NO: 18). In DNP (SEQID NO: 488): ISSGLLSGRSANP (SEQ ID NO: Some embodiments, CM2 comprises the amino acid 489): ISSGLLSGRSANI (SEQ ID NO: 490); AVGLLAP sequence GGWHTGRN (SEQ ID NO: 373). In some PGGLSGRSDDH (SEQ ID NO. 515); AVGLLAPPGGLS embodiments, CM2 comprises the amino acid sequence GRSDIH (SEQ ID NO:516); AVGLLAPPGGLSGRSDQH HTGRSGAL (SEQ ID NO: 374). In some embodiments, (SEQ ID NO: 517); AVGLLAPPGGLSGRSDTH (SEQ ID CM2 comprises the amino acid sequence PLTGRSGG (SEQ NO:518); AVGLLAPPGGLSGRSDYH (SEQID NO:519); ID NO: 375). In some embodiments, CM2 comprises the AVGLLAPPGGLSGRSDNP (SEQ ID NO: 520); AVGL amino acid sequence AARGPAIH (SEQ ID NO: 376). In LAPPGGLSGRSANP (SEQ ID NO. 521); AVGLLAPPG Some embodiments, CM2 comprises the amino acid GLSGRSANI (SEQID NO:522); ISSGLLSGRSDNI (SEQ sequence RGPAFNPM (SEQID NO:377). In some embodi ID NO. 555); and/or AVGLLAPPGGLSGRSDNI (SEQ ID ments, CM2 comprises the amino acid sequence SSRG NO:557). PAYL (SEQ ID NO: 378). In some embodiments, CM2 0024. In some embodiments, the CM1-CM2 substrate comprises the amino acid sequence RGPATPIM (SEQ ID includes the sequence ISSGLLSGRSDNH (SEQID NO: 1). NO: 379). In some embodiments, CM2 comprises the amino In some embodiments, the CM1-CM2 substrate includes the acid sequence RGPA (SEQ ID NO: 380). In some embodi sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: ments, CM2 comprises the amino acid sequence LSGRS 2). In some embodiments, the CM1-CM2 substrate includes GNH (SEQ ID NO: 412). In some embodiments, CM2 the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID comprises the amino acid sequence TSTSGRSANPRG NO: 3). In some embodiments, the CM1-CM2 substrate (SEQ ID NO: 413). In some embodiments, the CM2 com includes the sequence TSTSGRSANPRGGGAVGLLAPP prises the amino acid sequence SGRSANPRG (SEQID NO: (SEQ ID NO: 4). In some embodiments, the CM1-CM2 468). In some embodiments, CM2 comprises the amino acid substrate includes the sequence VHMPLGFLGPGGTSTS sequence TSGRSANP (SEQID NO: 414). In some embodi GRSANPRG (SEQ ID NO. 5). In some embodiments, the ments, CM2 comprises the amino acid sequence CM1-CM2 substrate includes the sequence TSTSGRSAN VAGRSMRP (SEQ ID NO: 415). In some embodiments, PRGGGVHMPLGFLGP (SEQID NO: 6). In some embodi CM2 comprises the amino acid sequence LSGRSDDH ments, the CM1-CM2 substrate includes the sequence (SEQ ID NO: 547). In some embodiments, CM2 comprises AVGLLAPPGGLSGRSDNH (SEQ ID NO: 7). In some the amino acid sequence LSGRSDIH (SEQID NO: 548). In embodiments, the CM1-CM2 substrate includes the Some embodiments, CM2 comprises the amino acid sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 8). In sequence LSGRSDQH (SEQID NO. 549). In some embodi some embodiments, the CM1-CM2 substrate includes the ments, CM2 comprises the amino acid sequence LSGRS sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: DTH (SEQ ID NO: 550). In some embodiments, CM2 9). In some embodiments, the CM1-CM2 substrate includes comprises the amino acid sequence LSGRSDYH (SEQ ID the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 551). In some embodiments, CM2 comprises the amino NO: 10). In some embodiments, the CM1-CM2 substrate acid sequence LSGRSDNP (SEQ ID NO. 552). In some includes the sequence LSGRSDNHGGSGGSISSGLLSS embodiments, CM2 comprises the amino acid sequence (SEQ ID NO: 11). In some embodiments, the CM1-CM2 LSGRSANP (SEQ ID NO: 553). In some embodiments, substrate includes the sequence LSGRSGNHGGSGGSISS CM2 comprises the amino acid sequence LSGRSANI (SEQ GLLSS (SEQ ID NO: 12). In some embodiments, the ID NO: 554). In some embodiments, CM2 comprises the CM1-CM2 substrate includes the sequence ISS amino acid sequence LSGRSDNI (SEQ ID NO: 71). GLLSSGGSGGSLSGRSGNH (SEQ ID NO: 13). In some 0023. In some embodiments, the CM1-CM2 substrate embodiments, the CM1-CM2 substrate includes the includes the sequence ISSGLLSGRSDNH (SEQID NO: 1); sequence LSGRSDNHGGSGGSQNQALRMA (SEQ ID ISSGLLSSGGSGGSLSGRSDNH (SEQID NO: 2); AVGL NO: 14). In some embodiments, the CM1-CM2 substrate LAPPGGTSTSGRSANPRG (SEQ ID NO:3); TSTSGR includes the sequence QNQALRMAGGSGGSLSGRSDNH SANPRGGGAVGLLAPP (SEQ ID NO: 4); VHMPLGFL (SEQ ID NO: 15). In some embodiments, the CM1-CM2 GPGGTSTSGRSANPRG (SEQ ID NO: 5); substrate includes the sequence LSGRSGNHGGSGGSQN TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 6): QALRMA (SEQ ID NO: 16). In some embodiments, the AVGLLAPPGGLSGRSDNH (SEQ ID NO: 7); LSGRSD CM1-CM2 substrate includes the sequence QNQALRMA NHGGAVGLLAPP (SEQ ID NO: 8); VHMPLGFLGPG GGSGGSLSGRSGNH (SEQ ID NO: 17). In some embodi GLSGRSDNH (SEQ ID NO: 9); LSGRSDNHGGVHMPL ments, the CM1-CM2 substrate includes the sequence ISS US 2016/0289.324 A1 Oct. 6, 2016

GLLSGRSGNH (SEQ ID NO: 22). In some embodiments, includes a linking peptide (LP") between CM2 and AB. In the CM1-CM2 substrate includes the sequence ISSGLLS Some embodiments, the activatable antibody includes a GRSANPRG (SEQID NO: 469). In some embodiments, the linking peptide (LP") between the MM and CM1 and a CM1-CM2 substrate includes the sequence AVGLLAPPTS linking peptide (LP") between CM2 and AB. In some GRSANPRG (SEQID NO: 470). In some embodiments, the embodiments, the activatable antibody includes a linking CM1-CM2 substrate includes the sequence AVGLLAPPS peptide between the MM and CM1 (LP") and a linking GRSANPRG (SEQID NO: 471). In some embodiments, the peptide between CM1 and CM2 (LP). In some embodi CM1-CM2 substrate includes the sequence ISSGLLS ments, the activatable antibody includes a linking peptide GRSDDH (SEQ ID NO: 483). In some embodiments, the (LP") between CM1 and CM2 and a linking peptide (LP") CM1-CM2 substrate includes the sequence ISSGLLSGRS between CM2 and AB. In some embodiments, the activat DIH (SEQ ID NO: 484). In some embodiments, the CM1 able antibody includes a linking peptide (LP") between the CM2 substrate includes the sequence ISSGLLSGRSDQH MM and CM1, a linking peptide (LP) between CM1 and (SEQ ID NO: 485). In some embodiments, the CM1-CM2 CM2, and a linking peptide (LP") between CM2 and AB. Substrate includes the sequence. In some embodiments, the 0028. In some embodiments, the activatable antibody CM1-CM2 substrate includes the sequence ISSGLLSGRS includes a linking peptide (LP) between CM1 and CM2. In DTH (SEQ ID NO: 486). In some embodiments, the CM1 Some embodiments, the activatable antibody includes a CM2 substrate includes the sequence ISSGLLSGRSDYH linking peptide (LP") between the AB and CM1. In some (SEQ ID NO: 487). In some embodiments, the CM1-CM2 embodiments, the activatable antibody includes a linking substrate includes the sequence ISSGLLSGRSDNP (SEQ peptide (LP") between CM2 and the masking moiety (MM). ID NO: 488). In some embodiments, the CM1-CM2 sub In some embodiments, the activatable antibody includes a strate includes the sequence ISSGLLSGRSANP (SEQ ID linking peptide (LP") between the AB and CM1 and a NO: 489). In some embodiments, the CM1-CM2 substrate linking peptide (LP") between CM2 and MM. In some includes the sequence ISSGLLSGRSANI (SEQ ID NO: embodiments, the activatable antibody includes a linking 490). In some embodiments, the CM1-CM2 substrate peptide between the AB and CM1 (LP") and a linking includes the sequence AVGLLAPPGGLSGRSDDH (SEQ peptide between CM1 and CM2 (LP). In some embodi ID NO: 515). In some embodiments, the CM1-CM2 sub ments, the activatable antibody includes a linking peptide strate includes the sequence AVGLLAPPGGLSGRSDIH (LP") between CM1 and CM2 and a linking peptide (LP") (SEQ ID NO: 516). In some embodiments, the CM1-CM2 between CM2 and MM. In some embodiments, the activat substrate includes the sequence AVGLLAPPGGLSGRS able antibody includes a linking peptide (LP") between the DQH (SEQ ID NO: 517). In some embodiments, the CM1 AB and CM1, a linking peptide (LP") between CM1 and CM2 substrate includes the sequence AVGLLAPPGGLS CM2, and a linking peptide (LP") between CM2 and MM. GRSDTH (SEQ ID NO: 518). In some embodiments, the 0029. In some embodiments, LP' is G.G. In some embodi CM1-CM2 substrate includes the sequence AVGLLAPPG ments, LP is GGSGGS (SEQ ID NO:350). GLSGRSDYH (SEQ ID NO: 519). In some embodiments, 0030. In some embodiments, at least one of LP1 or LP2 the CM1-CM2 substrate includes the sequence AVGLLAP comprises an amino acid sequence selected from the group PGGLSGRSDNP (SEQ ID NO: 520). In some embodi consisting of (GS), (GGS), (GSGGS), (SEQID NO:381) ments, the CM1-CM2 substrate includes the sequence and (GGGS), (SEQ ID NO:382), where n is an integer of AVGLLAPPGGLSGRSANP (SEQ ID NO. 521). In some at least one. embodiments, the CM1-CM2 substrate includes the 0031. In some embodiments, at least one of LP1 or LP2 sequence AVGLLAPPGGLSGRSANI (SEQ ID NO: 522). comprises an amino acid sequence selected from the group In some embodiments, the CM1-CM2 substrate includes the consisting of GGSG (SEQ ID NO: 383), GGSGG (SEQ ID sequence ISSGLLSGRSDNI (SEQ ID NO. 555). In some NO: 384), GSGSG (SEQ ID NO: 385), GSGGG (SEQ ID embodiments, the CM1-CM2 substrate includes the NO:386), GGGSG (SEQ ID NO:387), and GSSSG (SEQ sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 557). ID NO:388). 0025. In some embodiments, the activatable antibody in 0032. In some embodiments, LP1 comprises the amino the uncleaved State has the structural arrangement from acid sequence GSSGGSGGSGGSG (SEQ ID NO: 389), N-terminus to C-terminus as follows: MM-CM1-CM2-AB, GSSGGSGGSGG (SEQ ID NO: 390), GSSGGSGGSGGS AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1 (SEQ ID NO: 391), GSSGGSGGSGGSGGGS (SEQ ID CM2-MM. NO: 392), GSSGGSGGSG (SEQ ID NO: 393), or 0026. In some embodiments, the activatable antibody GSSGGSGGSGS (SEQ ID NO: 394). comprises a first linking peptide (LP1) and a second linking 0033. In some embodiments, LP2 comprises the amino peptide (LP2), and the antibody in the uncleaved state has acid sequence GSS, GGS, GGGS (SEQ ID NO: 395), the structural arrangement from N-terminus to C-terminus as GSSGT (SEQ ID NO: 396) or GSSG (SEQ ID NO:397). follows: MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2 0034. In some embodiments, the AB has a dissociation CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB, O constant of about 100 nM or less for binding to the target. AB-LP2-CM1-CM2-LP-MM. In some embodiments, each 0035. In some embodiments, the activatable antibody of LP1 and LP2 is a peptide of about 1 to 20 amino acids in includes an antibody or antigen-binding fragment (AB) length. In some embodiments, the two linking peptides need thereof that specifically binds a target. In some embodi not be identical to each other. ments, the AB is a full-length antibody. In some embodi 0027. In some embodiments, the activatable antibody ments, the AB is an immunologically active fragment. In includes a linking peptide (LP) between CM1 and CM2. In Some embodiments, the AB is an antigen-binding fragment. Some embodiments, the activatable antibody includes a In some embodiments, the AB is a monoclonal antibody, linking peptide (LP") between the masking moiety (MM) domain antibody, single chain, Fab fragment, a F(ab') and CM1. In some embodiments, the activatable antibody fragment, a scFv, a scab, a dAb, a single domain heavy chain US 2016/0289.324 A1 Oct. 6, 2016

antibody, or a single domain light chain antibody. In some In some embodiments, the CM2 substrate sequence of the embodiments, such an AB is a mouse, other rodent, chime CM1-CM2 substrate is a substrate for at least one SP and ric, humanized or fully human monoclonal antibody. comprises a polypeptide sequence that is not substantially 0036. In some embodiments, the MMP protease is co identical to any human polypeptide sequence that is natu localized with the target in a tissue, and the MMP protease rally cleaved by the same SP protease. In some embodi cleaves the CM1 in the antibody when the antibody is ments, the CM2 substrate sequence of the CM1-CM2 sub exposed to the protease. In some embodiments, the SP strate is a substrate for at least one SP and comprises a protease is co-localized with the target in a tissue, and the SP polypeptide sequence that is no more than 90% or more protease cleaves the CM2 substrate in the antibody when the identical to any polypeptide sequence, e.g., any animal antibody is exposed to the protease. In some embodiments, polypeptide sequence, that is naturally cleaved by the same the MMP protease and/or the SP protease are co-localized SP protease. In some embodiments, the CM2 substrate with the target in a tissue, and the MMP protease and/or the sequence of the CM1-CM2 substrate is a substrate for at SP protease cleave the CM1-CM2 substrate in the antibody least one SP and comprises a polypeptide sequence that is no when the antibody is exposed to the protease. In some more than 90% or more identical to any mammalian poly embodiments, the MMP protease and the SP protease are peptide sequence that is naturally cleaved by the same SP co-localized with the target in a tissue, and at least one of the protease. In some embodiments, the CM2 substrate MMP protease and the SP protease cleave the CM1-CM2 sequence of the CM1-CM2 substrate is a substrate for at substrate in the antibody when the antibody is exposed to the least one SP and comprises a polypeptide sequence that is no protease. more than 90% or more identical to any human polypeptide 0037. In some embodiments, each of the CM1 substrate sequence that is naturally cleaved by the same SP protease. sequence and the CM2 substrate sequence of the CM1-CM2 0040. In some embodiments, the CM1-CM2 substrate Substrate is independently a polypeptide of up to 15 amino comprises an amino acid sequence selected from the group acids in length. consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 0038. In some embodiments, the CM1 substrate sequence 515-522, 555, and 557. In some embodiments, an activatable of the CM1-CM2 substrate is a substrate for at least one antibody comprises a CM1-CM2 substrate comprising an MMP and comprises a polypeptide sequence that is not amino acid sequence selected from the group consisting of Substantially identical to any polypeptide sequence, e.g., any SEQ ID NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, animal polypeptide sequence, that is naturally cleaved by the and 557, as well as an antibody or antigen binding fragment same MMP protease. In some embodiments, the CM1 sub thereof (AB) that binds a target and a masking moiety (MM) strate sequence of the CM1-CM2 substrate is a substrate for that reduces the ability of the antigen- or epitope-binding at least one MMP and comprises a polypeptide sequence that domain of the AB to bind its target. is not substantially identical to any mammalian polypeptide 0041. In some embodiments, an activatable antibody sequence that is naturally cleaved by the same MMP pro comprises a CM1-CM2 Substrate comprising an amino acid tease. In some embodiments, the CM1 substrate sequence of sequence selected from the group consisting of SEQ ID the CM1-CM2 Substrate is a Substrate for at least one MMP NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, and comprises a polypeptide sequence that is not Substan and an anti-Jagged antibody comprising an amino acid tially identical to any human polypeptide sequence that is sequence of an anti-Jagged antibody disclosed herein. In naturally cleaved by the same MMP protease. In some Some embodiments, an activatable antibody comprises a embodiments, the CM1 substrate sequence of the CM1 CM1-CM2 Substrate comprising an amino acid sequence CM2 substrate is a substrate for at least one MMP and selected from the group consisting of SEQID NOs: 1-17, 22. comprises a polypeptide sequence that is no more than 90% 469-471, 483-490, 515-522, 555, and 557, and an antibody or more identical to any polypeptide sequence, e.g., any having a light chain comprising amino acid sequence SEQ animal polypeptide sequence, that is naturally cleaved by the ID NO: 162 or SEQ ID NO: 164 and a heavy chain same MMP protease. In some embodiments, the CM1 sub comprising amino acid sequence SEQID NO: 67 or SEQID strate sequence of the CM1-CM2 substrate is a substrate for NO: 163. at least one MMP and comprises a polypeptide sequence that 0042. In some embodiments, the CM1-CM2 is included is no more than 90% or more identical to any mammalian in an activatable antibody having a light chain amino acid polypeptide sequence that is naturally cleaved by the same sequence selected from the group consisting of SEQ ID MMP protease. In some embodiments, the CM1 substrate NOs: 420, 422,424, 426,428, 430, 432, 434, 436, 439, 477, sequence of the CM1-CM2 substrate is a substrate for at 479, 507-514, 539-546, 561, and 562, and a heavy chain least one MMP and comprises a polypeptide sequence that amino acid sequence of SEQ ID NO: 67. is no more than 90% or more identical to any human 0043. In some embodiments, the CM1-CM2 is included polypeptide sequence that is naturally cleaved by the same in an activatable antibody having a light chain amino acid MMP protease. sequence selected from the group consisting of SEQ ID 0039. In some embodiments, the CM2 substrate sequence NOs: 420, 422,424, 426,428, 430, 432, 434, 436, 439, 477, of the CM1-CM2 substrate is a substrate for at least one SP 479, 507-514, 539-546, 561, and 562, and a heavy chain and comprises a polypeptide sequence that is not Substan amino acid sequence of SEQ ID NO: 163. tially identical to any polypeptide sequence, e.g., any animal 0044. In some embodiments, an activatable antibody polypeptide sequence, that is naturally cleaved by the same comprises a CM1-CM2 Substrate comprising an amino acid SP protease. In some embodiments, the CM2 substrate sequence selected from the group consisting of SEQ ID sequence of the CM1-CM2 substrate is a substrate for at NOs: 1-17, 22, 469-471, 483-490, 515-522, 555, and 557, least one SP and comprises a polypeptide sequence that is and an anti-EGFR antibody comprising an amino acid not Substantially identical to any mammalian polypeptide sequence of an anti-EGFR antibody disclosed herein. In sequence that is naturally cleaved by the same SP protease. Some embodiments, an activatable antibody comprises a US 2016/0289.324 A1 Oct. 6, 2016

CM1-CM2 Substrate comprising an amino acid sequence TABLE 6-continued selected from the group consisting of SEQID NOs: 1-17, 22. 469-471, 483-490, 515-522, 555, and 557, and an antibody Exemplary Proteases and/or Enzymes having a light chain comprising amino acid sequence SEQ Aspartate proteases, e.g., ID NO: 111 and a heavy chain comprising amino acid BACE sequence SEQID NO: 108, SEQ ID NO: 109, or SEQ ID Renin NO: 110. Aspartic cathepsins, e.g., Cathepsin D 0045. In some embodiments, the CM1-CM2 is included Cathepsin E in an activatable antibody having a light chain amino acid Caspases, e.g., Caspase 1 sequence selected from the group consisting of SEQ ID Caspase 2 NOs: 449, 451, 453, 455, 457, 459, 461, 463,465, 467, 472, Caspase 3 474, 499-506, 531-538, 559, and 560, and a heavy chain Caspase 4 amino acid sequence of SEQ ID NO: 108. Caspase 5 Caspase 6 0046. In some embodiments, the CM1-CM2 is included Caspase 7 in an activatable antibody having a light chain amino acid Caspase 8 sequence selected from the group consisting of SEQ ID Caspase 9 NOs: 449, 451, 453, 455, 457, 459, 461, 463,465, 467, 472, Caspase 10 Caspase 14 474, 499-506, 531-538, 559, and 560, and a heavy chain Cysteine cathepsins, e.g., amino acid sequence of SEQ ID NO: 109. Cathepsin B 0047. In some embodiments, the CM1-CM2 is included Cathepsin C in an activatable antibody having a light chain amino acid Cathepsin K Cathepsin L sequence selected from the group consisting of SEQ ID Cathepsin S NOs: 449, 451, 453, 455, 457, 459, 461, 463,465, 467, 472, Cathepsin V/L2 474, 499-506, 531-538, 559, and 560, and a heavy chain Cathepsin X/Z/P amino acid sequence of SEQ ID NO: 110. Cysteine proteinases, e.g., Cruzipain 0.048. In some embodiments, the CM1-CM2 substrate is Legumain also a Substrate for at least one additional protease. Otubain-2 0049. In some embodiments, the at least one additional KLKS, e.g., protease is a different MMP protease than the MMP protease that cleaves the CM1. In some embodiments, the at least one KLKS additional protease is an MMP protease selected from the KLK7 group consisting of MMP1; MMP2: MMP3; MMP7; KLK8 MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; KLK10 KLK11 MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; KLK13 MMP23; MMP24; MMP26; and MMP27. KLK14 0050. In some embodiments, the at least one additional Metallo proteinases, e.g., Meprin protease is a different SP protease than the SP protease that cleaves CM2. In some embodiments, the at least one addi PSMA tional SP protease is selected from the group consisting of BMP-1 uPA; matriptase; activated protein C: Cathepsin A; Cathep sin G. Chymase; a coagulation factor protease Such as, e.g., FVIIa, FIXa, FXa. FXIa, FXIIa; Elastase; Granzyme B; 0.052 The disclosure also provides an antibody includes Guanidinobenzoatase: Htra1; Human Neutrophil Elastase: at least a first CM1 and a second CM2 and is conjugated to Lactoferrin; Marapsin; NS3/4A; PACE4: Plasmin; PSA; an agent. In some embodiments, the first CM1 and the tPA: Thrombin; Tryptase; a Type II Transmembrane Serine second CM2 are each polypeptides of no more than 15 Protease (TTSP) such as, e.g., DESC1, DPP-4, FAP. Hepsin, amino acids long. In some embodiments, the activatable Matriptase-2, TMPRSS2, TMPRSS3, and TMPRSS4. antibody is a conjugated activatable antibody that, in the 0051. In some embodiments, the at least one additional uncleaved state has the structural arrangement from N-ter protease is selected from the group consisting of those minus to C-terminus as follows: MM-CM1-CM2-AB shown in Table 6. Agent, Agent-AB-CM2-CM1-MM, MM-CM2-CM1-AB Agent, or Agent-AB-CM1-CM2-MM. In some TABLE 6 embodiments, the activatable antibody is a conjugated acti vatable antibody that, in the uncleaved state has the struc Exemplary Proteases and/or Enzymes tural arrangement from N-terminus to C-terminus as fol ADAMS, ADAMTS, e.g. lows: Agent-MM-CM1-CM2-AB, AB-CM2-CM1-MM ADAM8 Agent, Agent-MM-CM2-CM1-AB, or AB-CM1-CM2-MM ADAM9 Agent. In some embodiments, the activatable antibody is a ADAM10 ADAM12 conjugated activatable antibody that, in the uncleaved State ADAM15 has the structural arrangement from N-terminus to C-termi ADAM17 TACE nus as follows: Agent-MM-CM11-CM2-AB-Agent, Agent ADAMDEC1 AB-CM2-CM11-MM-Agent, Agent-MM-CM2-CM1-AB ADAMTS1 Agent, or Agent-AB-CM1-CM2-MM-Agent. ADAMTS4 0053. In some embodiments, the activatable antibody is a conjugated activatable antibody that comprises a masking US 2016/0289.324 A1 Oct. 6, 2016 moiety (MM), a first linking peptide (LP1) and a second gated activatable antibody includes a linking peptide linking peptide (LP2), and the antibody in the uncleaved between the AB and CM1 (LP") and a linking peptide state has the structural arrangement from N-terminus to between CM1 and CM2 (LP). In some embodiments, the C-terminus as follows: MM1-LP1-CM1-CM2-LP2-AB conjugated activatable antibody includes a linking peptide Agent, Agent-AB-LP2-CM2-CM1-LP1-MM, MM1-LP1 (LP") between CM1 and CM2 and a linking peptide (LP") CM2-CM1-LP2-AB-Agent, or Agent-AB-LP2-CM1-CM2 between CM2 and MM. In some embodiments, the conju LP1-MM. In some embodiments, each of LP1 and LP2 is a gated activatable antibody includes a linking peptide (LP") peptide of about 1 to 20 amino acids in length. In some between the AB and CM1, a linking peptide (LP) between embodiments, the two linking peptides need not be identical CM1 and CM2, and a linking peptide (LP") between CM2 to each other. and MM. 0054. In some embodiments, the activatable antibody is a 0058. In some embodiments, LP' is G.G. In some embodi conjugated activatable antibody that comprises a masking ments, LP is GGSGGS (SEQ ID NO:350). moiety (MM), a first linking peptide (LP1) and a second 0059. In some embodiments, at least one of LP1 or LP2 linking peptide (LP2), and the antibody in the uncleaved comprises an amino acid sequence selected from the group state has the structural arrangement from N-terminus to consisting of (GS), (GGS), (GSGGS), (SEQID NO:381) C-terminus as follows: Agent-MM1-LP1-CM1-CM2-LP2 and (GGGS), (SEQ ID NO:382), where n is an integer of AB, AB-LP2-CM2-CM1-LP I-MM-Agent, Agent-MM1 at least one. LP1-CM2-CM11-LP2-AB, or AB-LP2-CM1-CM2-LP1 0060. In some embodiments, at least one of LP1 or LP2 MM-Agent. In some embodiments, each of LP and LP2 is a comprises an amino acid sequence selected from the group peptide of about 1 to 20 amino acids in length. In some consisting of GGSG (SEQ ID NO: 383), GGSGG (SEQ ID embodiments, the two linking peptides need not be identical NO: 384), GSGSG (SEQ ID NO: 385), GSGGG (SEQ ID to each other. NO:386), GGGSG (SEQ ID NO:387), and GSSSG (SEQ 0055. In some embodiments, the activatable antibody is a ID NO:388). conjugated activatable antibody that comprises a masking 0061. In some embodiments, LP1 comprises the amino moiety (MM), a first linking peptide (LP1) and a second acid sequence GSSGGSGGSGGSG (SEQ ID NO: 389), linking peptide (LP2), and the antibody in the uncleaved GSSGGSGGSGG (SEQ ID NO: 390), GSSGGSGGSGGS state has the structural arrangement from N-terminus to (SEQ ID NO: 391), GSSGGSGGSGGSGGGS (SEQ ID C-terminus as follows: Agent-MM1-LP1-CM1-CM2-LP2 NO: 392), GSSGGSGGSG (SEQ ID NO: 393), or AB-Agent, Agent-AB-LP2-CM2-CM1-LP1-MM-Agent, GSSGGSGGSGS (SEQ ID NO: 394). Agent-MM1-LP1-CM2-CM1-LP2-AB-Agent, or Agent 0062. In some embodiments, LP2 comprises the amino AB-LP2-CM1-CM2-LP1-MM-Agent. In some embodi acid sequence GSS, GGS, GGGS (SEQ ID NO: 395), ments, each of LP1 and LP2 is a peptide of about 1 to 20 GSSGT (SEQ ID NO: 396) or GSSG (SEQ ID NO:397). amino acids in length. In some embodiments, the two 0063. In some embodiments, the CM1-CM2 substrate is linking peptides need not be identical to each other. linked or otherwise attached to an antibody. For example, the 0056. In some embodiments, the conjugated activatable CM1-CM2 is used to link one or more agents to the antibody antibody includes a linking peptide (LP") between CM1 and or antigen binding fragment thereof (AB) that binds a given CM2. In some embodiments, the conjugated activatable target, such that the CM1-CM2 is cleaved when exposed to antibody includes a linking peptide (LP") between the the MMP and/or the SP and the agent is released from the masking moiety (MM) and CM1. In some embodiments, the AB. Exemplary targets include, but are not limited to the conjugated activatable antibody includes a linking peptide targets shown in Table 1. Exemplary ABs include, but are (LP") between CM2 and AB. In some embodiments, the conjugated activatable antibody includes a linking peptide not limited to, the antibodies shown in Table 2. (LP") between the MM and CM1 and a linking peptide 0064. In some embodiments, the AB has a dissociation (LP") between CM2 and AB. In some embodiments, the constant of about 100 nM or less for binding to the target. conjugated activatable antibody includes a linking peptide 0065. In some embodiments, the antibody includes an between the MM and CM1 (LP") and a linking peptide antibody or antigen-binding fragment thereof that specifi between CM1 and CM2 (LP"). In some embodiments, the cally binds a target. In some embodiments, the antibody or conjugated activatable antibody includes a linking peptide immunologically active fragment thereof that binds the (LP") between CM1 and CM2 and a linking peptide (LP") target is a monoclonal antibody, domain antibody, single between CM2 and AB. In some embodiments, the conju chain, Fab fragment, a F(ab')2 fragment, a Scv, a Scab, a gated activatable antibody includes a linking peptide (LP") dAb, a single domain heavy chain antibody, or a single between the MM and CM1, a linking peptide (LP") between domain light chain antibody. In some embodiments, such an CM1 and CM2, and a linking peptide (LP") between CM2 antibody or immunologically active fragment thereof that and AB. binds the target is a mouse, other rodent, chimeric, human 0057. In some embodiments, the conjugated activatable ized or fully human monoclonal antibody. antibody includes a linking peptide (LP") between CM1 and 0066. In some embodiments, the MM has a dissociation CM2. In some embodiments, the conjugated activatable constant for binding to the AB that is no more than the antibody includes a linking peptide (LP") between the AB dissociation constant of the AB to the target. and CM1. In some embodiments, the conjugated activatable 0067. In some embodiments, the MM does not interfere antibody includes a linking peptide (LP") between CM2 and or compete with the AB for binding to the target in a cleaved the masking moiety (MM). In some embodiments, the State. conjugated activatable antibody includes a linking peptide 0068. In some embodiments, the MM is a polypeptide of (LP") between the AB and CM1 and a linking peptide (LP") about 2 to 40 amino acids in length. For example, the MM between CM2 and MM. In some embodiments, the conju is a polypeptide of up to about 40 amino acids in length. US 2016/0289.324 A1 Oct. 6, 2016

0069. In some embodiments, the MM polypeptide 0074. In some embodiments, the antibody and/or the AB sequence is different from that of any natural binding partner of the activatable antibody naturally contains one or more of the AB. In some embodiments, the MM polypeptide disulfide bonds. In some embodiments, the AB can be sequence is no more than 50% identical to any natural engineered to include one or more disulfide bonds. binding partner of the AB. In some embodiments, the MM 0075. In some embodiments, the antibody and/or conju polypeptide sequence is no more than 40%, 30%, 25%, 20%, gated antibody is monospecific. In some embodiments, the 15%, or 10% identical to any natural binding partner of the antibody and/or conjugated antibody is multispecific, AB. referred to herein as multispecific antibodies and/or conju 0070. In some embodiments, the agent conjugated to the gated multispecific antibodies. In some embodiments, the AB or the AB of an activatable antibody is a therapeutic multispecific antibody and/or conjugated multispecific anti agent. In some embodiments, the agent is an antineoplastic body is bispecific or trifunctional. In some embodiments, the agent. In some embodiments, the agent is a toxin or fragment antibody and/or conjugated antibody is formulated as part of thereof. As used herein, a fragment of a toxin is a fragment a pro-Bispecific T Cell Engager (pro-BITE) molecule. In that retains toxic activity. In some embodiments, the agent is Some embodiments, the antibody and/or conjugated anti conjugated to the AB via a cleavable linker. In some body is formulated as part of a pro-Chimeric Antigen embodiments, the agent is conjugated to the AB via a linker Receptor (pro-CAR) modified T cell or other engineered that includes at least one CM1-CM2 substrate sequence. In receptor or other immune effector cell, such as a CAR Some embodiments, the agent is conjugated to the AB via a modified NK cell. In some embodiments, the activatable noncleavable linker. In some embodiments, the agent is a antibody and/or conjugated activatable antibody is formu microtubule inhibitor. In some embodiments, the agent is a lated as part of a pro-Chimeric Antigen Receptor (CAR) nucleic acid damaging agent, such as a DNA alkylator or modified T cell. In some embodiments, the activatable DNA intercalator, or other DNA damaging agent. In some antibody and/or conjugated activatable antibody is formu embodiments, the agent is an agent selected from the group lated as part of a pro-Chimeric Antigen Receptor (CAR) listed in Table 3. In some embodiments, the agent is a modified NK cell. dolastatin. In some embodiments, the agent is an auristatin 0076. In some embodiments, the activatable antibody or derivative thereof. In some embodiments, the agent is and/or conjugated activatable antibody is monospecific. In auristatin E or a derivative thereof. In some embodiments, Some embodiments, the activatable antibody and/or conju the agent is monomethyl auristatin E (MMAE). In some gated activatable antibody is multispecific, referred to herein embodiments, the agent is monomethyl auristatin D as multispecific activatable antibodies and/or conjugated (MMAD). In some embodiments, the agent is a maytansi multispecific activatable antibodies. As used herein, terms noid or maytansinoid derivative. In some embodiments, the Such as “activatable antibody' and all grammatical varia agent is DM1 or DM4. In some embodiments, the agent is tions thereof, unless otherwise noted, are intended to encom a duocarmycin or derivative thereof. In some embodiments, pass, but are not limited to embodiments where the activat the agent is a calicheamicin or derivative thereof. In some able antibody is a multispecific activatable antibody of the embodiments, the agent is a pyrrolobenzodiazepine. In some disclosure. As used herein, terms such as "conjugated acti embodiments, the agent is a pyrrolobenzodiazepine dimer. vatable antibody” and all grammatical variations thereof, unless otherwise noted, are intended to encompass, but are 0071. In some embodiments, the agent is an anti-inflam not limited to embodiments where the conjugated activat matory agent. able antibody is a conjugated multispecific activatable anti 0072. In some embodiments, the antibody and/or activat body of the disclosure. In some embodiments, the multispe able antibody also includes a detectable moiety. In some cific activatable antibody and/or conjugated multispecific embodiments, the detectable moiety is a diagnostic agent. activatable antibody is bispecific or trifunctional. In some 0073. In some embodiments, the conjugated antibody embodiments, the activatable antibody and/or conjugated and/or conjugated activatable antibody includes a detectable activatable antibody is formulated as part of a pro-Bispecific label. In some embodiments, the detectable label includes an T Cell Engager (pro-BITE) molecule. In some embodi imaging agent, a contrasting agent, an , a fluorescent ments, the activatable antibody and/or conjugated activat label, a chromophore, a dye, one or more metal ions, or a able antibody is formulated as part of a pro-Chimeric ligand-based label. In some embodiments, the imaging agent Antigen Receptor (pro-CAR) modified T cell or other engi comprises a radioisotope. In some embodiments, the radio neered receptor. isotope is indium or technetium. In some embodiments, the 0077. In some embodiments, the antibodies, antibody contrasting agent comprises iodine, gadolinium or iron conjugates, activatable antibodies, conjugated activatable oxide. In some embodiments, the enzyme comprises horse antibodies, multispecific activatable antibodies, and/or con radish peroxidase, alkaline phosphatase, or 3-galactosidase. jugated multispecific activatable antibodies described herein In some embodiments, the fluorescent label comprises yel are used in conjunction with one or more additional agents low fluorescent protein (YFP), cyan fluorescent protein or a combination of additional agents. Suitable additional (CFP), green fluorescent protein (GFP), modified red fluo agents include current pharmaceutical and/or Surgical thera rescent protein (mRFP), red fluorescent protein tdimer2 pies for an intended application, such as, for example, (RFP tdimer2), HCRED, or a europium derivative. In some cancer. For example, the activatable antibodies, conjugated embodiments, the luminescent label comprises an N-methy activatable antibodies, multispecific activatable antibodies, lacrydium derivative. In some embodiments, the label com and/or conjugated multispecific activatable antibodies can prises an Alexa Fluor label, such as Alex Fluor R 680 or be used in conjunction with an additional chemotherapeutic Alexa Fluor R 750. In some embodiments, the ligand-based or anti-neoplastic agent. label comprises biotin, avidin, Streptavidin or one or more 0078. In some embodiments, the activatable antibody is a haptens. multispecific activatable antibody. The multispecific activat US 2016/0289.324 A1 Oct. 6, 2016 able antibodies provided herein are multispecific antibodies 398); a VHCD2 sequence that includes a sequence that is at that recognize two or more different antigens or epitopes and least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, that include at least one masking moiety (MM) linked to at 99%0 or more identical to the amino acid sequence SID least one antigen- or epitope-binding domain of the multi PEGRQTYYADSVKG (SEQ ID NO: 399); a VH CDR3 specific antibody such that coupling of the MM reduces the sequence that includes a sequence that is at least 90%, 91%, ability of the antigen- or epitope-binding domain to bind its 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more target. In some embodiments, the MM is coupled to the identical to the amino acid sequence DIGGRSAFDY (SEQ antigen- or epitope-binding domain of the multispecific ID NO: 400), and combinations thereof. antibody via a CM11-CM2 substrate that functions as a I0082 In some embodiments, the activatable antibody substrate for at least one MMP protease and at least one SP and/or conjugated activatable antibody provided herein, protease. The activatable multispecific antibodies provided including but not limited to a multispecific activatable herein are stable in circulation, activated at intended sites of antibody and/or conjugated multispecific activatable anti therapy and/or diagnosis but not in normal, i.e., healthy body of the disclosure, includes at least a first antibody or tissue, and, when activated, exhibit binding to a target that antigen binding fragment thereof (AB1) that specifically is at least comparable to the corresponding, unmodified binds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and multispecific antibody. that contains a combination of a VLCDR1 sequence, a VL 0079. In some embodiments, the activatable antibody CDR2 sequence, and a VLCDR3 sequence, wherein at least and/or conjugated activatable antibody provided herein, one of the VLCDR1 sequence, the VLCDR2 sequence, and including but not limited to a multispecific activatable the VL CDR3 sequence is selected from a VL CDR1 antibody and/or conjugated multispecific activatable anti sequence that includes a sequence that is at least 90%, 91%, body of the disclosure, includes at least a first antibody or 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more antigen binding fragment thereof (AB1) that specifically identical to the amino acid sequence RASQSISSY (SEQ ID binds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and NO: 401); a VL CDR2 sequence that includes a sequence that contains a combination of a VHCDR1 sequence, a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, CDR2 sequence, and a VHCDR3 sequence, wherein at least 98%, 99% or more identical to the amino acid sequence one of the VHCDR1 sequence, the VHCDR2 sequence, and AASSLQS (SEQ ID NO: 402); and a VL CDR3 sequence the VHCDR3 sequence is selected from a VHCDR1 that that includes a sequence that is at least 90%, 91%, 92%, sequence includes at least the amino acid sequence SYAMS 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to (SEQ ID NO: 398); a VH CD2 sequence that includes at the amino acid sequence QQTVVAPPL (SEQID NO. 403), least the amino acid sequence SIDPEGROTYYADSVKG and combinations thereof. (SEQ ID NO: 399); a VHCDR3 sequence that includes at I0083. In some embodiments, the activatable antibody least the amino acid sequence DIGGRSAFDY (SEQID NO: and/or conjugated activatable antibody provided herein, 400), and combinations thereof. including but not limited to a multispecific activatable 0080. In some embodiments, the activatable antibody antibody and/or conjugated multispecific activatable anti and/or conjugated activatable antibody provided herein, body of the disclosure, includes at least a first antibody or including but not limited to a multispecific activatable antigen binding fragment thereof (AB1) that specifically antibody and/or conjugated multispecific activatable anti binds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and body of the disclosure, includes at least a first antibody or that contains a combination of a VHCDR1 sequence, a VH antigen binding fragment thereof (AB1) that specifically CDR2 sequence, a VH CDR3 sequence, a VL CDR1 binds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and sequence, a VLCDR2 sequence, and a VLCDR3 sequence, that contains a combination of a VLCDR1 sequence, a VL wherein the VHCDR1 sequence includes at least the amino CDR2 sequence, and a VLCDR3 sequence, wherein at least acid sequence SYAMS (SEQ ID NO: 398); the VH CD2 one of the VLCDR1 sequence, the VLCDR2 sequence, and sequence includes at least the amino acid sequence SIDPE the VL CDR3 sequence is selected from a VL CDR1 GRQTYYADSVKG (SEQ ID NO: 399); the VH CDR3 sequence that includes at least the amino acid sequence sequence includes at least the amino acid sequence DIG RASQSISSY (SEQ ID NO: 401); a VLCDR2 sequence that GRSAFDY (SEQ ID NO: 400); the VL CDR1 sequence includes at least the amino acid sequence AASSLQS (SEQ includes at least the amino acid sequence RASQSISSY ID NO: 402); a VLCDR3 sequence that includes at least the (SEQ ID NO: 401); the VLCDR2 sequence includes at least amino acid sequence QQTVVAPPL (SEQID NO: 403), and the amino acid sequence AASSLQS (SEQID NO: 402); and combinations thereof. the VL CDR3 sequence includes at least the amino acid 0081. In some embodiments, the activatable antibody sequence QQTVVAPPL (SEQ ID NO: 403). and/or conjugated activatable antibody provided herein, I0084. In some embodiments, the activatable antibody including but not limited to a multispecific activatable and/or conjugated activatable antibody provided herein, antibody and/or conjugated multispecific activatable anti including but not limited to a multispecific activatable body of the disclosure, includes at least a first antibody or antibody and/or conjugated multispecific activatable anti antigen binding fragment thereof (AB1) that specifically body of the disclosure, includes at least a first antibody or binds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and antigen binding fragment thereof (AB1) that specifically that contains a combination of a VHCDR1 sequence, a VH binds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and CDR2 sequence, and a VHCDR3 sequence, wherein at least that contains a combination of a VHCDR1 sequence, a VH one of the VHCDR1 sequence, the VHCDR2 sequence, and CDR2 sequence, a VH CDR3 sequence, a VL CDR1 the VH CDR3 sequence is selected from a VH CDR1 sequence, a VLCDR2 sequence, and a VLCDR3 sequence, sequence that includes a sequence that is at least 90%, 91%, wherein the VHCDR1 sequence includes a sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, identical to the amino acid sequence SYAMS (SEQ ID NO: 99% or more identical to the amino acid sequence SYAMS US 2016/0289.324 A1 Oct. 6, 2016

