Matrix metalloproteinase cleavable and serine or cysteine protease cleavable substrates and methods of use thereof

Abstract

The present invention relates generally to polypeptides comprising at least a first cleavable moiety (CM1) as a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) as a substrate for at least one serine protease (SP) or at least one cysteine protease (CP); to activatable antibodies and other larger molecules comprising these polypeptides comprising at least CM1 as a substrate for at least one MMP protease and at least CM2 as a substrate for at least one SP protease or at least one cysteine protease (CP); the present invention relates to polypeptides comprising at least CM1 as a substrate for at least one MMP protease and at least CM2 as a substrate for at least one SP protease or at least one cysteine protease (CP), and to methods of making and using these polypeptides in a variety of therapeutic, diagnostic and prophylactic indications.

Description

[0001] This application is a divisional application of a Chinese patent application with application number 201980088092.4, filing date December 05, 2019, and invention title "Matrix metalloprotease-cleavable and serine or cysteine protease-cleavable substrates and methods of use thereof".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 776,409, filed December 6, 2018, and U.S. Provisional Application No. 62 / 778,062, filed December 11, 2018, the contents of which are incorporated herein by reference in their entirety. Technical field

[0004] The present invention generally relates to polypeptides that comprise at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine protease (CP); to activatable antibodies and other larger molecules that comprise such polypeptides, the polypeptides comprising at least CM1 that is a substrate for at least one MMP protease and at least CM2 that is a substrate for at least one SP protease and / or at least one CP protease; and to methods of making and using such polypeptides in a variety of therapeutic, diagnostic, and prophylactic indications, the polypeptides comprising at least CM1 that is a substrate for at least one MMP protease and at least CM2 that is a substrate for at least one SP protease and / or at least one CP protease.

[0005] Reference to the Sequence Listing

[0006] The "Sequence Listing" submitted electronically via EFS-Web as the file named "CYTX-058-PCT_ST25" in computer-readable form (CFR) in accordance with 37 C.F.R. § 1.821 is incorporated herein by reference. The electronic copy of the Sequence Listing was created on November 26, 2019, and is 159 kilobytes in size. Background art

[0007] Proteases are enzymes that degrade proteins by cleaving peptide bonds between amino acid residues. Proteases are naturally present in all organisms and are involved in a variety of physiological reactions from simple degradation to highly regulated pathways. Some proteases are known to break specific peptide bonds based on the presence of specific amino acid sequences in proteins.

[0008] Accordingly, there is a need to identify new substrates for proteases and use such substrates in a variety of therapeutic, diagnostic, and prophylactic indications. SUMMARY OF THE INVENTION

[0009] In one aspect of the present invention, there is provided an isolated polypeptide comprising a tandem substrate, wherein the tandem substrate comprises at least a first cleavable moiety (CM1) that is a substrate of at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) that is a substrate of at least one serine protease (SP) or cysteine protease (CP), wherein CM1 comprises the amino acid sequence AHGL or PRQV, and wherein the N-terminus to C-terminus arrangement of the tandem substrate is CM1-CM2 or CM2-CM1. In some embodiments, CM1 of the isolated polypeptide comprises an amino acid sequence selected from the group consisting of: ALAHGLF (SEQ ID NO:1), ALAHGL (SEQ ID NO:52), LAHGLF (SEQ ID NO:50), LAHGL (SEQ ID NO:53), and AHGLF (SEQ ID NO:51). In some embodiments, CM1 of the isolated polypeptide comprises an amino acid sequence selected from the group consisting of HVPRQV (SEQ ID NO:8) and VPRQV (SEQ ID NO:60). In some embodiments, the isolated polypeptide of the present disclosure comprises CM1 and CM2 linked by a linker peptide. In some embodiments, CM1 and CM2 of the isolated polypeptide are directly linked to each other. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 that is a substrate of a CP enzyme, and wherein the CP enzyme is legumain. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 that comprises a substrate of an SP enzyme selected from the group consisting of urokinase, matriptase, and neutrophil elastase. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 that comprises a substrate of an SP enzyme selected from the group consisting of urokinase, matriptase, and neutrophil elastase; and a substrate of a CP enzyme, and wherein the CP enzyme is legumain. In some embodiments, the isolated polypeptide of the present disclosure comprises CM1 that comprises a substrate of an MMP enzyme selected from the group consisting of MMP2, MMP9, or MMP14.

[0010] In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises an amino acid sequence selected from the group consisting of: SGR, LSGR (SEQ ID NO:73), ARG, PRS, TFVH (SEQ ID NO:141), AAN, SAN, and GPTN (SEQ ID NO:152). In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises an amino acid sequence selected from the group consisting of: SGR, LSGR (SEQ ID NO:73), LSGRS (SEQ ID NO:72), LSGRSD (SEQ IDNO:71), LSGRSA (SEQ ID NO:110), LSGRSDN (SEQ ID NO:70), LSGRSAN (SEQ ID NO:109), LSGRSDNH (SEQ ID NO:20), LSGRSGNH (SEQ ID NO:78), LSGRSDNP (SEQ ID NO:90), LSGRSDNI (SEQ ID NO:84), LSGRSANI (SEQ ID NO:108), LSGRSANP (SEQ ID NO:114), LSGRSDYH (SEQID NO:86), LSGRSDTH (SEQ ID NO:92), LSGRSDQH (SEQ ID NO:96), LSGRSDIH (SEQ ID NO:100), and LSGRSDDH (SEQ ID NO:104). In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises an amino acid sequence selected from the group consisting of: ARGP (SEQ ID NO:128), TARG (SEQ ID NO:125), and TARGP (SEQ ID NO:124). In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises an amino acid sequence selected from the group consisting of: APRS (SEQ ID NO:131), APRSF (SEQ ID NO:130), and PRSF(SEQ ID NO:132). In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises an amino acid sequence selected from the group consisting of: GLPTFVHL (SEQ ID NO:135), GLPTFVH (SEQ ID NO:136), GLPTFV (SEQ IDNO:137), LPTFVHL (SEQ ID NO:138), LPTFVH (SEQ ID NO:139), and LPTFV (SEQ ID NO:140).In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises an amino acid sequence selected from the group consisting of: AAN, SAN, and GPTN (SEQ ID NO: 152). In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises an amino acid sequence selected from the group consisting of: AAN, SAN, and GPTN (SEQ ID NO: 152); and an amino acid sequence selected from the group consisting of: SGR, LSGR (SEQ ID NO: 73), LSGRS (SEQ ID NO: 72), LSGRSD (SEQ ID NO: 71), LSGRSA (SEQ ID NO: 110), LSGRSDN (SEQ ID NO: 70), LSGRSAN (SEQ ID NO: 109), LSGRSDNH (SEQ ID NO: 20), LSGRSGNH (SEQ ID NO: 78), LSGRSDNP (SEQ ID NO: 90), LSGRSDNI (SEQ ID NO: 84), LSGRSANI (SEQ ID NO: 108), LSGRSANP (SEQ ID NO: 114), LSGRSDYH (SEQ ID NO: 86), LSGRSDTH (SEQ ID NO: 92), LSGRSDQH (SEQ ID NO: 96), LSGRSDIH (SEQ ID NO: 100), and LSGRSDDH (SEQ ID NO: 104).

[0011] In some embodiments, the N-terminal to C-terminal arrangement of the tandem substrates of the isolated polypeptide of the present disclosure is CM1-CM2. In some embodiments, the N-terminal to C-terminal arrangement of the tandem substrates of the isolated polypeptide of the present disclosure is CM2-CM1.