(SEQ ID NO: 398); the VH CD2 sequence includes a 99% or more identical to the amino acid sequence NYGVH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%6, (SEQ ID NO: 404); a VH CD2 sequence that includes a 96%, 97%, 98%, 99% or more identical to the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, sequence SIDPEGROTYYADSVKG (SEQ ID NO: 399); 96%, 97%, 98%, 99% or more identical to the amino acid the VHCDR3 sequence includes a sequence that is at least sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 405); a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or VH CDR3 sequence that includes a sequence that is at least more identical to the amino acid sequence DIGGRSAFDY 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or (SEQ ID NO: 400); the VL CDR1 sequence includes a more identical to the amino acid sequence ALTYYDYEFAY sequence that is at least 90%, 91%, 920%, 93%, 94%, 95%, (SEQ ID NO: 406); and combinations thereof. 96%, 97%, 98%, 990% or more identical to the amino acid I0088. In some embodiments, the activatable antibody sequence RASQSISSY (SEQ ID NO: 401); the VLCDR2 and/or conjugated activatable antibody provided herein, sequence includes a sequence that is at least 90%, 91%, including but not limited to a multispecific activatable 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more antibody and/or conjugated multispecific activatable anti identical to the amino acid sequence AASSLQS (SEQ ID body of the disclosure, includes at least a first antibody or NO: 402); and the VLCDR3 sequence includes a sequence antigen binding fragment thereof (AB1) that specifically that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, binds EGFR and that contains a combination of a VLCDR1 98%, 99% or more identical to the amino acid sequence sequence, a VLCDR2 sequence, and a VLCDR3 sequence, QQTVVAPPL (SEQ ID NO. 403). wherein at least one of the VL CDR1 sequence, the VL 0085. In some embodiments, the activatable antibody CDR2 sequence, and the VL CDR3 sequence is selected and/or conjugated activatable antibody provided herein, from a VLCDR1 sequence that includes a sequence that is including but not limited to a multispecific activatable at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, antibody and/or conjugated multispecific activatable anti 99% or more identical to the amino acid sequence body of the disclosure, includes at least a first antibody or RASQSIGTNIH (SEQ ID NO: 407); a VLCDR2 sequence antigen binding fragment thereof (AB1) that specifically that includes a sequence that is at least 90%, 91%, 92%, binds Epidermal Growth Factor Receptor (EGFR) and that 93%, 94%, 95%, 960%, 97%, 98%, 99% or more identical contains a combination of a VH CDR1 sequence, a VH to the amino acid sequence KYASESIS (SEQID NO: 408): CDR2 sequence, and a VHCDR3 sequence, wherein at least and a VLCDR3 sequence that includes a sequence that is at one of the VHCDR1 sequence, the VHCDR2 sequence, and least 90%, 910%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the VH CDR3 sequence is selected from a VH CDR1 99% or more identical to the amino acid sequence sequence that includes at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 409), and combinations NYGVH (SEQ ID NO: 404); a VH CD2 sequence that thereof. includes at least the amino acid sequence VIWSGGNT I0089. In some embodiments, the activatable antibody DYNTPFTS (SEQID NO: 405); a VHCDR3 sequence that and/or conjugated activatable antibody provided herein, includes at least the amino acid sequence ALTYYDYEFAY including but not limited to a multispecific activatable (SEQ ID NO: 406); and combinations thereof. antibody and/or conjugated multispecific activatable anti I0086. In some embodiments, the activatable antibody body of the disclosure, includes at least a first antibody or and/or conjugated activatable antibody provided herein, antigen binding fragment thereof (AB1) that specifically including but not limited to a multispecific activatable binds EGFR and that contains a combination of a VHCDR1 antibody and/or conjugated multispecific activatable anti sequence, a VHCDR2 sequence, a VH CDR3 sequence, a body of the disclosure, includes at least a first antibody or VLCDR1 sequence, a VLCDR2 sequence, and a VLCDR3 antigen binding fragment thereof (AB1) that specifically sequence, wherein the VHCDR1 sequence includes at least binds EGFR and that contains a combination of a VLCDR1 the amino acid sequence NYGVH (SEQ ID NO: 404); the sequence, a VLCDR2 sequence, and a VLCDR3 sequence, VHCD2 sequence includes at least the amino acid sequence wherein at least one of the VL CDR1 sequence, the VL VIWSGGNTDYNTPFTS (SEQ ID NO: 405); the VH CDR2 sequence, and the VL CDR3 sequence is selected CDR3 sequence includes at least the amino acid sequence from a VLCDR1 sequence that includes at least the amino ALTYYDYEFAY (SEQ ID NO: 406); the VL CDR1 acid sequence RASQSIGTNIH (SEQ ID NO: 407); a VL sequence includes at least the amino acid sequence CDR2 sequence that includes at least the amino acid RASQSIGTNIH (SEQ ID NO. 407); the VL CDR2 sequence KYASESIS (SEQ ID NO: 408); and a VL CDR3 sequence includes at least the amino acid sequence KYAS sequence that includes at least the amino acid sequence ESIS (SEQ ID NO: 408); and the VL CDR3 sequence QQNNNWPTT (SEQ ID NO: 409), and combinations includes at least the amino acid sequence QQNNNWPTT thereof. (SEQ ID NO: 409). 0087. In some embodiments, the activatable antibody 0090. In some embodiments, the activatable antibody and/or conjugated activatable antibody provided herein, and/or conjugated activatable antibody provided herein, including but not limited to a multispecific activatable including but not limited to a multispecific activatable antibody and/or conjugated multispecific activatable anti antibody and/or conjugated multispecific activatable anti body of the disclosure, includes at least a first antibody or body of the disclosure, includes at least a first antibody or antigen binding fragment thereof (AB1) that specifically antigen binding fragment thereof (AB1) that specifically binds EGFR and that contains a combination of a VHCDR1 binds EGFR and that contains a combination of a VHCDR1 sequence, a VHCDR2 sequence, and a VHCDR3 sequence, sequence, a VHCDR2 sequence, a VH CDR3 sequence, a wherein at least one of the VH CDR1 sequence, the VH VLCDR1 sequence, a VLCDR2 sequence, and a VLCDR3 CDR2 sequence, and the VHCDR3 sequence is selected sequence, wherein the VH CDR1 sequence includes a from a VHCDR1 sequence that includes a sequence that is sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 96%, 97%, 98%, 99% or more identical to the amino acid US 2016/0289.324 A1 Oct. 6, 2016

sequence NYGVH (SEQ ID NO: 404); the VH CD2 comprising amino acid sequence SEQ ID NO: 111 and a sequence includes a sequence that is at least 900%, 91%, heavy chain comprising amino acid sequence SEQ ID NO: 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more 108, SEQ ID NO: 109, or SEQ ID NO: 110. identical to the amino acid sequence VIWSGGNTDYNTP 0094. In some embodiments, the activatable antibody FTS (SEQ ID NO: 405); the VHCDR3 sequence includes and/or conjugated activatable antibody provided herein, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, including but not limited to a multispecific activatable 96%, 97%, 98%, 99% or more identical to the amino acid antibody and/or conjugated multispecific activatable anti sequence ALTYYDYEFAY (SEQ ID NO: 406); the VL body of the disclosure, includes at least a heavy chain amino CDR1 sequence includes a sequence that is at least 900%, acid sequence of SEQID NO: 108 and a light chain amino 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more acid sequence selected from the group consisting of SEQID identical to the amino acid sequence RASQSIGTNIH (SEQ NOs: 449, 451, 453, 455, 457, 459, 461, 463,465, 467, 472, ID NO: 407); the VLCDR2 sequence includes a sequence 474, 499-506, 531-538, 559, and 560. that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 0095. In some embodiments, the activatable antibody 98%, 99% or more identical to the amino acid sequence and/or conjugated activatable antibody provided herein, KYASESIS (SEQID NO: 408); and the VLCDR3 sequence including but not limited to a multispecific activatable includes a sequence that is at least 90%, 91%, 92%, 93%, antibody and/or conjugated multispecific activatable anti 94%, 95%, 96%, 97%, 98%, 99% or more identical to the body of the disclosure, includes at least a heavy chain amino amino acid sequence QQNNNWPTT (SEQ ID NO: 409). acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 0091. In some embodiments, the activatable antibody 95%, 96%, 97%, 98%, 99% or more identical to the amino and/or conjugated activatable antibody provided herein, acid sequence of SEQ ID NO: 67 and a light chain amino including but not limited to a multispecific activatable acid sequence that is at least 90%, 91%, 92%, 93%, 94%, antibody and/or conjugated multispecific activatable anti 95%, 96%, 97%, 98%, 99% or more identical to an amino body of the disclosure, comprises a CM1-CM2 substrate acid sequence selected from the group consisting of SEQID comprising an amino acid sequence selected from the group NOs: 420, 422,424, 426,428, 430, 432, 434, 436, 439, 477, consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, 479, 507-514, 539-546, 561, and 562. 515-522, 555, and 557, and an anti-Jagged antibody com 0096. In some embodiments, the activatable antibody prising an amino acid sequence of an anti-Jagged antibody and/or conjugated activatable antibody provided herein, disclosed herein. In some embodiments, the activatable including but not limited to a multispecific activatable antibody and/or conjugated activatable antibody provided antibody and/or conjugated multispecific activatable anti herein, including but not limited to a multispecific activat body of the disclosure, includes at least a heavy chain amino able antibody and/or conjugated multispecific activatable acid sequence that is at least 90%, 91%, 92%, 93%, 94%, antibody of the disclosure, comprises a CM1-CM2 substrate 95%, 96%, 97%, 98%, 99% or more identical to the amino comprising an amino acid sequence selected from the group acid sequence of SEQID NO: 108 and a light chain amino consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 515-522, 555, and 557, and an antibody having a light chain 95%, 96%, 97%, 98%, 99% or more identical to an amino comprising amino acid sequence SEQ ID NO: 162 or SEQ acid sequence selected from the group consisting of SEQID ID NO: 164 and a heavy chain comprising amino acid NOs: 449, 451, 453, 455, 457, 459, 461, 463,465, 467, 472, sequence SEQ ID NO: 67 or SEQ ID NO: 163. 474, 499-506, 531-538, 559, and 560. 0092. In some embodiments, the activatable antibody 0097. In some embodiments, the activatable antibody and/or conjugated activatable antibody provided herein, also includes an agent conjugated to the AB. In some including but not limited to a multispecific activatable embodiments, the agent is a therapeutic agent. In some antibody and/or conjugated multispecific activatable anti embodiments, the agent is an antineoplastic agent. In some body of the disclosure, includes at least a heavy chain amino embodiments, the agent is a toxin or a fragment thereof. In acid sequence of SEQ ID NO: 67 and a light chain amino Some embodiments, the agent is conjugated to the AB via a acid sequence selected from the group consisting of SEQID linker. In some embodiments, the linker is a cleavable linker. NOs: 420, 422,424, 426,428, 430, 432, 434, 436, 439, 477, In some embodiments, the agent is a microtubule inhibitor. 479, 507-514, 539-546, 561, and 562. In some embodiments, the agent is a nucleic acid damaging 0093. In some embodiments, the activatable antibody agent, such as a DNA alkylator or DNA intercalator, or other and/or conjugated activatable antibody provided herein, DNA damaging agent. In some embodiments, the linker is a including but not limited to a multispecific activatable cleavable linker. In some embodiments, the agent is conju antibody and/or conjugated multispecific activatable anti gated to the AB via a linker that includes at least one body of the disclosure, comprises a CM1-CM2 substrate CM1-CM2 substrate sequence. In some embodiments, the comprising an amino acid sequence selected from the group agent is an agent selected from the group listed in Table 3. consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, In some embodiments, the agent is a dolastatin. In some 515-522, 555, and 557, and an anti-EGFR antibody com embodiments, the agent is an auristatin or derivative thereof. prising an amino acid sequence of an anti-EGFR antibody In some embodiments, the agent is auristatin E or a deriva disclosed herein. In some embodiments, the activatable tive thereof. In some embodiments, the agent is monomethyl antibody and/or conjugated activatable antibody provided auristatin E (MMAE). In some embodiments, the agent is herein, including but not limited to a multispecific activat monomethyl auristatin D (MMAD). In some embodiments, able antibody and/or conjugated multispecific activatable the agent is a maytansinoid or maytansinoid derivative. In antibody of the disclosure, comprises a CM1-CM2 substrate some embodiments, the agent is DM1 or DM4. In some comprising an amino acid sequence selected from the group embodiments, the agent is a duocarmycin or derivative consisting of SEQ ID NOs: 1-17, 22, 469-471, 483-490, thereof. In some embodiments, the agent is a calicheamicin 515-522, 555, and 557, and an antibody having a light chain or derivative thereof. In some embodiments, the agent is a US 2016/0289.324 A1 Oct. 6, 2016

pyrrolobenzodiazepine. In some embodiments, the agent is embodiments, the serum half-life of the activatable antibody a pyrrolobenzodiazepine dimer. is at least 15 days when administered to an organism. In 0098. In some embodiments, the agent is an anti-inflam some embodiments, the serum half-life of the activatable matory agent. antibody is at least 12 days when administered to an organ 0099. In some embodiments, the activatable antibody ism. In some embodiments, the serum half-life of the also includes a detectable moiety. In some embodiments, the activatable antibody is at least 11 days when administered to detectable moiety is a diagnostic agent. an organism. In some embodiments, the serum half-life of 0100. In some embodiments, the conjugated antibody the activatable antibody is at least 10 days when adminis includes a detectable label. In some embodiments, the tered to an organism. In some embodiments, the serum detectable label includes an imaging agent, a contrasting half-life of the activatable antibody is at least 9 days when agent, an enzyme, a fluorescent label, a chromophore, a dye, administered to an organism. In some embodiments, the one or more metal ions, or a ligand-based label. In some serum half-life of the activatable antibody is at least 8 days embodiments, the imaging agent comprises a radioisotope. when administered to an organism. In some embodiments, In some embodiments, the radioisotope is indium or tech the serum half-life of the activatable antibody is at least 7 netium. In some embodiments, the contrasting agent com days when administered to an organism. In some embodi prises iodine, gadolinium or iron oxide. In some embodi ments, the serum half-life of the activatable antibody is at ments, the enzyme comprises horseradish peroxidase, least 6 days when administered to an organism. In some alkaline phosphatase, or 3-galactosidase. In some embodi embodiments, the serum half-life of the activatable antibody ments, the fluorescent label comprises yellow fluorescent is at least 5 days when administered to an organism. In some protein (YFP), cyan fluorescent protein (CFP), green fluo embodiments, the serum half-life of the activatable antibody rescent protein (GFP), modified red fluorescent protein is at least 4 days when administered to an organism. In some (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), embodiments, the serum half-life of the activatable antibody HCRED, or a europium derivative. In some embodiments, is at least 3 days when administered to an organism. In some the luminescent label comprises an N-methylacrydium embodiments, the serum half-life of the activatable antibody derivative. In some embodiments, the label comprises an is at least 2 days when administered to an organism. In some Alexa Fluor R label, such as Alex Fluor R 680 or Alexa embodiments, the serum half-life of the activatable antibody Fluorr 750. In some embodiments, the ligand-based label is at least 24 hours when administered to an organism. In comprises biotin, avidin, Streptavidin or one or more hap some embodiments, the serum half-life of the activatable tenS. antibody is at least 20 hours when administered to an 0101. In some embodiments, the activatable antibody organism. In some embodiments, the serum half-life of the also includes a signal peptide. In some embodiments, the activatable antibody is at least 18 hours when administered signal peptide is conjugated to the activatable antibody via to an organism. In some embodiments, the serum half-life of a spacer. In some embodiments, the spacer is conjugated to the activatable antibody is at least 16 hours when adminis the activatable antibody in the absence of a signal peptide. tered to an organism. In some embodiments, the serum In some embodiments, the spacer is joined directly to the half-life of the activatable antibody is at least 14 hours when MM of the activatable antibody. In some embodiments, the administered to an organism. In some embodiments, the spacer is joined directly to the MM of the activatable serum half-life of the activatable antibody is at least 12 hours antibody in the structural arrangement from N-terminus to when administered to an organism. In some embodiments, C-terminus of spacer-MM-CM1-CM2 substrate-AB. An the serum half-life of the activatable antibody is at least 10 example of a spacer joined directly to the N-terminus of MM hours when administered to an organism. In some embodi of the activatable antibody is an amino acid sequence ments, the serum half-life of the activatable antibody is at selected from the group consisting of QGQSGQ (SEQ ID least 8 hours when administered to an organism. In some NO: 410), GQSGQ (SEQ ID NO: 416), QSGQ (SEQ ID embodiments, the serum half-life of the activatable antibody NO: 417), SGQ (SEQ ID NO: 418), GQ, and Q. In some is at least 6 hours when administered to an organism. In embodiments, the spacer includes at least the amino acid some embodiments, the serum half-life of the activatable sequence QGQSGQ (SEQ ID NO: 410). In some embodi antibody is at least 4 hours when administered to an organ ments, the spacer includes at least the amino acid sequence ism. In some embodiments, the serum half-life of the GQSGQ (SEQ ID NO: 416). In some embodiments, the activatable antibody is at least 3 hours when administered to spacer includes at least the amino acid sequence QSGQ an organism. (SEQ ID NO: 417). In some embodiments, the spacer 0104. The disclosure also provides compositions and includes at least the amino acid sequence SGQ (SEQID NO: methods that include an activatable antibody that includes an 418). In some embodiments, the spacer includes at least the antibody or antibody fragment (AB) that specifically binds amino acid sequence GQ. In some embodiments, the spacer a given target, where the AB is coupled to a masking moiety includes at least the amino acid sequence Q. (MM) that decreases the ability of the AB to bind its target. 0102. In some embodiments, the AB of the activatable In some embodiments, the activatable antibody further antibody naturally contains one or more disulfide bonds. In includes a CM1-CM2 substrate that is a substrate for at least Some embodiments, the AB can be engineered to include one one MMP and at least one SP. The compositions and or more disulfide bonds. methods provided herein enable the attachment of one or 0103. In some embodiments, the serum half-life of the more agents to one or more cysteine residues in the AB activatable antibody is longer than that of the corresponding without compromising the activity (e.g., the masking, acti antibody; e.g., the pK of the activatable antibody is longer vating or binding activity) of the activatable antibody. In than that of the corresponding antibody. In some embodi Some embodiments, the compositions and methods provided ments, the serum half-life of the activatable antibody is herein enable the attachment of one or more agents to one or similar to that of the corresponding antibody. In some more cysteine residues in the AB without reducing or US 2016/0289.324 A1 Oct. 6, 2016

otherwise disturbing one or more disulfide bonds within the and efficiently mask the AB of the activatable antibody in an MM. The compositions and methods provided herein pro uncleaved state. The ratio of reduction agent to activatable duce an activatable antibody that is conjugated to one or antibody will vary depending on the activatable antibody. In more agents, e.g., any of a variety of therapeutic, diagnostic Some embodiments, the ratio of reducing agent to activat and/or prophylactic agents, for example, in Some embodi able antibody will be in a range from about 20:1 to 1:1, from ments, without any of the agent(s) being conjugated to the about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to MM of the activatable antibody. The compositions and 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about methods provided herein produce conjugated activatable 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from antibodies in which the MM retains the ability to effectively about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 and efficiently mask the AB of the activatable antibody in an to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, uncleaved State. The compositions and methods provided from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about herein produce conjugated activatable antibodies in which 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, the activatable antibody is still activated, i.e., cleaved, in the from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from presence of a MMP that can cleave the CM1-CM2 substrate. about 1:1 to 1:1.5. In some embodiments, the ratio is in a 0105. The activatable antibodies have at least one point range of from about 5:1 to 1:1. In some embodiments, the of conjugation for an agent, but in the methods and com ratio is in a range of from about 5:1 to 1.5:1. In some positions provided herein less than all possible points of embodiments, the ratio is in a range of from about 4:1 to 1:1. conjugation are available for conjugation to an agent. In In some embodiments, the ratio is in a range from about 4:1 Some embodiments, the one or more points of conjugation to 1.5:1. In some embodiments, the ratio is in a range from are sulfur atoms involved in disulfide bonds. In some about 8:1 to about 1:1. In some embodiments, the ratio is in embodiments, the one or more points of conjugation are a range of from about 2.5:1 to 1:1. sulfur atoms involved in interchain disulfide bonds. In some 0108. In some embodiments, a method of reducing inter embodiments, the one or more points of conjugation are chain disulfide bonds in the AB of an activatable antibody sulfur atoms involved in interchain sulfide bonds, but not and conjugating an agent, e.g., a thiol-containing agent Such sulfur atoms involved in intrachain disulfide bonds. In some as a drug, to the resulting interchain thiols to selectively embodiments, the one or more points of conjugation are locate agent(s) on the AB is provided. The method generally Sulfur atoms of cysteine or other amino acid residues con includes partially reducing the AB with a reducing agent to taining a Sulfur atom. Such residues may occur naturally in form at least two interchain thiols without forming all the antibody structure or may be incorporated into the possible interchain thiols in the activatable antibody; and antibody by site-directed mutagenesis, chemical conversion, conjugating the agent to the interchain thiols of the partially or mis-incorporation of non-natural amino acids. reduced AB. For example, the AB of the activatable anti 0106 Also provided are methods of preparing a conju body is partially reduced for about 1 hour at about 37° C. at gate of an activatable antibody having one or more inter a desired ratio of reducing agent:activatable antibody. In chain disulfide bonds in the AB and one or more intrachain Some embodiments, the ratio of reducing agent to activat disulfide bonds in the MM, and a drug reactive with free able antibody will be in a range from about 20:1 to 1:1, from thiols is provided. The method generally includes partially about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to reducing interchain disulfide bonds in the activatable anti 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about body with a reducing agent, Such as, for example, TCEP; and 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from conjugating the drug reactive with free thiols to the partially about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 reduced activatable antibody. As used herein, the term to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, partial reduction refers to situations where an activatable from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about antibody is contacted with a reducing agent and less than all 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, disulfide bonds, e.g., less than all possible sites of conjuga from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from tion are reduced. In some embodiments, less than 99%, 98%, about 1:1 to 1:1.5. In some embodiments, the ratio is in a 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, range of from about 5:1 to 1:1. In some embodiments, the 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or ratio is in a range of from about 5:1 to 1.5:1. In some less than 5% of all possible sites of conjugation are reduced. embodiments, the ratio is in a range of from about 4:1 to 1:1. 0107. In some embodiments, a method of reducing and In some embodiments, the ratio is in a range from about 4:1 conjugating an agent, e.g., a drug, to an activatable antibody to 1.5:1. In some embodiments, the ratio is in a range from resulting in selectivity in the placement of the agent is about 8:1 to about 1:1. In some embodiments, the ratio is in provided. The method generally includes partially reducing a range of from about 2.5:1 to 1:1. the activatable antibody with a reducing agent Such that any 0109 The thiol-containing reagent can be, for example, conjugation sites in the masking moiety or other non-AB cysteine or N-acetyl cysteine. The reducing agent can be, for portion of the activatable antibody are not reduced, and example, TCEP. In some embodiments, the reduced activat conjugating the agent to interchain thiols in the AB. The able antibody can be purified prior to conjugation, using for conjugation site(s) are selected so as to allow desired example, column chromatography, dialysis, or diafiltration. placement of an agent to allow conjugation to occur at a In some embodiments, the reduced antibody is not purified desired site. The reducing agent is, for example, TCEP. The after partial reduction and prior to conjugation. reduction reaction conditions such as, for example, the ratio 0110. The disclosure also provides partially reduced acti of reducing agent to activatable antibody, the length of vatable antibodies in which at least one interchain disulfide incubation, the temperature during the incubation, the pH of bond in the activatable antibody has been reduced with a the reducing reaction solution, etc., are determined by iden reducing agent without disturbing any intrachain disulfide tifying the conditions that produce a conjugated activatable bonds in the activatable antibody, wherein the activatable antibody in which the MM retains the ability to effectively antibody includes an antibody or an antigen binding frag US 2016/0289.324 A1 Oct. 6, 2016

ment thereof (AB) that specifically binds to the target, a linker. In some embodiments, the MMAD payload is con masking moiety (MM) that inhibits the binding of the AB of jugated to a lysine in the AB via a linker. In some embodi the activatable antibody in an uncleaved state to the target, ments, the MMAD payload is conjugated to another residue and a CM1-CM2 substrate coupled to the AB, wherein the of the AB via a linker, such as those residues disclosed CM1-CM2 substrate is a polypeptide that functions as a herein. In some embodiments, the linker is a thiol-containing substrate for at least one MMP and one SP. In some linker. In some embodiments, the linker is a cleavable linker. embodiments, the MM is coupled to the AB via the CM1 In some embodiments, the linker is a non-cleavable linker. CM2 substrate. In some embodiments, one or more intrac In some embodiments, the linker is selected from the group hain disulfide bond(s) of the activatable antibody is not consisting of the linkers shown in Tables 5 and 6. In some disturbed by the reducing agent. In some embodiments, one embodiments, the activatable antibody and the MMAD or more intrachain disulfide bond(s) of the MM within the payload are linked via a maleimide caproyl-valine-citrulline activatable antibody is not disturbed by the reducing agent. linker. In some embodiments, the activatable antibody and In some embodiments, the activatable antibody in the the MMAD payload are linked via a maleimide PEG-valine uncleaved state has the structural arrangement from N-ter citruline linker. In some embodiments, the activatable anti minus to C-terminus as follows: MM-CM1-CM2 substrate body and the MMAD payload are linked via a maleimide AB or AB-CM1-CM2 Substrate-MM. In some embodi caproyl-valine-citrulline-para-aminobenzyloxycarbonyl ments, the reducing agent is TCEP. linker. In some embodiments, the activatable antibody and 0111. The disclosure also provides partially reduced acti the MMAD payload are linked via a maleimide PEG-valine vatable antibodies, including but not limited to multispecific citrulline-para-aminobenzyloxycarbonyl linker. In some activatable antibodies of the disclosure, in which at least one embodiments, the MMAD payload is conjugated to the AB interchain disulfide bond in the activatable antibody has using the partial reduction and conjugation technology dis been reduced with a reducing agent without disturbing or closed herein. otherwise compromising the activity and/or efficacy of the 0115 The disclosure also provides polypeptides and activatable antibody, wherein the activatable antibody other larger molecules that include one or more of the includes an antibody or an antigen binding fragment thereof CM1-CM2 substrate sequences presented herein. By way of (AB) that specifically binds to a target, a masking moiety non-limiting example, the CM1-CM2 substrate sequences (MM) that inhibits the binding of the AB of the activatable presented herein are useful in prodrug compositions and antibody in an uncleaved state to the target, and a CM1-CM2 methods of use thereof. These CM1-CM2 substrate substrate coupled to the AB, and the CM1-CM2 substrate is sequences presented herein are also useful in probes and a polypeptide that functions as a Substrate for a protease. The other detection agents and methods of use thereof. For activity and/or efficacy of the activatable antibody is, by way example, the CM1-CM2 substrate sequences presented of nonlimiting example, masking activity, activation of the herein can be used in conjunction with fluors and other activatable antibody, and/or binding activity of the activated quenchers to produce detection agents. Such as imaging activatable antibody. In some embodiments, one or more agents and/or other diagnostic agents. Those of ordinary intrachain disulfide bond(s) of the activatable antibody is not skill in the art will appreciate that the CM1-CM2 substrate disturbed by the reducing agent. In some embodiments, one sequences presented herein are useful in any composition or more intrachain disulfide bond(s) of the MM within the and/or method in the art that would use a substrate that is activatable antibody is not disturbed by the reducing agent. cleavable by at least one MMP and at least one SP. In some embodiments, the activatable antibody in the 0116. The disclosure also provides an isolated nucleic uncleaved state has the structural arrangement from N-ter acid molecule encoding an antibody and/or an activatable minus to C-terminus as follows: MM-CM1-CM2 substrate antibody described herein, as well as vectors that include AB or AB-CM1-CM2 Substrate-MM. In some embodi these isolated nucleic acid sequences. The disclosure pro ments, the reducing agent is TCEP. vides methods of producing an antibody and/or activatable 0112 The disclosure also provides conjugated activatable antibody by culturing a cell under conditions that lead to antibodies that include an activatable antibody linked to expression of the antibody and/or activatable antibody, monomethyl auristatin D (MMAD) payload, wherein the wherein the cell comprises Such a vector. activatable antibody includes an antibody or an antigen 0117 The disclosure provides a method of manufacturing binding fragment thereof (AB) that specifically binds to a a conjugated antibody of the disclosure that bind a given target, a masking moiety (MM) that inhibits the binding of target by (a) culturing a cell comprising a nucleic acid the AB of the activatable antibody in an uncleaved state to construct that encodes the antibody under conditions that the target, and CM1-CM2 substrate coupled to the AB, and lead to expression of the antibody, (i) wherein the antibody the CM1-CM2 substrate is a polypeptide that functions as a includes a CM1-CM2 substrate, and (ii) wherein the CM1 substrate for at least one MMP protease and at least one SP CM2 substrate is a polypeptide that functions as a substrate protease. for a matrix metalloprotease and a serine protease; (b) 0113. In some embodiments, the MMAD-conjugated recovering the antibody; and (c) conjugating the recovered activatable antibody can be conjugated using any of several antibody to one or more additional agents. methods for attaching agents to ABS: (a) attachment to the 0118. The disclosure also provides a method of manu carbohydrate moieties of the AB, or (b) attachment to facturing the activatable antibodies of the disclosure that Sulfhydryl groups of the AB, or (c) attachment to amino bind in an activated State a given target by (a) culturing a cell groups of the AB, or (d) attachment to carboxylate groups of comprising a nucleic acid construct that encodes the acti the AB. vatable antibody under conditions that lead to expression of 0114. In some embodiments, the MMAD payload is the activatable antibody, wherein the activatable antibody conjugated to the AB via a linker. In some embodiments, the comprises a masking moiety (MM), a CM1-CM2 substrate, MMAD payload is conjugated to a cysteine in the AB via a and an antibody or an antigenbinding fragment thereof (AB) US 2016/0289.324 A1 Oct. 6, 2016

that specifically binds the target, (i) wherein the CM1-CM2 animal. In some embodiments, the Subject is a rodent. In Substrate is a polypeptide that functions as a Substrate for a Some embodiments, the Subject is a human. In some embodi MMP and a SP: and (ii) wherein the CM1-CM2 substrate is ments, the Subject is a companion animal. In some embodi positioned in the activatable antibody Such that, in an ments, the Subject is an animal in the care of a veterinarian. uncleaved state, the MM interferes with specific binding of 0.124. The conjugated antibody, activatable antibody and/ the AB to the target and in a cleaved state the MM does not or conjugated activatable antibody and therapeutic formu interfere or compete with specific binding of the AB to the lations thereof are administered to a subject suffering from target; and (b) recovering the activatable antibody. or Susceptible to a disease or disorder associated with 0119 The disclosure also provides a method of manu aberrant target expression and/or activity. A Subject Suffering facturing the conjugated activatable antibodies of the dis from or susceptible to a disease or disorder associated with closure that bind in an activated State a given target by (a) aberrant target expression and/or activity is identified using culturing a cell comprising a nucleic acid construct that any of a variety of methods known in the art. For example, encodes the activatable antibody under conditions that lead Subjects suffering from cancer or other neoplastic condition to expression of the activatable antibody, wherein the acti are identified using any of a variety of clinical and/or Vatable antibody comprises a masking moiety (MM), a laboratory tests such as, physical examination and blood, CM1-CM2 substrate, and an antibody or an antigen binding urine and/or stool analysis to evaluate health status. For fragment thereof (AB) that specifically binds the target, (i) example, Subjects Suffering from inflammation and/or an wherein the CM1-CM2 substrate is a polypeptide that func inflammatory disorder are identified using any of a variety of tions as a substrate for a MMP and a SP; and (ii) wherein the clinical and/or laboratory tests Such as physical examination CM1-CM2 substrate is positioned in the activatable anti and/or bodily fluid analysis, e.g., blood, urine and/or stool body such that, in an uncleaved state, the MM interferes with analysis, to evaluate health status. specific binding of the AB to the target and in a cleaved state 0.125 Administration of a conjugated antibody, an acti the MM does not interfere or compete with specific binding Vatable antibody and/or a conjugated activatable antibody to of the AB to the target; (b) recovering the activatable a patient Suffering from a disease or disorder associated with antibody; and (c) conjugating the recovered antibody to one aberrant target expression and/or activity is considered Suc or more additional agents. cessful if any of a variety of laboratory or clinical objectives 0120. The disclosure provides methods of preventing, is achieved. For example, administration of a conjugated delaying the progression of treating, alleviating a symptom antibody, an activatable antibody and/or a conjugated acti of, or otherwise ameliorating a target-related disease in a Vatable antibody to a patient suffering from a disease or Subject by administering a therapeutically effective amount disorder associated with aberrant target expression and/or of a conjugated antibody, an activatable antibody and/or a activity is considered successful if one or more of the conjugated activatable antibody described herein to a subject symptoms associated with the disease or disorder is allevi in need thereof. ated, reduced, inhibited or does not progress to a further, i.e., 0121 The disclosure provides methods of preventing, worse, State. Administration of a conjugated antibody, an delaying the progression of treating, alleviating a symptom activatable antibody and/or a conjugated activatable anti of or otherwise ameliorating inflammation and/or an body to a patient Suffering from a disease or disorder inflammatory disorder in a Subject by administering a thera associated with aberrant target expression and/or activity is peutically effective amount of a conjugated antibody, an considered successful if the disease or disorder enters remis activatable antibody and/or a conjugated activatable anti sion or does not progress to a further, i.e., worse, state. body described herein to a subject in need thereof. The I0126. In some embodiments, the antibodies, conjugated disclosure also provides methods of preventing, delaying the antibodies, activatable antibodies, and/or conjugated acti progression of treating, alleviating a symptom of, or oth vatable antibodies described herein are used in conjunction erwise ameliorating cancer in a subject by administering a with one or more additional agents or a combination of therapeutically effective amount of a conjugated antibody, additional agents. Suitable additional agents include current an activatable antibody and/or a conjugated activatable pharmaceutical and/or Surgical therapies for an intended antibody described herein to a subject in need thereof. The application, such as, for example, cancer. For example, the disclosure also provides methods of preventing, delaying the antibodies, conjugated antibodies, activatable antibodies, progression of treating, alleviating a symptom of, or oth and/or conjugated activatable antibodies can be used in erwise ameliorating an autoimmune disease in a subject by conjunction with an additional chemotherapeutic or anti administering a therapeutically effective amount a conju neoplastic agent. gated antibody, an activatable antibody and/or a conjugated I0127. In some embodiments, the additional agent(s) is a activatable antibody described herein to a subject in need chemotherapeutic agent, such as a chemotherapeutic agent thereof. selected from the group consisting of docetaxel, paclitaxel, 0122. A conjugated antibody, an activatable antibody abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, and/or a conjugated activatable antibody used in any of the oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcit embodiments of these methods and uses can be administered abine. at any stage of the disease. For example, such a conjugated I0128. In some embodiments, the additional agent(s) is a antibody, activatable antibody and/or conjugated activatable checkpoint inhibitor, a kinase inhibitor, an agent targeting antibody can be administered to a patient Suffering cancer of inhibitors in the tumor microenvironment, and/or a T cell or any stage, from early to metastatic. The terms subject and NKagonist. In some embodiments, the additional agent(s) is patient are used interchangeably herein. radiation therapy, alone or in combination with another 0123. In some embodiments, the subject is a mammal, additional agent(s) Such as a chemotherapeutic or anti Such as a human, non-human primate, companion animal neoplastic agent. In some embodiments, the additional agent (e.g., cat, dog, horse), farm animal, work animal, or Zoo (s) is a vaccine, an oncovirus, and/or a DC-activating agent US 2016/0289.324 A1 Oct. 6, 2016

Such as, by way of non-limiting example, a toll-like receptor ferent times during a treatment regimen. For example, the (TLR) agonist and/or C-CD40. In some embodiments, the antibody, conjugated antibody, activatable antibody, and/or additional agent(s) is a tumor-targeted antibody designed to conjugated activatable antibody is administered prior to the kill the tumor via ADCC or via direct conjugation to a toxin administration of the additional agent, the antibody, conju (e.g., an antibody drug conjugate (ADC). gated antibody, activatable antibody, and/or conjugated acti 0129. In some embodiments, the checkpoint inhibitor is Vatable antibody is administered Subsequent to the admin an inhibitor of a target selected from the group consisting of istration of the additional agent, or the antibody, conjugated CTLA-4, LAG-3, PD-1, PD-1, TIGIT, TIM-3, B7H4, antibody, activatable antibody, and/or conjugated activatable BTLA, and Vista. In some embodiments, the kinase inhibitor antibody and the additional agent are administered in an is selected from the group consisting of B-RAFi, MEKi, and alternating fashion. As described herein, the antibody, con Btk inhibitors, such as ibrutinib. In some embodiments, the jugated antibody, activatable antibody, and/or conjugated kinase inhibitor is crizotinib. In some embodiments, the activatable antibody and additional agent are administered in tumor microenvironment inhibitor is selected from the group single doses or in multiple doses. consisting of an IDO inhibitor, an O.-CSF1R inhibitor, an I0133. In some embodiments, the antibody, conjugated C.-CCR4 inhibitor, a TGF-beta, a myeloid-derived suppres antibody, activatable antibody, and/or conjugated activatable Sor cell, or a T-regulatory cell. In some embodiments, the antibody and the additional agent(s) are administered simul agonist is selected from the group consisting of Ox40, taneously. For example, the antibody, conjugated antibody, GITR, CD137, ICOS, CD27, and HVEM. activatable antibody, and/or conjugated activatable antibody 0130. In some embodiments, the inhibitor is a CTLA-4 and the additional agent(s) can be formulated in a single inhibitor. In some embodiments, the inhibitor is a LAG-3 composition or administered as two or more separate com inhibitor. In some embodiments, the inhibitor is a PD-1 positions. In some embodiments, the antibody, conjugated inhibitor. In some embodiments, the inhibitor is a PD-1 antibody, activatable antibody, and/or conjugated activatable inhibitor. In some embodiments, the inhibitor is a TIGIT antibody and the additional agent(s) are administered inhibitor. In some embodiments, the inhibitor is a TIM-3 sequentially, or the antibody, conjugated antibody, activat inhibitor. In some embodiments, the inhibitor is a B7H4 able antibody, and/or conjugated activatable antibody and inhibitor. In some embodiments, the inhibitor is a Vista the additional agent are administered at different times inhibitor. In some embodiments, the inhibitor is a B-RAFi during a treatment regimen. inhibitor. In some embodiments, the inhibitor is a MEKi I0134. In some embodiments, the conjugated antibody, inhibitor. In some embodiments, the inhibitor is a Btk activatable antibody and/or conjugated activatable antibody inhibitor. In some embodiments, the inhibitor is ibrutinib. In is administered during and/or after treatment in combination some embodiments, the inhibitor is crizotinib. In some with one or more additional agents such as, by way of embodiments, the inhibitor is an IDO inhibitor. In some non-limiting example, an anti-inflammatory agent, an embodiments, the inhibitor is an O.-CSF1R inhibitor. In immunosuppressive agent, a chemotherapeutic agent, Such some embodiments, the inhibitor is an O.-CCR4 inhibitor. In as an alkylating agent, an anti-metabolite, an anti-microtu some embodiments, the inhibitor is a TGF-beta. In some bule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, embodiments, the inhibitor is a myeloid-derived suppressor and/or any other nucleic acid damaging agent. In some cell. In some embodiments, the inhibitor is a T-regulatory embodiments, the additional agent is a taxane, such as cell. paclitaxel (e.g., AbraxaneR). In some embodiments, the 0131. In some embodiments, the agonist is Ox40. In additional agent is an anti-metabolite, Such as gemcitabine. Some embodiments, the agonist is GITR. In some embodi In some embodiments, the additional agent is an alkylating ments, the agonist is CD137. In some embodiments, the agent, such as platinum-based chemotherapy, such as car agonist is ICOS. In some embodiments, the agonist is CD27. boplatin or cisplatin. In some embodiments, the additional In some embodiments, the agonist is HVEM. agent is a targeted agent, such as a kinase inhibitor, e.g., 0.132. In some embodiments, the antibody, conjugated Sorafenib or erlotinib. In some embodiments, the additional antibody, activatable antibody, and/or conjugated activatable agent is a targeted agent. Such as another antibody, e.g., a antibody is administered during and/or after treatment in monoclonal antibody (e.g., bevacizumab), a bispecific anti combination with one or more additional agents such as, for body, or a multispecific antibody. In some embodiments, the example, a chemotherapeutic agent, an anti-inflammatory additional agent is a proteosome inhibitor, Such as bort agent, and/or an immunosuppressive agent. In some embodi eZomib or carfilzomib. In some embodiments, the additional ments, the antibody, conjugated antibody, activatable anti agent is an immune modulating agent, Such as lenolido body, and/or conjugated activatable antibody and the addi minde or IL-2. In some embodiments, the additional agent is tional agent are formulated into a single therapeutic radiation. In some embodiments, the additional agent is an composition, and the antibody, conjugated antibody, acti agent considered Standard of care by those skilled in the art. Vatable antibody, and/or conjugated activatable antibody and In some embodiments, the additional agent is a chemothera additional agent are administered simultaneously. Alterna peutic agent well known to those skilled in the art. tively, the antibody, conjugated antibody, activatable anti I0135) In some embodiments, the additional agent is an body, and/or conjugated activatable antibody and additional antibody, another conjugated antibody, another activatable agent are separate from each other, e.g., each is formulated antibody and/or another conjugated activatable antibody. In into a separate therapeutic composition, and the antibody, Some embodiments the additional agent is an antibody, conjugated antibody, activatable antibody, and/or conju another conjugated antibody, another activatable antibody gated activatable antibody and the additional agent are and/or another conjugated activatable antibody against the administered simultaneously, or the antibody, conjugated same target as the first conjugated antibody, activatable antibody, activatable antibody, and/or conjugated activatable antibody and/or a conjugated activatable antibody. In some antibody and the additional agent are administered at dif embodiments the additional agent is an antibody, another US 2016/0289.324 A1 Oct. 6, 2016 conjugated antibody, another activatable antibody and/or in the anti-Jagged-SPDB-DM4 ADC treated group showed another conjugated activatable antibody against a target significant body weight loss, whereas the PBS, Isotype different than the target of the first conjugated antibody, SPDB-DM4 ADC, and anti-Jagged 2001 activatable anti activatable antibody and/or a conjugated activatable anti body-SPDB-DM4 treated animals showed no significant body. weight loss. 0136. In some embodiments, the conjugated antibody, 0.143 FIG. 5 is a graph depicting in vivo activation of activatable antibody and/or conjugated activatable antibody EGFR activatable antibodies measured in plasma 8 days and the additional agent(s) are administered simultaneously. post-dose of 12.5 mg/kg of such EGFR activatable antibod For example, the conjugated antibody, activatable antibody ies in H292 Xenograft tumor-bearing mice. and/or conjugated activatable antibody and the additional 014.4 FIG. 6 is a graph depicting in vivo efficacy of agent(s) can be formulated in a single composition or EGFR activatable antibodies in H292 xenograft tumor administered as two or more separate compositions. In some bearing mice. embodiments, the conjugated antibody, activatable antibody 0145 FIG. 7 is a graph depicting in situ evaluation of and/or conjugated activatable antibody and the additional EGFR activatable antibody activation in a H292 xenograft agent(s) are administered sequentially, or the antibody and/ tumor microenvironment. or conjugated antibodies and the additional agent are admin 0146 FIGS. 8A, 8B, 8C, 8D, 8E, and 8F are a series of istered at different times during a treatment regimen. For graphs depicting the tumor volume of H292 Xenograft example, the antibody and/or conjugated antibodies is tumors in nu/nu mice at various time points following administered prior to the administration of the additional administration with a control intravenous immunoglobulin agent, the antibody and/or conjugated antibodies is admin (IVIG, FIG. 8A), the anti-EGFR antibody cetuximab (FIG. istered subsequent to the administration of the additional 8B), the anti-EGFR activatable antibody referred to herein agent, or the antibody and/or conjugated antibodies and the as anti-EGFR 2001 activatable antibody, which includes the additional agent are administered in an alternating fashion. heavy chain sequence of SEQ ID NO: 108, and the light As described herein, the antibody and/or conjugated anti chain sequence of SEQ ID NO: 449 (FIG. 8C); the anti bodies and additional agent are in single doses or in multiple EGFR activatable antibody referred to herein as anti-EGFR doses. 2003 activatable antibody, which includes the heavy chain 0.137 The disclosure also provides methods and kits for sequence of SEQID NO: 108, and the light chain sequence using the conjugated antibodies, activatable antibodies and/ of SEQID NO: 472 (FIG.8D); or the anti-EGFR activatable or conjugated activatable antibodies in a variety of diagnos antibody referred to herein as anti-EGFR 2005 activatable tic and/or prophylactic indications. antibody, which includes the heavy chain sequence of SEQ 0138 Pharmaceutical compositions according to the dis ID NO: 108, and the light chain sequence of SEQ ID NO: closure can include an antibody, conjugated antibody, acti 474 (FIG.8E). For the data shown in FIGS. 8C and 8D, each Vatable antibody and/or a conjugated activatable antibody of group lost one animal each due to body weight loss. FIG. 8F the disclosure and a carrier. These pharmaceutical compo is a graph that plots and compares the data presented in sitions can be included in kits, such as, for example, diag FIGS. 8A-8E in a single graph. nostic kits. 0147 FIG. 9 is a graph depicting toxicity as measured by body weight (BW) loss following administration with an BRIEF DESCRIPTION OF THE DRAWINGS isotype control intravenous immunoglobulin (IVIG), a 20 0139 FIGS. 1A and 1B are a series of graphs depicting mg/kg dose of the anti-Jagged antibody referred to herein as the results of human Jagged 1 binding ELISA assays that 4D11, which includes the heavy chain sequence of SEQ ID demonstrate the binding of the anti-Jagged antibody and the NO: 67 and the light chain sequence of SEQ ID NO: 162, a masked and activated activatable antibodies. Both the (A) 10 mg/kg dose of the 4D11 antibody, a 5 mg/kg dose of the 2001 and (B) 1001/LP/0001 substrate-containing activat 4D11 antibody, the anti-Jagged activatable antibody referred able antibodies were activated by uPA (a serine protease), to herein as anti-Jagged 2001 activatable antibody, which MMP14 (an MMP), and uPA in combination with MMP14. includes the heavy chain sequence of SEQ ID NO: 67, and The activated activatable antibodies showed binding equiva the light chain sequence of SEQID NO: 420, the anti-Jagged lent to the anti-Jagged antibody. activatable antibody referred to herein as anti-Jagged 1004/ 0140 FIG. 2 is a graph depicting human IgG serum LP/0001 activatable antibody, which includes the heavy concentrations for anti-Jagged antibody and the 1001/LP/ chain sequence of SEQ ID NO: 67, and the light chain 0001 and 2001 activatable antibodies following a single 5 sequence of SEQ ID NO. 432, the anti-Jagged activatable mg/kg intravenous dose. The anti-Jagged antibody is rapidly antibody referred to herein as anti-Jagged 2003 activatable cleared due to target-mediated clearance. In contrast, the antibody, which includes the heavy chain sequence of SEQ anti-Jagged activatable antibodies remain masked in circu ID NO: 67, and the light chain sequence of SEQID NO: 477, lation. and the anti-Jagged activatable antibody referred to herein as 0141 FIG. 3 is a graph depicting HCC1806 tumor vol anti-Jagged 2005 activatable antibody, which includes the ume (group meant-SEM n=8) plotted vs time post initial heavy chain sequence of SEQID NO: 67, and the light chain dose. Groups were dosed on day 1 and day 8 of the study. sequence of SEQ ID NO: 479. The results are shown as The anti-Jagged-SPDB-DM4 antibody and anti-Jagged 2001 relative body weight (BW) change percent (%) at various activatable antibody-SPDB-DM4 groups both showed time points during the study. tumor regression while the isotype-SPDB-DM4 group did 0148 FIG. 10 is a graph depicting toxicity as measured not show tumor growth inhibition. by body weight (BW) loss following administration with an 0142 FIG. 4 is a graph depicting percent initial body isotype control intravenous immunoglobulin (IVIG), a 20 weight (group mean n=8) plotted VS time post initial dose. mg/kg dose of the anti-Jagged antibody referred to herein as Groups were dosed on day 1 and day 8 of the study. Animals 4D11, which includes the heavy chain sequence of SEQ ID US 2016/0289.324 A1 Oct. 6, 2016

NO: 67 and the light chain sequence of SEQID NO: 162, a binds a target. Exemplary classes of targets of an AB 10 mg/kg dose of the 4D11 antibody, a 5 mg/kg dose of the include, but are not necessarily limited to, cell Surface 4D11 antibody, the anti-Jagged activatable antibody referred receptors and secreted binding proteins (e.g., growth fac to herein as anti-Jagged 2001 activatable antibody (2001), tors), Soluble enzymes, structural proteins (e.g. collagen, which includes the heavy chain sequence of SEQID NO: 67. fibronectin) and the like. In some embodiments, conjugated and the light chain sequence of SEQ ID NO: 420, the antibodies and/or activatable antibodies have an AB that anti-Jagged activatable antibody referred to herein as anti binds an extracellular target, usually an extracellular protein Jagged 1004/LP/0001 activatable antibody, which includes target. In some embodiments, conjugated antibodies and/or the heavy chain sequence of SEQID NO: 67, and the light activatable antibodies are designed for cellular uptake and chain sequence of SEQ ID NO: 432, and the anti-Jagged are switchable inside a cell. activatable antibody referred to herein as anti-Jagged 1004/ 0156. As a non-limiting example, the AB is a binding LP/0003 activatable antibody, which includes the heavy partner for any target listed in Table 1. chain sequence of SEQ ID NO: 67, and the light chain sequence of SEQ ID NO: 424. The results are shown as TABLE 1. relative body weight (BW) change percent (%) at various time points during the study. Exemplary Targets 0149 FIGS. 11A, 11B, 11C, 11D, 11E, 11F, 11G, and 11H 1-92-LFA-3 and FIG. 12 are a series of graphs depicting the efficacy of Alpha-4 integrin Alpha-V integrin various substrates of the disclosure when incorporated in alpha4beta1 integrin activatable anti-EGFR antibodies of the disclosure. Efficacy alpha4beta7 integrin of the Substrates was evaluated by measuring tumor volume AGR2 (TV mm) at various time points post-administration. Anti-Lewis-Y 0150 FIG. 13 is a graph depicting toxicity as measured Apelin J receptor as a function of body weight (BW) loss in DBA/1 mice APRIL following administration with a control intravenous immu BAFF noglobulin (IVIG), with anti-Jagged antibodies of the dis BTLA C5 complement closure, and with activatable anti-Jagged antibodies of the C-242 disclosure that include various substrates of the disclosure. CA9 0151 FIG. 14 is a graph depicting 3D reconstruction of CA19-9 (Lewis a) FLIT-LCT imaging data for cetuximab and two EGFR Carbonic anhydrase 9 activatable antibodies containing tandem substrates in H292 D2 (open arrows) and Faldu (filled arrows) co-implanted xeno graft tumor model. DETAILED DESCRIPTION OF THE D19 INVENTION D22 D24 0152 The disclosure provides amino acid sequences that D25 include at least a first cleavable moiety (CM1) that is a D27 substrate for at least one matrix metalloprotease (MMP) and D28 at least a second cleavable moiety (CM2) that is a substrate D30 for at least one serine protease (SP). These CM1-CM2 D33 Substrates are useful in a variety of therapeutic, diagnostic D38 and prophylactic indications. For example, these CM1-CM2 D40 substrates are useful in activatable antibodies that include D41 antibodies or antigen-binding fragments thereof (AB) that D44 include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the AB Such that D47 coupling of the MM reduces the ability of the AB to bind its D52 target. D64 0153. The working examples provided herein demon D70 strate that these CM1-CM2 substrates exhibit a number of desirable cleavage characteristics when exposed to at least one MMP protease and/or at least one SP protease under D80 specified conditions. D86 0154 The disclosure also provides antibodies that D95 include one or more of these CM1-CM2 substrates. For D117 D12S example, these CM1-CM2 substrates are useful when con D132 (IL-2RG) jugating antibodies to one or more additional agents to D133 produce conjugated antibodies. These CM1-CM2 substrates D137 are also useful in activatable antibodies and/or activatable D138 D166 antibody conjugates. D172A 0155 The conjugated antibodies, activatable antibodies, D248 and/or conjugated activatable antibodies include an antibody or antigen-binding fragment thereof (AB) that specifically

US 2016/0289.324 A1 Oct. 6, 2016 20

TABLE 1-continued TABLE 2-continued Exemplary Targets Exemplary sources for Abs TRAIL-R2 Antibody Trade Name (antibody name) Target Transferrin Transferrin receptor RAV12 RAAG12 TRK-A hu591 PSMA TRK-B Enbrel TM (etanercept) TNF-R uPAR Amevive TM (alefacept) -92-LFA-3 AP1 Antril TM, Kineret TM (ankinra) L-1Ra GC1008 TGFbeta Notch, e.g., Notch 1 agged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) GF1R (tocilizumab) L-6 receptor Stelara TM (ustekinumab) L-12. IL-23 Prolia TM (denosumab) RANKL

0158 Exemplary conjugated antibodies, activatable anti bodies and/or conjugated activatable antibodies of the dis closure include, for example, antibodies that bind interleukin 6 receptor (IL-6R) and that include a heavy chain and a light 0157. As a non-limiting example, the AB is or is derived chain that are, or are derived from, the antibody referred to from an antibody listed in Table 2. herein as the “AV1 antibody, which binds interleukin-6 receptor (IL-6R). The amino acid sequences for the AV1 TABLE 2 heavy chain and the AV 1 light chain are shown below in SEQ ID NO: 100 and SEQ ID NO: 101, respectively. Exemplary sources for Abs

Antibody Trade Name (antibody name) Target Av1. Antibody Heavy Chain Amino Acid Sequence: Avastin TM (bevacizumab) VEGF (SEQ ID NO: 1.OO) Lucentis TM (ranibizumab) VEGF OVOLOESGPGLVRPSOTLSLTCTVSGYSITSDHAWSWVROPPGRGLEWIG ErbituxTM (cetuximab) EGFR VectibixTM (panitumumab) EGFR YISYSGITTYNPSLKSRVTISRDNSKNTLYLOMNSLRAEDTAVYYCARSL Remicade TM (infliximab) TNFC Humira TM (adalimumab) TNFC ARTTAMDYWGOGSLWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD Tysabri TM (natalizumab) Integrino.4 Simulect TM (basiliximab) IL2R YFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVYTVPSSSLGTOTY Soliris TM (eculizumab) Complement C5 Raptiva TM (efalizumab) CD11a ICNWNHKPSNTKWDKKWEPKSCDKTHTCPPCPAPELLGGPSWFLFPPKPK Bexxar TM (tositumomab) CD2O Zevalin TM (ibritumomab tiuxetan) CD2O DTLMISRTPEVTCVVVDVSHEDPEVKFNWYWDGVEVHNAKTKPREEOYNS Rituxan TM (rituximab) CD2O Ocerlizumab CD2O TYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOV Arzerra TM (ofatumumab) CD2O Gazyva TM (Obinutuzumab) CD2O YTLPPSREEMTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVL ZenapaxTM (daclizumab) CD25 Adcetris TM (brentuximab vedotin) CD30 DSDGSFFLYSKLTVDKSRWOOGNWFSCSVMHEALHNHYTOKSLSLSPGK Myelotarg TM (gemtuzumab) CD33 Mylotarg TM (gemtuzumab ozogamicin) CD33 Av1. Antibody Light Chain Amino Acid Sequence: Campath TM (alemtuzumab) CD52 (SEQ ID NO: 101) ReoPro TM (abiciximab) Glycoprotein receptor IIb.IIIa DIOMTOSPSSLSASVGDRVTITCRASODISSYLNWYOOKPGKAPKLLIYY XolairTM (omalizumab) gE Herceptin TM (trastuzumab) Her2 TSRLHSGVPSRFSGSGSGTDFTFTISSLOPEDIATYYCOOGNTLPYTFGO Kadcyla TM (trastuzumab emtansine) Her2 Synagis TM (palivizumab) F protein of RSV GTKVEIKRTVAAPSVFIFPPSDEOLKSGTASVWCLLNNFYPREAKVOWKV (ipilimumab) CTLA-4 (tremelimumab) CTLA-4 DNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEWTHOG HuSc8 CD4OL (pertuzumab) Her2-neu LSSPWTKSFNRGEC (ertumaXomab) CD3. Her2-neu Orencia TM (abatacept) CTLA-4 0159 Exemplary activatable antibodies and/or conju (tanezumab) NGF gated activatable antibodies of the disclosure include, for (bavituximab) Phosphatidylserine example, antibodies that bind interleukin 6 receptor (IL-6R)

(Zalutumumab) EGFR (mapatumumab) EGFR and that include a heavy chain and a light chain that are, or (matuzumab) FR are derived from, the AV 1 antibody and a masking moiety. (nimotuzumab) FR Exemplary activatable antibodies and/or conjugated activat ICR62 FR able antibodies of the disclosure include an amino acid mAb 528 CH806 FR sequence attached to the N-terminus of the AV1 light chain. MDX-447 FRfCD64 These N-terminal amino acid sequences include, for (edrecolomab) EpCAM example, YGSCSWNYVHIFMDC (SEQ ID NO: 102); QGDFDIPFPAHWVPIT (SEQ ID NO: 103); MGV US 2016/0289.324 A1 Oct. 6, 2016

PAGCVWNYAHIFMDC (SEQ ID NO: 104); QGQS - Continued GQYGSCSWNYVHIFMDC (SEQ ID NO: 105); QGQS GQGDFDIPFPAHWVPIT (SEQ ID NO: 106); or YYDYEFAYWGOGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 107). It is also to be appreciated that such amino acid YFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSWWTVPSSSLGTOTY sequences can be attached to the N-terminus of the AV1 ICNWNHKPSNTKWDKKWEPKSCDKTHTCPPCPAPELLGGPSWFLFPPKPK heavy chain or to the C-terminus of the AV1 heavy or light chain. DTLMISRTPEVTCVVVDVSHEDPEVKFNWYWDGVEVHNAKTKPREEOYAS 0160 Exemplary activatable antibodies of the disclosure TYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOV include, for example, antibodies that bind Epidermal Growth Factor Receptor (EGFR) and that include a heavy chain and YTLPPSRDELTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVL a light chain that are, or are derived from, an antibody selected from the group consisting of the antibody referred DSDGSFFLYSKLTVDKSRWOOGNWFSCSVMHEALHNHYTOKSLSLSPGK to herein as the “c225v5' antibody (also referred to herein C225 Antibody Light Chain Amino Acid Sequence: as the C225v5 antibody), the antibody referred to herein as (SEQ ID NO: 111) the “c225v4 antibody (also referred to herein as the C225v4 OILLTOSPVILSVSPGERVSFSCRASOSIGTNIHWYOORTNGSPRLLIKY antibody), and the antibody referred to herein as the ASESISGIPSRFSGSGSGTDFTLSINSWESEDIADYYCOONNNWPTTFGA “c225v6' antibody (also referred to herein as the C225v6 antibody), each of which binds EGFR. The c225v5 antibody, GTKLELKRTVAAPSVFIFPPSDEOLKSGTASVWCLLNNFYPREAKVOWKV the c225v4 antibody, and the c225v6 antibody share the same light chain sequence, referred to herein as "c225 light DNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKWYACEWTHOG chain.” The amino acid sequences for the c225v5 heavy LSSPWTKSFNRGEC chain, the c225v4 antibody, the c225v6 antibody, and the c225 light chain are shown below. 0.161 Exemplary conjugated antibodies and/or activat able antibodies of the disclosure include, for example, antibodies that bind a Jagged target, e.g., Jagged-1, Jagged-2 C225v5 Antibody Heavy Chain Amino Acid Sequence: and/or both Jagged-1 and Jagged-2, and that include a (SEQ ID NO: 108) combination of a variable heavy chain region and a variable OVOLKOSGPGLVOPSOSLSITCTVSGFSLTNYGVHWVROSPGKGLEWLGV light chain region that are, or are derived from, the variable heavy chain and variable light chain sequences shown IWSGGNTDYNTPFTSRLSINKDNSKSOVFFKMNSLOSODTAIYYCARALT below. YYDYEFAYWGOGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSWWTVPSSSLGTOTY Variable Light Chain Amino Sequence Lic4 (SEQ ID NO: 112) ICNWNHKPSNTKWDKKWEPKSCDKTHTCPPCPAPELLGGPSWFLFPPKPK DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYWDGVEVHNAKTKPREEOYNS ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

TYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOV GTKWEIKR YTLPPSRDELTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVL Variable Heavy Chain Amino Sequence Ho4 (SEQ ID NO: 113) DSDGSFFLYSKLTVDKSRWOOGNWFSCSVMHEALHNHYTOKSLSLSPG EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS

K IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDI

C225v4 Antibody Heavy Chain Amino Acid Sequence: GGRSAFDYWGOGTLVTVSS (SEQ ID NO: 109) OVOLKOSGPGLVOPSOSLSITCTVSGFSLTNYGVHWVROSPGKGLEWLGV Variable Light Chain Amino Sequence Lic5 (SEQ ID NO: 114) IWSGGNTDYNTPFTSRLSINKDNSKSOVFFKMNSLOSNDTAIYYCARALT DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA

YYDYEFAYWGOGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

YFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSWWTVPSSSLGTOTY GTKWEIKR

ICNWNHKPSNTKWDKKWEPKSCDKTHTCPPCPAPELLGGPSWFLFPPKPK Variable Heavy Chain Amino Sequence Ho5 (SEQ ID NO: 115) DTLMISRTPEVTCVVVDVSHEDPEVKFNWYWDGVEVHNAKTKPREEOYNS EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS

TYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGOPREPOV IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKSP

YTLPPSRDELTKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVL PYHGOFDYWGOGTLVTVSS

DSDGSFFLYSKLTVDKSRWOOGNWFSCSVMHEALHNHYTOKSLSLSPGK Variable Light Chain Amino Sequence Lic7 (SEQ ID NO: 116) C225v6 Antibody Heavy Chain Amino Acid Sequence: DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA (SEQ ID NO: 110) OVOLKOSGPGLVOPSOSLSITCTVSGFSLTNYGVHWVROSPGKGLEWLGV ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

IWSGGNTDYNTPFTSRLSINKDNSKSOVFFKMNSLOSODTAIYYCARALT GTKWEIKR US 2016/0289.324 A1 Oct. 6, 2016 22

- Continued - Continued Variable Heavy Chain Amino Sequence Ho 7 Variable Light Chain Amino Sequence Lic21 (SEO ID NO : 117) (SEQ ID NO: 126) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA

IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKSP ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

PFFGOFDYWGOGTLVTVSS GTKWEIKR Variable Light Chain Amino Sequence Lic8 Variable Heavy Chain Amino Sequence Ho:21 (SEQ ID NO: 118) (SEO ID NO: 127) DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS

ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDI

GTKWEIKR GGRSAFDYWGOGTLVTVSS Variable Heavy Chain Amino Sequence Ho 8 Variable Light Chain Amino Sequence Lic24 (SEQ ID NO: 119) (SEQ ID NO: 128) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA

IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKHI ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

GRTNPFDYWGOGTLVTVSS GTKWEIKR Variable Light Chain Amino Sequence Lic13 Variable Heavy Chain Amino Sequence Ho24 (SEQ ID NO: 12O) (SEQ ID NO: 129) DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS

ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO IEEMGWOTLYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKSA

GTKWEIKR AAFDYWGOGTLWTVSS Variable Heavy Chain Amino Sequence Ho13 Variable Light Chain Amino Sequence Lic26 (SEQ ID NO: 121) (SEQ ID NO: 130) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA

IEOMGWOTEYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKSA ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

AAFDYWGOGTLVTVSS GTKWEIKR Variable Light Chain Amino Sequence Lic16 Variable Heavy Chain Amino Sequence Ho:26 (SEQ ID NO: 122) (SEQ ID NO: 131) DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS

ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDI

GTKWEIKR GGRSAFDYWGOGTLVTVSS Variable Heavy Chain Amino Sequence Ho16 Variable Light Chain Amino Sequence Lic27 (SEQ ID NO: 123) (SEQ ID NO: 132) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA

IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKSP ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

PYYGOFDYWGOGTLVTVSS GTKWEIKR Variable Light Chain Amino Sequence Lic 19 Variable Heavy Chain Amino Sequence Ho27 (SEQ ID NO: 124) (SEQ ID NO: 133) DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS

ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKSP

GTKWEIKR PFYGOFDYWGOGTLVTVSS Variable Heavy Chain Amino Sequence Hol9 Variable Light Chain Amino Sequence Lic28 (SEQ ID NO: 125) (SEQ ID NO: 134) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSS DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYA

IEOMGWOTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKSP ASSLOSGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCOOSVVAPLTFGO

PFFGOFDYWGOGTLVTVSS GTKWEIKR

US 2016/0289.324 A1 Oct. 6, 2016 26

NO: 298); DGGPAGCSWNYVHIFMEC (SEQ ID NO: of the parental AB to the target. When compared to the 299); AVGPAGCWWNYVHIFMEC (SEQ ID NO: 300); binding of the AB not modified with an MM or the binding CTWNYVHIFMDCGEGEGP (SEQ ID NO: 301); of the parental AB to the target the AB’s ability to bind the GGVPEGCTWNYAHIFMEC (SEQ ID NO: 302); AEV target when modified with an MM can be reduced by at least PAGCW WNYVHIFMEC (SEQ ID NO: 303); AGV 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, PAGCTWNYVHIFMEC (SEQID NO:304); SGASGGCK 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, WNYWHIFMDC (SEQ ID NO: 305); 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, TPGCRWNYVHIFMECEAL (SEQ ID NO: 306); 60,90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, VGVPNGCVWNYAHIFMEC (SEQ ID NO:307); PGAF 11, or 12 months or more when measured in Vivo or in an in DIPFPAHWVPNT (SEQ ID NO:308); RGACDIPFPAH vitro assay. WIPNT (SEQID NO:309); QGDFDIPFPAHWVPIT (SEQ (0171 The MM inhibits the binding of the AB to the ID NO:310); XGafDIPFPAHWvPnT (SEQ ID NO:311): target. The MM binds the antigen binding domain of the AB RGDGNDSDIPFPAHWVPRT (SEQ ID NO. 312); and inhibits binding of the AB to the target. The MM can SGVGRDRDIPFPAHWVPRT (SEQ ID NO:313); WAG sterically inhibit the binding of the AB to the target. The MM GNDCDIPFPAHWIPNT (SEQ ID NO:314); WGDGMD can allosterically inhibit the binding of the AB to its target. VDIPFPAHWVPVT (SEQ ID NO: 315): AGSGNDSDIP In these embodiments when the AB is modified or coupled FPAHWVPRT (SEQ ID NO: 316): to a MM and in the presence of target there is no binding or ESRSGYADIPFPAHWVPRT (SEQ ID NO: 317); and/or substantially no binding of the AB to the target, or no more RECGRCGDIPFPAHWVPRT (SEQ ID NO:318). than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, (0167. When the AB is modified with a MM and is in the 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% presence of the target, specific binding of the AB to its target binding of the AB to the target, as compared to the binding is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM, the parental AB, or the of the AB not modified with an MM or the specific binding AB not coupled to an MM to the target, for at least 2, 4, 6, of the parental AB to the target. 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, (0168 The K of the AB modified with a MM towards the 15, 30, 45, 60,90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 7, 8, 9, 10, 11, or 12 months or longer when measured in 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000, Vivo or in an in vitro assay. 000, 10,000,000, 50,000,000 or greater, or between 5-10, (0172. When an AB is coupled to or modified by a MM, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, the MM masks or reduces or otherwise inhibits the specific 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100 binding of the AB to the target. When an AB is coupled to 1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, or modified by a MM, such coupling or modification can 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, effect a structural change that reduces or inhibits the ability 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, of the AB to specifically bind its target. or 100,000-10,000,000 times greater than the K of the AB (0173 An AB coupled to or modified with an MM can be not modified with an MM or of the parental AB towards the represented by the following formulae (in order from an target. Conversely, the binding affinity of the AB modified amino (N) terminal region to carboxyl (C) terminal region: with a MM towards the target is at least 2, 3, 4, 5, 10, 20, 0.174 (MM)-(AB) 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, (0175 (AB)-(MM) 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000, (0176 (MM)-L-(AB) 000, 50,000,000 or greater, or between 5-10, 10-100, 10-1, 0177 (AB)-L-(MM) 000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, where MM is a masking moiety, the AB is an antibody or 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100 antibody fragment thereof, and the L is a linker. In many 10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, embodiments, it may be desirable to insert one or more 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, linkers, e.g., flexible linkers, into the composition so as to 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000, provide for flexibility. 000 times lower than the binding affinity of the AB not 0.178 In certain embodiments, the MM is not a natural modified with an MM or of the parental AB towards the binding partner of the AB. In some embodiments, the MM target. contains no or Substantially no homology to any natural 0169. The dissociation constant (K) of the MM towards binding partner of the AB. In some embodiments, the MM the AB is generally greater than the K of the AB towards the is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, target. The K of the MM towards the AB can be at least 5, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, any natural binding partner of the AB. In some embodi 100,000, 1,000,000 or even 10,000,000 times greater than ments, the MM is no more than 5%, 10%, 15%, 20%, 25%, the K of the AB towards the target. Conversely, the binding 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or affinity of the MM towards the AB is generally lower than 80% identical to any natural binding partner of the AB. In the binding affinity of the AB towards the target. The binding some embodiments, the MM is no more than 25% identical affinity of MM towards the AB can be at least 5, 10, 25, 50, to any natural binding partner of the AB. In some embodi 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000, ments, the MM is no more than 50% identical to any natural 000 or even 10,000,000 times lower than the binding affinity binding partner of the AB. In some embodiments, the MM of the AB towards the target. is no more than 20% identical to any natural binding partner (0170 When the AB is modified with a MM and is in the of the AB. In some embodiments, the MM is no more than presence of the target specific binding of the AB to its target 10% identical to any natural binding partner of the AB. is reduced or inhibited, as compared to the specific binding 0179. In some embodiments, the activatable antibodies of the AB not modified with an MM or the specific binding include an AB that is modified by an MM and also includes US 2016/0289.324 A1 Oct. 6, 2016 27 at least one cleavable moiety (CM1) that is a substrate for at tease. The term uncleaved state or fully uncleaved, as used least one matrix metalloprotease (MMP) and at least a herein, refers to the condition of the activatable antibodies in second cleavable moiety (CM2) that is a subject for at least the absence of cleavage of the CM1-CM2 substrate by a one serine protease (SP). Such activatable antibodies exhibit MMP and/or a SP. As discussed above, the term “activatable activatable/switchable binding, to the AB’s target. Activat antibodies’ is used herein to refer to an activatable antibody able antibodies generally include an antibody or antibody in both its uncleaved (native) state, as well as in its cleaved fragment (AB), modified by or coupled to a masking moiety state. An activatable antibody in its cleaved State is also (MM) and a CM1-CM2 substrate. referred to herein as an activated antibody and/or activated 0180. The elements of the activatable antibodies are activatable antibody. It will be apparent to the ordinarily arranged so that the MM and CM1-CM2 substrate are skilled artisan that in Some embodiments, a cleaved activat positioned such that in a cleaved (or relatively active) state able antibody may lack an MM due to cleavage of the and in the presence of a target, the AB binds a target while CM1-CM2 substrate by protease, resulting in release of at in an uncleaved (or relatively inactive) state in the presence least the MM. of the target, specific binding of the AB to its target is 0184. By activatable or switchable is meant that the reduced or inhibited. The specific binding of the AB to its activatable antibody exhibits a first level of binding to a target can be reduced due to the inhibition or masking of the target when in a inhibited, masked or uncleaved State (i.e., AB’s ability to specifically bind its target by the MM. a first conformation), and a second level of binding to the 0181. The K of the AB modified with a MM and a target in the uninhibited, unmasked and/or cleaved state (i.e., CM1-CM2 substrate towards the target is at least 5, 10, 20, a second conformation), where the second level of target 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, binding is greater than the first level of binding. In general, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000, the access of target to the AB of the activatable antibody is 000, 50,000,000 or greater, or between 5-10, 10-100, 10-1, greater in the presence of a cleaving agent capable of 000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, cleaving the CM1-CM2 substrate than in the absence of such 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100 a cleaving agent. Thus, when the activatable antibody is in 10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, the uncleaved state, the AB is inhibited from target binding 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, and can be masked from target binding (i.e., the first 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000, conformation is such the AB cannot bind the target), and in 000 times greater than the K of the AB not modified with the cleaved state the AB is not inhibited or is unmasked to an MM and a CM1-CM2 substrate or of the parental AB target binding. towards the target. Conversely, the binding affinity of the AB 0185. The CM1-CM2 substrate and AB of the activatable modified with a MM and a CM1-CM2 substrate towards the antibodies are selected so that the AB represents a binding target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, moiety for a given target, and the CM1-CM2 substrate 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, represents a substrate for a MMP and a SP, where the MMP 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or and/or the SP are co-localized with the target at a treatment between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, site or diagnostic site in a subject. The activatable antibodies 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100 disclosed herein find particular use where, for example, a 100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, MMP and a SP, each capable of cleaving a site in the 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000 CM1-CM2 substrate, are present at relatively higher levels 100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1, in target-containing tissue of a treatment site or diagnostic 000,000, or 100,000-10,000,000 times lower than the bind site than in tissue of non-treatment sites (for example in ing affinity of the AB not modified with an MM and a healthy tissue). CM1-CM2 substrate or of the parental AB towards the 0186. In some embodiments, activatable antibodies pro target. vide for reduced toxicity and/or adverse side effects that 0182. When the AB is modified with a MM and a could otherwise result from binding of the AB at non CM1-CM2 substrate and is in the presence of the target but treatment sites if the AB were not masked or otherwise not in the presence of a modifying agent (for example a inhibited from binding to the target. MMP and a SP), specific binding of the AB to its target is 0187. In general, an activatable antibody can be designed reduced or inhibited, as compared to the specific binding of by selecting an AB of interest and constructing the remain the AB not modified with an MM and a CM1-CM2 Substrate der of the activatable antibody so that, when conformation or of the parental AB to the target. When compared to the ally constrained, the MM provides for masking of the AB or binding of the parental AB or the binding of an AB not reduction of binding of the AB to its target. Structural design modified with an MM and a CM1-CM2 substrate to its criteria can be to be taken into account to provide for this target, the AB’s ability to bind the target when modified with functional feature. an MM and a CM1-CM2 substrate can be reduced by at least 0188 Activatable antibodies exhibiting a switchable phe 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, notype of a desired dynamic range for target binding in an 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, inhibited versus an uninhibited conformation are provided. 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45, Dynamic range generally refers to a ratio of (a) a maximum 60,90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, detected level of a parameter under a first set of conditions 11, or 12 months or longer when measured in vivo or in an to (b) a minimum detected value of that parameter under a in vitro assay. second set of conditions. For example, in the context of an 0183. As used herein, the term cleaved state refers to the activatable antibody, the dynamic range refers to the ratio of condition of the activatable antibodies following modifica (a) a maximum detected level of target protein binding to an tion, i.e., cleavage, of the CM1-CM2 substrate by at least activatable antibody in the presence of a MMP and a SP that one matrix metalloprotease and/or at least one serine pro are capable of cleaving the CM1-CM2 substrate of the US 2016/0289.324 A1 Oct. 6, 2016 28 activatable antibodies to (b) a minimum detected level of 80% identical to any natural binding partner of the AB. In target protein binding to an activatable antibody in the some embodiments, the MM is no more than 50% identical absence of the protease. The dynamic range of an activatable to any natural binding partner of the AB. In some embodi antibody can be calculated as the ratio of the dissociation ments, the MM is no more than 25% identical to any natural constant of an activatable antibody cleaving agent (e.g., binding partner of the AB. In some embodiments, the MM enzyme) treatment to the dissociation constant of the acti is no more than 20% identical to any natural binding partner Vatable antibodies cleaving agent treatment. The greater the of the AB. In some embodiments, the MM is no more than dynamic range of an activatable antibody, the better the 10% identical to any natural binding partner of the AB. switchable phenotype of the activatable antibody. Activat 0194 In many embodiments, it may be desirable to insert able antibodies having relatively higher dynamic range one or more linkers, e.g., flexible linkers, into the activatable values (e.g., greater than 1) exhibit more desirable Switching antibody construct so as to provide for flexibility at one or phenotypes such that target protein binding by the activat more of the MM-CM1-CM2 substrate junction, the CM1 able antibodies occurs to a greater extent (e.g., predomi CM2 substrate-AB junction, or both. For example, the AB, nantly occurs) in the presence of a cleaving agent (e.g., MM, and/or CM1-CM2 substrate may not contain a suffi enzyme) capable of cleaving the CM1-CM2 substrate of the cient number of residues (e.g., Gly, Ser, Asp, ASn, especially activatable antibodies than in the absence of a cleaving Gly and Ser, particularly Gly) to provide the desired flex agent. ibility. As such, the switchable phenotype of such activatable 0189 Activatable antibodies can be provided in a variety antibody constructs may benefit from introduction of one or of structural configurations. Exemplary formulae for acti more amino acids to provide for a flexible linker. In addition, vatable antibodies are provided below. It is specifically as described below, where the activatable antibody is pro contemplated that the N- to C-terminal order of the AB, MM vided as a conformationally constrained construct, a flexible and CM1-CM2 substrate may be reversed within an acti linker can be operably inserted to facilitate formation and vatable antibody. It is also specifically contemplated that the maintenance of a cyclic structure in the uncleaved activat CM and MM may overlap in amino acid sequence, e.g., Such able antibody. that the CM1-CM2 substrate is at least partially contained 0.195 For example, in certain embodiments, an activat within the MM. able antibody comprises one of the following formulae 0190. For example, activatable antibodies can be repre (where the formula below represent an amino acid sequence sented by the following formula (in order from an amino (N) in either N- to C-terminal direction or C- to N-terminal terminal region to carboxyl (C) terminal region: direction): (0191 (MM)-(CM1-CM2 substrate)-(AB) (0196) (MM)-L1-(CM1-CM2 substrate)-(AB) (0192 (AB)-(CM1-CM2 substrate)-(MM) (0197) (MM)-(CM1-CM2 substrate)-L2-(AB) where MM is a masking moiety, the CM1-CM2 substrate is a cleavable moiety, and AB is an antibody or fragment (0198 (MM)-L1-(CM1-CM2 substrate)-L2-(AB) thereof. As noted above, the term “CM1-CM2 substrate” is wherein MM, CM1-CM2 substrate, and AB are as defined not intended to convey any requirement regarding the ori above; wherein L1 and L2 are each independently and entation or other structural arrangement of the first cleavable optionally present or absent, are the same or different moiety (CM1) that is a substrate for at least one matrix flexible linkers that include at least 1 flexible amino acid metalloprotease (MMP) and at least a second cleavable (e.g., Gly). In addition, the formulae above provide for moiety (CM2) that is a substrate for at least one serine additional amino acid sequences that may be positioned protease (SP). Thus, the term “CM1-CM2 substrates' N-terminal or C-terminal to the activatable antibodies ele encompasses CM1-CM2 substrates having the structural ments. Examples include, but are not limited to, targeting arrangement from N-terminus to C-terminus as follows: moieties (e.g., a ligand for a receptor of a cell present in a CM1-CM2 or CM2-CM1. The term “CM1-CM2 Substrates’ target tissue) and serum half-life extending moieties (e.g., also encompasses substrates where at least a portion of the polypeptides that bind serum proteins, such as immuno CM1 sequence overlaps with at least a portion of the CM2 globulin (e.g., IgG) or serum albumin (e.g., human serum sequence. It should also be noted that although MM and albumin (HAS)). CM1-CM2 substrate are indicated as distinct components in (0199 The CM1-CM2 substrate is specifically cleaved by the formulae above, in all exemplary embodiments (includ at least one MMP at a rate of about 0.001-1500x10" M'S' ing formulae) disclosed herein it is contemplated that the or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, amino acid sequences of the MM and the CM1-CM2 sub 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, strate could overlap, e.g., such that the CM1-CM2 substrate 1250, or 1500x10" M'S' and is specifically cleaved by at is completely or partially contained within the MM. In least one SP at a rate of about 0.001-1500x10" M'S' or at addition, the formulae above provide for additional amino least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, acid sequences that may be positioned N-terminal or C-ter 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, minal to the activatable antibodies elements. 1250, or 1500x10 MS. 0193 In certain embodiments, the MM is not a natural 0200 For specific cleavage by an enzyme, contact binding partner of the AB. In some embodiments, the MM between the enzyme and CM1-CM2 substrate is made. contains no or Substantially no homology to any natural When the activatable antibody comprising an AB coupled to binding partner of the AB. In some embodiments, the MM a MM and a CM1-CM2 substrate is in the presence of target is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, and sufficient enzyme activity, the CM1-CM2 substrate can 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to be cleaved. Sufficient enzyme activity can refer to the ability any natural binding partner of the AB. In some embodi of the enzyme to make contact with the CM1-CM2 substrate ments, the MM is no more than 5%, 10%, 15%, 20%, 25%, and effect cleavage. It can readily be envisioned that an 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or enzyme may be in the vicinity of the CM1-CM2 substrate US 2016/0289.324 A1 Oct. 6, 2016 29 but unable to cleave because of other cellular factors or 0205. In some embodiments, the agent is a detectable protein modification of the enzyme. moiety Such as, for example, a label or other marker. For 0201 Linkers suitable for use in compositions described example, the agent is or includes a radiolabeled amino acid, herein are generally ones that provide flexibility of the one or more biotinyl moieties that can be detected by marked modified AB or the activatable antibodies to facilitate the avidin (e.g., Streptavidin containing a fluorescent marker or inhibition of the binding of the AB to the target. Such linkers enzymatic activity that can be detected by optical or calo are generally referred to as flexible linkers. Suitable linkers rimetric methods), one or more radioisotopes or radionu can be readily selected and can be of any of a suitable of clides, one or more fluorescent labels, one or more enzy different lengths, such as from 1 amino acid (e.g., Gly) to 20 matic labels, and/or one or more chemiluminescent agents. amino acids, from 2 amino acids to 15 amino acids, from 3 In some embodiments, detectable moieties are attached by amino acids to 12 amino acids, including 4 amino acids to spacer molecules. 10 amino acids, 5 amino acids to 9 amino acids, 6 amino 0206. The disclosure also pertains to immunoconjugates acids to 8 amino acids, or 7 amino acids to 8 amino acids, comprising an antibody conjugated to a cytotoxic agent Such and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, as a toxin (e.g., an enzymatically active toxin of bacterial, 17, 18, 19, or 20 amino acids in length. fungal, plant, or animal origin, or fragments thereof), or a 0202) Exemplary flexible linkers include glycine poly radioactive isotope (i.e., a radioconjugate). Suitable cyto mers (G)n, glycine-serine polymers (including, for example, toxic agents include, for example, dolastatins and deriva (GS)n, (GSGGS)n (SEQ ID NO: 381) and (GGGS)n (SEQ tives thereof (e.g. auristatin E, AFP MMAF, MMAE, ID NO: 382), where n is an integer of at least one), MMAD, DMAF, DMAE). For example, the agent is glycine-alanine polymers, alanine-serine polymers, and monomethyl auristatin E (MMAE) or monomethyl auristatin other flexible linkers known in the art. Glycine and glycine D (MMAD). In some embodiments, the agent is an agent serine polymers are relatively unstructured, and therefore selected from the group listed in Table 3. In some embodi may be able to serve as a neutral tether between components. ments, the agent is a dolastatin. In some embodiments, the Glycine accesses significantly more phi-psi space than even agent is an auristatin or derivative thereof. In some embodi alanine, and is much less restricted than residues with longer ments, the agent is auristatin E or a derivative thereof. In side chains (see Scheraga, Rev. Computational Chem. Some embodiments, the agent is monomethyl auristatin E 11173-142 (1992)). Exemplary flexible linkers include, but (MMAE). In some embodiments, the agent is monomethyl are not limited to Gly-Gly-Ser-Gly (SEQ ID NO: 383), auristatin D (MMAD). In some embodiments, the agent is a Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 384, Gly-Ser-Gly-Ser maytansinoid or maytansinoid derivative. In some embodi Gly (SEQID NO:385), Gly-Ser-Gly-Gly-Gly (SEQID NO: ments, the agent is DM1 or DM4. In some embodiments, the 386), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 387), Gly-Ser agent is a duocarmycin or derivative thereof. In some Ser-Ser-Gly (SEQID NO:388), and the like. The ordinarily embodiments, the agent is a calicheamicin or derivative skilled artisan will recognize that design of an activatable thereof. In some embodiments, the agent is a pyrroloben antibodies can include linkers that are all or partially flex Zodiazepine. In some embodiments, the agent is a pyr ible, such that the linker can include a flexible linker as well rolobenzodiazepine dimer. as one or more portions that confer less flexible structure to provide for a desired activatable antibodies structure. 0207. In some embodiments, the agent is linked to the AB 0203. In some embodiments, the activatable antibodies using a maleimide caproyl-valine-citrulline linker or a described herein also include an agent conjugated to the maleimide PEG-valine-citrulline linker. In some embodi activatable antibody. In some embodiments, the conjugated ments, the agent is linked to the AB using a maleimide agent is a therapeutic agent, such as an anti-inflammatory caproyl-valine-citrulline linker. In some embodiments, the and/or an antineoplastic agent. In Such embodiments, the agent is linked to the AB using a maleimide PEG-valine agent is conjugated to a carbohydrate moiety of the activat citrulline linker In some embodiments, the agent is monom able antibody, for example, in some embodiments, where the ethyl auristatin D (MMAD) linked to the AB using a carbohydrate moiety is located outside the antigen-binding maleimide PEG-valine-citruline-para-aminobenzyloxycar region of the antibody or antigen-binding fragment in the bonyl linker, and this linker payload construct is referred to activatable antibody. In some embodiments, the agent is herein as “vc-MMAD. In some embodiments, the agent is conjugated to a sulfhydryl group of the antibody or antigen monomethyl auristatin E (MMAE) linked to the AB using a binding fragment in the activatable antibody. maleimide PEG-valine-citruline-para-aminobenzyloxycar 0204. In some embodiments, the agent is a cytotoxic bonyl linker, and this linker payload construct is referred to agent Such as a toxin (e.g., an enzymatically active toxin of herein as “vc-MMAE. The structures of vic-MMAD and bacterial, fungal, plant, or animal origin, or fragments vc-MMAE are shown below: thereof), or a radioactive isotope (i.e., a radioconjugate). vc-MMAD:

e O o-N NHS- N O M O O I 3 r is 8 -NH -- NH--- s NH t 2

Orals NH US 2016/0289.324 A1 Oct. 6, 2016 30 vc-MMAE:

0208. Enzymatically active toxins and fragments thereof TABLE 3-continued that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, eXotoxin A chain (from Exemplary Pharmaceutical Agents for Conjugation Pseudomonas aeruginosa), ricin A chain, abrin A chain, Dolastatin 16 Dm modeccin. A chain, alpha-sarcin, Aleurites fordii proteins, Dolastatin 16 Dpv dianthin proteins, Phytolaca americana proteins (PAPI, Maytansinoids, e.g. DM-1: DM-4 Maytansinoid derivatives PAPII, and PAP-S), momordica charantia inhibitor, curcin, Duocarmycin crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, Duocarmycin derivatives restrictocin, phenomycin, enomycin, and the tricothecenes. Alpha-amanitin A variety of radionuclides are available for the production of Anthracyclines Doxorubicin radioconjugated antibodies. Examples include 'Bi, ''I, Daunorubicin In, Y, and Re. Bryostatins 0209 Conjugates of the antibody and cytotoxic agent are Camptothecin Camptothecin derivatives made using a variety of bifunctional protein-coupling agents 7-substituted Camptothecin such as N-succinimidyl-3-(2-pyridyldithiol) propionate 0,11-Difluoromethylenedioxycamptothecin (SPDP), iminothiolane (IT), bifunctional derivatives of imi Combretastatins doesters (such as dimethyl adipimidate HCL), active esters Debromoaplysiatoxin Kahalalide-F (such as disuccinimidyl Suberate), aldehydes (such as glu Discodermollide tareldehyde), bis-azido compounds (such as bis (p-azido Ecteinascidins benzoyl) hexanediamine), bis-diazonium derivatives (such ANTIVIRALS as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocya Acyclovir nates (such as tolyene 2,6-diisocyanate), and bis-active Vira A fluorine compounds (such as 1,5-difluoro-2,4-dinitroben Symmetrel Zene). For example, a ricin immunotoxin can be prepared as ANTIFUNGALS described in Vitetta et al., Science 238: 1098 (1987). Car bon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene Nystatin triaminepentaacetic acid (MX-DTPA) is an exemplary ADDITIONAL ANTI-NEOPLASTICS chelating agent for conjugation of radionucleotide to the Adriamycin Cerubidine antibody. (See WO94/11026). Bleomycin 0210 Table 3 lists some of the exemplary pharmaceutical Alkeran agents that may be employed in the herein described dis Welban Oncovin closure but in no way is meant to be an exhaustive list. Fluorouracil Methotrexate TABLE 3 Thiotepa Bisantrene Exemplary Pharmaceutical Agents for Conjugation Nowantrone Thioguanine CYTOTOXICAGENTS Procarabizine Cytarabine Auristatins ANTI-BACTERIALS Auristatin E Monomethyl auristatin D (MMAD) Aminoglycosides Monomethyl auristatin E (MMAE) Streptomycin Desmethyl auristatin E (DMAE) Auristatin F Neomycin Monomethyl auristatin F (MMAF) Kanamycin Desmethyl auristatin F (DMAF) Amikacin Auristatin derivatives, e.g., amides thereof Gentamicin Auristatin tyramine Tobramycin Auristatin quinolone Streptomycin B Dolastatins Spectinomycin Dolastatin derivatives Ampicillin US 2016/0289.324 A1 Oct. 6, 2016 31

TABLE 3-continued Press, New York, (1989), the entire contents of which are incorporated herein by reference). Exemplary Pharmaceutical Agents for Conjugation 0212 Coupling may be accomplished by any chemical Sulfanilamide reaction that will bind the two molecules so long as the Polymyxin antibody and the other moiety retain their respective activi Chloramphenicol ties. This linkage can include many chemical mechanisms, Turbostatin for instance covalent binding, affinity binding, intercalation, Phenstatins coordinate binding and complexation. In some embodi Hydroxyphenstatin ments, the binding is, however, covalent binding. Covalent Spongistatin 5 Spongistatin 7 binding can be achieved either by direct condensation of Halistatin 1 existing side chains or by the incorporation of external Halistatin 2 bridging molecules. Many bivalent or polyvalent linking Halistatin 3 agents are useful in coupling protein molecules, such as the Modified Bryostatins antibodies of the present disclosure, to other molecules. For HalocomStatins example, representative coupling agents can include organic Pyrrolobenzimidazoles (PBI) compounds such as thioesters, carbodiimides, succinimide Cibrostatinó esters, diisocyanates, glutaraldehyde, diazobenzenes and Doxaliform Anthracyclins analogues hexamethylene diamines. This listing is not intended to be Cemadotin analogue (CemcH2-SH) exhaustive of the various classes of coupling agents known Pseudomonas toxin A (PE38) variant in the art but, rather, is exemplary of the more common Pseudomonas toxin A (ZZ-PE38) variant coupling agents. (See Killen and Lindstrom, Jour. Immun. ZJ-101 133: 1335-2549 (1984); Jansen et al., Immunological OSW-1 4-Nitrobenzyloxycarbonyl Derivatives of O6-Benzylguanine Reviews 62:185-216 (1982); and Vitetta et al., Science Topoisomerase inhibitors 238:1098 (1987). Hemiasterlin 0213. In some embodiments, in addition to the compo Cephalotaxine sitions and methods provided herein, the conjugated acti Homoharringtonine vatable antibody can also be modified for site-specific Pyrrolobenzodiazepine (PBD) conjugation through modified amino acid sequences inserted Pyrrolobenzodiazepine (PBD) dimers or otherwise included in the activatable antibody sequence. Functionalized pyrrolobenzodiazepenes These modified amino acid sequences are designed to allow Functionalized pyrrolobenzodiazepene dimers Calicheamicins for controlled placement and/or dosage of the conjugated Podophyllotoxins agent within a conjugated activatable antibody. For example, Taxanes the activatable antibody can be engineered to include cys Vinca alkaloids teine Substitutions at positions on light and heavy chains that CONUGATABLE DETECTION REAGENTS provide reactive thiol groups and do not negatively impact protein folding and assembly, nor alter antigen binding. In Fluorescein and derivatives thereof Some embodiments, the activatable antibody can be engi Fluorescein isothiocyanate (FITC) neered to include or otherwise introduce one or more RADIOPHARMACEUTICALS non-natural amino acid residues within the activatable anti body to provide Suitable sites for conjugation. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce enzymatically activatable peptide sequences within the activatable antibody sequence. 0214 Suitable linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201 208 (1984) describing use of MBS (M-maleimidobenzoyl N-hydroxysuccinimide ester). See also, U.S. Pat. No. 5,030, 719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. In some embodiments, suitable linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-al pha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. 82Rb Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 3-(2- 'mTc (Technetium) pyridyldithio) propionamidohexanoate (Pierce Chem. Co., HEAVYMETALS Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 Barium 3-(2-pyridyldithio)-propianamide hexanoate (Pierce Chem. Gold Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo Platinum succinimide: Pierce Chem. Co., Cat. #24510) conjugated to ANTI-MYCOPLASMALS EDC. Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB. Tylosine 0215. The linkers described above contain components Spectinomycin that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, Sulfo NHS esters of alkyl carboxylates are more stable than 0211 Those of ordinary skill in the art will recognize that sulfo-NHS esters of aromatic carboxylates. NHS-ester con a large variety of possible moieties can be coupled to the taining linkers are less soluble than sulfo-NHS esters. Fur resultant antibodies of the disclosure. (See, for example, ther, the linker SMPT contains a sterically hindered disulfide “Conjugate Vaccines”. Contributions to Microbiology and bond, and can form conjugates with increased stability. Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Disulfide linkages, are in general, less stable than other US 2016/0289.324 A1 Oct. 6, 2016 32 linkages because the disulfide linkage is cleaved in vitro, with Suitable reagents. Suitable activating reagents include resulting in less conjugate available. Sulfo-NHS, in particu EDC, with or without added NHS or sulfo-NHS, and other lar, can enhance the stability of carbodimide couplings. dehydrating agents utilized for carboxamide formation. In Carbodimide couplings (such as EDC) when used in con these instances, the functional groups present in the Suitable junction with sulfo-NHS, forms esters that are more resistant linkers would include primary and secondary amines, hydra to hydrolysis than the carbodimide coupling reaction alone. Zines, hydroxylamines, and hydrazides. 0216. In some embodiments, the linkers are cleavable. In 0221) The agent may be attached to the linker before or Some embodiments, the linkers are non-cleavable. In some after the linker is attached to the AB. In certain applications embodiments, two or more linkers are present. The two or it may be desirable to first produce an AB-linker interme more linkers are all the same, i.e., cleavable or non-cleav diate in which the linker is free of an associated agent. able, or the two or more linkers are different, i.e., at least one Depending upon the particular application, a specific agent cleavable and at least one non-cleavable. may then be covalently attached to the linker. In some 0217. The present disclosure utilizes several methods for embodiments, the AB is first attached to the MM, CM1 attaching agents to ABS: (a) attachment to the carbohydrate CM2 substrate and associated linkers and then attached to moieties of the AB, or (b) attachment to sulfhydryl groups the linker for conjugation purposes. of the AB, or (c) attachment to amino groups of the AB, or 0222 Branched Linkers: (d) attachment to carboxylate groups of the AB. According 0223) In specific embodiments, branched linkers that to the disclosure, ABs may be covalently attached to an have multiple sites for attachment of agents are utilized. For agent through an intermediate linker having at least two multiple site linkers, a single covalent attachment to an AB reactive groups, one to react with AB and one to react with would result in an AB-linker intermediate capable of binding the agent. The linker, which may include any compatible an agent at a number of sites. The sites may be aldehyde or organic compound, can be chosen such that the reaction with Sulfhydryl groups or any chemical site to which agents can AB (or agent) does not adversely affect AB reactivity and be attached. selectivity. Furthermore, the attachment of linker to agent 0224. In some embodiments, higher specific activity (or might not destroy the activity of the agent. Suitable linkers higher ratio of agents to AB) can be achieved by attachment for reaction with oxidized antibodies or oxidized antibody of a single site linker at a plurality of sites on the AB. This fragments include those containing an amine selected from plurality of sites may be introduced into the AB by either of the group consisting of primary amine, secondary amine, two methods. First, one may generate multiple aldehyde hydrazine, hydrazide, hydroxylamine, phenylhydrazine, groups and/or sulfhydryl groups in the same AB. Second, semicarbazide and thiosemicarbazide groups. Such reactive one may attach to an aldehyde or sulfhydryl of the AB a functional groups may exist as part of the structure of the “branched linker having multiple functional sites for sub linker, or may be introduced by suitable chemical modifi sequent attachment to linkers. The functional sites of the cation of linkers not containing Such groups. branched linker or multiple site linker may be aldehyde or 0218. According to the present disclosure, suitable link Sulfhydryl groups, or may be any chemical site to which ers for attachment to reduced ABs include those having linkers may be attached. Still higher specific activities may certain reactive groups capable of reaction with a Sulfhydryl be obtained by combining these two approaches, that is, group of a reduced antibody or fragment. Such reactive attaching multiple site linkers at several sites on the AB. groups include, but are not limited to: reactive haloalkyl 0225 Cleavable Linkers: groups (including, for example, haloacetyl groups), p-mer 0226 Peptide linkers that are susceptible to cleavage by curibenzoate groups and groups capable of Michael-type enzymes of the complement system, such as but not limited addition reactions (including, for example, maleimides and to urokinase, tissue plasminogen activator, trypsin, plasmin, groups of the type described by Mitra and Lawton, 1979, J. or another enzyme having proteolytic activity may be used Amer. Chem. Soc. 101: 3097-3110). in one embodiment of the present disclosure. According to 0219. According to the present disclosure, suitable link one method of the present disclosure, an agent is attached ers for attachment to neither oxidized nor reduced Abs via a linker Susceptible to cleavage by complement. The include those having certain functional groups capable of antibody is selected from a class that can activate comple reaction with the primary amino groups present in unmodi ment. The antibody-agent conjugate, thus, activates the fied lysine residues in the Ab. Such reactive groups include, complement cascade and releases the agent at the target site. but are not limited to, NHS carboxylic or carbonic esters, According to another method of the present disclosure, an sulfo-NHS carboxylic or carbonic esters, 4-nitrophenyl car agent is attached via a linker Susceptible to cleavage by boxylic or carbonic esters, pentafluorophenyl carboxylic or enzymes having a proteolytic activity Such as a urokinase, a carbonic esters, acyl imidazoles, isocyanates, and isothio tissue plasminogen activator, plasmin, or trypsin. These cyanates. cleavable linkers are useful in conjugated activatable anti 0220 According to the present disclosure, suitable link bodies that include an extracellular toxin, e.g., by way of ers for attachment to neither oxidized nor reduced Abs non-limiting example, any of the extracellular toxins shown include those having certain functional groups capable of in Table 3. reaction with the carboxylic acid groups present in aspartate 0227 Non-limiting examples of cleavable linker or glutamate residues in the Ab, which have been activated sequences are provided in Table 4. TABLE 4 Exemplary Linker Sequences for Conjugation Types of Cleavable Sequences Amino Acid Sequence Plasmin cleavable sequences

Pro - urokinase PRFKIIGG (SEO ID NO : 319) PRFRIIGG (SEO ID NO : 32O) US 2016/0289.324 A1 Oct. 6, 2016 33

TABLE 4- Continued Exemplary Linker Sequences for Coniugation Types of Cleavable Sequences Amino Acid Sequence TGFB SSRHRRALD (SEQ ID NO: 321) Plasminogen RKSSIIIRMRDVVL (SEO ID NO: 322) Staphyllokinase SSSFDKGKYKKGDDA (SEO ID NO : 323) SSSFDKGKYKRGDDA (SEO ID NO : 324) Factor Xa cleavable sequences IEGR (SEO ID NO : 325) IDGR (SEQ ID NO: 326) GGSIDGR (SEQ ID NO: 327) MMP cleavable sequences A PLGLWA (SEQ ID NO: 328) cleavable sequences Calf skin collagen (C.1 (I) chain) GPOGIAGQ (SEQ ID NO: 329) Calf skin collagen (C2 (I) chain) GPOGLLGA (SEQ ID NO: 330) Bovine cartilage collagen (C.1 (III) chain) GIAGQ (SEQ ID NO : 331) Human liver collagen (C.1 (III) chain) GPLGIAGI (SEQ ID NO: 332) Human CM GPEGLRWG (SEQ ID NO: 333) Human PZP YGAGLGVW (SEQ ID NO: 334) AGLGWWER (SEQ ID NO: 335) AGLGISST (SEQ ID NO: 336) Rat CM EPOALAMS (SEQ ID NO: 337) QALAMSAI (SEQ ID NO: 338) Rat CM AAYHLWSQ (SEQ ID NO: 339) MDAFLESS (SEQ ID NO: 340) Rat C.I. (2 J) ESLPVVAV (SEO ID NO. 341) Rat CI (27J) SAPAVESE (SEQ ID NO: 342) Human fibroblast collagenase DVAOFVLT (SEQ ID NO. 343) (autolytic cleavages) VAOFVLTE (SEO ID NO : 344) AOFWLTEG (SEQ ID NO: 345) PWOPIGPO (SEQ ID NO: 346)

0228 In addition, agents may be attached via disulfide serves to position the cleavable element away from the core bonds (for example, the disulfide bonds on a cysteine of the AB such that the cleavable element is more accessible molecule) to the AB. Since many tumors naturally release to the enzyme responsible for cleavage. Certain of the high levels of glutathione (a reducing agent) this can reduce branched linkers described above may serve as spacer the disulfide bonds with subsequent release of the agent at elements. the site of delivery. In certain specific embodiments, the reducing agent that would modify a CM1-CM2 substrate 0232 Throughout this discussion, it should be under would also modify the linker of the conjugated activatable stood that the attachment of linker to agent (or of spacer antibody. element to cleavable element, or cleavable element to agent) 0229 Spacers and Cleavable Elements: need not be particular mode of attachment or reaction. Any 0230. In some embodiments, it may be necessary to reaction providing a product of Suitable stability and bio construct the linker in Such a way as to optimize the spacing logical compatibility is acceptable. between the agent and the AB of the activatable antibody. This may be accomplished by use of a linker of the general 0233 Serum Complement and Selection of Linkers: Structure: 0234. According to one method of the present disclosure, when release of an agent is desired, an AB that is an antibody of a class that can activate complement is used. The resulting wherein conjugate retains both the ability to bind antigen and activate W is either —NH-CH or —CH2—, the complement cascade. Thus, according to this embodi Q is an amino acid, peptide; and ment of the present disclosure, an agent is joined to one end n is an integer from 0 to 20. of the cleavable linker or cleavable element and the other 0231. In some embodiments, the linker may comprise a end of the linker group is attached to a specific site on the spacer element and a cleavable element. The spacer element AB. For example, if the agent has an hydroxy group or an US 2016/0289.324 A1 Oct. 6, 2016 34 amino group, it may be attached to the carboxy terminus of desired since activation of the complement cascade will a peptide, amino acid or other Suitably chosen linker via an ultimately lyse the target cell. Hence, this approach is useful ester or amide bond, respectively. For example, such agents when delivery and release of the agent should be accom may be attached to the linker peptide via a carbodimide plished without killing the target cell. Such is the goal when reaction. If the agent contains functional groups that would delivery of cell mediators such as hormones, enzymes, interfere with attachment to the linker, these interfering corticosteroids, neurotransmitters, or enzymes to tar functional groups can be blocked before attachment and get cells is desired. These conjugates may be prepared by deblocked once the product conjugate or intermediate is attaching the agent to an AB that is not capable of activating made. The opposite or amino terminus of the linker is then complement via a linker that is mildly susceptible to cleav used either directly or after further modification for binding age by serum proteases. When this conjugate is administered to an AB that is capable of activating complement. to an individual, antigen-antibody complexes will form 0235 Linkers (or spacer elements of linkers) may be of quickly whereas cleavage of the agent will occur slowly, any desired length, one end of which can be covalently thus resulting in release of the compound at the target site. 0239 Biochemical Cross Linkers: attached to specific sites on the AB of the activatable 0240. In some embodiments, the activatable antibody antibody. The other end of the linker or spacer element may may be conjugated to one or more therapeutic agents using be attached to an amino acid or peptide linker. certain biochemical cross-linkers. Cross-linking reagents 0236. Thus when these conjugates bind to antigen in the form molecular bridges that tie together functional groups of presence of complement the amide or ester bond that two different molecules. To link two different proteins in a attaches the agent to the linker will be cleaved, resulting in step-wise manner, hetero-bifunctional cross-linkers can be release of the agent in its active form. These conjugates, used that eliminate unwanted homopolymer formation. when administered to a subject, will accomplish delivery 0241 Peptidyl linkers cleavable by lysosomal proteases and release of the agent at the target site, and are particularly are also useful, for example, Val-Cit, Val-Ala or other effective for the in vivo delivery of pharmaceutical agents, dipeptides. In addition, acid-labile linkers cleavable in the antibiotics, antimetabolites, antiproliferative agents and the low-pH environment of the lysosome may be used, for like as presented in but not limited to those in Table 3. example: bis-sialyl ether. Other suitable linkers include 0237 Linkers for Release without Complement Activa cathepsin-labile Substrates, particularly those that show opti tion: mal function at an acidic pH. 0238. In yet another application of targeted delivery, 0242 Exemplary hetero-bifunctional cross-linkers are release of the agent without complement activation is referenced in Table 5. TABLE 5 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length after cross-linking Linker Reactive Toward Advantages and Applications (Angstroms) SMPT Primary amines Greater stability 11.2 A Sulfhydryls SPDP Primary amines Thiolation 6.8 A Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC-SPDP Primary amines Extender spacer arm 15.6 A Sulfhydryls Water-soluble SMCC Primary amines Stable maleimide reactive group 11.6 A Sulfhydryls Enzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo-SMCC Primary amines Stable maleimide reactive group 11.6 A Sulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary amines Enzyme-antibody conjugation 9.9 A Sulfhydryls Hapten-carrier protein conjugation Sulfo-MBS Primary amines Water-soluble 9.9 A Sulfhydryls SIAB Primary amines Enzyme-antibody conjugation 10.6 A Sulfhydryls Sulfo-SIAB Primary amines Water-soluble 10.6 A Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 A Sulfhydryls Water-soluble EDEASlfo-NHS Primary amines Hapten-Carrier conjugation O Carboxyl groups ABH Carbohydrates Reacts with Sugar groups 11.9 A Nonselective US 2016/0289.324 A1 Oct. 6, 2016 35

0243. Non-Cleavable Linkers or Direct Attachment: described herein. The foregoing techniques and procedures 0244. In some embodiments of the disclosure, the con are generally performed according to conventional methods jugate may be designed so that the agent is delivered to the well known in the art and as described in various general and target but not released. This may be accomplished by more specific references that are cited and discussed attaching an agent to an AB either directly or via a non throughout the present specification. See e.g., Sambrook et cleavable linker. al. Molecular Cloning: A Laboratory Manual (2d ed., Cold 0245. These non-cleavable linkers may include amino Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. acids, peptides, D-amino acids or other organic compounds (1989)). The nomenclatures utilized in connection with, and that may be modified to include functional groups that can the laboratory procedures and techniques of analytical subsequently be utilized in attachment to ABs by the meth chemistry, synthetic organic chemistry, and medicinal and ods described herein. A-general formula for Such an organic pharmaceutical chemistry described herein are those well linker could be known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, phar maceutical preparation, formulation, and delivery, and treat wherein ment of patients. W is either —NH-CH or —CH2—, 0251. As utilized in accordance with the present disclo Q is an amino acid, peptide; and Sure, the following terms, unless otherwise indicated, shall n is an integer from 0 to 20. be understood to have the following meanings: 0246 Non-Cleavable Conjugates: 0252. As used herein, the term “antibody” refers to 0247. In some embodiments, a compound may be immunoglobulin molecules and immunologically active attached to ABs that do not activate complement. When portions of immunoglobulin (Ig) molecules, i.e., molecules using ABS that are incapable of complement activation, this that contain an antigen binding site that specifically binds attachment may be accomplished using linkers that are (immunoreacts with) an antigen. By “specifically bind’ or Susceptible to cleavage by activated complement or using “immunoreacts with or “immunospecifically bind' is linkers that are not Susceptible to cleavage by activated meant that the antibody reacts with one or more antigenic complement. determinants of the desired antigen and does not react with 0248. The antibodies disclosed herein can also be formu other polypeptides or binds at much lower affinity (KC-10 lated as immunoliposomes. Liposomes containing the anti 6). Antibodies include, but are not limited to, polyclonal, body are prepared by methods known in the art, such as monoclonal, chimeric, domain antibody, single chain, Fab, described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: and F(ab')2 fragments, ScPVs, and an Fab expression library. 3688 (1985): Hwang et al., Proc. Natl Acad. Sci. USA, 77: 0253) The basic antibody structural unit is known to 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544.545. comprise a tetramer. Each tetramer is composed of two Liposomes with enhanced circulation time are disclosed in identical pairs of polypeptide chains, each pair having one U.S. Pat. No. 5,013,556. “light” (about 25 kDa) and one “heavy” chain (about 50-70 0249 Particularly useful liposomes can be generated by kDa). The amino-terminal portion of each chain includes a the reverse-phase evaporation method with a lipid compo variable region of about 100 to 110 or more amino acids sition comprising phosphatidylcholine, cholesterol, and primarily responsible for antigen recognition. The carboxy PEG-derivatized phosphatidylethanolamine (PEG-PE). terminal portion of each chain defines a constant region Liposomes are extruded through filters of defined pore size primarily responsible for effector function. In general, anti to yield liposomes with the desired diameter. Fab' fragments body molecules obtained from humans relate to any of the of the antibody of the present disclosure can be conjugated classes IgG, IgM, IgA, IgE and Ig), which differ from one to the liposomes as described in Martin et al., J. Biol. Chem. another by the nature of the heavy chain present in the 257; 286-288 (1982) via a disulfide-interchange reaction. molecule. Certain classes have subclasses as well. Such as IgG, IgG, and others. Furthermore, in humans, the light DEFINITIONS chain may be a kappa chain or a lambda chain. 0250. Unless otherwise defined, scientific and technical 0254 The term “monoclonal antibody” (mAb) or “mono terms used in connection with the present disclosure shall clonal antibody composition', as used herein, refers to a have the meanings that are commonly understood by those population of antibody molecules that contain only one of ordinary skill in the art. The term “a” entity or “an entity molecular species of antibody molecule consisting of a refers to one or more of that entity. For example, a com unique light chain product and a unique heavy chain pound refers to one or more compounds. As such, the terms gene product. In particular, the complementarity determin “a”, “an”, “one or more' and “at least one' can be used ing regions (CDRS) of the monoclonal antibody are identical interchangeably. Further, unless otherwise required by con in all the molecules of the population. MAbs contain an text, singular terms shall include pluralities and plural terms antigen binding site capable of immunoreacting with a shall include the singular. Generally, nomenclatures utilized particular epitope of the antigen characterized by a unique in connection with, and techniques of cell and tissue culture, binding affinity for it. molecular biology, and protein and oligo- or polynucleotide 0255. The term “antigen-binding site' or “binding por chemistry and hybridization described herein are those well tion” refers to the part of the immunoglobulin molecule that known and commonly used in the art. Standard techniques participates in antigen binding. The antigen binding site is are used for recombinant DNA, oligonucleotide synthesis, formed by amino acid residues of the N-terminal variable and tissue culture and transformation (e.g., electroporation, (“V”) regions of the heavy (“H”) and light (“L) chains. lipofection). Enzymatic reactions and purification tech Three highly divergent stretches within the V regions of the niques are performed according to manufacturer's specifi heavy and light chains, referred to as “hypervariable cations or as commonly accomplished in the art or as regions.” are interposed between more conserved flanking US 2016/0289.324 A1 Oct. 6, 2016 36 stretches known as “framework regions,” or “FRs'. Thus, with all or a portion of a polynucleotide in which the the term “FR refers to amino acid sequences that are "isolated polynucleotide' is found in nature, (2) is operably naturally found between, and adjacent to, hyperVariable linked to a polynucleotide that it is not linked to in nature, regions in immunoglobulins. In an antibody molecule, the or (3) does not occur in nature as part of a larger sequence. three hypervariable regions of a light chain and the three Polynucleotides in accordance with the disclosure include hyperVariable regions of a heavy chain are disposed relative the nucleic acid molecules encoding the heavy chain immu to each other in three dimensional space to form an antigen noglobulin molecules shown herein, and nucleic acid mol binding Surface. The antigen-binding Surface is complemen ecules encoding the light chain immunoglobulin molecules tary to the three-dimensional Surface of a bound antigen, and shown herein. the three hypervariable regions of each of the heavy and 0260. The term "isolated protein” referred to herein light chains are referred to as "complementarity-determining means a protein of cDNA, recombinant RNA, or synthetic regions,” or “CDRs.” The assignment of amino acids to each origin or some combination thereof, which by virtue of its domain is in accordance with the definitions of Kabat origin, or source of derivation, the "isolated protein’ (1) is Sequences of Proteins of Immunological Interest (National not associated with proteins found in nature, (2) is free of Institutes of Health, Bethesda, Md. (1987 and 1991)), or other proteins from the same source, e.g., free of murine Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia proteins, (3) is expressed by a cell from a different species, et al. Nature 342:878-883 (1989). or (4) does not occur in nature. 0256. As used herein, the term “epitope' includes any 0261 The term “polypeptide' is used herein as a generic protein determinant capable of specific binding to an immu term to refer to native protein, fragments, or analogs of a noglobulin, a schv, or a T-cell receptor. The term “epitope' polypeptide sequence. Hence, native protein fragments, and includes any protein determinant capable of specific binding analogs are species of the polypeptide genus. Polypeptides to an immunoglobulin or T-cell receptor. in accordance with the disclosure comprise the heavy chain 0257 Epitopic determinants usually consist of chemi immunoglobulin molecules shown herein, and the light cally active Surface groupings of molecules such as amino chain immunoglobulin molecules shown herein, as well as acids or Sugar side chains and usually have specific three antibody molecules formed by combinations comprising the dimensional structural characteristics, as well as specific heavy chain immunoglobulin molecules with light chain charge characteristics. For example, antibodies may be immunoglobulin molecules, such as kappa light chain raised against N-terminal or C-terminal peptides of a poly immunoglobulin molecules, and Vice versa, as well as peptide. An antibody is said to specifically bind an antigen fragments and analogs thereof. when the dissociation constant is s1 uM; in Some embodi 0262 The term “naturally-occurring” as used herein as ments, s100 nM and in some embodiments, s10 nM. applied to an object refers to the fact that an object can be 0258 As used herein, the terms “specific binding.” found in nature. For example, a polypeptide or polynucle “immunological binding,” and “immunological binding otide sequence that is present in an organism (including properties’ refer to the non-covalent interactions of the type viruses) that can be isolated from a source in nature and that that occur between an immunoglobulin molecule and an has not been intentionally modified by man in the laboratory antigen for which the immunoglobulin is specific. The or otherwise is naturally-occurring. strength, or affinity of immunological binding interactions 0263. The term “operably linked as used herein refers to can be expressed in terms of the dissociation constant (K) positions of components so described are in a relationship of the interaction, wherein a smaller K represents a greater permitting them to function in their intended manner. A affinity. Immunological binding properties of selected poly control sequence “operably linked to a coding sequence is peptides can be quantified using methods well known in the ligated in Such a way that expression of the coding sequence art. One Such method entails measuring the rates of antigen is achieved under conditions compatible with the control binding site/antigen complex formation and dissociation, Sequences. wherein those rates depend on the concentrations of the 0264. The term “control sequence' as used herein refers complex partners, the affinity of the interaction, and geo to polynucleotide sequences that are necessary to effect the metric parameters that equally influence the rate in both expression and processing of coding sequences to which directions. Thus, both the “on rate constant” (K) and the they are ligated. The nature of such control sequences differs “off rate constant” (K) can be determined by calculation of depending upon the host organism in prokaryotes, such the concentrations and the actual rates of association and control sequences generally include promoter, ribosomal dissociation. (See Nature 361:186-87 (1993)). The ratio of binding site, and transcription termination sequence in K/K, enables the cancellation of all parameters not eukaryotes, generally, Such control sequences include pro related to affinity, and is equal to the dissociation constant moters and transcription termination sequence. The term K. (See, generally, Davies et al. (1990) Annual Rev Bio “control sequences” is intended to include, at a minimum, all chem 59:439-473). An antibody of the present disclosure is components whose presence is essential for expression and said to specifically bind to the target, when the binding processing, and can also include additional components constant (K) is s1 LM, in Some embodiments s100 nM, in whose presence is advantageous, for example, leader Some embodiments s10 nM, and in some embodiments sequences and fusion partner sequences. The term “poly s100 pM to about 1 pM, as measured by assays such as nucleotide' as referred to herein means nucleotides of at radioligand binding assays or similar assays known to those least 10 bases in length, either ribonucleotides or deoxy skilled in the art. nucleotides or a modified form of either type of nucleotide. 0259. The term "isolated polynucleotide' as used herein The term includes single and double stranded forms of shall mean a polynucleotide of genomic, cDNA, or synthetic DNA origin or some combination thereof, which by virtue of its 0265. The term oligonucleotide referred to herein origin the "isolated polynucleotide' (1) is not associated includes naturally occurring, and modified nucleotides US 2016/0289.324 A1 Oct. 6, 2016 37 linked together by naturally occurring, and non-naturally identity, in Some embodiments, at least 95 percent sequence occurring oligonucleotide linkages. Oligonucleotides are a identity, and in some embodiments, at least 99 percent polynucleotide Subset generally comprising a length of 200 sequence identity. bases or fewer. In some embodiments, oligonucleotides are 0270. In some embodiments, residue positions that are 10 to 60 bases in length and in some embodiments, 12, 13, not identical differ by conservative amino acid substitutions. 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligo 0271. As discussed herein, minor variations in the amino nucleotides are usually single stranded, e.g., for probes, acid sequences of antibodies or immunoglobulin molecules although oligonucleotides may be double stranded, e.g., for are contemplated as being encompassed by the present use in the construction of a gene mutant. Oligonucleotides of disclosure, providing that the variations in the amino acid the disclosure are either sense or antisense oligonucleotides. sequence maintain at least 75%, in Some embodiments, at 0266 The term “naturally occurring nucleotides’ least 80%, 90%, 95%, and in some embodiments, 99%. In referred to herein includes deoxyribonucleotides and ribo particular, conservative amino acid replacements are con nucleotides. The term “modified nucleotides' referred to templated. Conservative replacements are those that take herein includes nucleotides with modified or substituted place within a family of amino acids that are related in their Sugar groups and the like. The term "oligonucleotide link side chains. Genetically encoded amino acids are generally ages' referred to herein includes oligonucleotide linkages divided into families: (1) acidic amino acids are aspartate, Such as phosphorothioate, phosphorodithioate, phospho glutamate; (2) basic amino acids are lysine, arginine, histi roselerloate, phosphorodiselenoate, phosphoroanilothioate, dine; (3) non-polar amino acids are alanine, Valine, leucine, phoshoraniladate, phosphoronimidate, and the like. See e.g., isoleucine, proline, phenylalanine, methionine, tryptophan, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. and (4) uncharged polar amino acids are glycine, asparagine, J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids glutamine, cysteine, serine, threonine, tyrosine. The hydro Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design philic amino acids include arginine, asparagine, aspartate, 6:539 (1991); Zonet al. Oligonucleotides and Analogues: A glutamine, glutamate, histidine, lysine, serine, and threo Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford nine. The hydrophobic amino acids include alanine, cyste University Press, Oxford England (1991)); Stec et al. U.S. ine, isoleucine, leucine, methionine, phenylalanine, proline, Pat. No. 5,151,510; Uhlmann and Peyman Chemical tryptophan, tyrosine and valine. Other families of amino Reviews 90:543 (1990). An oligonucleotide can include a acids include (i) serine and threonine, which are the ali label for detection, if desired. phatic-hydroxy family; (ii) asparagine and glutamine, which 0267 As used herein, the twenty conventional amino are the amide containing family; (iii) alanine, valine, leucine acids and their abbreviations follow conventional usage. See and isoleucine, which are the aliphatic family; and (iv) Immunology—A Synthesis (2nd Edition, E. S. Golub and D. phenylalanine, tryptophan, and tyrosine, which are the aro R. Gren, Eds. Sinauer Associates. Sunderland 7 Mass. matic family. For example, it is reasonable to expect that an (1991)). Stereoisomers (e.g., D-amino acids) of the twenty isolated replacement of a leucine with an isoleucine or conventional amino acids, unnatural amino acids such as C.-, valine, an aspartate with a glutamate, a threonine with a C.-disubstituted amino acids, N-alkyl amino acids, lactic serine, or a similar replacement of an amino acid with a acid, and other unconventional amino acids may also be structurally related amino acid will not have a major effect Suitable components for polypeptides of the present disclo on the binding or properties of the resulting molecule, Sure. Examples of unconventional amino acids include: 4 especially if the replacement does not involve an amino acid hydroxyproline, Y-carboxyglutamate, e-N.N.N-trimethylly within a framework site. Whether an amino acid change sine, e-N-acetylysine, O-phosphoserine, N-acetylserine, results in a functional peptide can readily be determined by N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, assaying the specific activity of the polypeptide derivative. O-N-methylarginine, and other similar amino acids and Assays are described in detail herein. Fragments or analogs imino acids (e.g., 4-hydroxyproline). In the polypeptide of antibodies or immunoglobulin molecules can be readily notation used herein, the left-hand direction is the amino prepared by those of ordinary skill in the art. Suitable amino terminal direction and the right-hand direction is the car and carboxy-termini of fragments or analogs occur near boxy-terminal direction, in accordance with standard usage boundaries of functional domains. Structural and functional and convention. domains can be identified by comparison of the nucleotide 0268 Similarly, unless specified otherwise, the left-hand and/or amino acid sequence data to public or proprietary end of single-stranded polynucleotide sequences is the 5' end sequence databases. In some embodiments, computerized the left-hand direction of double-stranded polynucleotide comparison methods are used to identify sequence motifs or sequences is referred to as the 5' direction. The direction of predicted protein conformation domains that occur in other 5' to 3' addition of nascent RNA transcripts is referred to as proteins of known structure and/or function. Methods to the transcription direction sequence regions on the DNA identify protein sequences that fold into a known three Strand having the same sequence as the RNA and that are 5' dimensional structure are known. Bowie et al. Science to the 5' end of the RNA transcript are referred to as 253:164 (1991). Thus, the foregoing examples demonstrate "upstream sequences', sequence regions on the DNA strand that those of skill in the art can recognize sequence motifs having the same sequence as the RNA and that are 3' to the and structural conformations that may be used to define 3' end of the RNA transcript are referred to as “downstream structural and functional domains in accordance with the sequences'. disclosure. 0269. As applied to polypeptides, the term “substantial 0272 Suitable amino acid substitutions are those that: (1) identity” means that two peptide sequences, when optimally reduce susceptibility to proteolysis, (2) reduce susceptibility aligned, such as by the programs GAP or BESTFIT using to oxidation, (3) alter binding affinity for forming protein default gap weights, share at least 80 percent sequence complexes, (4) alter binding affinities, and (5) confer or identity, in Some embodiments, at least 90 percent sequence modify other physicochemical or functional properties of US 2016/0289.324 A1 Oct. 6, 2016