[0012] In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptide of the present disclosure with respect to the first cleavable portion of CM1 is at least 80% each. In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptide of the present disclosure with respect to the first cleavable portion of CM1 is at least 85% each. In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptide of the present disclosure with respect to the first cleavable portion of CM1 is at least 90% each.

[0013] In some embodiments, the in vivo stability of the first cleavable portion of CM1 of the isolated polypeptide of the present disclosure is less than 30% activation. In some embodiments, the in vivo stability of the first cleavable portion of CM1 of the isolated polypeptide of the present disclosure is less than 25% activation.

[0014] In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptides of the present disclosure for the tandem substrate is at least 30% each. In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptides of the present disclosure for the tandem substrate is at least 50% each. In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptides of the present disclosure for the tandem substrate is at least 70% each.

[0015] In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptides of the present disclosure for the tandem substrate is at least 15% each, and the cleavability of the proteolytic enzyme for the tandem substrate is at least 30%. In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptides of the present disclosure for the tandem substrate is at least 30% each, and the cleavability of the proteolytic enzyme for the tandem substrate is at least 30%. In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptides of the present disclosure for the tandem substrate is at least 50% each, and the cleavability of the proteolytic enzyme for the tandem substrate is at least 50%. In some embodiments, the cleavability of MMP9 and MMP14 of the isolated polypeptides of the present disclosure for the tandem substrate is at least 70% each, and the cleavability of the proteolytic enzyme for the tandem substrate is at least 70%.

[0016] In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability lower than 40% activation. In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability lower than 30% activation. In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability lower than 25% activation. In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability lower than 20% activation.

[0017] In some embodiments, the isolated polypeptides of the present disclosure comprise a tandem substrate that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43. In some embodiments, the isolated polypeptides of the present disclosure comprise a tandem substrate that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 29, 31, 32, 34, 36, and 37. In some embodiments, the isolated polypeptides of the present disclosure comprise a tandem substrate that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35. In some embodiments, the isolated polypeptides of the present disclosure comprise a tandem substrate that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 29, 31, 36, and 37. In some embodiments, the isolated polypeptides of the present disclosure comprise a tandem substrate that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, and 34. In some embodiments, the isolated polypeptides of the present disclosure comprise a tandem substrate that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 33. In some embodiments, the isolated polypeptides of the present disclosure comprise a tandem substrate that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35.

[0018] In some embodiments, provided herein is an isolated polypeptide of the present disclosure that comprises an antibody or an antigen-binding fragment (AB) thereof that specifically binds a target; at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP); and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) or cysteine protease (CP), wherein CM1 comprises the amino acid sequence AHGL or PRQV, and wherein the N-terminus to C-terminus arrangement of the tandem substrates is CM1-CM2 or CM2-CM1. In some embodiments, at least one of the MMP, CP, and SP is co-localized with the target in a tissue. In some embodiments, the isolated polypeptide of the present disclosure comprises an antigen-binding fragment selected from the group consisting of: Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single-domain heavy-chain antibody, and single-domain light-chain antibody. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB linked to CM1. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB directly linked to CM1. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB linked to CM1 via a linker peptide. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB linked to CM2. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB directly linked to CM2. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB linked to CM2 via a linker peptide. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB that comprises the light-chain variable region of an antibody or an antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the light-chain variable region of the AB. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB that comprises the heavy-chain variable region of an antibody or an antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the heavy-chain variable region of the AB.

[0019] In some embodiments, the isolated polypeptides of the present disclosure comprise a masking moiety (MM). In some embodiments, the isolated polypeptides of the present disclosure comprise an MM having a dissociation constant for binding to AB that is greater than the dissociation constant for AB binding to the target. In some embodiments, the isolated polypeptides of the present disclosure comprise an MM that is a polypeptide having a length of no more than 40 amino acids. In some embodiments, the isolated polypeptides of the present disclosure comprise an MM that is linked to CM1 such that the isolated polypeptide in the uncleaved state comprises the following structural arrangement from the N-terminus to the C-terminus: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, the isolated polypeptides of the present disclosure comprise a linker peptide between MM and CM1. In some embodiments, the isolated polypeptides of the present disclosure comprise a linker peptide between CM2 and AB. In some embodiments, the isolated polypeptides of the present disclosure comprise a linker peptide between MM and CM1 and a linker peptide between CM2 and AB. In some embodiments, the isolated polypeptides of the present disclosure comprise an MM that is linked to CM1 such that the isolated polypeptide in the uncleaved state comprises the following structural arrangement from the N-terminus to the C-terminus: MM-CM2-CM1-AB or AB-CM1-CM2-MM. In some embodiments, the isolated polypeptides of the present disclosure comprise a linker peptide between MM and CM2. In some embodiments, the isolated polypeptides of the present disclosure comprise a linker peptide between CM1 and AB. In some embodiments, the isolated polypeptides of the present disclosure comprise a linker peptide between MM and CM2 and a linker peptide between CM1 and AB.

[0020] In some embodiments, the isolated polypeptides of the present disclosure comprise a first linker peptide (LP1) and a second linker peptide (LP2), and wherein the isolated polypeptide in the uncleaved state has the following structural arrangement from the N-terminus to the C-terminus: MM-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM. In some embodiments, the two linker peptides are different from each other. In some embodiments, each of LP1 and LP2 is a peptide having a length of about 1 to 20 amino acids. In some embodiments, the isolated polypeptide comprises a third linker peptide (LP') between CM1 and CM2. In some embodiments, the isolated polypeptides of the present disclosure comprise an AB that comprises the light chain variable region of an antibody or an antigen-binding fragment thereof, and wherein LP2 is linked to the N-terminus of the light chain variable region of AB. In some embodiments, the isolated polypeptides of the present disclosure comprise an AB that comprises the heavy chain variable region of an antibody or an antigen-binding fragment thereof, and wherein LP2 is linked to the N-terminus of the heavy chain variable region of AB.

[0021] In some embodiments, the isolated polypeptide of the present disclosure comprises MM, wherein the amino acid sequence of MM is different from the amino acid sequence of the target and is no more than 10% identical to the amino acid sequence of the natural binding partner of AB. In some embodiments, the isolated polypeptide of the present disclosure comprises MM that does not interfere with or compete with the binding of AB to the target in the cleaved state.

[0022] In some embodiments, the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450 - 462. In some embodiments, the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450 - 462 and a heavy chain amino acid sequence comprising SEQ ID NO: 400.

[0023] In another aspect of the invention, provided herein is an activatable antibody that specifically binds to a target in an activated state, the activatable antibody comprising an antibody or an antigen - binding fragment thereof (AB) that specifically binds to the target; a masking moiety (MM) coupled to AB, wherein MM inhibits the binding of AB to the target when the activatable antibody is in the uncleaved state; and a cleavable moiety (CM) that comprises a tandem substrate according to any of the tandem substrates provided herein. In some embodiments, the activatable antibody comprises MM that has a dissociation constant for binding to AB that is greater than the dissociation constant for binding of AB to the target. In some embodiments, the activatable antibody comprises MM that is a polypeptide having a length of no more than 40 amino acids. In some embodiments, the activatable antibody comprises an antigen - binding fragment selected from the group consisting of: Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single - domain heavy - chain antibody, and single - domain light - chain antibody. In some embodiments, the activatable antibody comprises MM that has a dissociation constant for binding to AB that is greater than the dissociation constant for binding of AB to the target.

[0024] In some embodiments, the activatable antibody comprises MM that is linked to CM1 such that the activatable antibody in the uncleaved state has the following structural arrangement from the N - terminus to the C - terminus: MM - CM1 - CM2 - AB or AB - CM2 - CM1 - MM. In some embodiments, the activatable antibody comprises a linker peptide between MM and CM1. In some embodiments, the activatable antibody comprises a linker peptide between CM2 and AB. In some embodiments, the activatable antibody comprises a linker peptide between MM and CM1 and a linker peptide between CM2 and AB.