Such analogs. Analogs can include various muteins of a reduce potential steric hindrance. The term “pharmaceutical sequence other than the naturally-occurring peptide agent or drug as used herein refers to a chemical compound sequence. For example, single or multiple amino acid Sub or composition capable of inducing a desired therapeutic stitutions (for example, conservative amino acid substitu effect when properly administered to a patient. tions) may be made in the naturally-occurring sequence (for 0276. Other chemistry terms herein are used according to example, in the portion of the polypeptide outside the conventional usage in the art, as exemplified by The domain(s) forming intermolecular contacts. A conservative McGraw-Hill Dictionary of Chemical Terms (Parker, S., amino acid Substitution should not substantially change the Ed., McGraw-Hill, San Francisco (1985)). structural characteristics of the parent sequence (e.g., a 0277 As used herein, “substantially pure” means an replacement amino acid should not tend to break a helix that object species is the predominant species present (i.e., on a occurs in the parent sequence, or disrupt other types of molar basis it is more abundant than any other individual secondary structure that characterizes the parent sequence). species in the composition), and in Some embodiments, a Examples of art-recognized polypeptide secondary and ter substantially purified fraction is a composition wherein the tiary structures are described in Proteins, Structures and object species comprises at least about 50 percent (on a Molecular Principles (Creighton, Ed., W. H. Freeman and molar basis) of all macromolecular species present. Company, New York (1984)); Introduction to Protein Struc 0278 Generally, a substantially pure composition will ture (C. Branden and J. Tooze, eds. Garland Publishing, comprise more than about 80 percent of all macromolecular New York, N.Y. (1991)); and Thornton et at. Nature 354:105 species present in the composition, in Some embodiments, (1991). more than about 85%, 90%, 95%, and 99%. In some 0273. The term “polypeptide fragment as used herein embodiments, the object species is purified to essential refers to a polypeptide that has an amino terminal and/or homogeneity (contaminant species cannot be detected in the carboxy-terminal deletion and/or one or more internal dele composition by conventional detection methods) wherein tion(s), but where the remaining amino acid sequence is the composition consists essentially of a single macromo identical to the corresponding positions in the naturally lecular species. occurring sequence deduced, for example, from a full length 0279. The term patient includes human and veterinary cDNA sequence. Fragments typically are at least 5, 6, 8 or Subjects. 10 amino acids long, in some embodiments, at least 14 0280 Activatable antibodies of the disclosure specifi amino acids long, in Some embodiments, at least 20 amino cally bind a given target, e.g., a human target protein. Also acids long, usually at least 50 amino acids long, and in some included in the disclosure are activatable antibodies that embodiments, at least 70 amino acids long. The term “ana bind to the same epitope as the activatable antibodies log as used herein refers to polypeptides that are comprised described herein. of a segment of at least 25 amino acids that has substantial 0281 Those skilled in the art will recognize that it is identity to a portion of a deduced amino acid sequence and possible to determine, without undue experimentation, if a that has specific binding to the target, under Suitable binding monoclonal antibody (e.g., a murine monoclonal or human conditions. Typically, polypeptide analogs comprise a con ized antibody) has the same specificity as a monoclonal servative amino acid substitution (or addition or deletion) antibody used in the methods described herein by ascertain with respect to the naturally-occurring sequence. Analogs ing whether the former prevents the latter from binding to typically are at least 20 amino acids long, in Some embodi the target. If the monoclonal antibody being tested competes ments, at least 50 amino acids long or longer, and can often with the monoclonal antibody of the disclosure, as shown by be as long as a full-length naturally-occurring polypeptide. a decrease in binding by the monoclonal antibody of the 0274 The term "agent' is used herein to denote a chemi disclosure, then the two monoclonal antibodies bind to the cal compound, a mixture of chemical compounds, a biologi same, or a closely related, epitope. A method for determining cal macromolecule, or an extract made from biological whether a monoclonal antibody has the specificity of a materials. monoclonal antibody of the disclosure is to pre-incubate the 0275. As used herein, the terms “label' or “labeled monoclonal antibody of the disclosure with the target and refers to incorporation of a detectable marker, e.g., by then add the monoclonal antibody being tested to determine incorporation of a radiolabeled amino acid or attachment to if the monoclonal antibody being tested is inhibited in its a polypeptide of biotinyl moieties that can be detected by ability to bind the target. If the monoclonal antibody being marked avidin (e.g., Streptavidin containing a fluorescent tested is inhibited then, in all likelihood, it has the same, or marker or enzymatic activity that can be detected by optical functionally equivalent, epitopic specificity as the monoclo or calorimetric methods). In certain situations, the label or nal antibody of the disclosure. marker can also be therapeutic. Various methods of labeling 0282 Multispecific Activatable Antibodies polypeptides and glycoproteins are known in the art and may 0283. The disclosure also provides multispecific activat be used. Examples of labels for polypeptides include, but are able antibodies. The multispecific activatable antibodies not limited to, the following: radioisotopes or radionuclides provided herein are multispecific antibodies that recognize (e.g., H, 14C, 15N, 35S, 90Y. 99Tc, Ill In, 125I, 13|I), fluores two or more different antigens or epitopes and that include cent labels (e.g., FITC, rhodamine, lanthanide phosphors), at least one masking moiety (MM) linked to at least one enzymatic labels (e.g., horseradish peroxidase, p-galactosi antigen- or epitope-binding domain of the multispecific dase, luciferase, alkaline phosphatase), chemiluminescent, antibody such that coupling of the MM reduces the ability of biotinyl groups, predetermined polypeptide epitopes recog the antigen- or epitope-binding domain to bind its target. In nized by a secondary reporter (e.g., leucine Zipper pair some embodiments, the MM is coupled to the antigen- or sequences, binding sites for secondary antibodies, metal epitope-binding domain of the multispecific antibody via a binding domains, epitope tags). In some embodiments, CM1-CM2 substrate that functions as a substrate for at least labels are attached by spacer arms of various lengths to one MMP protease and at least one SP. The activatable US 2016/0289.324 A1 Oct. 6, 2016 39 multispecific antibodies provided herein are stable in circu antibody is a cancer targeting antibody. In some embodi lation, activated at intended sites of therapy and/or diagnosis ments the non-immune cell effector antibody is an IgG. In but not in normal, i.e., healthy tissue, and, when activated, Some embodiments the immune effector cell engaging anti exhibit binding to a target that is at least comparable to the body is a scFv. In some embodiments the targeting antibody corresponding, unmodified multispecific antibody. (e.g., non-immune cell effector antibody) is an IgG and the 0284. In some embodiments, the multispecific activatable immune effector cell engaging antibody is a ScPV. In some antibodies are designed to engage immune effector cells, embodiments, the immune effector cell is a leukocyte. In also referred to herein as immune-effector cell engaging some embodiments, the immune effector cell is a T cell. In multispecific activatable antibodies. In some embodiments, some embodiments, the immune effector cell is a NK cell. In the multispecific activatable antibodies are designed to Some embodiments, the immune effector cell is a myeloid engage leukocytes, also referred to herein as leukocyte mononuclear cell. engaging multispecific activatable antibodies. In some 0286. In some embodiments, T-cell engaging multispe embodiments, the multispecific activatable antibodies are cific activatable antibodies of the disclosure include a tar designed to engage T cells, also referred to herein as T-cell geting antibody or antigen-binding fragment thereof and a engaging multispecific activatable antibodies. In some T-cell engaging antibody or antigen-binding portion thereof, embodiments, the multispecific activatable antibodies where at least one of the targeting antibody or antigen engage a surface antigen on a leukocyte, Such as on a T cell, binding fragment thereof and/or the T-cell engaging anti on a natural killer (NK) cell, on a myeloid mononuclear cell, body or antigen-binding portion thereof is masked. In some on a macrophage, and/or on another immune effector cell. In embodiments, the T-cell engaging antibody or antigen bind Some embodiments, the immune effector cell is a leukocyte. ing fragment thereof includes a first antibody or antigen In some embodiments, the immune effector cell is a T cell. binding fragment thereof (AB1) that binds a first, T-cell In some embodiments, the immune effector cell is a NK cell. engaging target, where the AB1 is attached to a masking In some embodiments, the immune effector cell is a mono moiety (MM1) such that coupling of the MM1 reduces the nuclear cell. Such as a myeloid mononuclear cell. In some ability of the AB1 to bind the first target. In some embodi embodiments, the multispecific activatable antibodies are ments, the targeting antibody or antigen-binding fragment designed to bind or otherwise interact with more than one thereof includes a second antibody or fragment thereof that target and/or more than one epitope, also referred to herein includes a second antibody or antigen-binding fragment as multi-antigen targeting activatable antibodies. As used thereof (AB2) that binds a second target, where the AB2 is herein, the terms “target” and “antigen” are used inter attached to a masking moiety (MM2) such that coupling of changeably. the MM2 reduces the ability of the AB2 to bind the second 0285. In some embodiments, immune effector cell engag target. In some embodiments, the T-cell engaging antibody ing multispecific activatable antibodies of the disclosure or antigen binding fragment thereof includes a first antibody include a targeting antibody or antigen-binding fragment or antigen-binding fragment thereof (AB1) that binds a first, thereof and an immune effector cell engaging antibody or T-cell engaging target, where the AB1 is attached to a antigen-binding portion thereof, where at least one of the masking moiety (MM1) such that coupling of the MM1 targeting antibody or antigen-binding fragment thereof and/ reduces the ability of the AB1 to bind the first target, and the or the immune effector cell engaging antibody or antigen targeting antibody or antigen-binding fragment thereof binding portion thereof is masked. In some embodiments, includes a second antibody or fragment thereof that includes the immune effector cell engaging antibody or antigen a second antibody or antigen-binding fragment thereof binding fragment thereof includes a first antibody or anti (AB2) that binds a second target, where the AB2 is attached gen-binding fragment thereof (AB1) that binds a first, to a masking moiety (MM2) such that coupling of the MM2 immune effector cell engaging target, where the AB1 is reduces the ability of the AB2 to bind the second target. attached to a masking moiety (MM1) Such that coupling of 0287. In some embodiments, the T-cell engaging multi the MM1 reduces the ability of the AB1 to bind the first specific activatable antibodies include a cancer targeting target. In some embodiments, the targeting antibody or antibody or antigen-binding fragment thereof and a T-cell antigen-binding fragment thereof includes a second anti engaging antibody or antigen-binding portion thereof, where body or fragment thereof that includes a second antibody or at least one of the cancer targeting antibody or antigen antigen-binding fragment thereof (AB2) that binds a second binding fragment thereof and/or the T-cell engaging anti target, where the AB2 is attached to a masking moiety body or antigen-binding portion thereof is masked. In some (MM2) such that coupling of the MM2 reduces the ability of embodiments, the T-cell engaging antibody or antigen bind the AB2 to bind the second target. In some embodiments, the ing fragment thereof includes a first antibody or antigen immune effector cell engaging antibody or antigen binding binding fragment thereof (AB1) that binds a first, T-cell fragment thereof includes a first antibody or antigen-binding engaging target, where the AB1 is attached to a masking fragment thereof (AB1) that binds a first, immune effector moiety (MM1) such that coupling of the MM1 reduces the cell engaging target, where the AB1 is attached to a masking ability of the AB1 to bind the first target. In some embodi moiety (MM1) such that coupling of the MM1 reduces the ments, the cancer targeting antibody or antigen-binding ability of the AB1 to bind the first target, and the targeting fragment thereof includes a second antibody or fragment antibody or antigen-binding fragment thereof includes a thereof that includes a second antibody or antigen-binding second antibody or fragment thereof that includes a second fragment thereof (AB2) that binds a second, cancer-related antibody or antigen-binding fragment thereof (AB2) that target, where the AB2 is attached to a masking moiety binds a second target, where the AB2 is attached to a (MM2) such that coupling of the MM2 reduces the ability of masking moiety (MM2) such that coupling of the MM2 the AB2 to bind the second, cancer-related target. In some reduces the ability of the AB2 to bind the second target. In embodiments, the T-cell engaging antibody or antigen bind Some embodiments, the non-immune effector cell engaging ing fragment thereof includes a first antibody or antigen US 2016/0289.324 A1 Oct. 6, 2016 40 binding fragment thereof (AB1) that binds a first, T-cell tory receptors include, but are not limited to, BTLA, CTLA engaging target, where the AB1 is attached to a masking 4, LAG3, PD-1, TIGIT TIM3, and NK-expressed KIRs. The moiety (MM1) such that coupling of the MM1 reduces the antibody domain conferring specificity to the T-cell Surface ability of the AB1 to bind the first target, and the cancer antigen may also be substituted by a ligand or ligand domain targeting antibody or antigen-binding fragment thereof that binds to a T-cell receptor, a NK-cell receptor, a macro includes a second antibody or fragment thereof that includes phage receptor, and/or other immune effector cell receptor, a second antibody or antigen-binding fragment thereof such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, (AB2) that binds a second, cancer-related target, where the PD-L2, or TNFSF9. AB2 is attached to a masking moiety (MM2) such that 0290. One embodiment of the disclosure is a multispe coupling of the MM2 reduces the ability of the AB2 to bind cific activatable antibody that is activatable in a cancer the second, cancer-related target. microenvironment and that includes an antibody, for 0288. In some embodiments, the T-cell engaging multi example a IgG or Scv, directed to a tumor target and an specific activatable antibodies include a cancer targeting IgG agonist antibody, for example an IgG or scFv, directed to a antibody or antigen-binding fragment thereof and a T-cell co-stimulatory receptor expressed on the Surface of an engaging scFv, where at least one of the cancertargeting IgG activated T cell or NK cell, wherein at least one of the cancer antibody or antigen-binding fragment thereof and/or the target antibody and/or agonist antibody is masked. T-cell engaging antibody or antigen-binding portion thereof Examples of co-stimulatory receptors include, but are not is masked. In some embodiments, the T-cell engaging anti limited to, CD27, CD137, GITR, HVEM, NKG2D, and body or antigen binding fragment thereof includes a first OX40. In this embodiment, the multispecific activatable antibody or antigen-binding fragment thereof (AB1) that antibody, once activated by tumor-associated proteases, binds a first, T-cell engaging target, where the AB1 is would effectively crosslink and activate the T cell or NK cell attached to a masking moiety (MM1) Such that coupling of expressed co-stimulatory receptors in a tumor-dependent the MM1 reduces the ability of the AB1 to bind the first manner to enhance the activity of T cells that are responding target. In some embodiments, the cancer targeting IgG to any tumor antigen via their endogenous T cell antigen or antibody or antigen-binding fragment thereof includes a NK-activating receptors. The activation-dependent nature of second antibody or fragment thereof that includes a second these T cell or NK cell costimulatory receptors would focus antibody or antigen-binding fragment thereof (AB2) that the activity of the activated multispecific activatable anti binds a second, cancer-related target, where the AB2 is body to tumor-specific T cells, without activating all T cells attached to a masking moiety (MM2) such that coupling of independent of their antigen specificity. In one embodiment, the MM2 reduces the ability of the AB2 to bind the second, at least the co-stimulatory receptor antibody of the multi cancer-related target. In some embodiments, the T-cell specific activatable antibody is masked to prevent activation engaging antibody or antigen binding fragment thereof of auto-reactive T cells that may be present in tissues that includes a first antibody or antigen-binding fragment thereof also express the antigen recognized by the tumor target (AB1) that binds a first, T-cell engaging target, where the directed antibody in the multispecific activatable antibody, AB1 is attached to a masking moiety (MM1) such that but whose activity is restricted by lack of co-receptor coupling of the MM1 reduces the ability of the AB1 to bind engagement. the first target, and the cancer targeting IgG antibody or 0291. One embodiment of the disclosure is a multispe antigen-binding fragment thereof includes a second anti cific activatable antibody that is activatable in a disease body or fragment thereof that includes a second antibody or characterized by T cell overstimulation, such as, but not antigen-binding fragment thereof (AB2) that binds a second, limited to, an autoimmune disease or inflammatory disease cancer-related target, where the AB2 is attached to a mask microenvironment. Such a multispecific activatable anti ing moiety (MM2) such that coupling of the MM2 reduces body includes an antibody, for example a IgG or scFv, the ability of the AB2 to bind the second, cancer-related directed to a target comprising a surface antigen expressed target. in a tissue targeted by a T cell in autoimmune or inflamma 0289. In some embodiments of an immune effector cell tory disease and an antibody, for example a IgG or scFv, engaging multispecific activatable antibody, one antigen is directed to an inhibitory receptor expressed on the surface of typically an antigen present on the Surface of a tumor cell or a T cell or NK cell, wherein at least one of the disease tissue other cell type associated with disease, such as, but not target antibody and/or T cell inhibitory receptor antibody is limited to, any target listed in Table 1. Such as, but not masked. Examples of inhibitory receptors include, but are limited to, EGFR, erbB2, EpCAM, Jagged, PD-L, B7H3, or not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, CD71 (transferrin receptor), and another antigen is typically and NK-expressed KIRs. Examples of a tissue antigen a stimulatory or inhibitory receptor present on the surface of targeted by T cells in autoimmune disease include, but are a T-cell, natural killer (NK) cell, myeloid mononuclear cell, not limited to, a Surface antigen expressed on myelin or macrophage, and/or other immune effector cell. Such as, but nerve cells in multiple Sclerosis or a surface antigen not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, expressed on pancreatic islet cells in Type 1 diabetes. In this CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, embodiment, the multispecific activatable antibody when GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, localized in the tissue under autoimmune attack or inflam TIM3, or VISTA. In some embodiments, the antigen is a mation is activated and co-engages the T cell or NK cell stimulatory receptor present on the surface of a T cell or NK inhibitory receptor to suppress the activity of autoreactive T cell; examples of Such stimulatory receptors include, but are cells responding to any disease tissue-targeted antigens via not limited to, CD3, CD27, CD28, CD137 (also referred to their endogenous TCR or activating receptors. In one as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In embodiment, at least one or multiple antibodies are masked Some embodiments, the antigen is an inhibitory receptor to prevent Suppression of T cell responses in non-disease present on the surface of a T-cell; examples of such inhibi tissues where the target antigen may also be expressed. US 2016/0289.324 A1 Oct. 6, 2016

0292. In some embodiments, the T-cell engaging multi reduces the ability of the AB1 to bind CD3e. In some specific activatable antibody includes an anti-CD3 epsilon embodiments, the cancer targeting IgG antibody or antigen (CD3e, also referred to herein as CD3e and CD3) scFv and binding fragment thereof includes a second antibody or a targeting antibody or antigen-binding fragment thereof, fragment thereof that includes a second antibody or antigen where at least one of the anti-CD3e scFv and/or the targeting binding fragment thereof (AB2) that binds a second, cancer antibody or antigen-binding portion thereof is masked. In related target, where the AB2 is attached to a masking some embodiments, the CD3e scFv includes a first antibody moiety (MM2) such that coupling of the MM2 reduces the or antigen-binding fragment thereof (AB1) that binds CD3e, ability of the AB2 to bind the second, cancer-related target. where the AB1 is attached to a masking moiety (MM1) such In some embodiments, the CD3e scEv includes a first that coupling of the MM1 reduces the ability of the AB1 to antibody or antigen-binding fragment thereof (AB1) that bind CD3e. In some embodiments, the targeting antibody or binds CD3e, where the AB1 is attached to a masking moiety antigen-binding fragment thereof includes a second anti (MM1) such that coupling of the MM1 reduces the ability of body or fragment thereof that includes a second antibody or the AB1 to bind CD3e, and the cancer targeting antibody antigen-binding fragment thereof (AB2) that binds a second IgG or antigen-binding fragment thereof includes a second target, where the AB2 is attached to a masking moiety antibody or fragment thereofthat includes a second antibody (MM2) such that coupling of the MM2 reduces the ability of or antigen-binding fragment thereof (AB2) that binds a the AB2 to bind the second target. In some embodiments, the second, cancer-related target, where the AB2 is attached to CD3e scFv includes a first antibody or antigen-binding a masking moiety (MM2) such that coupling of the MM2 fragment thereof (AB1) that binds CD3e, where the AB1 is reduces the ability of the AB2 to bind the second, cancer attached to a masking moiety (MM1) Such that coupling of related target. the MM1 reduces the ability of the AB1 to bind CD3e, and 0295. In some embodiments, the T-cell engaging multi the targeting antibody or antigen-binding fragment thereof specific activatable antibody includes an anti-CD3 epsilon includes a second antibody or fragment thereof that includes (CD3e) schv that is derived from OKT3, where at least one a second antibody or antigen-binding fragment thereof of the targeting antibody or antigen-binding fragment (AB2) that binds a second target, where the AB2 is attached thereof and/or the OKT3 ScFv or OKT3-derived scFv is to a masking moiety (MM2) such that coupling of the MM2 masked. In some embodiments, the OKT3 scv or OKT3 reduces the ability of the AB2 to bind the second target. derived scEv includes a first antibody or antigen-binding 0293. In some embodiments, the T-cell engaging multi fragment thereof (AB1) that binds CD3e, where the AB1 is specific activatable antibody includes an anti-CD3e scFV attached to a masking moiety (MM1) such that coupling of and a cancer targeting antibody or antigen-binding fragment the MM1 reduces the ability of the AB1 to bind CD3e. In thereof, where at least one of the anti-CD3e scEv and/or the Some embodiments, the targeting antibody or antigen-bind cancer targeting antibody or antigen-binding portion thereof ing fragment thereof includes a second antibody or fragment is masked. In some embodiments, the CD3e scEv includes a thereof that includes a second antibody or antigen-binding first antibody or antigen-binding fragment thereof (AB1) fragment thereof (AB2) that binds a second target, where the that binds CD3e, where the AB1 is attached to a masking AB2 is attached to a masking moiety (MM2) such that moiety (MM1) such that coupling of the MM1 reduces the coupling of the MM2 reduces the ability of the AB2 to bind ability of the AB1 to bind CD3e. In some embodiments, the the second target. In some embodiments, the OKT3 schvor cancer targeting antibody or antigen-binding fragment OKT3-derived scFv includes a first antibody or antigen thereof includes a second antibody or fragment thereof that binding fragment thereof (AB1) that binds CD3e, where the includes a second antibody or antigen-binding fragment AB1 is attached to a masking moiety (MM1) such that thereof (AB2) that binds a second, cancer-related target, coupling of the MM1 reduces the ability of the AB1 to bind where the AB2 is attached to a masking moiety (MM2) such CD3e, and the targeting antibody or antigen-binding frag that coupling of the MM2 reduces the ability of the AB2 to ment thereof includes a second antibody or fragment thereof bind the second, cancer-related target. In some embodi that includes a second antibody or antigen-binding fragment ments, the CD3e scFv includes a first antibody or antigen thereof (AB2) that binds a second target, where the AB2 is binding fragment thereof (AB1) that binds CD3e, where the attached to a masking moiety (MM2) Such that coupling of AB1 is attached to a masking moiety (MM1) such that the MM2 reduces the ability of the AB2 to bind the second coupling of the MM1 reduces the ability of the AB1 to bind target. CD3e, and the cancer targeting antibody or antigen-binding 0296. In some embodiments, the T-cell engaging multi fragment thereof includes a second antibody or fragment specific activatable antibody includes an OKT3 scFv or thereof that includes a second antibody or antigen-binding OKT3-derived scFv and a cancer targeting antibody or fragment thereof (AB2) that binds a second, cancer-related antigen-binding fragment thereof, where at least one of the target, where the AB2 is attached to a masking moiety OKT3 ScFv or OKT3-derived scFv and/or the cancer tar (MM2) such that coupling of the MM2 reduces the ability of geting antibody or antigen-binding portion thereof is the AB2 to bind the second, cancer-related target. masked. In some embodiments, the OKT3 scv or OKT3 0294. In some embodiments, the T-cell engaging multi derived scEv includes a first antibody or antigen-binding specific activatable antibody includes an anti-CD3e scFV fragment thereof (AB1) that binds CD3e, where the AB1 is and a cancer targeting IgG antibody or antigen-binding attached to a masking moiety (MM1) Such that coupling of fragment thereof, where at least one of the anti-CD3e schv the MM1 reduces the ability of the AB1 to bind CD3e. In and/or the cancer targeting IgG antibody or antigen-binding Some embodiments, the cancer targeting antibody or anti portion thereof is masked. In some embodiments, the CD3 gen-binding fragment thereof includes a second antibody or ScFv includes a first antibody or antigen-binding fragment fragment thereof that includes a second antibody or antigen thereof (AB1) that binds CD3e, where the AB1 is attached binding fragment thereof (AB2) that binds a second, cancer to a masking moiety (MM1) such that coupling of the MM1 related target, where the AB2 is attached to a masking US 2016/0289.324 A1 Oct. 6, 2016 42 moiety (MM2) such that coupling of the MM2 reduces the and the targeting antibody or antigen-binding fragment ability of the AB2 to bind the second, cancer-related target. thereof includes a second antibody or fragment thereof that In some embodiments, the OKT3 scFv or OKT3-derived includes a second antibody or antigen-binding fragment ScFv includes a first antibody or antigen-binding fragment thereof (AB2) that binds a second target, where the AB2 is thereof (AB1) that binds CD3e, where the AB1 is attached attached to a masking moiety (MM2) Such that coupling of to a masking moiety (MM1) such that coupling of the MM1 the MM2 reduces the ability of the AB2 to bind the second reduces the ability of the AB1 to bind CD3e, and the cancer target. targeting antibody or antigen-binding fragment thereof 0299. In some embodiments, the T-cell engaging multi includes a second antibody or fragment thereof that includes specific activatable antibody includes an anti-CTLA-4 scFV a second antibody or antigen-binding fragment thereof and a targeting IgG antibody or antigen-binding fragment (AB2) that binds a second, cancer-related target, where the thereof, where at least one of the anti-CTLA-4 scEv and/or AB2 is attached to a masking moiety (MM2) such that the targeting IgG antibody or antigen-binding portion coupling of the MM2 reduces the ability of the AB2 to bind thereof is masked. In some embodiments, the anti-CTLA-4 the second, cancer-related target. ScFv includes a first antibody or antigen-binding fragment 0297. In some embodiments, the T-cell engaging multi thereof (AB1) that binds CTLA-4, where the AB1 is specific activatable antibody includes an OKT3 scFv or attached to a masking moiety (MM1) Such that coupling of OKT3-derived scFv and a cancer targeting IgG antibody or the MM1 reduces the ability of the AB1 to bind CTLA-4. In antigen-binding fragment thereof, where at least one of the Some embodiments, the targeting IgG antibody or antigen OKT3 ScFv or OKT3-derived ScFv and/or the cancer tar binding fragment thereof includes a second antibody or geting IgG antibody or antigen-binding portion thereof is fragment thereof that includes a second antibody or antigen masked. In some embodiments, the OKT3 scv or OKT3 binding fragment thereof (AB2) that binds a second target, derived scEv includes a first antibody or antigen-binding where the AB2 is attached to a masking moiety (MM2) such fragment thereof (AB1) that binds CD3e, where the AB1 is that coupling of the MM2 reduces the ability of the AB2 to attached to a masking moiety (MM1) Such that coupling of bind the second target. In some embodiments, the anti the MM1 reduces the ability of the AB1 to bind CD3e. In CTLA-4 schv includes a first antibody or antigen-binding Some embodiments, the cancer targeting IgG antibody or fragment thereof (AB1) that binds CTLA-4, where the AB1 antigen-binding fragment thereof includes a second anti is attached to a masking moiety (MM1) Such that coupling body or fragment thereof that includes a second antibody or of the MM1 reduces the ability of the AB1 to bind CTLA-4, antigen-binding fragment thereof (AB2) that binds a second, and the targeting antibody IgG or antigen-binding fragment cancer-related target, where the AB2 is attached to a mask thereof includes a second antibody or fragment thereof that ing moiety (MM2) such that coupling of the MM2 reduces includes a second antibody or antigen-binding fragment the ability of the AB2 to bind the second, cancer-related thereof (AB2) that binds a second target, where the AB2 is target. In some embodiments, the OKT3 schv or OKT3 attached to a masking moiety (MM2) Such that coupling of derived scEv includes a first antibody or antigen-binding the MM2 reduces the ability of the AB2 to bind the second fragment thereof (AB1) that binds CD3e, where the AB1 is target. attached to a masking moiety (MM1) Such that coupling of 0300. In some embodiments, the multi-antigen targeting the MM1 reduces the ability of the AB1 to bind CD3e, and antibodies and/or multi-antigen targeting activatable anti the cancer targeting antibody IgG or antigen-binding frag bodies include at least a first antibody or antigen-binding ment thereof includes a second antibody or fragment thereof fragment thereof that binds a first target and/or first epitope that includes a second antibody or antigen-binding fragment and a second antibody or antigen-binding fragment thereof thereof (AB2) that binds a second, cancer-related target, that binds a second target and/or a second epitope. In some where the AB2 is attached to a masking moiety (MM2) such embodiments, the multi-antigen targeting antibodies and/or that coupling of the MM2 reduces the ability of the AB2 to multi-antigen targeting activatable antibodies bind two or bind the second, cancer-related target. more different targets. In some embodiments, the multi 0298. In some embodiments, the T-cell engaging multi antigen targeting antibodies and/or multi-antigen targeting specific activatable antibody includes an anti-CTLA-4 schv, activatable antibodies bind two or more different epitopes on where at least one of the targeting antibody or antigen the same target. In some embodiments, the multi-antigen binding fragment thereof and/or the anti-CTLA-4 schv is targeting antibodies and/or multi-antigen targeting activat masked. In some embodiments, the anti-CTLA-4 scEv able antibodies bind a combination of two or more different includes a first antibody or antigen-binding fragment thereof targets and two or more different epitopes on the same target. (AB1) that binds CTLA-4, where the AB1 is attached to a 0301 In some embodiments, a multispecific activatable masking moiety (MM1) such that coupling of the MM1 antibody comprising an IgG has the IgG variable domains reduces the ability of the AB1 to bind CTLA-4. In some masked. In some embodiments, a multispecific activatable embodiments, the targeting antibody or antigen-binding antibody comprising a scFv has the scFv domains masked. fragment thereof includes a second antibody or fragment In some embodiments, a multispecific activatable antibody thereof that includes a second antibody or antigen-binding has both IgG variable domains and scFv domains, where at fragment thereof (AB2) that binds a second target, where the least one of the IgG variable domains is coupled to a AB2 is attached to a masking moiety (MM2) such that masking moiety. In some embodiments, a multispecific coupling of the MM2 reduces the ability of the AB2 to bind activatable antibody has both IgG variable domains and the second target. In some embodiments, the anti-CTLA-4 scFv domains, where at least one of the scEv domains is ScFv includes a first antibody or antigen-binding fragment coupled to a masking moiety. In some embodiments, a thereof (AB1) that binds CTLA-4, where the AB1 is multispecific activatable antibody has both IgG variable attached to a masking moiety (MM11) Such that coupling of domains and ScHv domains, where at least one of the IgG the MM1 reduces the ability of the AB1 to bind CTLA-4, variable domains is coupled to a masking moiety and at least US 2016/0289.324 A1 Oct. 6, 2016

one of the scFv domains is coupled to a masking moiety. In L4-VH-CH1-CH2-CH3); (VL-CL-L4-VL*-L3-VH*-L2 Some embodiments, a multispecific activatable antibody has CM1-CM2 substrate-L1-MM): (MM-L1-CM1-CM2 sub both IgG variable domains and scFv domains, where each of strate-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3); (VL the IgG variable domains and the scFv domains is coupled CL-L4-VL*-L3-VH*-L2-CM1-CM2 substrate-L1-MM): to its own masking moiety. In some embodiments, one (MM-L1-CM1-CM2 substrate-L2-VH*-L3-VL*-L4-VH antibody domain of a multispecific activatable antibody has CH1-CH2-CH3); (VL-CL-L4-VH*-L3-VL*): (MM-L1 specificity for a target antigen and another antibody domain CM1-CM2 Substrate-L2-VL-L3-VH-L4-VH-CH1-CH2 has specificity for a T-cell Surface antigen. In some embodi CH3); (VL-CL-L4-VH*-L3-VL*): (MM-L1-CM1-CM2 ments, one antibody domain of a multispecific activatable substrate-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3); (VL antibody has specificity for a target antigen and another CL-L4-VL*-L3-VH*: (MM-L1-CM1-CM2 substrate-L2 antibody domain has specificity for another target antigen. In VL*-L3-VH*-L4-VH-CH1-CH2-CH3), (VL-CL-L4-VL*- Some embodiments, one antibody domain of a multispecific L3-VH*): (MM-L1-CM1-CM2 substrate-L2-VH*-L3 activatable antibody has specificity for an epitope of a target VL*-L4-VH-CH1-CH2-CH3); (VL-CL-L4-VH*-L3-VL*- antigen and another antibody domain has specificity for L2-CM1-CM2 substrate-L-MM): (VL*-L3-VH*-L4-VH another epitope of the target antigen. CH1-CH2-CH3); (VL-CL-L4-VH*-L3-VL*-L2-CM1 0302) In a multispecific activatable antibody, a scFv can CM2 substrate-L1-MM). (VH*-L3-VL*-L4-VH-CH1 be fused to the carboxyl terminus of the heavy chain of an CH2-CH3); (VL-CL-L4-VL*-L3-VH*-L2-CM1-CM2 IgG activatable antibody, to the carboxyl terminus of the substrate-L-MM): (VL*-L3-VH*-L4-VH-CH1-CH2-CH3) light chain of an IgG activatable antibody, or to the carboxyl ; or (VL-CL-L4-VL*-L3-VH*-L2-CM1-CM2 substrate termini of both the heavy and light chains of an IgG L1-MM): (VH*-L3-VL*-L4-VH-CH1-CH2-CH3), activatable antibody. In a multispecific activatable antibody, wherein: VL and VH represent the light and heavy variable a schv can be fused to the amino terminus of the heavy chain domains of the first specificity, contained in the IgG: VL* of an IgG activatable antibody, to the amino terminus of the and VH represent the variable domains of the second light chain of an IgG activatable antibody, or to the amino specificity, contained in the sclv; L1 is a linker peptide termini of both the heavy and light chains of an IgG connecting the masking moiety (MM) and the CM1-CM2 activatable antibody. In a multispecific activatable antibody, substrate; L2 is a linker peptide connecting the CM1-CM2 a scV can be fused to any combination of one or more Substrate, and the antibody, L3 is a linker peptide connecting carboxyl termini and one or more amino termini of an IgG the variable domains of the schv: L4 is a linker peptide activatable antibody. In some embodiments, a masking connecting the antibody of the first specificity to the anti moiety (MM) linked to a CM1-CM2 substrate is attached to body of the second specificity; CL is the light-chain constant and masks an antigen binding domain of the IgG. In some domain; and CH1, CH2, CH3 are the heavy chain constant embodiments, a masking moiety (MM) linked to a CM1 domains. The first and second specificities may be toward CM2 Substrate is attached to and masks an antigen binding any antigen or epitope. domain of at least one Sclv. In some embodiments, a 0304. In some embodiments of a T-cell engaging multi masking moiety (MM) linked to a CM1-CM2 substrate is specific activatable antibody, one antigen is typically an attached to and masks an antigen binding domain of an IgG antigen present on the Surface of a tumor cell or other cell and a masking moiety (MM) linked to a CM1-CM2 sub type associated with disease, such as, but not limited to, any strate is attached to and masks an antigen binding domain of target listed in Table 1, such as, but not limited to, EGFR, at least one scEv. erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transfer rin receptor), and another antigen is typically a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain con ferring specificity to the T-cell Surface antigen may also be Substituted by a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9. In some embodiments of a multi-antigen targeting activatable anti body, one antigen is selected from the group of targets listed in Table 1, and another antigen is selected from the group of targets listed in Table 1. 0305. 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 US 2016/0289.324 A1 Oct. 6, 2016 44