[0025] In some embodiments, the activatable antibody comprises MM, which is linked to CM1 such that the activatable antibody in the uncleaved state has the following structural arrangement from the N-terminus to the C-terminus: MM-CM2-CM1-AB or AB-CM1-CM2-MM. In some embodiments, the activatable antibody comprises a linker peptide between MM and CM2. In some embodiments, the activatable antibody comprises a linker peptide between CM1 and AB. In some embodiments, the activatable antibody comprises a linker peptide between MM and CM2 and a linker peptide between CM1 and AB.

[0026] In some embodiments, the activatable antibody comprises AB, which comprises the light chain variable region of an antibody or an antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the light chain variable region of AB. In some embodiments, the activatable antibody comprises AB, which comprises the heavy chain variable region of an antibody or an antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the heavy chain variable region of AB.

[0027] In some embodiments, the activatable antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2), and wherein the activatable antibody in the uncleaved state has the following structural arrangement from the N-terminus to the C-terminus: MM-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM-LP1-CM2-CM1-LP2-AB or AB-LP2-CM1-CM2-LP1-MM. In some embodiments, the two linker peptides are different from each other. In some embodiments, each of LP1 and LP2 is a peptide having a length of about 1 to 20 amino acids. In some embodiments, the activatable antibody comprises a third linker peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises AB, which comprises the light chain variable region of an antibody or an antigen-binding fragment thereof, and wherein LP2 is linked to the N-terminus of the light chain variable region of AB. In some embodiments, the activatable antibody comprises AB, which comprises the heavy chain variable region of an antibody or an antigen-binding fragment thereof, and wherein LP2 is linked to the N-terminus of the heavy chain variable region of AB.

[0028] In some embodiments, the activatable antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 450-462. In some embodiments, the activatable antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 450-462 and a heavy chain amino acid sequence comprising SEQ ID NO: 400.

[0029] In some embodiments, the activatable antibody comprises an MM that is different from the target and is no more than 10% identical to the amino acid sequence of the native binding partner of the AB. In some embodiments, the activatable antibody comprises an MM that does not interfere with or compete with the binding of the AB to the target in the cleaved state.

[0030] In another aspect of the invention, provided herein is a conjugated activatable antibody comprising an activatable antibody conjugated to an agent. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker.

[0031] In some embodiments, the conjugated activatable antibody comprises an agent that is a toxin or a fragment thereof. In some embodiments, the conjugated activatable antibody comprises an agent that is a microtubule inhibitor. In some embodiments, the conjugated activatable antibody comprises an agent that is a nucleic acid damaging agent. In some embodiments, the conjugated activatable antibody comprises an agent that is dolastatin or a derivative thereof, auristatin or a derivative thereof, maytansinoid or a derivative thereof, duocarmycin or a derivative thereof, calicheamicin or a derivative thereof, auristatin E or a derivative thereof, monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD) or a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the conjugated activatable antibody comprises an agent that is a detectable moiety or a diagnostic agent.

[0032] In another aspect of the invention, provided herein is a pharmaceutical composition comprising the isolated polypeptide of the present disclosure, or the activatable antibody of the present disclosure, or the conjugated activatable antibody of the present disclosure, and a carrier. In some embodiments, the pharmaceutical composition comprises an additional agent. In some embodiments, the pharmaceutical composition comprises an additional agent as a therapeutic agent.

[0033] In another aspect of the invention, provided herein is an isolated nucleic acid molecule encoding the isolated polypeptide of the present disclosure, or the activatable antibody of the present disclosure. In some embodiments, the vector comprises the isolated nucleic acid molecule of the present disclosure.

[0034] In another aspect of the invention, provided herein is a method of producing an antibody or an activatable antibody, which is carried out by culturing cells under conditions that result in the expression of the isolated polypeptide of the present disclosure, or the activatable antibody of the present disclosure. In another aspect of the invention, provided herein is a method of making an activatable antibody that binds a target in an activated state, the method comprising: culturing cells comprising a nucleic acid construct encoding the activatable antibody of the present disclosure, and recovering the activatable antibody.

[0035] In another aspect of the present invention, provided herein is a method of treating a disorder or disease, alleviating the symptoms of the disorder or disease, or delaying the progression of the disorder or disease, which comprises administering to a subject in need thereof a therapeutically effective amount of the isolated polypeptide of the present disclosure, or the activatable antibody of the present disclosure, or the conjugated activatable antibody of the present disclosure, or the pharmaceutical composition of the present disclosure. In some embodiments, the disorder or disease is cancer. In some embodiments, the method comprises administering an additional agent to the subject. In some embodiments, the method comprises administering an additional therapeutic agent to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a graph showing exemplary results of the percentage of in vivo cleavage observed after administration of the indicated activatable anti-EGFR antibody of the present disclosure to nu / nu mice. These exemplary results show that several of the tested activatable antibodies exhibit a higher degree of stability than other substrates cleavable by multiple enzymes.

[0037] Figure 2A and Figure 2B is a graph showing exemplary results of the in vitro binding of the indicated anti-EGFR activatable antibody of the present disclosure to EGFR. These exemplary results show that the MMP substrate affects the masking efficiency of the prodomain of the activatable antibody.

[0038] Figure 3A and Figure 3B is a graph showing exemplary results of the in vivo efficacy of the indicated activatable anti-EGFR antibody of the present disclosure obtained using a murine H292 xenograft model. These exemplary results show that the activatable antibodies of the present disclosure having certain MMP substrates exhibit efficacy comparable to that of unmasked anti-EGFR cetuximab in this xenograft model.

[0039] Figure 4 is a graph showing exemplary results of the percentage of activation within tumor tissue after administration of the indicated activatable anti-EGFR antibody of the present disclosure to a murine H292 xenograft model.

[0040] Figure 5 is a graph showing exemplary results of the percentage of in vivo cleavage observed after administration of the indicated activatable anti-EGFR antibody of the present disclosure having a single MMP or tandem substrate to nu / nu mice. These exemplary results show that several of the tested activatable antibodies having tandem substrates exhibit a higher degree of in vivo stability than other substrates cleavable by multiple enzymes.

[0041] Figure 6FIG. is an exemplary result showing the in vivo efficacy of the indicated activatable anti-EGFR antibody of the present disclosure obtained using a murine H292 xenograft model. These exemplary results indicate that the activatable antibody of the present disclosure having certain MMP substrates exhibits efficacy comparable to that of the unmasked anti-EGFR cetuximab antibody in this xenograft model.

[0042] Figure 7 FIG. is an exemplary result showing the percentage of the indicated activatable anti-EGFR antibody of the present disclosure that is activated within intratumoral tissue after administration to a murine H292 xenograft model.

[0043] Figure 8 FIG. is an exemplary result showing the calculated masking efficiency calculated from the in vitro binding of the indicated anti-EGFR activatable antibody of the present disclosure to EGFR. These exemplary results indicate that the tandem substrate affects the masking efficiency of the pro-domain of the activatable antibody.

[0044] Figure 9 FIG. is an exemplary result showing the percentage of the indicated activatable anti-EGFR antibody of the present disclosure that is activated after incubation with human tumor tissue. DETAILED DESCRIPTION

[0045] The present disclosure provides amino acid sequences that include at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine protease (CP). These amino acid sequences are collectively referred to herein as "tandem substrates." This term is not intended to convey any requirement regarding the orientation or other structural arrangement of the first cleavable portion (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and the at least second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine protease (CP). Thus, the term "tandem substrate" encompasses CM1-CM2 substrates having the following structural arrangement from the N-terminus to the C-terminus: CM1-CM2 or CM2-CM1. The term "tandem substrate" also encompasses substrates in which at least a portion of the CM1 sequence overlaps with at least a portion of the CM2 sequence.