C225v6, which is specific for binding to EGFR. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the targeting antibody is an anti-Jagged anti embodiments, the agent is a toxin or fragment thereof. In body. In some embodiments, the targeting antibody is 4D11, Some embodiments, the agent is conjugated to the multispe which is specific for binding to human and mouse Jagged 1 cific activatable antibody via a linker. In some embodiments, and Jagged 2. In some embodiments, the targeting antibody the agent is conjugated to the AB via a cleavable linker. In is 4D11 V2, which is specific for binding to human and Some embodiments, the agent is conjugated to the AB via a mouse Jagged 1 and Jagged 2. linker that includes at least one CM1-CM2 substrate 0306 In some embodiments, the targeting antibody can sequence. In some embodiments, the linker is a non-cleav be in the form an activatable antibody. In some embodi able linker. In some embodiments, the agent is a microtubule ments, the schv(s) can be in the form of a Pro-scFv (see, e.g., inhibitor. In some embodiments, the agent is a nucleic acid WO 2009/025846, WO 2010/081173). damaging agent, Such as a DNA alkylator or DNA interca 0307. In some embodiments, the scFv is specific for lator, or other DNA damaging agent. In some embodiments, binding CD3e, and is or is derived from an antibody or the linker is a cleavable linker. In some embodiments, the fragment thereof that binds CD3e, e.g., CH2527, FN 18, agent is an agent selected from the group listed in Table 4. H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, In some embodiments, the agent is a dolastatin. In some the scFv is specific for binding CTLA-4 (also referred to embodiments, the agent is an auristatin or derivative thereof. herein as CTLA and CTLA4). In some embodiments, the agent is auristatin E or a deriva 0308. In some embodiments, the anti-CTLA-4 scFv tive thereof. In some embodiments, the agent is monomethyl includes the amino acid sequence: auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or maytansinoid derivative. In (SEO ID NO. 347) some embodiments, the agent is DM1 or DM4. In some GGGSGGGGSGSGGGSGGGGSGGGEIWLTOSPGTLSLSPGERATLSCRASQ embodiments, the agent is a duocarmycin or derivative SVSSSYLAWYOOKPGOAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS thereof. In some embodiments, the agent is a calicheamicin or derivative thereof. In some embodiments, the agent is a RLEPEDFAVYYCOOYGSSPLTFGGGTKVEIKRSGGSTITSYNWYYTKLSS pyrrolobenzodiazepine. In some embodiments, the agent is SGTOVOLVOTGGGVWOPGRSLRLSCAASGSTFSSYAMSWVROAPGKGLEW a pyrrolobenzodiazepine dimer. 0314. In some embodiments, the multispecific activatable WSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCA antibody also includes a detectable moiety. In some embodi ments, the detectable moiety is a diagnostic agent. TNSLYWYFDLWGRGTLWTWSSAS 0315. In some embodiments, the multispecific activatable 0309. In some embodiments, the anti-CTLA-4 scFv antibody naturally contains one or more disulfide bonds. In includes the amino acid sequence that is at least 90%, 91%, some embodiments, the multispecific activatable antibody 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more can be engineered to include one or more disulfide bonds. identical to the amino acid sequence of SEQ ID NO. 347. 0316 The disclosure also provides an isolated nucleic 0310. In some embodiments, the anti-CD3e scFv acid molecule encoding a multispecific activatable antibody includes the amino acid sequence: described herein, as well as vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing a multispecific activatable antibody (SEQ ID NO: 349) by culturing a cell under conditions that lead to expression GGGSGGGGSGSGGGSGGGGSGGGOVOLOOSGAELARPGASVKMSCKASGY of the activatable antibody, wherein the cell comprises such TFTRYTMHWWKORPGOGLEWIGYINPSRGYTNYNOKFKDKATLTTDKSSS a nucleic acid molecule. In some embodiments, the cell comprises such a vector. TAYMOLSSLTSEDSAVYYCARYYDDHYCLDYWGOGTTL TVSSGGGGSGGG 0317. The disclosure also provides a method of manu GSGGGGSOIVLTOSPAIMSASPGEKVTMTCSASSSVSYMNWYOOKSGTSP facturing multispecific activatable antibodies of the disclo Sure by (a) culturing a cell comprising a nucleic acid KRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCOOWSSN construct that encodes the multispecific activatable antibody under conditions that lead to expression of the multispecific PFTFGSGTKLEINR activatable, and (b) recovering the multispecific activatable 0311. In some embodiments, the anti-CD3e schv includes antibody. the amino acid sequence that is at least 90%, 91%, 92%, 0318. The disclosure also provides multispecific activat 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to able antibodies and/or multispecific activatable antibody the amino acid sequence of SEQ ID NO. 349. compositions that include at least a first antibody or antigen 0312. In some embodiments, the scFv is specific for binding fragment thereof (AB1) that specifically binds a first binding one or more T-cells, one or more NK-cells and/or target or first epitope and a second antibody or antigen one or more macrophages. In some embodiments, the Sclv biding fragment thereof (AB2) that binds a second target or is specific for binding a target selected from the group a second epitope, where at least AB1 is coupled or otherwise consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, attached to a masking moiety (MM1). Such that coupling of CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, the MM1 reduces the ability of AB1 to bind its target. In GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, some embodiments, the MM1 is coupled to AB1 via a TIM3, or VISTA. CM1-CM2 substrate for an MMP and a SP, where at least 0313. In some embodiments, the multispecific activatable one of the MMP and the SP is co-localized with the target antibody also includes an agent conjugated to the AB. In of AB1 at a treatment site or a diagnostic site in a Subject. Some embodiments, the agent is a therapeutic agent. In some The multispecific activatable antibodies provided herein are US 2016/0289.324 A1 Oct. 6, 2016

stable in circulation, activated at intended sites of therapy 0328. In some embodiments, MM1 has a dissociation and/or diagnosis but not in normal, i.e., healthy tissue, and, constant for binding to its corresponding AB that is greater when activated, exhibit binding to the target of AB1 that is than the dissociation constant of the AB to its corresponding at least comparable to the corresponding, unmodified mul target or epitope. tispecific antibody. 0329. In some embodiments, MM1 has a dissociation 0319. In some embodiments, the multispecific activatable constant for binding to its corresponding AB that is no more antibody comprises a linking peptide between the MM1 and than the dissociation constant of the AB to its corresponding the CM1-CM2 Substrate. target or epitope. 0330. In some embodiments, MM1 does not interfere or 0320 In some embodiments, the multispecific activatable compete with its corresponding AB for binding to the antibody comprises a linking peptide between the CM1 corresponding target or epitope when the multispecific acti CM2 Substrate and the AB1. vatable antibody is in a cleaved state. 0321. In some embodiments, the activatable antibody 0331. In some embodiments, MM1 is a polypeptide of comprises a first linking peptide (LP1) and a second linking about 2 to 40 amino acids in length. In some embodiments, peptide (LP2), and at least a portion of the multispecific each of the MM in the multispecific activatable antibody is activatable antibody has the structural arrangement from a polypeptide of no more than 40 amino acids in length. N-terminus to C-terminus as follows in the uncleaved state: 0332. In some embodiments, MM1 has a polypeptide MM1-LP1-CM1-CM2 Substrate-LP2-AB1 or AB1-LP2 sequence that is different from that of target of the corre CM1-CM2 substrate-LP1-MM1. In some embodiments, the sponding AB. two linking peptides need not be identical to each other. 0333. In some embodiments, MM1 has a polypeptide 0322. In some embodiments, at least one of LP1 or LP2 sequence that is no more than 50% identical to any natural includes an amino acid sequence selected from the group binding partner of the corresponding AB. In some embodi consisting of (GS), (GGS), (GSGGS), (SEQID NO:381) ments, MM1 has a polypeptide sequence that is no more and (GGGS), (SEQ ID NO:382), where n is an integer of than 25% identical to any natural binding partner of the at least one. In some embodiments, at least one of LP1 or corresponding AB. In some embodiments, MM1 has a LP2 includes an amino acid sequence selected from the polypeptide sequence that is no more than 100% identical to group consisting of GGSG (SEQ ID NO: 383), GGSGG any natural binding partner of the corresponding AB. (SEQ ID NO:384), GSGSG (SEQ ID NO: 385), GSGGG 0334. In some embodiments, the coupling of MM1 (SEQ ID NO: 386), GGGSG (SEQ ID NO: 387), and reduces the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (K) of the AB GSSSG (SEQ ID NO:388). when coupled to the MM1 towards its corresponding target 0323. In some embodiments, the activatable antibody or epitope is at least 20 times greater than the K of the AB includes a linking peptide (LP) between CM1 and CM2. when not coupled to the MM1 towards its corresponding 0324. In some embodiments, the activatable antibody target or epitope. comprises a first linking peptide (LP1), a second linking 0335. In some embodiments, the coupling of MM1 peptide (LP2), and a linking peptide (LP") between CM1 and reduces the ability of the corresponding AB to bind its target CM2, and at least a portion of the multispecific activatable or epitope such that the dissociation constant (K) of the AB antibody has the structural arrangement from N-terminus to when coupled to the MM1 towards its corresponding target C-terminus as follows in the uncleaved state: MM1-LP1 or epitope is at least 40 times greater than the K of the AB CM1-CM2 Substrate-LP2-AB1 or AB1-LP2-CM1-CM2 when not coupled to the MM1 towards its corresponding substrate-LP1-MM1. In some embodiments, linking pep target or epitope. tides need not be identical to each other. 0336. In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind its target 0325 In some embodiments, LP' is G.G. In some embodi or epitope such that the dissociation constant (K) of the AB ments, LP is GGSGGS (SEQ ID NO:350). when coupled to the MM1 towards its corresponding target 0326 In some embodiments, the multispecific activatable or epitope is at least 100 times greater than the K of the AB antibody includes at least a first antibody or antigen-binding when not coupled to the MM1 towards its corresponding fragment thereof (AB1) that specifically binds a first target target or epitope. or first epitope and a second antibody or antigen-binding 0337. In some embodiments, the coupling of MM1 fragment thereof (AB2) that specifically binds a second reduces the ability of the corresponding AB to bind its target target or second epitope. In some embodiments, each of the or epitope such that the dissociation constant (K) of the AB AB in the multispecific activatable antibody is indepen when coupled to the MM1 towards its corresponding target dently selected from the group consisting of a monoclonal or epitope is at least 1000 times greater than the K of the AB antibody, domain antibody, single chain, Fab fragment, a when not coupled to the MM1 towards its corresponding F(ab')2 fragment, a scFv, a ScAb, a dAb, a single domain target or epitope. heavy chain antibody, and a single domain light chain 0338. In some embodiments, the coupling of MM1 antibody. In some embodiments, each of the AB in the reduces the ability of the corresponding AB to bind its target multispecific activatable antibody is a rodent (e.g., mouse or or epitope such that the dissociation constant (K) of the AB rat), chimeric, humanized or fully human monoclonal anti when coupled to the MM1 towards its corresponding target body. or epitope is at least 10,000 times greater than the K of the 0327. In some embodiments, each of the AB in the AB when not coupled to the MM1 towards its corresponding multispecific activatable antibody has a dissociation con target or epitope. stant of about 100 nM or less for binding to its correspond 0339. In some embodiments, MM1 is an amino acid ing target or epitope. sequence selected from a MM disclosed herein. US 2016/0289.324 A1 Oct. 6, 2016 46

0340. In some embodiments, the multispecific activatable multispecific activatable antibody has a polypeptide antibody includes at least a second masking moiety (MM2) sequence that is no more than 10% identical to any natural that inhibits the binding of the AB2 to its target when the binding partner of the corresponding AB. multispecific activatable antibody is in an uncleaved state, 0348. In some embodiments, the coupling of each of the and an additional cleavable moiety (CM) coupled to the MM reduces the ability of the corresponding AB to bind its AB2, wherein the CM' is either a CM1-CM2 substrate or a target or epitope Such that the dissociation constant (K) of polypeptide that functions as a Substrate for a second pro the AB when coupled to the MM towards its corresponding tease. In some embodiments, CM is a polypeptide of no target or epitope is at least 20 times greater than the K of more than 15 amino acids long. In some embodiments, CM' the AB when not coupled to the MM towards its correspond is a CM1-CM2 substrate, wherein each of CM1 and CM2 in ing target or epitope. the CM1-CM2 substrate is independently no more than 15 0349. In some embodiments, the coupling of each of the amino acids long. MM reduces the ability of the corresponding AB to bind its (0341. In some embodiments, the MMP protease, the SP target or epitope Such that the dissociation constant (K) of protease, and/or the second protease is co-localized with the the AB when coupled to the MM towards its corresponding second target or epitope in a tissue, and wherein the MMP target or epitope is at least 40 times greater than the K of protease, the SP protease, and/or the second protease cleaves the AB when not coupled to the MM towards its correspond the CM in the multispecific activatable antibody when the ing target or epitope. multispecific activatable antibody is exposed to the MMP 0350. In some embodiments, the coupling of each of the protease, the SP protease, and/or the second protease. In MM reduces the ability of the corresponding AB to bind its some embodiments, the MMP protease, the SP protease, target or epitope such that the dissociation constant (K) of and/or the second protease are co-localized with the first the AB when coupled to the MM towards its corresponding target or epitope and the second target or epitope in a tissue. target or epitope is at least 100 times greater than the K of In some embodiments, the MMP protease, the SP protease, the AB when not coupled to the MM towards its correspond and/or the second protease are the same MMP protease and ing target or epitope. the same SP protease. In some embodiments, the MMP protease, the SP protease, and/or the second protease are not 0351. In some embodiments, the coupling of each of the the same MMP protease and not the same SP protease. In MM reduces the ability of the corresponding AB to bind its some embodiments, the CM1-CM2 substrate and CM are target or epitope such that the dissociation constant (K) of different substrates for the same MMP protease and same SP the AB when coupled to the MM towards its corresponding protease. In some embodiments, the protease that cleaves target or epitope is at least 1000 times greater than the K of CM is selected from the group consisting of those shown in the AB when not coupled to the MM towards its correspond Table 6. ing target or epitope. 0342. In some embodiments, each of the MM in the 0352. In some embodiments, the coupling of each of the multispecific activatable antibody, e.g., MM1 and at least MM reduces the ability of the corresponding AB to bind its MM2, has a dissociation constant for binding to its corre target or epitope Such that the dissociation constant (K) of sponding AB that is greater than the dissociation constant of the AB when coupled to the MM towards its corresponding the AB to its corresponding target or epitope. target or epitope is at least 10,000 times greater than the K 0343. In some embodiments, each of the MM in the of the AB when not coupled to the MM towards its corre multispecific activatable antibody has a dissociation con sponding target or epitope. stant for binding to its corresponding AB that is no more 0353. In some embodiments, each of the MM is an amino than the dissociation constant of the AB to its corresponding acid sequence selected from a MM disclosed herein. target or epitope. 0354. In some embodiments, the protease that cleaves the 0344. In some embodiments, each of the MM in the CM1-CM2 substrate sequence is co-localized with the target multispecific activatable antibody does not interfere or com of the AB1 in the multispecific activatable antibody in a pete with its corresponding AB for binding to the corre tissue, and the MMP protease and/or SP protease, i.e., at sponding target or epitope when the multispecific activatable least one of the MMP protease and the SP protease, cleave antibody is in a cleaved state. the CM1-CM2 substrate in the multispecific activatable 0345. In some embodiments, each of the MM in the antibody when the multispecific activatable antibody is multispecific activatable antibody is a polypeptide of about exposed to the proteases. 2 to 40 amino acids in length. In some embodiments, each 0355. In some embodiments, the multispecific activatable of the MM in the multispecific activatable antibody is a antibody includes more than one CM1-CM2 substrate polypeptide of no more than 40 amino acids in length. sequence, and the MMP protease and/or the SP protease that 0346. In some embodiments, each of the MM in the cleaves at least one CM1-CM2 substrate sequence is co multispecific activatable antibody has a polypeptide localized with the target of at least one of the AB regions in sequence that is different from that of target of the corre the multispecific activatable antibody in a tissue, and the sponding AB. MMP protease and/or SP protease cleaves the CM1-CM2 0347 In some embodiments, each of the MM in the substrate in the multispecific activatable antibody when the multispecific activatable antibody has a polypeptide multispecific activatable antibody is exposed to the pro sequence that is no more than 50% identical to any natural teases. binding partner of the corresponding AB. In some embodi 0356. In some embodiments, each CM1-CM2 substrate, ments, each of the MM in the multispecific activatable is positioned in the multispecific activatable antibody such antibody has a polypeptide sequence that is no more than that in the uncleaved state, binding of the multispecific 25% identical to any natural binding partner of the corre activatable antibody to a target of one of the AB regions is sponding AB. In some embodiments, each of the MM in the reduced to occur with a dissociation constant that is at least US 2016/0289.324 A1 Oct. 6, 2016 47 twofold greater than the dissociation constant of an unmodi greater than the dissociation constant of an unmodified AB fied AB binding to its target, and whereas in the cleaved binding to its target, and whereas in the cleaved State, the AB state, the AB binds its target. binds its target. 0357. In some embodiments, each CM1-CM2 substrate, 0365. In some embodiments, each CM1-CM2 substrate is is positioned in the multispecific activatable antibody such positioned in the multispecific activatable antibody such that that in the uncleaved state, binding of the multispecific in the uncleaved State, binding of the multispecific activat activatable antibody to a target of one of the AB regions is able antibody to a target of one of the AB regions is reduced reduced to occur with a dissociation constant that is at least to occur with a dissociation constant that is at least 200-fold threefold greater than the dissociation constant of an greater than the dissociation constant of an unmodified AB unmodified AB binding to its target, and whereas in the binding to its target, and whereas in the cleaved State, the AB cleaved state, the AB binds its target. binds its target. 0358. In some embodiments, each CM1-CM2 substrate, 0366. The disclosure also provides compositions and is positioned in the multispecific activatable antibody such methods that include a multispecific activatable antibody that in the uncleaved state, binding of the multispecific that includes at least a first antibody or antibody fragment activatable antibody to a target of one of the AB regions is (AB1) that specifically binds a target and a second antibody reduced to occur with a dissociation constant that is at least or antibody fragment (AB2), where at least the first AB in fourfold greater than the dissociation constant of an unmodi the multispecific activatable antibody is coupled to a mask fied AB binding to its target, and whereas in the cleaved ing moiety (MM1) that decreases the ability of AB1 to bind state, the AB binds its target. its target. In some embodiments, each AB is coupled to a 0359. In some embodiments, each CM1-CM2 substrate, MM that decreases the ability of its corresponding AB to is positioned in the multispecific activatable antibody such each target. For example, in bispecific activatable antibody that in the uncleaved state, binding of the multispecific embodiments, AB1 is coupled to a first masking moiety activatable antibody to a target of one of the AB regions is (MM1) that decreases the ability of AB1 to bind its target, reduced to occur with a dissociation constant that is at least and AB2 is coupled to a second masking moiety (MM2) that fivefold greater than the dissociation constant of an unmodi decreases the ability of AB2 to bind its target. In some fied AB binding to its target, and whereas in the cleaved embodiments, the multispecific activatable antibody com state, the AB binds its target. prises more than two AB regions; in Such embodiments, 0360. In some embodiments, each CM1-CM2 substrate, AB1 is coupled to a first masking moiety (MM1) that is positioned in the multispecific activatable antibody such decreases the ability of AB1 to bind its target, AB2 is that in the uncleaved state, binding of the multispecific coupled to a second masking moiety (MM2) that decreases activatable antibody to a target of one of the AB regions is the ability of AB2 to bind its target, AB3 is coupled to a third reduced to occur with a dissociation constant that is at least masking moiety (MM3) that decreases the ability of AB3 to tenfold greater than the dissociation constant of an unmodi bind its target, and so on for each AB in the multispecific fied AB binding to its target, and whereas in the cleaved activatable antibody. state, the AB binds its target. 0367. In some embodiments, the multispecific activatable 0361. In some embodiments, each CM1-CM2 substrate, antibody further includes at least one CM1-CM2 substrate is positioned in the multispecific activatable antibody such that is a substrate for a MMP protease and a SP protease, that in the uncleaved state, binding of the multispecific where the CM1-CM2 Substrate links a MM to an AB. For activatable antibody to a target of one of the AB regions is example, in Some embodiments, the multispecific activat reduced to occur with a dissociation constant that is at least able antibody includes at least a first antibody or antibody 20-fold greater than the dissociation constant of an unmodi fragment (AB1) that specifically binds a target and a second fied AB binding to its target, and whereas in the cleaved antibody or antibody fragment (AB2), where at least the first state, the AB binds its target. AB in the multispecific activatable antibody is coupled via 0362. In some embodiments, each CM1-CM2 substrate is a first CM1-CM2 substrate to a masking moiety (MM1) that positioned in the multispecific activatable antibody such that decreases the ability of AB1 to bind its target. In some in the uncleaved State, binding of the multispecific activat bispecific activatable antibody embodiments, AB1 is able antibody to a target of one of the AB regions is reduced coupled via the first CM1-CM2 substrate to MM1, and AB2 to occur with a dissociation constant that is at least 40-fold is coupled via a second CM1-CM2 substrate to a second greater than the dissociation constant of an unmodified AB masking moiety (MM2) that decreases the ability of AB2 to binding to its target, and whereas in the cleaved State, the AB bind its target. In some embodiments, the multispecific binds its target. activatable antibody comprises more than two AB regions; 0363. In some embodiments, each CM1-CM2 substrate is in some of these embodiments, AB1 is coupled via the first positioned in the multispecific activatable antibody such that CM1-CM2 substrate to MM1, AB2 is coupled via the second in the uncleaved State, binding of the multispecific activat CM1-CM2 substrate to MM2, and AB3 is coupled via a third able antibody to a target of one of the AB regions is reduced CM1-CM2 substrate to a third masking moiety (MM3) that to occur with a dissociation constant that is at least 50-fold decreases the ability of AB3 to bind its target, and so on for greater than the dissociation constant of an unmodified AB each AB in the multispecific activatable antibody. binding to its target, and whereas in the cleaved State, the AB 0368 Activatable Antibodies Having Non-Binding Steric binds its target. Moieties or Binding Partners for Non-Binding Steric Moi 0364. In some embodiments, each CM1-CM2 substrate is eties positioned in the multispecific activatable antibody such that 0369. The disclosure also provides activatable antibodies in the uncleaved State, binding of the multispecific activat that include non-binding steric moieties (NB) or binding able antibody to a target of one of the AB regions is reduced partners (BP) for non-binding steric moieties, where the BP to occur with a dissociation constant that is at least 100-fold recruits or otherwise attracts the NB to the activatable US 2016/0289.324 A1 Oct. 6, 2016 48 antibody. The activatable antibodies provided herein cleavage of the CM1-CM2 substrate by the enzyme. For include, for example, an activatable antibody that includes a example, each of the CM substrate sequence and the CM2 non-binding steric moiety (NB), a CM1-CM2 substrate and substrate sequence in the CM1-CM2 substrate independent antibody or antibody fragment (AB) that binds a target; an has a length of up to 15 amino acids. activatable antibody that includes a binding partner for a 0373) In one embodiment, the activatable antibody non-binding steric moiety (BP), a CM1-CM2 substrate and includes a non-binding steric moiety (NB); a CM1-CM2 an AB; and an activatable antibody that includes a BP to substrate; and an antibody or antibody fragment (AB) that which an NB has been recruited, a CM1-CM2 substrate and binds specifically to the target, wherein (i) the NB includes an AB that binds the target. Activatable antibodies in which a polypeptide that does not bind specifically to the AB; (ii) the NB is covalently linked to the CM1-CM2 substrate and the CM1-CM2 substrate is a polypeptide that includes a AB of the activatable antibody or is associated by interaction substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is with a BP that is covalently linked to the CM1-CM2 positioned such that in an uncleaved state, the NB interferes substrate and AB of the activatable antibody are referred to with binding of the AB to the target and in a cleaved state, herein as “NB-containing activatable antibodies.” By acti the NB does not interfere with binding of the AB to the vatable or switchable is meant that the activatable antibody target; (iv) the NB does not inhibit cleavage of the CM1 exhibits a first level of binding to a target when the activat CM2 substrate by the enzyme; and (v) the activatable able antibody is in an inhibited, masked or uncleaved state antibody has the structural arrangement from N-terminus to (i.e., a first conformation), and a second level of binding to C-terminus as follows in the uncleaved state: NB-CM1 the target when the activatable antibody is in an uninhibited, CM2 Substrate-AB or AB-CM1-CM2 Substrate-NB. unmasked and/or cleaved state (i.e., a second conformation, 0374. In one embodiment, the activatable antibody i.e., activated antibody), where the second level of target includes a non-binding steric moiety (NB); a CM1-CM2 binding is greater than the first level of target binding. The substrate; and an antibody or antibody fragment (AB) that activatable antibody compositions can exhibit increased binds specifically to the target, wherein (i) the NB includes bioavailability and more favorable biodistribution compared a polypeptide that does not bind specifically to the AB; (ii) to conventional antibody therapeutics. the CM1-CM2 substrate is a polypeptide that includes a 0370. In some embodiments, activatable antibodies pro substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is vide for reduced toxicity and/or adverse side effects that positioned such that in an uncleaved state, the NB interferes could otherwise result from binding of the at non-treatment with binding of the AB to the target and in a cleaved state, sites and/or non-diagnostic sites if the AB were not masked the NB does not interfere with binding of the AB to the or otherwise inhibited from binding to such a site. target, and wherein the NB in the uncleaved activatable 0371. In one embodiment, the activatable antibody antibody reduces the ability of the AB to bind the target by includes a non-binding steric moiety (NB); a CM1-CM2 at least 50%, for example, by at least 60%, by at least 70%, substrate; and an antibody or antibody fragment (AB) that by at least 75%, by at least 80%, by at least 85%, by at least binds specifically to the target, wherein the NB is a poly 90%, by at least 95%, by at least 96%, by at least 97%, by peptide that does not bind specifically to the AB; the at least 98%, by at least 99%, by at least 100% as compared CM1-CM2 substrate is a polypeptide that includes a sub to the ability of the cleaved AB to bind the target; and (iv) strate (S) for an enzyme; the CM1-CM2 substrate is posi the NB does not inhibit cleavage of the CM1-CM2 substrate tioned such that in an uncleaved state, the NB interferes with by the enzyme. The reduction in the ability of the AB to bind binding of the AB to the target and in a cleaved state, the NB the target is determined, e.g., using an assay as described does not interfere with binding of the AB to the target; and herein or an in vitro target displacement assay Such as, for the NB does not inhibit cleavage of the CM1-CM2 substrate example, the assay described in PCT Publication Nos. WO by the enzyme. As used herein and throughout, the term 2009/O25846 and WO 2010/081173. polypeptide refers to any polypeptide that includes at least 0375. In one embodiment, the activatable antibody two amino acid residues, including larger polypeptides, includes a binding partner (BP) for a non-binding steric full-length proteins and fragments thereof, and the term moiety (NB); a CM1-CM2 substrate; and an antibody or polypeptide is not limited to single-chain polypeptides and antibody fragment (AB) that binds specifically to the target, can include multi-unit, e.g., multi-chain, polypeptides. In wherein the BP is a polypeptide that binds to the NB when cases where the polypeptide is of a shorter length, for exposed thereto; the NB does not bind specifically to the AB; example, less than 50 amino acids total, the terms peptide the CM1-CM2 substrate is a polypeptide that includes a and polypeptide are used interchangeably herein, and in substrate (S) for an enzyme; the CM1-CM2 substrate is cases where the polypeptide is of a longer length, e.g., 50 positioned such that in an uncleaved State in the presence of amino acids or greater, the terms polypeptide and protein are the NB, the NB interferes with binding of the AB to the used interchangeably herein. target and in a cleaved state, the NB does not interfere with 0372. In one embodiment, the activatable antibody binding of the AB to the target and the BP does not interfere includes a non-binding steric moiety (NB); a CM1-CM2 with binding of the AB to the target; and the NB and the BP substrate; and an antibody or antibody fragment (AB) that do not inhibit cleavage of the CM1-CM2 substrate by the binds specifically to the target, wherein (i) the NB includes enzyme. In some examples of this embodiment, the BP of a polypeptide that does not bind specifically to the AB; (ii) the activatable antibody is optionally bound to the NB. In CM1-CM2 substrate is a polypeptide of up to 50 amino acids one embodiment, the NB is recruited by the BP of the in length that includes a substrate (S) for an enzyme; (iii) the activatable antibody in vivo. CM1-CM2 substrate is positioned such that in an uncleaved 0376. In some examples of any of these activatable state, the NB interferes with binding of the AB to the target antibody embodiments, the activatable antibody is formu and in a cleaved state, the NB does not interfere with binding lated as a composition. In some of these embodiments, the of the AB to the target; and (iv) the NB does not inhibit composition also includes the NB, where the NB is co US 2016/0289.324 A1 Oct. 6, 2016 49 formulated with the activatable antibody that includes the includes a combination of a variable heavy chain region BP, the CM1-CM2 substrate, and the AB. In some examples comprising an amino acid sequence that is at least 90%, of this embodiment, the BP is selected from the group 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%0 or more consisting of an albumin binding peptide, a fibrinogen identical to an amino acid sequence presented herein, and a binding peptide, a fibronectin binding peptide, a hemoglobin variable light chain region comprising an amino acid binding peptide, a transferrin binding peptide, an immuno sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, globulin domain binding peptide, and other serum protein 96%, 97%, 98%, 99% or more identical to an amino acid binding peptides. sequence presented herein. 0377. In some examples of any of these activatable 0382. In some examples of any of these activatable antibody embodiments, the NB is a soluble, globular pro antibody embodiments, the activatable antibody also tein. In some examples of any of these activatable antibody includes an agent conjugated to the AB. In some embodi embodiments, the NB is a protein that circulates in the ments, the agent is a therapeutic agent. In some embodi bloodstream. In some examples of any of these activatable ments, the agent is an antineoplastic agent. In some embodi antibody embodiments, the NB is selected from the group ments, the agent is a toxin or fragment thereof. In some consisting of albumin, fibrinogen, fibronectin, hemoglobin, embodiments, the agent is conjugated to the AB via a linker. transferrin, an immunoglobulin domain, and other serum In some embodiments, the linker is a cleavable linker. In proteins. Some embodiments, the agent is conjugated to the AB via a 0378. In some examples of any of these activatable noncleavable linker. In some embodiments, the agent is an antibody embodiments, the CM1-CM2 substrate is a poly agent selected from the group listed in Table 3. In some peptide that includes a Substrate (S) for a protease. In some embodiments, the agent is a microtubule inhibitor. In some examples of any of these activatable antibody embodiments, embodiments, the agent is a nucleic acid damaging agent, the protease is co-localized with the in a tissue, and the such as a DNA alkylator or DNA intercalator, or other DNA protease cleaves the CM1-CM2 substrate in the activatable damaging agent. In some embodiments, the agent is a antibody when the activatable antibody is exposed to the dolastatin. In some embodiments, the agent is an auristatin protease. In some examples of any of these activatable or derivative thereof. In some embodiments, the agent is antibody embodiments, the CM1-CM2 substrate is a poly auristatin E or a derivative thereof. In some embodiments, peptide of up to 50 amino acids in length. In some examples the agent is monomethyl auristatin E (MMAE). In some of any of these activatable antibody embodiments, the embodiments, the agent is monomethyl auristatin D CM1-CM2 substrate is a polypeptide that includes a sub (MMAD). In some embodiments, the agent is a maytansi strate (S) having a length of up to 15 amino acids, e.g., 3 noid or maytansinoid derivative. In some embodiments, the amino acids long, 4 amino acids long. 5 amino acids long, agent is DM1 or DM4. In some embodiments, the agent is 6 amino acids long, 7 amino acids long, 8 amino acids long, a duocarmycin or derivative thereof. In some embodiments, 9 amino acids long, 10 amino acids long, 11 amino acids the agent is a calicheamicin or derivative thereof. In some long, 12 amino acids long, 13 amino acids long, 14 amino embodiments, the agent is a pyrrolobenzodiazepine. In some acids long, or 15 amino acids long. embodiments, the agent is a pyrrolobenzodiazepine dimer. 0379. In some examples of any of these activatable 0383. In some examples of any of these activatable antibody embodiments, the activatable antibody has the antibody embodiments, the activatable antibody also structural arrangement from N-terminus to C-terminus as includes a detectable moiety. In some embodiments, the follows in the uncleaved state: NB-CM1-CM2 substrate detectable moiety is a diagnostic agent. AB, AB-CM1-CM2 substrate-NB, BP-CM1-CM2 substrate 0384. In some examples of any of these activatable AB or AB-CM1-CM2 Substrate-BP. In embodiments where antibody embodiments, the activatable antibody also the activatable antibody includes a BP and the activatable includes a spacer. In some examples of any of these acti antibody is in the presence of the corresponding NB, the vatable antibody embodiments, the activatable antibody also activatable antibody has a structural arrangement from includes a signal peptide. In some embodiments, the signal N-terminus to C-terminus as follows in the uncleaved state: peptide is conjugated to the activatable antibody via a NB:BP-CM1-CM2-AB, NB:BP-CM2-CM1-AB, AB-CM1 spacer. In some examples of any of these activatable anti CM2-BP:NB or AB-CM2-CM1-BP:NB, where “:” repre body embodiments, the spacer is joined directly to the MM sents an interaction, e.g., binding, between the NB and BP. of the activatable antibody. 0380. In some examples of any of these activatable 0385. In some embodiments, the serum half-life of the antibody embodiments, the activatable antibody includes an activatable antibody is longer than that of the corresponding antibody or antigen-binding fragment thereof that specifi antibody; e.g., the pK of the activatable antibody is longer cally binds a given target and is a monoclonal antibody, than that of the corresponding antibody. In some embodi domain antibody, single chain, Fab fragment, a F(ab') ments, the serum half-life of the activatable antibody is fragment, a scFv, a scab, a dAb, a single domain heavy chain similar to that of the corresponding antibody. In some antibody, or a single domain light chain antibody. In some embodiments, the serum half-life of the activatable antibody embodiments, such an antibody or immunologically active is at least 15 days when administered to an organism. In fragment thereof that binds the target a mouse, other rodent, some embodiments, the serum half-life of the activatable chimeric, humanized or fully human monoclonal antibody. antibody is at least 12 days when administered to an organ 0381. In some examples of any of these activatable ism. In some embodiments, the serum half-life of the antibody embodiments, the activatable antibody includes a activatable antibody is at least 11 days when administered to combination of a variable heavy chain region comprising an an organism. In some embodiments, the serum half-life of amino acid sequence presented herein and a variable light the activatable antibody is at least 10 days when adminis chain region comprising an amino acid sequence presented tered to an organism. In some embodiments, the serum herein. In some embodiments, the activatable antibody half-life of the activatable antibody is at least 9 days when US 2016/0289.324 A1 Oct. 6, 2016 50 administered to an organism. In some embodiments, the the AB when it is not associated with the NB or NB:BP or serum half-life of the activatable antibody is at least 8 days the K of the parental AB towards the target. Conversely, the when administered to an organism. In some embodiments, binding affinity of the NB-containing activatable antibody the serum half-life of the activatable antibody is at least 7 towards the target is lower than the binding affinity of the AB days when administered to an organism. In some embodi when it is not associated with the NB or NB:BP or lower ments, the serum half-life of the activatable antibody is at than the binding affinity of the parental AB towards the least 6 days when administered to an organism. In some target. For example, the binding affinity of the NB-contain examples of any of these activatable antibody embodiments, ing activatable antibody toward the target is at least 5, 10. the serum half-life of the activatable antibody is at least 5 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, days when administered to an organism. In some embodi 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, ments, the serum half-life of the activatable antibody is at 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, least 4 days when administered to an organism. In some 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100 embodiments, the serum half-life of the activatable antibody 1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10, is at least 3 days when administered to an organism. In some 000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, embodiments, the serum half-life of the activatable antibody 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, is at least 2 days when administered to an organism. In some 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000, embodiments, the serum half-life of the activatable antibody 000 times lower than the binding affinity of the AB when it is at least 24 hours when administered to an organism. In is not associated with the NB or NB:BP or lower than the some embodiments, the serum half-life of the activatable binding affinity of the parental AB towards the target. antibody is at least 20 hours when administered to an 0388. When the NB-containing activatable antibody is in organism. In some embodiments, the serum half-life of the the presence of the target, specific binding of the AB to the activatable antibody is at least 18 hours when administered target is reduced or inhibited, as compared to the specific to an organism. In some embodiments, the serum half-life of binding of the AB when it is not associated with the NB or the activatable antibody is at least 16 hours when adminis NB:BP. When the NB-containing activatable antibody is in tered to an organism. In some embodiments, the serum the presence of the target, specific binding of the AB to the half-life of the activatable antibody is at least 14 hours when target is reduced or inhibited, as compared to the specific administered to an organism. In some embodiments, the binding of the parental AB to the target. When compared to serum half-life of the activatable antibody is at least 12 hours the binding of the AB not associated with an NB or NB:BP when administered to an organism. In some embodiments, or the binding of the parental AB to the target, the ability of the serum half-life of the activatable antibody is at least 10 the NB-containing activatable antibody to bind the target is hours when administered to an organism. In some embodi reduced, for example, by at least 50%, 60%, 70%, 80%, ments, the serum half-life of the activatable antibody is at 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even least 8 hours when administered to an organism. In some 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, embodiments, the serum half-life of the activatable antibody 84, or 96 hours, or 5, 10, 15, 30, 45, 60,90, 120, 150, or 180 is at least 6 hours when administered to an organism. In days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer some embodiments, the serum half-life of the activatable when measured in vitro and/or in vivo. antibody is at least 4 hours when administered to an organ 0389. When the NB-containing activatable antibody is in ism. In some embodiments, the serum half-life of the the presence of the target but not in the presence of a activatable antibody is at least 3 hours when administered to modifying agent (for example a protease or other enzyme), an organism. specific binding of the AB to the target is reduced or 0386 The disclosure also provides an isolated nucleic inhibited, as compared to the specific binding of the AB acid molecule encoding any of these activatable antibodies, when it is not associated with the NB or NB:BP. When the as well as vectors that include these isolated nucleic acid NB-containing activatable antibody is in the presence of the sequences. The disclosure provides methods of producing an target but not in the presence of a modifying agent (for activatable antibody by culturing a cell under conditions that example a protease, other enzyme, reduction agent, or light), lead to expression of the activatable antibody, wherein the specific binding of the AB to the target is reduced or cell comprises such a nucleic acid sequence. In some inhibited, as compared to the specific binding of the parental embodiments, the cell comprises such a vector. AB to the target. When compared to the binding of the AB 0387. The dissociation constant (K) of the NB-contain not associated with an NB or NB:BP or the binding of the ing activatable antibody toward the target is greater than the parental AB to the target, the ability of the NB-containing K of the AB towards the target when it is not associated activatable antibody to bind the target is reduced, for with the NB or NB:BP. The dissociation constant (K) of the example, by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, NB-containing activatable antibody toward the target is 94%. 95%, 96%, 97%, 98%, 99%, or even 100% for at least greater than the K of the parental AB towards the target. For 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or example, the K of the NB-containing activatable antibody 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, toward the target is at least 5, 10, 25, 50, 100, 250, 500, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, in vitro and/or in vivo. 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or 0390. In some examples of any of these activatable between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, antibody embodiments, the activatable antibody includes an 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100 agent conjugated to the AB to produce an activatable anti 100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, body conjugate. In some embodiments of the activatable 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000 antibody conjugate, the agent is a therapeutic agent. In some 100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1, embodiments, the agent is a diagnostic agent. In some 000,000, or 100,000-10,000,000 times greater than the K of embodiments, the agent is a detectable marker. In some US 2016/0289.324 A1 Oct. 6, 2016

embodiments of the activatable antibody conjugate, the body under conditions that lead to expression of the acti agent is an antineoplastic agent. In some embodiments of the vatable antibody, wherein the activatable antibody includes activatable antibody conjugate, the agent is a toxin or (i) a non-binding steric moiety (NB); (ii) a CM1-CM2 fragment thereof. In some embodiments of the activatable Substrate; and (iii) an antibody or an antigen binding frag antibody conjugate, the agent is conjugated to the AB via a ment thereof (AB) that specifically binds a target, wherein linker. In some embodiments of the activatable antibody (1) the NB does not bind specifically to the AB; (2) the conjugate, the linker is a cleavable linker. In some embodi CM1-CM2 substrate is a polypeptide that includes a sub ments, the agent is conjugated to the AB via a noncleavable strate (S) for an enzyme; (3) the CM1-CM2 substrate is linker. In some embodiments, the agent is a microtubule positioned such that in an uncleaved state, the NB interferes inhibitor. In some embodiments, the agent is a nucleic acid with binding of the AB to the target and in a cleaved state, damaging agent, Such as a DNA alkylator or DNA interca the NB does not interfere with binding of the AB to the lator, or other DNA damaging agent. In some embodiments, target; and (4) the NB does not inhibit cleavage of the the agent is an agent selected from the group listed in Table CM1-CM2 substrate by the enzyme; and (b) recovering the 3. In some embodiments, the agent is a dolastatin. In some activatable antibody. embodiments, the agent is an auristatin or derivative thereof. 0395. In some embodiments, the method includes the In some embodiments, the agent is auristatin E or a deriva steps of (a) culturing a cell that includes a nucleic acid tive thereof. In some embodiments, the agent is monomethyl construct that encodes the activatable antibody under con auristatin E (MMAE). In some embodiments, the agent is ditions that lead to expression of the activatable antibody, monomethyl auristatin D (MMAD). In some embodiments, wherein the activatable antibody includes (i) a binding the agent is a maytansinoid or maytansinoid derivative. In partner (BP) for a non-binding steric moiety (NB); (ii) a some embodiments, the agent is DM1 or DM4. In some CM1-CM2 substrate; and (iii) an antibody or an antigen embodiments, the agent is a duocarmycin or derivative binding fragment thereof (AB) that specifically binds a thereof. In some embodiments, the agent is a calicheamicin target, wherein (1) the NB does not bind specifically to the or derivative thereof. In some embodiments, the agent is a AB; (2) the CM1-CM2 substrate is a polypeptide that pyrrolobenzodiazepine. In some embodiments, the agent is includes a substrate (S) for an enzyme; (3) the CM1-CM2 a pyrrolobenzodiazepine dimer. Substrate is positioned Such that in an uncleaved state in the 0391. In some examples of any of these activatable presence of the NB, the NB interferes with binding of the AB antibody embodiments, the activatable antibodies are dual to the target and in a cleaved state, the NB does not interfere target binding activatable antibodies. Such dual target bind with binding of the AB to the target and the BP does not ing activatable antibodies contain two Abs that may bind the interfere with binding of the AB to the target; and (4) the NB same or different targets. In specific embodiments, dual and the BP do not inhibit cleavage of the CM1-CM2 targeting activatable antibodies contain bispecific antibodies substrate by the enzyme; and (b) recovering the activatable or antibody fragments. antibody. In some examples of this embodiment, the BP of 0392 Dual target binding activatable antibodies are the activatable antibody is bound to the NB. designed so as to have a CM1-CM2 substrate cleavable by 0396 Use of Activatable Antibodies and Conjugated a cleaving agent that is co-localized in a target tissue with Activatable Antibodies one or both of the targets capable of binding to the ABs of 0397. It will be appreciated that administration of thera the activatable antibodies. Dual target binding activatable peutic entities in accordance with the disclosure will be antibodies with more than one AB to the same or different administered with Suitable carriers, excipients, and other targets can be designed so as to have more than one agents that are incorporated into formulations to provide CM1-CM2 substrate, wherein the first CM1-CM2 substrate improved transfer, delivery, tolerance, and the like. A mul is cleavable by a cleaving agent in a first target tissue and titude of appropriate formulations can be found in the wherein the second CM1-CM2 substrate is cleavable by a formulary known to all pharmaceutical chemists: Reming cleaving agent in a second target tissue, with one or more of ton's Pharmaceutical Sciences (15th ed, Mack Publishing the targets binding to the ABs of the activatable antibodies. Company, Easton, Pa. (1975)), particularly Chapter 87 by In one embodiment, the first and second target tissues are Blaug, Seymour, therein. These formulations include, for spatially separated, for example, at different sites in the example, powders, pastes, ointments, jellies, waxes, oils, organism. In one embodiment, the first and second target lipids, lipid (cationic oranionic) containing vesicles (such as tissues are the same tissue temporally separated, for example LipofectinTM), DNA conjugates, anhydrous absorption the same tissue at two different points in time, for example pastes, oil-in-water and water-in-oil emulsions, emulsions the first time point is when the tissue is an early stage tumor, carbowax (polyethylene glycols of various molecular and the second time point is when the tissue is a late stage weights), semi-solid gels, and semi-solid mixtures contain tumor. ing carbowax. Any of the foregoing mixtures may be appro 0393. The disclosure also provides nucleic acid mol priate in treatments and therapies in accordance with the ecules encoding the activatable antibodies described herein. present disclosure, provided that the active ingredient in the The disclosure also provides vectors that include these formulation is not inactivated by the formulation and the nucleic acids. The activatable antibodies described herein formulation is physiologically compatible and tolerable with are produced by culturing a cell under conditions that lead the route of administration. See also Baldrick P. "Pharma to expression of the activatable antibody, wherein the cell ceutical excipient development: the need for preclinical includes these nucleic acid molecules or vectors. guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), 0394 The disclosure also provides methods of manufac Wang W. “Lyophilization and development of solid protein turing activatable antibodies. In one embodiment, the pharmaceuticals.” Int. J. Pharm. 203(1-2): 1-60 (2000), method includes the steps of (a) culturing a cell that includes Charman WN“Lipids, lipophilic drugs, and oral drug deliv a nucleic acid construct that encodes the activatable anti ery-some emerging concepts. J Pharm Sci.89(8):967-78 US 2016/0289.324 A1 Oct. 6, 2016 52