[0046] In some embodiments, CM2 includes at least two substrates. In some embodiments, CM2 includes a substrate for a first serine protease and a substrate for a second serine protease. In some embodiments, CM2 includes a substrate for a serine protease and a substrate for a cysteine protease.

[0047] The CM1-CM2 substrates described herein can be used in a variety of therapeutic, diagnostic, and prophylactic indications. For example, these CM1-CM2 substrates can be used in activatable antibodies that comprise an antibody or an antigen-binding fragment (AB) thereof that contains a prodomain. The prodomain comprises at least one masking moiety (MM) that is linked to at least one antigen or epitope-binding domain of the AB such that coupling of the MM reduces the ability of the AB to bind its target.

[0048] In some embodiments, the activatable antibody comprises at least a first CM (CM1) and at least a second CM (CM2). In some embodiments, at least a portion of the CM1 substrate sequence overlaps with at least a portion of the CM2 sequence. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least one common amino acid residue. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least two common amino acid residues. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least three common amino acid residues. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share three or more common amino acid residues.

[0049] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together.

[0050] In some embodiments, CM1 and CM2 are separate polypeptides that are directly linked together, i.e., the N-terminus of one substrate is directly linked to the C-terminus of the other substrate polypeptide. In some embodiments, the N-terminus of CM1 is directly linked to the C-terminus of CM2. In some embodiments, the N-terminus of CM2 is directly linked to the C-terminus of CM1.

[0051] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together by at least one linking moiety.

[0052] In some embodiments, the first cleavable moiety CM1 and the second cleavable moiety CM2 in the activatable antibody have the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM.

[0053] In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP″) between a masking moiety (MM) and CM1. In some embodiments, the activatable antibody comprises a linker peptide (LP″′) between CM2 and AB. In some embodiments, the activatable antibody comprises a linker peptide (LP″) between MM and CM1 and a linker peptide (LP″′) between CM2 and AB. In some embodiments, the activatable antibody comprises a linker peptide (LP″) between MM and CM1 and a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2 and a linker peptide (LP″′) between CM2 and AB. In some embodiments, the activatable antibody comprises a linker peptide (LP″) between MM and CM1, a linker peptide (LP′) between CM1 and CM2, and a linker peptide (LP′") between CM2 and AB.

[0054] In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP″) between AB and CM1. In some embodiments, the activatable antibody comprises a linker peptide (LP″′) between CM2 and a masking moiety (MM). In some embodiments, the activatable antibody comprises a linker peptide (LP") between AB and CM1 and a linker peptide (LP′") between CM2 and MM. In some embodiments, the activatable antibody comprises a linker peptide (LP") between AB and CM1 and a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2 and a linker peptide (LP″′) between CM2 and MM. In some embodiments, the activatable antibody comprises a linker peptide (LP″) between AB and CM1, a linker peptide (LP′) between CM1 and CM2, and a linker peptide (LP″′) between CM2 and MM.

[0055] In some embodiments, LP′ is GG. In some embodiments, LP′ is GGSGGS (SEQ ID NO:218).

[0056] In some embodiments, CM1 is a substrate for at least one matrix metalloproteinase (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.

[0057] In some embodiments, CM1 is a substrate of MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and / or MMP19. In some embodiments, CM1 is a substrate of MMP2. In some embodiments, CM1 is a substrate of MMP9. In some embodiments, CM1 is a substrate of MMP14. In some embodiments, CM1 is a substrate of two or more MMPs. In some embodiments, CM1 is a substrate of at least MMP9 and MMP14. In some embodiments, CM1 is a substrate of at least MMP2 and MMP9. In some embodiments, CM1 is a substrate of at least MMP2 and MMP14. In some embodiments, CM1 is a substrate of three or more MMPs. In some embodiments, CM1 is a substrate of at least MMP2, MMP9, and MMP14. In some embodiments, CM1 contains two or more substrates of the same MMP. In some embodiments, CM1 contains at least two or more MMP2 substrates. In some embodiments, CM1 contains at least two or more MMP9 substrates. In some embodiments, CM1 contains at least two or more MMP14 substrates.

[0058] In some embodiments, CM1 is a substrate of MMP and contains at least the sequences ALAHGLF (SEQ ID NO:1); DLAHPLL (SEQ ID NO:2); AFRHLR (SEQ ID NO:3); PHGFFQ (SEQ ID NO:4); SVHHLI (SEQ ID NO:5); RGPKLYW (SEQ ID NO:6); RFPYGVW (SEQ ID NO:7); HVPRQV (SEQ ID NO:8); SNPFKY (SEQ ID NO:9); RFPLKV (SEQ ID NO:10); PFHLSR (SEQ ID NO:11); STVFHM (SEQ ID NO:12); MGPWFM (SEQ ID NO:13); RHLAKL (SEQ ID NO:14); PLGVRGK (SEQ ID NO:15); and QNQALRIA (SEQ ID NO:16).

[0059] In some embodiments, CM1 comprises the amino acid sequence ALAHGLF (SEQ ID NO:1). In some embodiments, CM1 comprises the amino acid sequence DLAHPLL (SEQ ID NO:2). In some embodiments, CM1 comprises the amino acid sequence AFRHLR (SEQ ID NO:3). In some embodiments, CM1 comprises the amino acid sequence PHGFFQ (SEQ ID NO:4). In some embodiments, CM1 comprises the amino acid sequence SVHHLI (SEQ ID NO:5). In some embodiments, CM1 comprises the amino acid sequence RGPKLYW (SEQ ID NO:6). In some embodiments, CM1 comprises the amino acid sequence RFPYGVW (SEQID NO:7). In some embodiments, CM1 comprises the amino acid sequence HVPRQV (SEQ ID NO:8). In some embodiments, CM1 comprises the amino acid sequence SNPFKY (SEQ ID NO:9). In some embodiments, CM1 comprises the amino acid sequence RFPLKV (SEQ ID NO:10). In some embodiments, CM1 comprises the amino acid sequence PFHLSR (SEQ ID NO:11). In some embodiments, CM1 comprises the amino acid sequence STVFHM (SEQ ID NO:12). In some embodiments, CM1 comprises the amino acid sequence MGPWFM (SEQ ID NO:13). In some embodiments, CM1 comprises the amino acid sequence RHLAKL (SEQID NO:14). In some embodiments, CM1 comprises the amino acid sequence PLGVRGK (SEQ ID NO:15). In some embodiments, CM1 comprises the amino acid sequence QNQALRIA (SEQ ID NO:16).

[0060] In some embodiments, CM1 comprises the amino acid sequence LAHGLF (SEQ ID NO:50). In some embodiments, CM1 comprises the amino acid sequence AHGLF (SEQ ID NO:51). In some embodiments, CM1 comprises the amino acid sequence ALAHGL (SEQ ID NO:52). In some embodiments, CM1 comprises the amino acid sequence LAHGL (SEQ ID NO:53). In some embodiments, CM1 comprises the amino acid sequence AHGL (SEQ ID NO:54). In some embodiments, CM1 comprises the amino acid sequence ALAHG (SEQ ID NO:55). In some embodiments, CM1 comprises the amino acid sequence LAHG (SEQ ID NO:56). In some embodiments, CM1 comprises the amino acid sequence AHG.

[0061] In some embodiments, CM1 comprises the amino acid sequence VPRQV (SEQ ID NO:60). In some embodiments, CM1 comprises the amino acid sequence PRQV (SEQ ID NO:61). In some embodiments, CM1 comprises the amino acid sequence HVPRQ (SEQ ID NO:62). In some embodiments, CM1 comprises the amino acid sequence VPRQ (SEQ ID NO:63). In some embodiments, CM1 comprises the amino acid sequence PRQ.