(2000), Powell et al. “Compendium of excipients for par DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. enteral formulations' PDAJ Pharm SciTechnol. 52:238-311 Sci. USA, 90:7889-7893 (1993)). The formulation can also (1998) and the citations therein for additional information contain more than one active compounds as necessary for related to formulations, excipients and carriers well known the particular indication being treated, for example, in some to pharmaceutical chemists. embodiments, those with complementary activities that do 0398. Therapeutic formulations of the disclosure, which not adversely affect each other. In some embodiments, or in include a conjugated antibody, an activatable antibody and/ addition, the composition can comprise an agent that or a conjugated activatable antibody, are used to prevent, enhances its function, Such as, for example, a cytotoxic treat or otherwise ameliorate a disease or disorder associated agent, cytokine, chemotherapeutic agent, or growth-inhibi with aberrant target expression and/or activity. For example, tory agent. Such molecules are Suitably present in combi therapeutic formulations of the disclosure, which include a nation in amounts that are effective for the purpose intended. conjugated antibody, an activatable antibody and/or a con 0402. The active ingredients can also be entrapped in jugated activatable antibody, are used to treat or otherwise microcapsules prepared, for example, by coacervation tech ameliorate inflammation, an inflammatory disorder, an auto niques or by interfacial polymerization, for example, immune disease and/or a cancer or other neoplastic condi hydroxymethylcellulose or gelatin-microcapsules and poly tion. In some embodiments, the cancer is a solid tumor or a (methylmethacrylate) microcapsules, respectively, in colloi hematologic malignancy where the target is expressed. In dal drug delivery systems (for example, liposomes, albumin Some embodiments, the cancer is a Solid tumor where the microspheres, microemulsions, nano-particles, and nano target is expressed. In some embodiments, the cancer is a capsules) or in macroemulsions. hematologic malignancy where the target is expressed. In 0403. The formulations to be used for in vivo adminis Some embodiments, the target is expressed on parenchyma tration must be sterile. This is readily accomplished by (e.g., in cancer, the portion of an organ or tissue that often filtration through sterile filtration membranes. carries out function(s) of the organ or tissue). In some 04.04 Sustained-release preparations can be prepared. embodiments, the target is expressed on a cell, tissue, or Suitable examples of Sustained-release preparations include organ. In some embodiments, the target is expressed on semipermeable matrices of solid hydrophobic polymers con stroma (i.e., the connective Supportive framework of a cell, taining the antibody, which matrices are in the form of tissue, or organ). In some embodiments, the target is shaped articles, e.g., films, or microcapsules. Examples of expressed on an osteoblast. In some embodiments, the target Sustained-release matrices include polyesters, hydrogels (for is expressed on the endothelium (vasculature). In some example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl embodiments, the target is expressed on a cancer stem cell. alcohol)), polylactides (U.S. Pat. No. 3,773.919), copoly In some embodiments, the agent to which the activatable mers of L-glutamic acid and Y ethyl-L-glutamate, non antibody is conjugated is a microtubule inhibitor. In some degradable ethylene-vinyl acetate, degradable lactic acid embodiments, the agent to which the activatable antibody is glycolic acid copolymers such as the LUPRON DEPOTTM conjugated is a nucleic acid damaging agent. (injectable microspheres composed of lactic acid-glycolic 0399. Efficaciousness of prevention, amelioration or acid copolymer and leuprolide acetate), and poly-D-(-)-3- treatment is determined in association with any known hydroxybutyric acid. While polymers such as ethylene-vinyl method for diagnosing or treating the disease or disorder acetate and lactic acid-glycolic acid enable release of mol associated with target expression and/or activity, such as, for ecules for over 100 days, certain hydrogels release proteins example, aberrant target expression and/or activity. Prolong for shorter time periods. ing the Survival of a subject or otherwise delaying the 0405. In some embodiments, the conjugated antibody, progression of the disease or disorder associated with target activatable antibody and/or conjugated activatable antibody expression and/or activity, e.g., aberrant target expression contains a detectable label. An intact antibody, or a fragment and/or activity, in a Subject indicates that the conjugated thereof (e.g., Fab, sclv, or F(ab)) is used. The term antibody, activatable antibody and/or conjugated activatable “labeled, with regard to the probe or antibody, is intended antibody confers a clinical benefit. to encompass direct labeling of the probe or antibody by 0400. A conjugated antibody, an activatable antibody coupling (i.e., physically linking) a detectable Substance to and/or a conjugated activatable antibody can be adminis the probe or antibody, as well as indirect labeling of the tered in the form of pharmaceutical compositions. Principles probe or antibody by reactivity with another reagent that is and considerations involved in preparing Such compositions, directly labeled. Examples of indirect labeling include as well as guidance in the choice of components are pro detection of a primary antibody using a fluorescently-labeled vided, for example, in Remington: The Science And Practice secondary antibody and end-labeling of a DNA probe with Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) biotin such that it can be detected with fluorescently-labeled Mack Pub. Co., Easton, Pa...: 1995; Drug Absorption streptavidin. The term “biological sample' is intended to Enhancement: Concepts, Possibilities, Limitations, And include tissues, cells and biological fluids isolated from a Trends, Harwood Academic Publishers, Langhorne, Pa., Subject, as well as tissues, cells and fluids present within a 1994; and Peptide And Protein Drug Delivery (Advances. In subject. Included within the usage of the term “biological Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York. sample', therefore, is blood and a fraction or component of 04.01. In some embodiments where antibody fragments blood including blood serum, blood plasma, or lymph. That are used, the Smallest fragment that specifically binds to the is, the detection method of the disclosure can be used to binding domain of the target protein is selected. For detect an analyte mRNA, protein, or genomic DNA in a example, based upon the variable-region sequences of an biological sample in vitro as well as in vivo. For example, antibody, peptide molecules can be designed that retain the in vitro techniques for detection of an analyte mRNA ability to bind the target protein sequence. Such peptides can include Northern hybridizations and in situ hybridizations. be synthesized chemically and/or produced by recombinant In vitro techniques for detection of an analyte protein US 2016/0289.324 A1 Oct. 6, 2016

include enzyme linked immunosorbent assays (ELISAS), antigen in the test sample is determined by comparison with Western blots, immunoprecipitations, immunochemical a standard curve developed from the standard samples. staining, and immunofluorescence. In vitro techniques for 0411. It will be appreciated that based on the results detection of an analyte genomic DNA include Southern obtained using the antibodies of the disclosure, and conju hybridizations. Procedures for conducting immunoassays gated versions thereof, in an in vitro diagnostic assay, it is are described, for example in “ELISA: Theory and Practice: possible to stage a disease in a subject based on expression Methods in Molecular Biology”, Vol. 42, J. R. Crowther levels of the target antigen. For a given disease, Samples of (Ed.) Human Press, Totowa, N.J., 1995: “Immunoassay”. E. blood are taken from Subjects diagnosed as being at various Diamandis and T. Christopoulus, Academic Press, Inc., San stages in the progression of the disease, and/or at various Diego, Calif., 1996; and “Practice and Theory of Enzyme points in the therapeutic treatment of the disease. Using a Immunoassays, P. Tijssen, Elsevier Science Publishers, population of samples that provides statistically significant Amsterdam, 1985. Furthermore, in vivo techniques for results for each stage of progression or therapy, a range of detection of an analyte protein include introducing into a concentrations of the antigen that may be considered char Subject a labeled anti-analyte protein antibody. For example, acteristic of each stage is designated. the antibody can be labeled with a radioactive marker whose 0412. A conjugated antibody, an activatable antibody presence and location in a Subject can be detected by and/or a conjugated activatable antibody can also be used in Standard imaging techniques. diagnostic and/or imaging methods. In some embodiments, 0406. The conjugated antibodies, activatable antibodies Such methods are in vitro methods. In some embodiments, and/or conjugated activatable antibodies of the disclosure Such methods are in vivo methods. In some embodiments, are also useful in a variety of diagnostic and prophylactic Such methods are in situ methods. In some embodiments, formulations. In one embodiment, a conjugated antibody, an such methods are ex vivo methods. For example, activatable activatable antibody and/or a conjugated activatable anti antibodies having an enzymatically cleavable CM1-CM2 body is administered to patients that are at risk of developing Substrate can be used to detect the presence or absence of an one or more of the aforementioned disorders. A patient’s or enzyme that is capable of cleaving the CM1-CM2 substrate. organ's predisposition to one or more of the aforementioned Such activatable antibodies can be used in diagnostics, disorders can be determined using genotypic, serological or which can include in vivo detection (e.g., qualitative or biochemical markers. quantitative) of enzyme activity (or, in some embodiments, 0407. In some embodiments, a conjugated antibody, an an environment of increased reduction potential Such as that activatable antibody and/or a conjugated activatable anti which can provide for reduction of a disulfide bond) through body is administered to human individuals diagnosed with a measured accumulation of activated antibodies (i.e., anti clinical indication associated with one or more of the bodies resulting from cleavage of an activatable antibody) in aforementioned disorders. Upon diagnosis, a conjugated a given cell or tissue of a given host organism. Such antibody, an activatable antibody and/or a conjugated acti accumulation of activated antibodies indicates not only that vatable antibody is administered to mitigate or reverse the the tissue expresses enzymatic activity (or an increased effects of the clinical indication. reduction potential depending on the nature of the CM1 0408. A conjugated antibody, an activatable antibody CM2 substrate) but also that the tissue expresses target to and/or a conjugated activatable antibody of the disclosure is which the activated antibody binds. also useful in the detection of a target in patient samples and 0413 For example, the CM1-CM2 substrate can be accordingly are useful as diagnostics. For example, the selected to be substrate for a matrix metalloprotease (MMP) antibodies and/or activatable antibodies, and conjugated and a serine protease (SP) found at the site of a tumor, at the versions thereof, of the disclosure are used in in vitro assays. site of a viral or bacterial infection at a biologically confined e.g., ELISA, to detect target levels in a patient sample. site (e.g., Such as in an abscess, in an organ, and the like), and the like. The AB can be one that binds a target antigen. 04.09. In one embodiment, a conjugated antibody, an Using methods as disclosed herein, or when appropriate, activatable antibody and/or a conjugated activatable anti methods familiar to one skilled in the art, a detectable label body of the disclosure is immobilized on a solid support (e.g., a fluorescent label or radioactive label or radiotracer) (e.g., the well(s) of a microtiter plate). The immobilized can be conjugated to an AB or other region of an antibody conjugated antibody, activatable antibody and/or conjugated and/or activatable antibody. Suitable detectable labels are activatable antibody serves as a capture antibody for any discussed in the context of the above screening methods and target that may be present in a test sample. Prior to contact additional specific examples are provided below. Using an ing the immobilized antibody with a patient sample, the AB specific to a protein or peptide of the disease state, along Solid Support is rinsed and treated with a blocking agent Such with an MMP whose activity is elevated in the disease tissue as milk protein or albumin to prevent nonspecific adsorption of interest, activatable antibodies will exhibit an increased of the analyte. rate of binding to disease tissue relative to tissues where the 0410 Subsequently the wells are treated with a test CM1-CM2 substrate specific enzyme is not present at a sample Suspected of containing the antigen, or with a detectable level or is present at a lower level than in disease Solution containing a standard amount of the antigen. Such tissue or is inactive (e.g., in Zymogen form or in complex a sample is, e.g., a serum sample from a subject Suspected with an inhibitor). Since small proteins and peptides are of having levels of circulating antigen considered to be rapidly cleared from the blood by the renal filtration system, diagnostic of a pathology. After rinsing away the test sample and because the enzyme specific for the CM1-CM2 substrate or standard, the Solid Support is treated with a second is not present at a detectable level (or is present at lower antibody that is detectably labeled. The labeled second levels in non-disease tissues or is present in inactive con antibody serves as a detecting antibody. The level of detect formation), accumulation of activated antibodies in the able label is measured, and the concentration of target disease tissue is enhanced relative to non-disease tissues. US 2016/0289.324 A1 Oct. 6, 2016 54

0414. In another example, activatable antibodies can be 0419 Detection of the label in a sample that has been used to detect the presence or absence of a cleaving agent in incubated with the labeled, activatable antibody indicates a sample. For example, where the activatable antibodies that the sample contains the target and contains a matrix contain a CM1-CM2 substrate susceptible to cleavage by an metalloprotease (MMP) and one serine protease (SP) that enzyme, the activatable antibodies can be used to detect are specific for the CM1-CM2 substrate of the activatable (either qualitatively or quantitatively) the presence of an antibody. In some embodiments, the presence of the MMP enzyme in the sample. In another example, where the can be confirmed using broad spectrum protease inhibitors activatable antibodies contain a CM1-CM2 substrate sus Such as those described herein, and/or by using an agent that ceptible to cleavage by reducing agent, the activatable is specific for the protease, for example, an antibody Such as antibodies can be used to detect (either qualitatively or A11, which is specific for the protease matriptase (MT-SP1) quantitatively) the presence of reducing conditions in a and inhibits the proteolytic activity of matriptase; see e.g., sample. To facilitate analysis in these methods, the activat International Publication Number WO 2010/129609, pub able antibodies can be detectably labeled, and can be bound lished 11 Nov. 2010. The same approach of using broad to a Support (e.g., a solid Support, such as a slide or bead). spectrum protease inhibitors such as those described herein, The detectable label can be positioned on a portion of the and/or by using a more selective inhibitory agent can be used activatable antibody that is not released following cleavage, to identify a MMP and a SP specific for the CM1-CM2 for example, the detectable label can be a quenched fluo substrate of the activatable antibody. In some embodiments, rescent label or other label that is not detectable until the presence of the target can be confirmed using an agent cleavage has occurred. The assay can be conducted by, for that is specific for the target, e.g., another antibody, or the example, contacting the immobilized, detectably labeled detectable label can be competed with unlabeled target. In activatable antibodies with a sample Suspected of containing some embodiments, unlabeled activatable antibody could be an enzyme and/or reducing agent for a time sufficient for used, with detection by a labeled secondary antibody or cleavage to occur, then washing to remove excess sample more complex detection system. and contaminants. The presence or absence of the cleaving 0420 Similar techniques are also useful for in vivo agent (e.g., enzyme or reducing agent) in the sample is then imaging where detection of the fluorescent signal in a assessed by a change in detectable signal of the activatable Subject, e.g., a mammal, including a human, indicates that antibodies prior to contacting with the sample e.g., the the disease site contains the target and contains a MMP and presence of and/or an increase in detectable signal due to a SP that is specific for the CM1-CM2 substrate of the cleavage of the activatable antibody by the cleaving agent in activatable antibody. the sample. 0421. These techniques are also useful in kits and/or as 0415. Such detection methods can be adapted to also reagents for the detection, identification or characterization provide for detection of the presence or absence of a target of protease activity in a variety of cells, tissues, and organ that is capable of binding the AB of the activatable antibod isms based on the protease-specific CM1-CM2 substrate in ies when cleaved. Thus, the assays can be adapted to assess the activatable antibody. the presence or absence of a cleaving agent and the presence 0422 The disclosure provides methods of using the anti or absence of a target of interest. The presence or absence of bodies and/or activatable antibodies in a variety of diagnos the cleaving agent can be detected by the presence of and/or tic and/or prophylactic indications. For example, the disclo an increase in detectable label of the activatable antibodies Sure provides methods of detecting presence or absence of a as described above, and the presence or absence of the target cleaving agent and a target of interest in a Subject or a can be detected by detection of a target-AB complex e.g., by sample by (i) contacting a Subject or sample with an acti use of a detectably labeled anti-target antibody. vatable antibody, wherein the activatable antibody com 0416) Activatable antibodies are also useful in in situ prises a masking moiety (MM), a CM1-CM2 substrate that imaging for the validation of activatable antibody activation, is cleaved by the cleaving agent, and an antigen binding e.g., by protease cleavage, and binding to a particular target. domain or fragment thereof (AB) that specifically binds the In situ imaging is a technique that enables localization of target of interest, wherein the activatable antibody in an proteolytic activity and target in biological samples Such as uncleaved, non-activated State comprises a structural cell cultures or tissue sections. Using this technique, it is arrangement from N-terminus to C-terminus as follows: possible to confirm both binding to a given target and MM-CM1-CM2 Substrate-AB or AB-CM1-CM2 Substrate proteolytic activity based on the presence of a detectable MM; (a) wherein the MM is a peptide that inhibits binding label (e.g., a fluorescent label). of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding 0417. These techniques are useful with any frozen cells partner of the AB and is not a modified form of a natural or tissue derived from a disease site (e.g. tumor tissue) or binding partner of the AB; and (b) wherein, in an uncleaved, healthy tissues. These techniques are also useful with fresh non-activated state, the MM interferes with specific binding cell or tissue samples. of the AB to the target, and in a cleaved, activated state the 0418. In these techniques, an activatable antibody is MM does not interfere or compete with specific binding of labeled with a detectable label. The detectable label may be the AB to the target; and (ii) measuring a level of activated a fluorescent dye, (e.g. a fluorophore, Fluorescein Isothio activatable antibody in the subject or sample, wherein a cyanate (FITC), Rhodamine Isothiocyanate (TRITC), an detectable level of activated activatable antibody in the Alexa Fluor R label), a near infrared (NIR) dye (e.g., Qdot(R) Subject or sample indicates that the cleaving agent and the nanocrystals), a colloidal metal, a hapten, a radioactive target are present in the Subject or sample and wherein no marker, biotin and an amplification reagent such as Strepta detectable level of activated activatable antibody in the vidin, or an enzyme (e.g. horseradish peroxidase or alkaline Subject or sample indicates that the cleaving agent, the target phosphatase). or both the cleaving agent and the target are absent and/or US 2016/0289.324 A1 Oct. 6, 2016

not sufficiently present in the Subject or sample. In some wherein the MM does not have an amino acid sequence of embodiments, the activatable antibody is an activatable a naturally occurring binding partner of the AB and is not a antibody to which a therapeutic agent is conjugated. In some modified form of a natural binding partner of the AB; and (b) embodiments, the activatable antibody is not conjugated to wherein, in an uncleaved, non-activated state, the MM an agent. In some embodiments, the activatable antibody interferes with specific binding of the AB to the target, and comprises a detectable label. In some embodiments, the in a cleaved, activated state the MM does not interfere or detectable label is positioned on the AB. In some embodi compete with specific binding of the AB to the target; and ments, measuring the level of activatable antibody in the (ii) measuring a level of activated activatable antibody in the Subject or sample is accomplished using a secondary reagent subject or sample, wherein a detectable level of activated that specifically binds to the activated antibody, wherein the activatable antibody in the subject or sample indicates that reagent comprises a detectable label. In some embodiments, the cleaving agent is present in the Subject or sample and the secondary reagent is an antibody comprising a detectable wherein no detectable level of activated activatable antibody label. in the Subject or sample indicates that the cleaving agent is 0423. The disclosure also provides methods of detecting absent and/or not sufficiently present in the subject or presence or absence of a cleaving agent in a subject or a sample. In some embodiments, the activatable antibody is an sample by (i) contacting a subject or sample with an acti activatable antibody to which a therapeutic agent is conju Vatable antibody in the presence of a target of interest, e.g., gated. In some embodiments, the activatable antibody is not the target, wherein the activatable antibody comprises a conjugated to an agent. In some embodiments, the activat masking moiety (MM), a CM1-CM2 substrate that is able antibody comprises a detectable label. In some embodi cleaved by the cleaving agent, and an antigen binding ments, the detectable label is positioned on the AB. In some domain or fragment thereof (AB) that specifically binds the embodiments, measuring the level of activatable antibody in target of interest, wherein the activatable antibody in an the Subject or sample is accomplished using a secondary uncleaved, non-activated State comprises a structural reagent that specifically binds to the activated antibody, arrangement from N-terminus to C-terminus as follows: wherein the reagent comprises a detectable label. In some MM-CM1-CM2 Substrate-AB or AB-CM1-CM2 Substrate embodiments, the secondary reagent is an antibody com MM; (a) wherein the MM is a peptide that inhibits binding prising a detectable label. of the AB to the target, and wherein the MM does not have 0425 The disclosure also provides methods of detecting an amino acid sequence of a naturally occurring binding presence or absence of a cleaving agent in a Subject or a partner of the AB and is not a modified form of a natural sample by (i) contacting a subject or sample with an acti binding partner of the AB; and (b) wherein, in an uncleaved, vatable antibody, wherein the activatable antibody com non-activated state, the MM interferes with specific binding prises a masking moiety (MM), a CM1-CM2 substrate that of the AB to the target, and in a cleaved, activated state the is cleaved by the cleaving agent, an antigen binding domain MM does not interfere or compete with specific binding of (AB) that specifically binds the target, and a detectable label, the AB to the target; and (ii) measuring a level of activated wherein the activatable antibody in an uncleaved, non activatable antibody in the subject or sample, wherein a activated State comprises a structural arrangement from detectable level of activated activatable antibody in the N-terminus to C-terminus as follows: MM-CM1-CM2 sub Subject or sample indicates that the cleaving agent is present strate-AB or AB-CM1-CM2 substrate-MM; wherein the in the subject or sample and wherein no detectable level of MM is a peptide that inhibits binding of the AB to the target, activated activatable antibody in the subject or sample and wherein the MM does not have an amino acid sequence indicates that the cleaving agent is absent and/or not suffi of a naturally occurring binding partner of the AB and is not ciently present in the Subject or sample. In some embodi a modified form of a natural binding partner of the AB; ments, the activatable antibody is an activatable antibody to wherein, in an uncleaved, non-activated state, the MM which a therapeutic agent is conjugated. In some embodi interferes with specific binding of the AB to the target, and ments, the activatable antibody is not conjugated to an agent. in a cleaved, activated state the MM does not interfere or In some embodiments, the activatable antibody comprises a compete with specific binding of the AB to the target; and detectable label. In some embodiments, the detectable label wherein the detectable label is positioned on a portion of the is positioned on the AB. In some embodiments, measuring activatable antibody that is released following cleavage of the level of activatable antibody in the subject or sample is the CM1-CM2 substrate; and (ii) measuring a level of accomplished using a secondary reagent that specifically detectable label in the subject or sample, wherein a detect binds to the activated antibody, wherein the reagent com able level of the detectable label in the subject or sample prises a detectable label. In some embodiments, the second indicates that the cleaving agent is absent and/or not suffi ary reagent is an antibody comprising a detectable label. ciently present in the Subject or sample and wherein no 0424 The disclosure also provides kits for use in meth detectable level of the detectable label in the subject or ods of detecting presence or absence of a cleaving agent and sample indicates that the cleaving agent is present in the the target in a Subject or a sample, where the kits include at Subject or sample. In some embodiments, the activatable least an activatable antibody comprises a masking moiety antibody is an activatable antibody to which a therapeutic (MM), a CM1-CM2 substrate that is cleaved by the cleaving agent is conjugated. In some embodiments, the activatable agent, and an antigen binding domain or fragment thereof antibody is not conjugated to an agent. In some embodi (AB) that specifically binds the target of interest, wherein ments, the activatable antibody comprises a detectable label. the activatable antibody in an uncleaved, non-activated State In some embodiments, the detectable label is positioned on comprises a structural arrangement from N-terminus to the AB. In some embodiments, measuring the level of C-terminus as follows: MM-CM1-CM2 substrate-AB or activatable antibody in the Subject or sample is accom AB-CM1-CM2 substrate-MM; (a) wherein the MM is a plished using a secondary reagent that specifically binds to peptide that inhibits binding of the AB to the target, and the activated antibody, wherein the reagent comprises a US 2016/0289.324 A1 Oct. 6, 2016 56 detectable label. In some embodiments, the secondary In some embodiments, the measuring step includes use of a reagent is an antibody comprising a detectable label. secondary reagent comprising a detectable label. 0426. The disclosure also provides kits for use in meth 0428 The disclosure also provides kits for use in meth ods of detecting presence or absence of a cleaving agent and ods of detecting presence or absence of a cleaving agent and the target in a Subject or a sample, where the kits include at the target in a Subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or antibody (e.g., an activatable antibody to which a therapeu biological sample with an activatable antibody in the pres tic agent is conjugated) described herein for use in contact ence of the target, and measuring a level of activated ing a subject or biological sample and means for detecting activatable antibody in the Subject or biological sample, the level of activated activatable antibody and/or conjugated wherein a detectable level of activated activatable antibody activatable antibody in the Subject or biological sample, in the Subject or biological sample indicates that the cleaving wherein a detectable level of activated activatable antibody agent is present in the Subject or biological sample and in the Subject or biological sample indicates that the cleaving wherein no detectable level of activated activatable antibody agent and the target are present in the Subject or biological in the Subject or biological sample indicates that the cleaving sample and wherein no detectable level of activated activat agent is absent and/or not sufficiently present in the Subject able antibody in the Subject or biological sample indicates or biological sample at a detectable level. Such that protease that the cleaving agent, the target or both the cleaving agent cleavage of the activatable antibody cannot be detected in and the target are absent and/or not sufficiently present in the the Subject or biological sample. Such an activatable anti Subject or biological sample, Such that the target binding body includes a masking moiety (MM), a CM1-CM2 sub and/or protease cleavage of the activatable antibody cannot strate that is cleaved by the cleaving agent, and an antigen be detected in the subject or biological sample. binding domain or fragment thereof (AB) that specifically 0427. The disclosure also provides methods of detecting binds the target, wherein the activatable antibody in an presence or absence of a cleaving agent in a subject or a uncleaved (i.e., non-activated) state comprises a structural sample by (i) contacting a subject or biological sample with arrangement from N-terminus to C-terminus as follows: an activatable antibody in the presence of the target, and (ii) MM-CM1-CM2 Substrate-AB or AB-CM1-CM2 Substrate measuring a level of activated activatable antibody in the MM; (a) wherein the MM is a peptide that inhibits binding subject or biological sample, wherein a detectable level of of the AB to the target, and wherein the MM does not have activated activatable antibody in the subject or biological an amino acid sequence of a naturally occurring binding sample indicates that the cleaving agent is present in the partner of the AB; and (b) wherein the MM of the activatable subject or biological sample and wherein no detectable level antibody in an uncleaved state interferes with specific bind of activated activatable antibody in the subject or biological ing of the AB to the target, and wherein the MM of an sample indicates that the cleaving agent is absent and/or not activatable antibody in a cleaved (i.e., activated) state does Sufficiently present in the Subject or biological sample at a not interfere or compete with specific binding of the AB to detectable level, such that protease cleavage of the activat the target. In some embodiments, the activatable antibody is able antibody cannot be detected in the subject or biological an activatable antibody to which a therapeutic agent is sample. Such an activatable antibody includes a masking conjugated. In some embodiments, the activatable antibody moiety (MM), a CM1-CM2 substrate that is cleaved by the is not conjugated to an agent. In some embodiments, the cleaving agent, and an antigen binding domain or fragment detectable label is attached to the masking moiety. In some thereof (AB) that specifically binds the target, wherein the embodiments, the detectable label is attached to the cleav activatable antibody in an uncleaved (i.e., non-activated) able moiety N-terminal to the protease cleavage site. In state comprises a structural arrangement from N-terminus to Some embodiments, a single antigen binding site of the AB C-terminus as follows: MM-CM1-CM2 substrate-AB or is masked. In some embodiments wherein an antibody of the AB-CM1-CM2 substrate-MM; (a) wherein the MM is a disclosure has at least two antigen binding sites, at least one peptide that inhibits binding of the AB to the target, and antigen binding site is masked and at least one antigen wherein the MM does not have an amino acid sequence of binding site is not masked. In some embodiments, all a naturally occurring binding partner of the AB; and (b) antigen binding sites are masked. In some embodiments, the wherein the MM of the activatable antibody in an uncleaved measuring step includes use of a secondary reagent com state interferes with specific binding of the AB to the target, prising a detectable label. and wherein the MM of an activatable antibody in a cleaved 0429. The disclosure also provides kits for use in meth (i.e., activated) state does not interfere or compete with ods of detecting presence or absence of a cleaving agent in specific binding of the AB to the target. In some embodi a Subject or a sample, where the kits include at least an ments, the activatable antibody is an activatable antibody to activatable antibody and/or conjugated activatable antibody which a therapeutic agent is conjugated. In some embodi described herein for use in contacting a Subject or biological ments, the activatable antibody is not conjugated to an agent. sample and means for detecting the level of activated In some embodiments, the detectable label is attached to the activatable antibody and/or conjugated activatable antibody masking moiety. In some embodiments, the detectable label in the subject or biological sample, wherein the activatable is attached to the cleavable moiety N-terminal to the pro antibody includes a detectable label that is positioned on a tease cleavage site. In some embodiments, a single antigen portion of the activatable antibody that is released following binding site of the AB is masked. In some embodiments cleavage of the CM11-CM2 substrate, wherein a detectable wherein an antibody of the disclosure has at least two level of activated activatable antibody in the subject or antigen binding sites, at least one antigen binding site is biological sample indicates that the cleaving agent is absent masked and at least one antigen binding site is not masked. and/or not sufficiently present in the subject or biological In some embodiments, all antigen binding sites are masked. sample Such that the target binding and/or protease cleavage US 2016/0289.324 A1 Oct. 6, 2016 57 of the activatable antibody cannot be detected in the subject detectable level of activated activatable antibody in the or biological sample, and wherein no detectable level of Subject or biological sample indicates that the cleaving activated activatable antibody in the subject or biological agent, the target or both the cleaving agent and the target are sample indicates that the cleaving agent is present in the absent and/or not sufficiently present in the subject or subject or biological sample at a detectable level. biological sample, such that the target binding and/or pro 0430. The disclosure provides methods of detecting pres tease cleavage of the activatable antibody cannot be detected ence or absence of a cleaving agent and the target in a in the Subject or biological sample, and wherein a reduced Subject or a sample by (i) contacting a subject or biological detectable level of activated activatable antibody in the sample with an activatable antibody, wherein the activatable Subject or biological sample indicates that the cleaving agent antibody includes a detectable label that is positioned on a and the target are present in the Subject or biological sample. portion of the activatable antibody that is released following A reduced level of detectable label is, for example, a cleavage of the CM1-CM2 substrate and (ii) measuring a reduction of about 5%, about 10%, about 15%, about 20%, level of activated activatable antibody in the subject or about 25%, about 30%, about 35%, about 400%, about 45%, biological sample, wherein a detectable level of activated activatable antibody in the Subject or biological sample about 50%, about 55%, about 60%, about 65%, about 70%, indicates that the cleaving agent, the target or both the about 75%, about 80%, about 85%, about 90%, about 95% cleaving agent and the target are absent and/or not suffi and/or about 100%. ciently present in the Subject or biological sample, such that 0432. The disclosure also provides methods of detecting the target binding and/or protease cleavage of the activatable presence or absence of a cleaving agent in a Subject or a antibody cannot be detected in the subject or biological sample by (i) contacting a Subject or biological sample with sample, and wherein a reduced detectable level of activated an activatable antibody, wherein the activatable antibody activatable antibody in the Subject or biological sample includes a detectable label that is positioned on a portion of indicates that the cleaving agent and the target are present in the activatable antibody that is released following cleavage the subject or biological sample. A reduced level of detect of the CM1-CM2 substrate; and (ii) measuring a level of able label is, for example, a reduction of about 5%, about detectable label in the subject or biological sample, wherein 10%, about 15%, about 20%, about 25%, about 30%, about a detectable level of the detectable label in the subject or 35%, about 40%, about 45%, about 50%, about 55%, about biological sample indicates that the cleaving agent is absent 60%, about 65%, about 70%, about 75%, about 80%, about and/or not sufficiently present in the subject or biological 85%, about 90%, about 95% and/or about 100%. Such an activatable antibody includes a masking moiety (MM), a sample at a detectable level. Such that protease cleavage of CM1-CM2 substrate that is cleaved by the cleaving agent, the activatable antibody cannot be detected in the subject or and an antigen binding domain or fragment thereof (AB) biological sample, and wherein a reduced detectable level of that specifically binds the target, wherein the activatable the detectable label in the subject or biological sample antibody in an uncleaved (i.e., non-activated) state com indicates that the cleaving agent is present in the Subject or prises a structural arrangement from N-terminus to C-ter biological sample. A reduced level of detectable label is, for minus as follows: MM-CM1-CM2 substrate-AB or example, a reduction of about 5%, about 10%, about 15%, AB-CM1-CM2 substrate-MM; (a) wherein the MM is a about 20%, about 25%, about 30%, about 35%, about 40%, peptide that inhibits binding of the AB to the target, and about 45%, about 50%, about 55%, about 60%, about 65%, wherein the MM does not have an amino acid sequence of about 70%, about 75%, about 80%, about 85%, about 90%, a naturally occurring binding partner of the AB; and (b) about 95% and/or about 100%. Such an activatable antibody wherein the MM of the activatable antibody in an uncleaved includes a masking moiety (MM), a CM1-CM2 substrate state interferes with specific binding of the AB to the target, that is cleaved by the cleaving agent, and an antigen binding and wherein the MM of an activatable antibody in a cleaved domain or fragment thereof (AB) that specifically binds the (i.e., activated) state does not interfere or compete with target, wherein the activatable antibody in an uncleaved (i.e., specific binding of the AB to the target. In some embodi non-activated) state comprises a structural arrangement ments, the activatable antibody is an activatable antibody to from N-terminus to C-terminus as follows: MM-CM1-CM2 which a therapeutic agent is conjugated. In some embodi substrate-AB or AB-CM1-CM2 substrate-MM; (a) wherein ments, the activatable antibody is not conjugated to an agent. the MM is a peptide that inhibits binding of the AB to the In some embodiments, the activatable antibody comprises a target, and wherein the MM does not have an amino acid detectable label. In some embodiments, the detectable label sequence of a naturally occurring binding partner of the AB; is positioned on the AB. In some embodiments, measuring and (b) wherein the MM of the activatable antibody in an the level of activatable antibody in the subject or sample is uncleaved state interferes with specific binding of the AB to accomplished using a secondary reagent that specifically the target, and wherein the MM of an activatable antibody in binds to the activated antibody, wherein the reagent com a cleaved (i.e., activated) state does not interfere or compete prises a detectable label. In some embodiments, the second with specific binding of the AB to the target. In some ary reagent is an antibody comprising a detectable label. embodiments, the activatable antibody is an activatable 0431. The disclosure also provides kits for use in meth antibody to which a therapeutic agent is conjugated. In some ods of detecting presence or absence of a cleaving agent and embodiments, the activatable antibody is not conjugated to the target in a Subject or a sample, where the kits include at an agent. In some embodiments, the activatable antibody least an activatable antibody and/or conjugated activatable comprises a detectable label. In some embodiments, the antibody described herein for use in contacting a subject or detectable label is positioned on the AB. In some embodi biological sample and means for detecting the level of ments, measuring the level of activatable antibody in the activated activatable antibody and/or conjugated activatable Subject or sample is accomplished using a secondary reagent antibody in the Subject or biological sample, wherein a that specifically binds to the activated antibody, wherein the US 2016/0289.324 A1 Oct. 6, 2016