[0062] In some embodiments, CM2 is a substrate for at least one serine protease (SP). In some embodiments, the SP is selected from urokinase-type plasminogen activator (uPA, also known as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1), neutrophil elastase (e.g., human neutrophil elastase), and combinations thereof. By way of non-limiting example, examples of other SPs that cleave CM2 described herein include activated protein C; cathepsin A; cathepsin G; chymotrypsin; coagulation factor proteases such as FVIIa, FIXa, FXa, FXIa, FXIIa; elastase; granzyme B; Guanidinobenzoatase; HtrA1; lactoferrin; Marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine protease (TTSP) such as DESC1, DPP-4, FAP, Hepsin, matriptase-2, TMPRSS2, TMPRSS3, and / or TMPRSS4.

[0063] For example, a suitable CM2 is cleaved by at least one serine protease and may comprise at least one sequence selected from the following amino acid sequences: LSGRSDNH (SEQ ID NO:20), LSGRSDN (SEQ ID NO:70), LSGRSD (SEQ ID NO:71), LSGRS (SEQ ID NO:72), LSGR (SEQ ID NO:73), SGRSDN (SEQ ID NO:74), SGRSD (SEQ ID NO:75), SGRS (SEQ ID NO:76), SGR, LSGRSGNH (SEQ ID NO:78), LSGRSGN (SEQ ID NO:79), LSGRSG (SEQ ID NO:80), SGRSGNH (SEQ ID NO:81), SGRSGN (SEQ ID NO:82), SGRSG (SEQ ID NO:83), LSGRSDNI (SEQ ID NO:84), SGRSDNI (SEQ ID NO:85), LSGRSDYH (SEQ ID NO:86), LSGRSDY (SEQ ID NO:87), SGRSDYH (SEQ ID NO:88), SGRSDY (SEQ ID NO:89), LSGRSDNP (SEQ ID NO:90), SGRSDNP (SEQ ID NO:91), LSGRSDTH (SEQ ID NO:92), LSGRSDT (SEQ ID NO:93), SGRSDTH (SEQ ID NO:94), SGRSDT (SEQ ID NO:95), LSGRSDQH (SEQ ID NO:96), LSGRSDQ (SEQ ID NO:97), SGRSDQH (SEQ ID NO:98), SGRSDQ (SEQ ID NO:99), LSGRSDIH (SEQ ID NO:100), LSGRSDI (SEQ ID NO:101), SGRSDIH (SEQ ID NO:102), SGRSDI (SEQ ID NO:103), LSGRSDDH (SEQ ID NO:104), LSGRSDD (SEQ ID NO:105), SGRSDDH (SEQ ID NO:106), SGRSDD (SEQ ID NO:107), LSGRSANI (SEQ ID NO:108), LSGRSAN (SEQ ID NO:109), LSGRSA (SEQ ID NO:110), SGRSANI (SEQ ID NO:111), SGRSAN (SEQ ID NO:112), SGRSA (SEQID NO: 113), LSGRSANP (SEQ ID NO: 114), and SGRSANP (SEQ ID NO: 115).

[0064] In some embodiments, the suitable CM2 is cleaved by at least one serine protease and may comprise at least one sequence selected from the following amino acid sequences: TARGPSFK (SEQ ID NO: 120), ARGPSFK (SEQ ID NO: 121), TARGPSF (SEQ ID NO: 122), TARGPS (SEQ ID NO: 123), TARGP (SEQ ID NO: 124), TARG (SEQ ID NO: 125), ARGPSF (SEQ ID NO: 126), ARGPS (SEQ ID NO: 127), ARGP (SEQ ID NO: 128), and ARG.

[0065] In some embodiments, the suitable CM2 is cleaved by at least one serine protease and may comprise at least one sequence selected from the following amino acid sequences: APRSF (SEQ ID NO: 130), APRS (SEQ ID NO: 131), and PRSF (SEQ ID NO: 132).

[0066] In some embodiments, the suitable CM2 is cleaved by at least one serine protease and may comprise at least one sequence selected from the following amino acid sequences: GLPTFVHL (SEQ ID NO: 135), GLPTFVH (SEQ ID NO: 136), GLPTFV (SEQ ID NO: 137), LPTFVHL (SEQ ID NO: 138), LPTFVH (SEQ ID NO: 139), and LPTFV (SEQ ID NO: 140).

[0067] In some embodiments, CM2 is a substrate for at least one cysteine protease (CP). In some embodiments, the CP is papain. In some embodiments, the suitable CM2 is cleaved by at least one cysteine protease and may comprise at least one sequence selected from the following amino acid sequences: AAN, SAN, and GPTN (SEQ ID NO: 152).

[0068] In some embodiments, the CM1-CM2 substrate comprises an amino acid sequence selected from the group consisting of: LSGRSALAHGLF (SEQ ID NO:25), ALAHGLFSGRSAN (SEQ ID NO:26), HVPRQVLSGRS (SEQ ID NO:27), HVPRQVLSGRSAN (SEQ ID NO:28), TARGPALAHGLF (SEQ ID NO:29), TARGPVPRQV (SEQ ID NO:30), APRSALAHGLF (SEQ ID NO:31), ALAHGLFAPRSF (SEQ ID NO:32), HVPRQVAPRSF (SEQ ID NO:33), ALAHGLPTFVHL (SEQ ID NO:34), GLPTFVHLPRQV (SEQ ID NO:35), AANALAHGLF (SEQ ID NO:36), GPTNALAHGLF (SEQ ID NO:37), ISSGLLSGRSNI (SEQ ID NO:38), AVGLLAPPGGLSGRSNI (SEQ ID NO:39), ISSGLLSGRSNIGS (SEQ ID NO:40), AVGLLAPPGGLSGRSNIGS (SEQ ID NO:41), ISSGLLSGRSNIG (SEQ ID NO:42), and AVGLLAPPGGLSGRSNIG (SEQ ID NO:43).

[0069] In some embodiments, the activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM.

[0070] In some embodiments, the activatable antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2), and the antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide having a length of about 1 to 20 amino acids. In some embodiments, the two linker peptides need not be identical to each other.

[0071] In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP1) between a masking moiety (MM) and CM1. In some embodiments, the activatable antibody comprises a linker peptide (LP2) between CM2 and AB. In some embodiments, the activatable antibody comprises a linker peptide (LP1) between MM and CM1 and a linker peptide (LP2) between CM2 and AB. In some embodiments, the activatable antibody comprises a linker peptide (LP1) between MM and CM1 and a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2 and a linker peptide (LP2) between CM2 and AB. In some embodiments, the activatable antibody comprises a linker peptide (LP1) between MM and CM1, a linker peptide (LP′) between CM1 and CM2, and a linker peptide (LP2) between CM2 and AB.

[0072] In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP1) between AB and CM1. In some embodiments, the activatable antibody comprises a linker peptide (LP2) between CM2 and a masking moiety (MM). In some embodiments, the activatable antibody comprises a linker peptide (LP1) between AB and CM1 and a linker peptide (LP2) between CM2 and MM. In some embodiments, the activatable antibody comprises a linker peptide (LP1) between AB and CM1 and a linker peptide (LP′) between CM1 and CM2. In some embodiments, the activatable antibody comprises a linker peptide (LP′) between CM1 and CM2 and a linker peptide (LP2) between CM2 and MM. In some embodiments, the activatable antibody comprises a linker peptide (LP1) between AB and CM1, a linker peptide (LP′) between CM1 and CM2, and a linker peptide (LP2) between CM2 and MM.

[0073] In some embodiments, LP′ is GG. In some embodiments, LP′ is GGSGGS (SEQ ID NO:218).

[0074] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO:200) and (GGGS) n (SEQ ID NO:201), where n is an integer of at least 1.

[0075] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: GGSG (SEQ ID NO:202), GGSGG (SEQ ID NO:203), GSGSG (SEQ ID NO:204), GSGGG (SEQ ID NO:205), GGGSG (SEQ ID NO:206), and GSSSG (SEQ ID NO:207).

[0076] In some embodiments, LP1 comprises the amino acid sequences GSSGGSGGSGGSG (SEQ ID NO:208), GSSGGSGGSGG (SEQ ID NO:209), GSSGGSGGSGGS (SEQ ID NO:210), GSSGGSGGSGGSGGGS (SEQ ID NO:211), GSSGGSGGSG (SEQ ID NO:212), or GSSGGSGGSGS (SEQ ID NO:213), GGGSSGGS (SEQ ID NO:214), and GGGS (SEQ ID NO:215).

[0077] In some embodiments, LP2 comprises the amino acid sequences GSS, GGS, GGGS (SEQ ID NO:215), GSSGT (SEQ ID NO:216), or GSSG (SEQ ID NO:217). In some embodiments, LP2 comprises the amino acid sequence GGS. In some embodiments, LP2 comprises the amino acid sequence GGGS (SEQ ID NO:215).

[0078] In some embodiments, CM1 is a substrate of MMP and comprises at least the sequences ALAHGLF (SEQ ID NO:1); DLAHPLL (SEQ ID NO:2); AFRHLR (SEQ ID NO:3); PHGFFQ (SEQ ID NO:4); SVHHLI (SEQ ID NO:5); RGPKLYW (SEQ ID NO:6); RFPYGVW (SEQ ID NO:7); HVPRQV (SEQ ID NO:8); SNPFKY (SEQ ID NO:9); RFPLKV (SEQ ID NO:10); PFHLSR (SEQ ID NO:11); STVFHM (SEQ ID NO:12); MGPWFM (SEQ ID NO:13); RHLAKL (SEQ ID NO:14); PLGVRGK (SEQ ID NO:15), and QNQALRIA (SEQ ID NO:16).

[0079] In some embodiments, the isolated polypeptide of the present disclosure or the activatable antibody of the present disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 comprises an amino acid sequence selected from the group consisting of: LSGRSALAHGLF (SEQ ID NO: 25), ALAHGLFSGRSAN (SEQ ID NO: 26), HVPRQVLSGRS (SEQ ID NO: 27), HVPRQVLSGRSAN (SEQ ID NO: 28), TARGPALAHGLF (SEQ ID NO: 29), TARGPVPRQV (SEQ ID NO: 30), APRSALAHGLF (SEQ ID NO: 31), ALAHGLFAPRSF (SEQ ID NO: 32), HVPRQVAPRSF (SEQ ID NO: 33), ALAHGLPTFVHL (SEQ ID NO: 34), GLPTFVHLPRQV (SEQ ID NO: 35), AANALAHGLF (SEQ ID NO: 36), GPTNALAHGLF (SEQ ID NO: 37), ISSGLLSGRSNI (SEQ ID NO: 38), and AVGLLAPPGGLSGRSNI (SEQ ID NO: 39), and the LP2 linker comprises GGGS (SEQ ID NO: 215).

[0080] In some embodiments, the isolated polypeptide of the present disclosure or the activatable antibody of the present disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 comprises an amino acid sequence selected from the group consisting of: LSGRSALAHGLF (SEQ ID NO:25), ALAHGLFSGRSAN (SEQ ID NO:26), HVPRQVLSGRS (SEQ ID NO:27), HVPRQVLSGRSAN (SEQ ID NO:28), TARGPALAHGLF (SEQ ID NO:29), TARGPVPRQV (SEQ ID NO:30), APRSALAHGLF (SEQ ID NO:31), ALAHGLFAPRSF (SEQ ID NO:32), HVPRQVAPRSF (SEQ ID NO:33), ALAHGLPTFVHL (SEQ ID NO:34), GLPTFVHLPRQV (SEQ ID NO:35), AANALAHGLF (SEQ ID NO:36), GPTNALAHGLF (SEQ ID NO:37), ISSGLLSGRSNI (SEQ ID NO:38), and AVGLLAPPGGLSGRSNI (SEQ ID NO:39), and the LP2 linker comprises GGS.

[0081] In some embodiments, the isolated polypeptide of the present disclosure or the activatable antibody of the present disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 and LP2 comprise amino acid sequences selected from the group consisting of: LSGRSALAHGLFGGGS (SEQ ID NO: 226), ALAHGLFSGRSANGGGS (SEQ ID NO: 227), HVPRQVLSGRSGGGS (SEQ ID NO: 228), HVPRQVLSGRSANGGGS (SEQ ID NO: 229), TARGPALAHGLFGGGS (SEQ ID NO: 230), TARGPVPRQVGGGS (SEQ ID NO: 231), APRSALAHGLFGGGS (SEQ ID NO: 232), ALAHGLFAPRSFGGGS (SEQ ID NO: 233), HVPRQVAPRSFGGGS (SEQ ID NO: 234), ALAHGLPTFVHLGGGS (SEQ ID NO: 235), GLPTFVHLPRQVGGGS (SEQ ID NO: 236), AANALAHGLFGGGS (SEQ ID NO: 237), GPTNALAHGLFGGGS (SEQ ID NO: 238), ISSGLLSGRSNIGGGS (SEQ ID NO: 239), and AVGLLAPPGGLSGRSNIGGGS (SEQ ID NO: 240).

[0082] In some embodiments, the isolated polypeptide of the present disclosure or the activatable antibody of the present disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 and LP2 comprise amino acid sequences selected from the group consisting of: LSGRSALAHGLFGGS (SEQ ID NO:241), ALAHGLFSGRSANGGS (SEQ ID NO:242), HVPRQVLSGRSGGS (SEQ ID NO:243), HVPRQVLSGRSANGGS (SEQ ID NO:244), TARGPALAHGLFGGS (SEQ ID NO:245), TARGPVPRQVGGS (SEQ ID NO:246), APRSALAHGLFGGS (SEQ ID NO:247), ALAHGLFAPRSFGGS (SEQ ID NO:248), HVPRQVAPRSFGGS (SEQ ID NO:249), ALAHGLPTFVHLGGS (SEQ ID NO:250), GLPTFVHLPRQVGGS (SEQ ID NO:251), AANALAHGLFGGS (SEQ ID NO:252), GPTNALAHGLFGGS (SEQ ID NO:253), ISSGLLSGRSNIGGS (SEQ ID NO:254), and AVGLLAPPGGLSGRSNIGGS (SEQ ID NO:255).

[0083] In some embodiments, the dissociation constant of the AB binding to the target is about 100 nM or lower.

[0084] In some embodiments, the activatable antibody comprises an antibody or an antigen-binding fragment (AB) thereof that specifically binds to a target. In some embodiments, the AB is a full-length antibody. In some embodiments, the AB is an immunologically active fragment. In some embodiments, the AB is an antigen-binding fragment. In some embodiments, the AB is a monoclonal antibody, a domain antibody, a single-chain, a Fab fragment, an F(ab')2 fragment, an scFv, an scab, a dAb, a single-domain heavy-chain antibody, or a single-domain light-chain antibody. In some embodiments, such an AB is a murine antibody, other rodent antibody, chimeric antibody, humanized antibody, or fully human monoclonal antibody.