reagent comprises a detectable label. In some embodiments, methods, the method is an in situ method. In some embodi the secondary reagent is an antibody comprising a detectable ments of these methods, the method is an ex vivo method. label. In some embodiments of these methods, the method is an in 0433. The disclosure also provides kits for use in meth vitro method. ods of detecting presence or absence of a cleaving agent of 0437. In some embodiments, in situ imaging and/or in interest in a subject or a sample, where the kits include at Vivo imaging are useful in methods to identify which least an activatable antibody and/or conjugated activatable patients to treat. For example, in in situ imaging, the antibody described herein for use in contacting a subject or activatable antibodies are used to screen patient samples to biological sample and means for detecting the level of identify those patients having the appropriate protease(s) activated activatable antibody and/or conjugated activatable and target(s) at the appropriate location, e.g., at a tumor site. antibody in the subject or biological sample, wherein the 0438. In some embodiments, in situ imaging is used to activatable antibody includes a detectable label that is posi identify or otherwise refine a patient population suitable for tioned on a portion of the activatable antibody that is treatment with an activatable antibody of the disclosure. For released following cleavage of the CM1-CM2 substrate, example, patients that test positive for both the target (e.g., wherein a detectable level of the detectable label in the the target) and a protease that cleaves the Substrate in the Subject or biological sample indicates that the cleaving CM1-CM2 substrate of the activatable antibody being tested agent, the target, or both the cleaving agent and the target are (e.g., accumulate activated antibodies at the disease site) are absent and/or not sufficiently present in the subject or identified as suitable candidates for treatment with such an biological sample, Such that the target binding and/or pro activatable antibody comprising such a CM1-CM2 sub tease cleavage of the activatable antibody cannot be detected strate. Likewise, patients that test negative for either or both in the Subject or biological sample, and wherein a reduced of the target (e.g., the target) and the protease that cleaves detectable level of the detectable label in the subject or the substrate in the CM1-CM2 substrate in the activatable biological sample indicates that the cleaving agent and the antibody being tested using these methods might be identi target are present in the Subject or biological sample. A fied as suitable candidates for another form of therapy. In reduced level of detectable label is, for example, a reduction Some embodiments, such patients that test negative with of about 5%, about 10%, about 15%, about 200, about 25%, respect to a first activatable antibody can be tested with other about 300, about 35%, about 400, about 45%, about 500, activatable antibodies comprising different CM1-CM2 sub about 55%, about 60%, about 65%, about 70%, about 75%, strates until a suitable activatable antibody for treatment is about 80%, about 85%, about 90%, about 95% and/or about identified (e.g., an activatable antibody comprising a CM1 100%. CM2 substrate that is cleaved by the patient at the site of 0434. In some embodiments of these methods and kits, disease). In some embodiments, the patient is then admin the activatable antibody includes a detectable label. In some istered a therapeutically effective amount of the conjugated embodiments of these methods and kits, the detectable label activatable antibody for which the patient tested positive. includes an imaging agent, a contrasting agent, an enzyme, 0439. In some embodiments, in vivo imaging is used to a fluorescent label, a chromophore, a dye, one or more metal identify or otherwise refine a patient population suitable for ions, or a ligand-based label. In some embodiments of these treatment with an activatable antibody of the disclosure. For methods and kits, the imaging agent comprises a radioiso example, patients that test positive for both the target (e.g., tope. In some embodiments of these methods and kits, the the target) and a protease that cleaves the Substrate in the radioisotope is indium or technetium. In some embodiments CM1-CM2 substrate of the activatable antibody being tested of these methods and kits, the contrasting agent comprises (e.g., accumulate activated antibodies at the disease site) are iodine, gadolinium or iron oxide. In some embodiments of identified as suitable candidates for treatment with such an these methods and kits, the enzyme comprises horseradish activatable antibody comprising such a CM1-CM2 sub peroxidase, alkaline phosphatase, or 3-galactosidase. In strate. Likewise, patients that test negative might be identi some embodiments of these methods and kits, the fluores fied as suitable candidates for another form of therapy. In cent label comprises yellow fluorescent protein (YFP), cyan Some embodiments, such patients that test negative with fluorescent protein (CFP), green fluorescent protein (GFP), respect to a first activatable antibody can be tested with other modified red fluorescent protein (mRFP), red fluorescent activatable antibodies comprising different CM1-CM2 sub protein tdimer2 (RFP tdimer2), HCRED, or a europium strates until a suitable activatable antibody for treatment is derivative. In some embodiments of these methods and kits, identified (e.g., an activatable antibody comprising a CM1 the luminescent label comprises an N-methylacrydium CM2 substrate that is cleaved by the patient at the site of derivative. In some embodiments of these methods, the label disease). In some embodiments, the patient is then admin comprises an Alexa Fluor R label, such as Alex Fluor R 680 istered a therapeutically effective amount of the conjugated or Alexa FluorR 750. In some embodiments of these meth activatable antibody for which the patient tested positive. ods and kits, the ligand-based label comprises biotin, avidin, 0440. In some embodiments of the methods and kits, the streptavidin or one or more haptens. method or kit is used to identify or otherwise refine a patient 0435. In some embodiments of these methods and kits, population suitable for treatment with an activatable anti the Subject is a mammal. In some embodiments of these body of the disclosure. For example, patients that test methods and kits, the Subject is a human. In some embodi positive for both the target (e.g., the target) and a protease ments, the Subject is a non-human mammal. Such as a that cleaves the substrate in the CM1-CM2 substrate of the non-human primate, companion animal (e.g., cat, dog, activatable antibody being tested in these methods are horse), farm animal, work animal, or Zoo animal. In some identified as suitable candidates for treatment with such an embodiments, the Subject is a rodent. activatable antibody comprising such a CM1-CM2 sub 0436. In some embodiments of these methods, the strate. Likewise, patients that test negative for both of the method is an in vivo method. In some embodiments of these targets (e.g., the target) and the protease that cleaves the US 2016/0289.324 A1 Oct. 6, 2016 59 substrate in the CM1-CM2 substrate in the activatable amino acids. In some embodiments of these methods and antibody being tested using these methods might be identi kits, the activatable antibody comprises a linker peptide, fied as suitable candidates for another form of therapy. In wherein the linker peptide is positioned between the MM Some embodiments, such patients can be tested with other and the CM1-CM2 substrate. In some embodiments of these activatable antibodies until a suitable activatable antibody methods and kits, the activatable antibody comprises a for treatment is identified (e.g., an activatable antibody linker peptide, where the linker peptide is positioned comprising a CM1-CM2 substrate that is cleaved by the between the AB and the CM1-CM2 substrate. In some patient at the site of disease). In some embodiments, patients embodiments of these methods and kits, the activatable that test negative for either of the target (e.g., the target) are antibody comprises a first linker peptide (L1) and a second identified as suitable candidates for treatment with such an linker peptide (L2), wherein the first linker peptide is activatable antibody comprising such a CM1-CM2 sub positioned between the MM and the CM1-CM2 substrate strate. In some embodiments, patients that test negative for and the second linker peptide is positioned between the AB either of the target (e.g., the target) are identified as not being and the CM1-CM2 substrate. In some embodiments of these suitable candidates for treatment with such an activatable methods and kits, each of L1 and L2 is a peptide of about 1 antibody comprising such a CM1-CM2 substrate. In some to 20 amino acids in length, and wherein each of L1 and L2 embodiments, such patients can be tested with other acti need not be the same linker. In some embodiments of these vatable antibodies until a suitable activatable antibody for methods and kits, one or both of L1 and L2 comprises a treatment is identified (e.g., an activatable antibody com glycine-serine polymer. In some embodiments of these prising a CM1-CM2 substrate that is cleaved by the patient methods and kits, at least one of L1 and L2 comprises an at the site of disease). In some embodiments, the activatable amino acid sequence selected from the group consisting of antibody is an activatable antibody to which a therapeutic (GS)n, (GSGGS)n (SEQ ID NO: 381) and (GGGS)n (SEQ agent is conjugated. In some embodiments, the activatable ID NO: 382), where n is an integer of at least one. In some antibody is not conjugated to an agent. In some embodi embodiments of these methods and kits, at least one of L1 ments, the activatable antibody comprises a detectable label. and L2 comprises an amino acid sequence having the In some embodiments, the detectable label is positioned on formula (GGS)n, where n is an integer of at least one. In the AB. In some embodiments, measuring the level of Some embodiments of these methods and kits, at least one of activatable antibody in the Subject or sample is accom L1 and L2 comprises an amino acid sequence selected from plished using a secondary reagent that specifically binds to the group consisting of Gly-Gly-Ser-Gly (SEQID NO: 383), the activated antibody, wherein the reagent comprises a Gly-Gly-Ser-Gly-Gly (SEQID NO:384), Gly-Ser-Gly-Ser detectable label. In some embodiments, the secondary Gly (SEQID NO:385), Gly-Ser-Gly-Gly-Gly (SEQID NO: reagent is an antibody comprising a detectable label. 386), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 387), and Gly 0441. In some embodiments, a method or kit is used to Ser-Ser-Ser-Gly (SEQ ID NO:388). identify or otherwise refine a patient population suitable for 0443. In some embodiments of these methods and kits, treatment with an anti-the target activatable antibody and/or the AB comprises an antibody or antibody fragment conjugated activatable antibody (e.g., activatable antibody sequence selected from the cross-reactive antibody to which a therapeutic agent is conjugated) of the disclosure, sequences presented herein. In some embodiments of these followed by treatment by administering that activatable methods and kits, the AB comprises a Fab fragment, a schv antibody and/or conjugated activatable antibody to a subject or a single chain antibody (ScAb). in need thereof. For example, patients that test positive for 0444. In some embodiments of these methods and kits, both the targets (e.g., the target) and a protease that cleaves the cleaving agent is a protease that is co-localized in the the CM1-CM2 substrate of the activatable antibody and/or subject or sample with the target and the CM1-CM2 sub conjugated activatable antibody being tested in these meth strate is a polypeptide that functions as a Substrate for the ods are identified as suitable candidates for treatment with protease, wherein the protease cleaves the CM1-CM2 sub Such antibody and/or such a conjugated activatable antibody strate in the activatable antibody when the activatable anti comprising such a CM1-CM2 substrate, and the patient is body is exposed to the protease. In some embodiments of then administered a therapeutically effective amount of the these methods and kits, each of the CM1 substrate sequence activatable antibody and/or conjugated activatable antibody and the CM2 substrate sequence in the CM1-CM2 substrate that was tested. Likewise, patients that test negative for is independently a polypeptide of up to 15 amino acids in either or both of the target (e.g., the target) and the protease length. In some embodiments of these methods and kits, the that cleaves the substrate in the CM1-CM2 substrate in the CM1-CM2 substrate is coupled to the N-terminus of the AB. activatable antibody being tested using these methods might In some embodiments of these methods and kits, the CM1 be identified as suitable candidates for another form of CM2 substrate is coupled to the C-terminus of the AB. In therapy. In some embodiments, such patients can be tested some embodiments of these methods and kits, the CM1 with other antibody and/or conjugated activatable antibody CM2 substrate is coupled to the N-terminus of a VL chain until a Suitable antibody and/or conjugated activatable anti of the AB. body for treatment is identified (e.g., an activatable antibody 0445. The activatable antibodies and/or conjugated acti and/or conjugated activatable antibody comprising a CM1 Vatable antibodies of the disclosure are used in diagnostic CM2 substrate that is cleaved by the patient at the site of and prophylactic formulations. In one embodiment, an acti disease). In some embodiments, the patient is then admin vatable antibody is administered to patients that are at risk istered a therapeutically effective amount of the activatable of developing one or more of the aforementioned inflam antibody and/or conjugated for which the patient tested mation, inflammatory disorders, cancer or other disorders. positive. 0446. A patient’s or organ's predisposition to one or more 0442. In some embodiments of these methods and kits, of the aforementioned disorders can be determined using the MM is a peptide having a length from about 4 to 40 genotypic, serological or biochemical markers. US 2016/0289.324 A1 Oct. 6, 2016 60

0447. In some embodiments, an activatable antibody matic activity (or an increased reduction potential depending and/or conjugated activatable antibodies is administered to on the nature of the CM1-CM2 substrate) but also that the human individuals diagnosed with a clinical indication asso tissue expresses target to which the activated antibody binds. ciated with one or more of the aforementioned disorders. 0453 For example, the CM1-CM2 substrate can be Upon diagnosis, an activatable antibody and/or conjugated selected to be a protease substrate for a protease found at the activatable antibodies is administered to mitigate or reverse site of a tumor, at the site of a viral or bacterial infection at the effects of the clinical indication. a biologically confined site (e.g., Such as in an abscess, in an 0448 Activatable antibodies and/or conjugated activat organ, and the like), and the like. The AB can be one that able antibodies of the disclosure are also useful in the binds a target antigen. Using methods familiar to one skilled detection of the target in patient samples and accordingly are in the art, a detectable label (e.g., a fluorescent label or useful as diagnostics. For example, the activatable antibod radioactive label or radiotracer) can be conjugated to an AB ies and/or conjugated activatable antibodies of the disclosure or other region of an activatable antibody. Suitable detect are used in in vitro assays, e.g., ELISA, to detect target able labels are discussed in the context of the above screen levels in a patient sample. ing methods and additional specific examples are provided 0449 In one embodiment, an activatable antibody of the below. Using an AB specific to a protein or peptide of the disclosure is immobilized on a Solid Support (e.g., the well(s) disease state, along with a protease whose activity is of a microtiter plate). The immobilized activatable antibody elevated in the disease tissue of interest, activatable anti serves as a capture antibody for any target that may be bodies will exhibit an increased rate of binding to disease present in a test sample. Prior to contacting the immobilized tissue relative to tissues where the CM1-CM2 substrate antibody with a patient sample, the Solid Support is rinsed specific enzyme is not present at a detectable level or is and treated with a blocking agent such as milk protein or present at a lower level than in disease tissue or is inactive albumin to prevent nonspecific adsorption of the analyte. (e.g., in Zymogen form or in complex with an inhibitor). 0450 Subsequently the wells are treated with a test Since Small proteins and peptides are rapidly cleared from sample Suspected of containing the antigen, or with a the blood by the renal filtration system, and because the Solution containing a standard amount of the antigen. Such enzyme specific for the CM1-CM2 substrate is not present a sample is, e.g., a serum sample from a subject Suspected at a detectable level (or is present at lower levels in non of having levels of circulating antigen considered to be disease tissues or is present in inactive conformation), diagnostic of a pathology. After rinsing away the test sample accumulation of activated antibodies in the disease tissue is or standard, the solid support is treated with a second enhanced relative to non-disease tissues. antibody that is detectably labeled. The labeled second 0454. In another example, activatable antibodies can be antibody serves as a detecting antibody. The level of detect used to detect the presence or absence of a cleaving agent in able label is measured, and the concentration of target a sample. For example, where the activatable antibodies antigen in the test sample is determined by comparison with contain a CM1-CM2 substrate susceptible to cleavage by an a standard curve developed from the standard samples. enzyme, the activatable antibodies can be used to detect 0451. It will be appreciated that based on the results (either qualitatively or quantitatively) the presence of an obtained using the antibodies of the disclosure in an in vitro enzyme in the sample. In another example, where the diagnostic assay, it is possible to stage a disease in a subject activatable antibodies contain a CM1-CM2 substrate sus based on expression levels of the Target antigen. For a given ceptible to cleavage by reducing agent, the activatable disease, Samples of blood are taken from Subjects diagnosed antibodies can be used to detect (either qualitatively or as being at various stages in the progression of the disease, quantitatively) the presence of reducing conditions in a and/or at various points in the therapeutic treatment of the sample. To facilitate analysis in these methods, the activat disease. Using a population of samples that provides statis able antibodies can be detectably labeled, and can be bound tically significant results for each stage of progression or to a Support (e.g., a Solid Support, such as a slide or bead). therapy, a range of concentrations of the antigen that may be The detectable label can be positioned on a portion of the considered characteristic of each stage is designated. activatable antibody that is not released following cleavage, 0452 Activatable antibodies and/or conjugated activat for example, the detectable label can be a quenched fluo able antibodies can also be used in diagnostic and/or imag rescent label or other label that is not detectable until ing methods. In some embodiments, such methods are in cleavage has occurred. The assay can be conducted by, for vitro methods. In some embodiments. Such methods are in example, contacting the immobilized, detectably labeled Vivo methods. In some embodiments, such methods are in activatable antibodies with a sample Suspected of containing situ methods. In some embodiments, such methods are ex an enzyme and/or reducing agent for a time sufficient for Vivo methods. For example, activatable antibodies having an cleavage to occur, then washing to remove excess sample enzymatically cleavable CM1-CM2 substrate can be used to and contaminants. The presence or absence of the cleaving detect the presence or absence of an enzyme that is capable agent (e.g., enzyme or reducing agent) in the sample is then of cleaving the CM1-CM2 substrate. Such activatable anti assessed by a change in detectable signal of the activatable bodies can be used in diagnostics, which can include in vivo antibodies prior to contacting with the sample e.g., the detection (e.g., qualitative or quantitative) of enzyme activ presence of and/or an increase in detectable signal due to ity (or, in some embodiments, an environment of increased cleavage of the activatable antibody by the cleaving agent in reduction potential Such as that which can provide for the sample. reduction of a disulfide bond) through measured accumula 0455 Such detection methods can be adapted to also tion of activated antibodies (i.e., antibodies resulting from provide for detection of the presence or absence of a target cleavage of an activatable antibody) in a given cell or tissue that is capable of binding the AB of the activatable antibod of a given host organism. Such accumulation of activated ies when cleaved. Thus, the assays can be adapted to assess antibodies indicates not only that the tissue expresses enzy the presence or absence of a cleaving agent and the presence US 2016/0289.324 A1 Oct. 6, 2016

or absence of a target of interest. The presence or absence of identify those patients having the appropriate protease(s) the cleaving agent can be detected by the presence of and/or and target(s) at the appropriate location, e.g., at a tumor site. an increase in detectable label of the activatable antibodies 0463. In some embodiments, in situ imaging is used to as described above, and the presence or absence of the target identify or otherwise refine a patient population suitable for can be detected by detection of a target-AB complex e.g., by treatment with an activatable antibody of the disclosure. For use of a detectably labeled anti-target antibody. example, patients that test positive for both the target and a 0456 Activatable antibodies are also useful in in situ protease that cleaves the substrate in the cleavable moiety imaging for the validation of activatable antibody activation, (CM1-CM2 substrate) of the activatable antibody being e.g., by protease cleavage, and binding to a particular target. tested (e.g., accumulate activated antibodies at the disease In situ imaging is a technique that enables localization of site) are identified as suitable candidates for treatment with proteolytic activity and target in biological samples Such as such an activatable antibody comprising such a CM1-CM2 cell cultures or tissue sections. Using this technique, it is Substrate. Likewise, patients that test negative for either or possible to confirm both binding to a given target and both of the target and the protease that cleaves the substrate proteolytic activity based on the presence of a detectable in the CM1-CM2 substrate in the activatable antibody being label (e.g., a fluorescent label). tested using these methods are identified as Suitable candi 0457. These techniques are useful with any frozen cells dates for another form of therapy (i.e., not suitable for or tissue derived from a disease site (e.g. tumor tissue) or treatment with the activatable antibody being tested). In healthy tissues. These techniques are also useful with fresh Some embodiments, such patients that test negative with cell or tissue samples. respect to a first activatable antibody can be tested with other 0458 In these techniques, an activatable antibody is activatable antibodies comprising different CMs until a labeled with a detectable label. The detectable label may be suitable activatable antibody for treatment is identified (e.g., a fluorescent dye, (e.g. Fluorescein Isothiocyanate (FITC), an activatable antibody comprising a CM1-CM2 substrate Rhodamine Isothiocyanate (TRITC), a near infrared (NIR) that is cleaved by the patient at the site of disease). dye (e.g., Qdot(R) nanocrystals), a colloidal metal, a hapten, 0464. In some embodiments, in vivo imaging is used to a radioactive marker, biotin and an amplification reagent identify or otherwise refine a patient population suitable for Such as Streptavidin, or an enzyme (e.g. horseradish peroxi treatment with an activatable antibody of the disclosure. For dase or alkaline phosphatase). example, patients that test positive for both the target and a 0459 Detection of the label in a sample that has been protease that cleaves the substrate in the cleavable moiety incubated with the labeled, activatable antibody indicates (CM1-CM2 substrate) of the activatable antibody being that the sample contains the target and contains a protease tested (e.g., accumulate activated antibodies at the disease that is specific for the CM1-CM2 substrate of the activatable site) are identified as suitable candidates for treatment with antibody. In some embodiments, the presence of the protease such an activatable antibody comprising such a CM1-CM2 can be confirmed using broad spectrum protease inhibitors Substrate. Likewise, patients that test negative are identified Such as those described herein, and/or by using an agent that as Suitable candidates for another form of therapy (i.e., not is specific for the protease, for example, an antibody Such as suitable for treatment with the activatable antibody being A11, which is specific for the protease matriptase (MT-SP1) tested). In some embodiments, such patients that test nega and inhibits the proteolytic activity of matriptase; see e.g., tive with respect to a first activatable antibody can be tested International Publication Number WO 2010/129609, pub with other activatable antibodies comprising different CMs lished 11 Nov. 2010. The same approach of using broad until a suitable activatable antibody for treatment is identi spectrum protease inhibitors such as those described herein, fied (e.g., an activatable antibody comprising a CM1-CM2 and/or by using a more selective inhibitory agent can be used substrate that is cleaved by the patient at the site of disease). to identify a protease or class of proteases specific for the 0465 Pharmaceutical Compositions CM1-CM2 substrate of the activatable antibody. In some 0466. The conjugated antibodies, activatable antibodies embodiments, the presence of the target can be confirmed and/or conjugated activatable antibodies of the disclosure using an agent that is specific for the target, e.g., another (also referred to herein as “active compounds’), and deriva antibody, or the detectable label can be competed with tives, fragments, analogs and homologs thereof, can be unlabeled target. In some embodiments, unlabeled activat incorporated into pharmaceutical compositions Suitable for able antibody could be used, with detection by a labeled administration. Such compositions typically comprise the secondary antibody or more complex detection system. conjugated antibody, activatable antibody and/or conjugated 0460 Similar techniques are also useful for in vivo activatable antibody and a pharmaceutically acceptable car imaging where detection of the fluorescent signal in a rier. As used herein, the term “pharmaceutically acceptable Subject, e.g., a mammal, including a human, indicates that carrier is intended to include any and all solvents, disper the disease site contains the target and contains a protease sion media, coatings, antibacterial and antifungal agents, that is specific for the CM1-CM2 substrate of the activatable isotonic and absorption delaying agents, and the like, com antibody. patible with pharmaceutical administration. Suitable carriers 0461 These techniques are also useful in kits and/or as are described in the most recent edition of Remington's reagents for the detection, identification or characterization Pharmaceutical Sciences, a standard reference text in the of protease activity in a variety of cells, tissues, and organ field, which is incorporated herein by reference. Suitable isms based on the protease-specific CM1-CM2 substrate in examples of Such carriers or diluents include, but are not the activatable antibody. limited to, water, Saline, ringer's solutions, dextrose solu 0462. In some embodiments, in situ imaging and/or in tion, and 5% human serum albumin. Liposomes and non Vivo imaging are useful in methods to identify which aqueous vehicles Such as fixed oils may also be used. The patients to treat. For example, in in situ imaging, the use of Such media and agents for pharmaceutically active activatable antibodies are used to screen patient samples to Substances is well known in the art. Except insofar as any US 2016/0289.324 A1 Oct. 6, 2016 62 conventional media or agent is incompatible with the active plus any additional desired ingredient from a previously compound, use thereof in the compositions is contemplated. sterile-filtered solution thereof. Supplementary active compounds can also be incorporated 0470 Oral compositions generally include an inert into the compositions. diluent or an edible carrier. They can be enclosed in gelatin 0467. A pharmaceutical composition of the disclosure is capsules or compressed into tablets. For the purpose of oral formulated to be compatible with its intended route of therapeutic administration, the active compound can be administration. Examples of routes of administration include incorporated with excipients and used in the form of tablets, parenteral, e.g., intravenous, intradermal, Subcutaneous, oral troches, or capsules. Oral compositions can also be prepared (e.g., inhalation), transdermal (i.e., topical), transmucosal, using a fluid carrier for use as a mouthwash, wherein the and rectal administration. Solutions or Suspensions used for compound in the fluid carrier is applied orally and Swished parenteral, intradermal, or Subcutaneous application can and expectorated or Swallowed. Pharmaceutically compat include the following components: a sterile diluent Such as ible binding agents, and/or adjuvant materials can be water for injection, Saline solution, fixed oils, polyethylene included as part of the composition. The tablets, pills, glycols, glycerine, propylene glycol or other synthetic Sol capsules, troches and the like can contain any of the fol vents; antibacterial agents such as benzyl alcohol or methyl lowing ingredients, or compounds of a similar nature: a parabens; antioxidants such as ascorbic acid or sodium binder Such as microcrystalline cellulose, gum tragacanth or bisulfite; chelating agents such as ethylenediaminetet gelatin; an excipient Such as starch or lactose, a disintegrat raacetic acid (EDTA); buffers such as acetates, citrates or ing agent Such as alginic acid, Primogel, or corn starch; a phosphates, and agents for the adjustment of tonicity Such as lubricant such as magnesium Stearate or Sterotes; a glidant sodium chloride or dextrose. The pH can be adjusted with Such as colloidal silicon dioxide; a Sweetening agent Such as acids or bases, such as hydrochloric acid or sodium hydrox Sucrose or saccharin; or a flavoring agent Such as pepper ide. The parenteral preparation can be enclosed in ampoules, mint, methyl salicylate, or orange flavoring. disposable Syringes or multiple dose vials made of glass or 0471. For administration by inhalation, the compounds plastic. are delivered in the form of an aerosol spray from pressured 0468 Pharmaceutical compositions suitable for inject container or dispenser that contains a Suitable propellant, able use include sterile aqueous solutions (where water e.g., a gas such as carbon dioxide, or a nebulizer. soluble) or dispersions and sterile powders for the extem 0472 Systemic administration can also be by transmu poraneous preparation of sterile injectable solutions or dis cosal or transdermal means. For transmucosal or transder persion. For intravenous administration, suitable carriers mal administration, penetrants appropriate to the barrier to include physiological saline, bacteriostatic water. Cremo be permeated are used in the formulation. Such penetrants phor ELTM (BASF, Parsippany, N.J.) or phosphate buffered are generally known in the art, and include, for example, for saline (PBS). In all cases, the composition must be sterile transmucosal administration, detergents, bile salts, and and should be fluid to the extent that easy syringeability fusidic acid derivatives. Transmucosal administration can be exists. It must be stable under the conditions of manufacture accomplished through the use of nasal sprays or Supposito and storage and must be preserved against the contaminating ries. For transdermal administration, the active compounds action of microorganisms such as bacteria and fungi. The are formulated into ointments, salves, gels, or creams as carrier can be a solvent or dispersion medium containing, for generally known in the art. example, water, ethanol, polyol (for example, glycerol, 0473. The compounds can also be prepared in the form of propylene glycol, and liquid polyethylene glycol, and the Suppositories (e.g., with conventional Suppository bases like), and suitable mixtures thereof. The proper fluidity can Such as cocoa butter and other glycerides) or retention be maintained, for example, by the use of a coating Such as enemas for rectal delivery. lecithin, by the maintenance of the required particle size in 0474. In one embodiment, the active compounds are the case of dispersion and by the use of surfactants. Pre prepared with carriers that will protect the compound against vention of the action of microorganisms can be achieved by rapid elimination from the body, such as a controlled release various antibacterial and antifungal agents, for example, formulation, including implants and microencapsulated parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, delivery systems. Biodegradable, biocompatible polymers and the like. In some embodiments, it will be desirable to can be used. Such as ethylene vinyl acetate, polyanhydrides, include isotonic agents, for example, Sugars, polyalcohols polyglycolic acid, collagen, polyorthoesters, and polylactic Such as manitol, Sorbitol, Sodium chloride in the composi acid. Methods for preparation of such formulations will be tion. Prolonged absorption of the injectable compositions apparent to those skilled in the art. The materials can also be can be brought about by including in the composition an obtained commercially from Alza Corporation and Nova agent that delays absorption, for example, aluminum monos Pharmaceuticals, Inc. Liposomal Suspensions (including tearate and gelatin. liposomes targeted to infected cells with monoclonal anti 0469 Sterile injectable solutions can be prepared by bodies to viral antigens) can also be used as pharmaceuti incorporating the active compound in the required amount in cally acceptable carriers. These can be prepared according to an appropriate solvent with one or a combination of ingre methods known to those skilled in the art, for example, as dients enumerated above, as required, followed by filtered described in U.S. Pat. No. 4,522,811. sterilization. Generally, dispersions are prepared by incor 0475. It is especially advantageous to formulate oral or porating the active compound into a sterile vehicle that parenteral compositions in dosage unit form for ease of contains a basic dispersion medium and the required other administration and uniformity of dosage. Dosage unit form ingredients from those enumerated above. In the case of as used herein refers to physically discrete units Suited as sterile powders for the preparation of sterile injectable unitary dosages for the Subject to be treated; each unit Solutions, methods of preparation are vacuum drying and containing a predetermined quantity of active compound freeze-drying that yields a powder of the active ingredient calculated to produce the desired therapeutic effect in asso US 2016/0289.324 A1 Oct. 6, 2016 ciation with the required pharmaceutical carrier. The speci fication for the dosage unit forms of the disclosure are CM1 - CM2 dictated by and directly dependent on the unique character Substrate AA sequence SEO ID NO : istics of the active compound and the particular therapeutic 2OO1 ISSGLISGRSDNH 1. effect to be achieved, and the limitations inherent in the art of compounding Such an active compound for the treatment 1OO1/LP 'AOOO1 ISSGLLSSGGSGGSLSGRSDNH 2 of individuals. 1OO4/LP 'AOOO3 AVGLLAPPGGTSTSGRSANPRG 3 0476. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions OOO3/LP 'A1 OO4 TSTSGRSANPRGGGAVGLLAPP 4. for administration. 1OO3/LP 'A OOO3 VHMPLGFLGPGGTSTSGRSANPRG 5 0477. The invention will be further described in the following examples, which do not limit the scope of the OOO3/LP 'A1 OO3 TSTSGRSANPRGGGVHMPLGFLGP 6 invention described in the claims. 1OO4/LP 'AOOO1 AVGLLAPPGGLSGRSDNH 7 Examples OOO1/LP 'A1 OO4 LSGRSDNHGGAVGLLAPP 8 10O3/LP 'AOOO1 VHMPLGFLGPGGLSGRSDNH 9 Example 1 OOO1/LP 'A1 OO3 LSGRSDNHGGVHMPLGFLGP 10 Matrix Metalloprotease (MMP) and Serine Protease (SP) Cleavable Anti-Jagged Activatable Antibodies 0480 Construction of the Anti-Jagged activatable anti body light chains was performed as follows. 0478. This Example demonstrates the generation and 0481. The CM1-CM2 substrates were incorporated into evaluation of activatable antibodies that bind a Jagged the Jagged activatable antibody vector (described in PCT target, e.g., Jagged 1 and/or Jagged 2, where the activatable Publication No. WO2013/192550) as follows. Using stan antibodies are activated in the presence of at least one matrix dard molecular biology techniques, the forward (F) primers metalloprotease (MMP) and at least one serine protease. encoding the CM1-CM2 substrates (see Table A) and the reverse (R) primer CX1198 were used to amplify the sub 0479. The studies described herein used the following strate and VL domain of the Jagged activatable antibody and substrate sequences, where LP is a linking peptide between were subsequently cloned into the activatable antibody CM1 and CM2. For the CM1-CM2 substrate 1001/LP'/0001, vector using the XhoI and BsiWI restriction sites. The LP' is GGSGGS (SEQ ID NO: 350), and for all other resulting vectors encoded the following anti-Jagged activat CM1-CM2 in the Table below, LP is GG: able antibody light chains. TABL E A Primers used to construct the CM1 - CM2 expression vectors CX1198 Light chain R Gtgcago cacco tacgtttgattitccaccittggtc.cc (SEO ID NO : 37) CaggggggctcgagcGGCGGCTCTATCTCTTCCGGACTGCT GTCCGGCAGATCCGACAATCACGGCGGAGGCTCTGacatcc. agatgacccagt ct c (SEQ ID NO: 38 CX2O67 1001/LP'/0001 F CaggggggctcgagcGGCGGCTCTATCTCTTCTGGCCTGCT GTCTAGCGGCGGCTCCGGCGGATCTCTGTCTGGCAGATCTG ACAACCACGGCGGAGGCTCCGacatccagatgacccagtct c (SEO ID NO. 39) CX2190 1004/LP' /OOO3 F CaggggggctcgagcGGAGGATCTGCTGTGGGACTGCTGGC TCCTCCTGGCGGCACATCTACCTCTGGCAGATCCGCCAACC CTCGGGGCGGAGGATCTGacatccagatgaccoagtctic (SEQ ID NO: 40) CX2191 OOO3/LP '/100.4 F CaggggggctcgagcGGCGGCTCCACATCTACCTCTGGCAG ATCCGCCAACCCCAGAGGTGGCGGAGCTGTGGGACTGCTGG CTCCACCAGGCGGATCTGacatccagatgaccoagtctic (SEQ ID NO: 41) CX2192 1003/LP' /OOO3 F CaggggggctcgagcGGCGGCTCTGTGCATATGCCCCTGGG CTTTCTGGGCCCTGGCGGCACATCTAC CTCTGGCAGATCCG CCAACCCTCGGGGCGGAGGATCTGacatccagatgacccag tctic (SEQ ID NO: 42) CX2193 OOO3/LP '/1003 F CaggggggctcgagcGGCGGCTCCACATCTACCTCTGGCAG ATCCGCCAACCCCAGAGGCGGCGGAGTGCATATGCCTCTGG GCTTTCTGGGACCTGGCGGCTCTGacatccagatgacccag tctic (SEQ ID NO: 43)

US 2016/0289.324 A1 Oct. 6, 2016

- Continued Anti-Jagged activatable antibody Ho (SEO ID NO : 67) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSSIDPEGROTYYADSV

KGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDIGGRSAFDYWGOGTLVTVSSASTKGPSVF

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNEGALTSGVHTFPAVLOSSGLYSLSSWWTVPS

SSLGTOTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYWDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKE

YKCKVSNKALPAPIEKTISKAKGOPREPOVYTLPPSREEMTKNOWSLTCLVKGFYPSDIAVEWE

SNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNWFSCSVMHEALHNHYTOKSLSLSPG

K

0482 Anti-Jagged CM1-CM2 Activatable antibody in Jagged 1/2 in normal tissues. If the anti-Jagged CM1-CM2 vitro binding and activation was evaluated as follows. Activatable antibody remains masked (stable) in circulation, 0483 Anti-Jagged activatable antibodies were expressed then the activatable antibody should avoid target-mediated from transiently transfected HEK-293 cells and purified clearance and show prolonged serum half-life. from the culture Supernatant by Protein A chromatography. To verify that the anti-Jagged CM1-CM2 activatable anti 0488. The plasma pharmacokinetics of the anti-Jagged bodies could be activated by both MMPs and serine pro antibody and activatable antibodies were evaluated as fol teases, the purified activatable antibodies were digested with lows. As shown in Table B, each group consisted of 2 uPA and/or MMP14 and subsequently evaluated for their cohorts of 5 mice. Mice were given a single intravenous ability to bind to human Jagged 1-Fc by ELISA. ELISA dose of 5 mg/kg of the indicated compound. Lithium hepa plates (Greiner Bio-One #655061) were coated with human rinized plasma was collected from cohort 1 at 24 hours, 96 Jag1-Fc (R&D # 1277-JG-050) in Hank's Balanced Salt hours, and 10 days post dose while plasma was collected Solution pH 7.4 (HBSS) (Teknova #H8057) at 1 microgram/ from cohort 2 at 48 hours, 7 days, and 14 days by the mi overnight at 4° C.; as used herein, microgram(s) is also retro-orbital route with isoflurane anesthesia. Total plasma represented by ug and lug. Plates were blocked with 2% human IgG levels were detected using a human IgG sand Nonfat dry milk (NFDM) in HBSS for 1 hour at room wich ELISA. Briefly, ELISA plates (Costar 3590 Fisher temperature (RT). The block was removed and anti-Jagged Scientific Cat. #07-200-35) were coated with AffiniPure antibody 4D11, an anti-Jagged CM1-CM2 activatable anti Goat Anti-Human IgG F(ab')2 Fragment Specific, (Jackson body, or a digested activatable antibody was added to the ImmunoResearch Cat. #109-006-097) in phosphate buffered indicated concentration in 2% NFDM/HBSS and incubated saline (PBS) at 1 ug/ml overnight at 4° C. Plates were at RT for 1 hours. The ELISA plate was washed 3 times with blocked with Superblock (ScyTek Laboratories Cat. excess HBSS, 0.05% TWEEN (HBSS-T) before adding the #AAA500) for 1 hour at room temperature (RT). The block mouse anti-human IgG, F 2 specific, HRP conjugated was removed and an appropriate dilution of standard (test (Jackson ImmunoResearch #209-035-097) diluted to 1:30, article) and test samples (plasma samples) were added to the 000 in 2% NFDM/HBSS. The ELISA plate was subse quently washed 3x with HBSS-T and developed using plate and incubated at RT for 1 hour. The ELISA plate was 1-Step TMB Substrate (Pierce/Thermo Fisher washed 3 times with excess PBS, 0.05% TWEEN (PBS-T) #NC0140927). The plates were read at ODs and plotted before adding the AffiniPure Goat Anti-Human IgG F. using Prism Software. Fragment Specific Horseradish Peroxidase (HRP) (Jackson 0484 FIG. 1 demonstrates the anti-Jagged CM1-CM2 ImmunoResearch Cat. #109-035-097) diluted to 1:25,000. activatable antibodies (a) were effectively masked prior to The ELISA plate was subsequently washed 3x with PBS-T cleavage by uPA or MMP14 and (b) showed binding equiva and developed using 1-Step TMB Substrate (Pierce/Thermo lent to the antibody when cleaved by uPA or MMP14 or a Fisher iNCO140927) following the manufacturers protocol. combination of uPA and MMP14. Serum human IgG levels were calculated by comparing the 0485 Anti-Jagged activatable antibody containing CM1 test sample values to the standard curve. Pharmacokinetic CM2 Substrate pharmacokinetics were evaluated in non parameters were calculated using a noncompartmental tumor bearing nude mice as follows. analysis with sparse sampling (Phoenix WinNonlin V6.3). 0486. As a surrogate for the stability of the mask and Substrate, the pharmacokinetics of the anti-Jagged activat 0489. As shown in FIG. 2, the anti-Jagged antibody was able antibodies containing the CM1-CM2 substrates 2001 rapidly cleared and was below the detection limit of the and 1001/LP/0001 were compared to that of the anti-Jagged assay by day 10. In contrast, the anti-Jagged CM1-CM2 antibody in non-tumor bearing mice. activatable antibodies 1001/LP/0001 and 2001 showed sig 0487. The mouse/human cross-reactive anti-Jagged anti nificantly extended half-life indicating that the activatable body shows rapid clearance in mice due to the binding of antibodies remained stable and well masked in circulation. US 2016/0289.324 A1 Oct. 6, 2016 76

TABLE B both the anti-Jagged ADC and anti-Jagged 2001 activatable antibody drug conjugate groups showed tumor regression. Groups and dosing for the pharmacokinetic analysis of the The animals treated with the anti-Jagged ADC showed anti-Jagged CM1-CM2 Activatable antibodies 2001 and 1001/LP/0001. significant side effects as measured by weight loss, as shown Dose in FIG. 4. However, animals treated with anti-Jagged 2001 Dose volume activatable antibody drug conjugate showed no weight loss Group N Treatment (mg/kg) (mL/kg) Schedule Route (also shown in FIG. 4) demonstrating that the anti-Jagged 1 2 x 5 Anti-Jagged 5 10 Single IP CM1-CM2 activatable antibody drug conjugate’s activity 2 2 x 5 Anti-Jagged 5 10 Single IP was localized to the tumor. Activatable antibody 2001 3 2 x 5 Anti-Jagged 5 10 Single IP TABLE C Activatable antibody Groups and dose schedule for the HCC1806 study 1001 LP'OOO1 Dose Group Count Treatment (mg/kg) Schedule Route 0490. In vivo evaluation of the safety and efficacy of an 1 8 PBS 10 q7dx2 IV anti-Jagged CM1-CM2 Substrate containing activatable anti 3 8 Isotype-SPDB-DM4 10 q7dx2 IV body drug conjugate was performed as follows. 4 8 Anti-Jagged-SPDB-DM4 10 q7dx2 IV 5 8 Anti-Jagged 2001 10 q7dx2 IV 0491. The efficacy of the anti-Jagged CM1-CM2 sub activatable antibody strate containing 2001 activatable antibody drug conjugate, SPDB-DM4 which comprised the anti-Jagged 2001 activatable antibody conjugated to maytansinoid DM4 (see, e.g., U.S. Pat. No. 7.276,497) via a SPDB linker, was evaluated in the human Example 2 breast cancer cell line HCC1806 xenograft tumor model. HCC1806 cells were harvested during log phase growth and Matrix Metalloprotease (MMP) and Serine Protease resuspended in 50% Matrigel (BD Biosciences) in PBS at a (SP) Cleavable Anti-EGFR Activatable Antibodies concentration of 5x10" cells/ml. Mice were injected subcu 0493. This Example demonstrates the generation and taneously in the right flank with 5x10° cells and allowed to evaluation of activatable antibodies that bind Epidermal grow to a mean volume of 100-150 mm. Mice were Growth Factor Receptor (EGFR), where the activatable randomized and dosed as indicated in Table C. Tumor antibodies are activated in the presence of at least one matrix volume and body weight were measured twice weekly for metalloprotease (MMP) and at least one serine protease. the duration of the study, and measures of efficacy and 0494. The activatable anti-EGFR antibodies used in this safety, respectively were obtained. Example were generated using a method similar to the 0492 FIG. 3 shows the group mean tumor volumetthe methods used in Example 1 to generate activatable anti standard error of the mean (SEM) for the PBS, Isotype Jagged antibodies. SPDB-DM4, anti-Jagged-SPDB-DM4 drug conjugate 0495. The studies described herein used the following (ADC), and anti-Jagged 2001 activatable antibody drug Substrate sequences, where LP' is a linking peptide between conjugate treated animals. Neither control group (PBS nor CM1 and CM2. For all CM1-CM2 Substrates in the Table Isotype-SPDB-DM4) showed tumor growth inhibition while below, LP is GGSGGS (SEQ ID NO:350):

CM1 - CM2 Substrate Amino Acid Sequence Nucleotide Sequence

OOO1 LSGRSDNH TTAAGCGGGCGGTCGGACAACCAC (SEQ ID NO: 18) (SEQ ID NO: 23)

OOO2 LSGRSGNE CTTAGCGGGCGGAGCGGCAACCAC (SEQ ID NO: 19) (SEQ ID NO: 24)

1 OO1 ISSGLISS ATCTCCTCCGGGCTACTGAGTTCT (SEQ ID NO: 2O) (SEQ ID NO: 25)

1 OO2 QNOALRMA CAGAACCAGGCGCTCAGAATGGCA (SEQ ID NO: 21) (SEQ ID NO: 26)

2 OO1 ISSGLISGRSDNE ATATCATCCGGCCTCCTTAGCGGC (SEQ ID NO: 1) CGTTCCGACAATCAC (SEO ID NO: 27)

2 OO2 ISSGLISGRSGNH ATAAGTTCTGGGCTCCTGTCGGGC (SEQ ID NO: 22) CGGAGTGGAAATCAC (SEQ ID NO: 28) OOO1/LP' A1 OO1 LSGRSDNHGGSGSISSGLLSS CTGAGCGGGCGGTCCGATAATCAT (SEQ ID NO: 11) GGTGGTTCAGGAGGGAGTATTTCT TCCGGCTTACTGAGTAGC (SEQ ID NO: 29)