[0085] In some embodiments, the MMP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease cleaves CM1 in the antibody. In some embodiments, the SP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the SP protease cleaves the CM2 substrate in the antibody. In some embodiments, the CP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the CP protease cleaves the CM2 substrate in the antibody. In some embodiments, the MMP protease and / or the SP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease and / or the SP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and the SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, at least one of the MMP protease and the SP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and / or the CP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease and / or the CP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and the CP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, at least one of the MMP protease and the CP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and / or the CP protease and / or the SP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease and / or the CP protease and / or the SP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and the CP protease and the SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, at least one of the MMP protease, the SP protease, and the CP protease cleaves the CM1-CM2 substrate in the antibody.

[0086] In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide having a length of at most 15 amino acids.

[0087] In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any polypeptide sequence that is naturally cleaved by the same MMP protease (e.g., any animal polypeptide sequence). In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 90% or more identical to any polypeptide sequence (e.g., any animal polypeptide sequence) that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease.

[0088] In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP, and comprises a polypeptide sequence that is substantially different from any polypeptide sequence (e.g., any animal polypeptide sequence) that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP, and comprises a polypeptide sequence that is substantially different from any mammalian polypeptide sequence that is naturally cleaved by the same SP and / or CP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP, and comprises a polypeptide sequence that is substantially different from any human polypeptide sequence that is naturally cleaved by the same SP and / or CP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP, and comprises a polypeptide sequence that is not more than 90% or more identical to any polypeptide sequence (e.g., any animal polypeptide sequence) that is naturally cleaved by the same SP and / or CP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP, and comprises a polypeptide sequence that is not more than 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same SP and / or CP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP, and comprises a polypeptide sequence that is not more than 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same SP and / or CP protease.

[0089] In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 29, 31, 32, 34, 36, and 37. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 29, 31, 36, and 37. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, and 34. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 33. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35.

[0090] In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 29, 31, 32, 34, 36, and 37; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 29, 31, 36, and 37; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, and 34; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 33; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target.

[0091] In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43; and an anti-EGFR antibody comprising the amino acid sequence of the anti-EGFR antibody disclosed herein. In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43; and an antibody having a light chain comprising the amino acid sequence SEQ ID NO: 401 and a heavy chain comprising the amino acid sequence SEQ ID NO: 400.

[0092] In some embodiments, CM1-CM2 is included in an activatable antibody having a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450-462 and a heavy chain amino acid sequence of SEQ ID NO: 400.

[0093] In some embodiments, the isolated polypeptide of the present disclosure comprises a CM1 substrate that is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any polypeptide sequence (e.g., any animal polypeptide sequence) that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence that is substantially different from any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 90% or more identical to any polypeptide sequence (e.g., any animal polypeptide sequence) that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence that is not more than 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence selected from the group consisting of: ALAHGLF (SEQ ID NO: 1), DLAHPLL (SEQ ID NO: 2), RGPKLYW (SEQ ID NO: 6), RFPYGVW (SEQ ID NO: 7), and QNQALRIA (SEQ ID NO: 16).

[0094] In some embodiments, the activatable antibodies of the present disclosure comprise a CM1 substrate comprising an amino acid sequence selected from the group consisting of: ALAHGLF (SEQ ID NO:1), DLAHPLL (SEQ ID NO:2), RGPKLYW (SEQ ID NO:6), RFPYGVW (SEQ ID NO:7), and QNQALRIA (SEQ ID NO:16); and an antibody or an antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind to its target.

[0095] In some embodiments, the isolated polypeptide of the present disclosure comprises a CM2 substrate that is a substrate for at least one SP protease and comprises a polypeptide sequence that is substantially different from any polypeptide sequence that is naturally cleaved by the same SP protease (e.g., any animal polypeptide sequence). In some embodiments, the CM2 substrate sequence is a substrate for at least one SP protease and comprises a polypeptide sequence that is substantially different from any mammalian polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence is a substrate for at least one SP protease and comprises a polypeptide sequence that is substantially different from any human polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence is a substrate for at least one SP protease and comprises a polypeptide sequence that is not more than 90% or more identical to any polypeptide sequence that is naturally cleaved by the same SP protease (e.g., any animal polypeptide sequence). In some embodiments, the CM2 substrate sequence is a substrate for at least one SP protease and comprises a polypeptide sequence that is not more than 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence is a substrate for at least one SP protease and comprises a polypeptide sequence that is not more than 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence is a substrate for at least one SP protease and comprises a polypeptide sequence selected from the group consisting of: APRSF (SEQ ID NO:130) and GLPTFVHL (SEQ ID NO:135).

[0096] In some embodiments, the activatable antibodies of the present disclosure comprise a CM2 substrate comprising an amino acid sequence selected from the group consisting of APRSF (SEQ ID NO:130) and GLPTFVHL (SEQ ID NO:135); and an antibody or an antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind to its target.

[0097] In some embodiments, the activatable antibody of the present disclosure comprises a substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16; and an antibody that binds a target or an antigen-binding fragment (AB) thereof and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target.

[0098] In some embodiments, the CM1-CM2, CM1 or CM2 substrate is also a substrate for at least one additional protease.

[0099] In some embodiments, the at least one additional protease is a MMP protease different from the MMP protease that cleaves CM1. In some embodiments, the at least one additional protease is a MMP protease selected from the group consisting of: MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.

[0100] In some embodiments, the at least one additional protease is a SP protease different from the SP protease that cleaves CM2. In some embodiments, the at least one additional SP protease is selected from the group consisting of: uPA; proteolytic enzyme; activated protein C; cathepsin A; cathepsin G; chymotrypsin; coagulation factor protease, such as FVIIa, FIXa, FXa, FXIa, FXIIa; elastase; granzyme B; guanidobenzoylase; HtrA1; human neutrophil elastase; lactoferrin; Marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine protease (TTSP), such as DESC1, DPP-4, FAP, Hepsin, proteolytic enzyme-2, TMPRSS2, TMPRSS3 and TMPRSS4.

[0101] In some embodiments, the at least one additional protease is selected from the group consisting of those proteases shown in Table 1.

[0102] Table 1: Exemplary proteases and / or enzymes

[0103]

[0104]

[0105] The present disclosure also provides an antibody that comprises at least a first CM1 and at least a second CM2 and is conjugated to an agent. In some embodiments, each of the first CM1 and the second CM2 is a polypeptide no more than 15 amino acids in length. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM-CM1-CM2-AB-agent, agent-AB-CM2-CM1-MM, MM-CM2-CM1-AB-agent, or agent-AB-CM1-CM2-MM. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: agent-MM-CM1-CM2-AB, AB-CM2-CM1-MM-agent, agent-MM-CM2-CM1-AB, or AB-CM1-CM2-MM-agent. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: agent-MM-CM1-CM2-AB-agent, agent-AB-CM2-CM1-MM-agent, agent-MM-CM2-CM1-AB-agent, or agent-AB-CM1-CM2-MM-agent.

[0106] In some embodiments, the activatable antibody is a conjugated activatable antibody that comprises a masking moiety (MM), a first linker peptide (LP1), and a second linker peptide (LP2), and the antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM1-LP1-CM1-CM2-LP2-AB-agent, agent-AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB-agent, or agent-AB-LP2-CM1-CM2-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide about 1 to 20 amino acids in length. In some embodiments, the two linker peptides need not be identical to each other.

[0107] In some embodiments, the activatable antibody is a conjugated activatable antibody that comprises a masking moiety (MM), a first linker peptide (LP1), and a second linker peptide (LP2), and the antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: agent-MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM-agent, agent-MM1-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM-agent. In some embodiments, each of LP1 and LP2 is a peptide about 1 to 20 amino acids in length. In some embodiments, the two linker peptides need not be identical to each other.

[0108] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first linker peptide (LP1), and a second linker peptide (LP2), and the antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: agent-MM1-LP1-CM1-CM2-LP2-AB-agent, agent-AB-LP2-CM2-CM1-LP1-MM-agent, agent-MM1-LP1-CM2-CM1-LP2-AB-agent, or agent-AB-LP2-CM1-CM2-LP1-MM-agent. In some embodiments, each of LP1 and LP2 is a peptide having a length of about 1 to 20 amino acids. In some embodiments, the two linker peptides need not be identical to each other.

[0109] In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP′) between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between the masking moiety (MM) and CM1. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP2) between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between MM and CM1 and a linker peptide (LP2) between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between MM and CM1 and a linker peptide (LP′) between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP′) between CM1 and CM2 and a linker peptide (LP2) between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between MM and CM1, a linker peptide (LP′) between CM1 and CM2, and a linker peptide (LP2) between CM2 and AB.

[0110] In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP′) between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between AB and CM1. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP2) between CM2 and a masking moiety (MM). In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between AB and CM1 and a linker peptide (LP2) between CM2 and MM. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between AB and CM1 and a linker peptide (LP′) between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP′) between CM1 and CM2 and a linker peptide (LP2) between CM2 and MM. In some embodiments, the conjugated activatable antibody comprises a linker peptide (LP1) between AB and CM1, a linker peptide (LP′) between CM1 and CM2, and a linker peptide (LP2) between CM2 and MM.

[0111] In some embodiments, LP′ is GG. In some embodiments, LP′ is GGSGGS (SEQ ID NO:218).

[0112] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO:381) and (GGGS) n (SEQ ID NO:382), where n is an integer of at least 1.

[0113] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: GGSG (SEQ ID NO:202), GGSGG (SEQ ID NO:203), GSGSG (SEQ ID NO:204), GSGGG (SEQ IDNO:205), GGGSG (SEQ ID NO:206), and GSSSG (SEQ ID NO:207).

[0114] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO:208), GSSGGSGGSGG (SEQ ID NO:209), GSSGGSGGSGGS (SEQ ID NO:210), GSSGGSGGSGGSGGGS (SEQ ID NO:211), GSSGGSGGSG (SEQ ID NO:212), or GSSGGSGGSGS (SEQ ID NO:213) and GGGSSGGS (SEQ ID NO:214).

[0115] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO:215), GSSGT (SEQ ID NO:216), or GSSG (SEQ ID NO:217).

[0116] In some embodiments, the CM1-CM2 substrate is linked to or otherwise attached to an antibody. For example, CM1-CM2 is used to link one or more agents to an antibody or an antigen-binding fragment (AB) thereof that binds to a given target such that CM1-CM2 is cleaved upon exposure to MMP and / or SP and / or CP, and the agent is released from the AB. Exemplary targets include, but are not limited to, the targets shown in Table 2. Exemplary ABs include, but are not limited to, the antibodies shown in Table 3.

[0117] In some embodiments, the dissociation constant of the AB binding to the target is about 100 nM or lower.

[0118] In some embodiments, the antibody comprises an antibody or an antigen-binding fragment thereof that specifically binds to a target. In some embodiments, the antibody or an immunoreactive fragment thereof that binds to a target is a monoclonal antibody, a domain antibody, a single-chain, a Fab fragment, an F(ab')2 fragment, an scFv, an scab, a dAb, a single-domain heavy-chain antibody, or a single-domain light-chain antibody. In some embodiments, such an antibody or an immunoreactive fragment thereof that binds to a target is a murine antibody, an antibody of other rodents, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0119] In some embodiments, the dissociation constant of MM binding to the AB does not exceed the dissociation constant of the AB binding to the target.

[0120] In some embodiments, MM in the cleaved state does not interfere with or compete for the binding of the AB to the target.

[0121] In some embodiments, MM is a polypeptide having a length of about 2 to 40 amino acids. For example, MM is a polypeptide having a length of at most about 40 amino acids.

[0122] In some embodiments, the MM polypeptide sequence is different from the sequence of any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is not more than 50% identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is not more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of AB.

[0123] In some embodiments, the agent conjugated to AB or the activatable antibody of AB is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. As used herein, a fragment of a toxin is a fragment that retains the toxin activity. In some embodiments, the agent is conjugated to AB through a cleavable linker. In some embodiments, the agent is conjugated to AB through a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to AB through a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agents. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is rhizoxin or a derivative thereof. In some embodiments, the agent is rhizoxin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansine alkaloid or a maytansine alkaloid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine (pyrrolobenzodiazepine). In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0124] In some embodiments, the agent is an anti-inflammatory agent.

[0125] In some embodiments, the antibody and / or the activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0126] In some embodiments, the conjugated antibody and / or conjugated activatable antibody comprise a detectable label. In some embodiments, the detectable label includes an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent includes a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the contrast agent includes iodine, gadolinium, or iron oxide. In some embodiments, the enzyme includes horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label includes yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or an europium derivative. In some embodiments, the luminescent label includes an N-methylacrydinium derivative. In some embodiments, the label includes Alexa label, such as Alex 680 or Alexa 750. In some embodiments, the ligand-based label includes biotin, avidin, streptavidin, or one or more haptens.

[0127] In some embodiments, the AB of the antibody and / or activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0128] In some embodiments, the antibody and / or conjugated antibody is monospecific. In some embodiments, the antibody and / or conjugated antibody is multispecific, referred to herein as a multispecific antibody and / or conjugated multispecific antibody. In some embodiments, the multispecific antibody and / or conjugated multispecific antibody is bispecific or trifunctional. In some embodiments, the antibody and / or conjugated antibody is formulated as part of a pro-bispecific T cell engaging (pro-BITE) molecule. In some embodiments, the antibody and / or conjugated antibody is formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor or other immune effector cell (such as a CAR modified NK cell). In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (CAR) modified T cell. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (CAR) modified NK cell.

[0129] In some embodiments, the activatable antibody and / or conjugated activatable antibody is monospecific. In some embodiments, the activatable antibody and / or conjugated activatable antibody is multispecific, referred to herein as a multispecific activatable antibody and / or conjugated multispecific activatable antibody. Unless otherwise indicated, as used herein, terms such as "activatable antibody" and all of its grammatical variations are intended to cover, but are not limited to, embodiments in which the activatable antibody is a multispecific activatable antibody of the present disclosure. Unless otherwise indicated, as used herein, terms such as "conjugated activatable antibody" and all of its grammatical variations are intended to cover, but are not limited to, embodiments in which the conjugated activatable antibody is a conjugated multispecific activatable antibody of the present disclosure. In some embodiments, the multispecific activatable antibody and / or conjugated multispecific activatable antibody is bispecific or trifunctional. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor.

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

[0131] In some embodiments, the activatable antibody is a multispecific activatable antibody. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize two or more different antigens or epitopes and comprise at least one masking moiety (MM) that is linked to at least one antigen or epitope binding domain of the multispecific antibody such that the coupling of the MM reduces the ability of the antigen or epitope binding domain to bind its target. In some embodiments, the MM is coupled to the antigen or epitope binding domain of the multispecific antibody via a CM1-CM2 substrate that serves as a substrate for at least one MMP protease and at least one SP protease. The activatable multispecific antibodies provided herein are stable in circulation, are activated at the intended therapeutic and / or diagnostic site, but are not activated in normal tissue (i.e., healthy tissue), and upon activation exhibit binding to the target that is at least comparable to that of the corresponding unmodified multispecific antibody.

[0132] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to epidermal growth factor receptor (EGFR) and contains a combination of VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence is selected from VH CDR1 sequences that at least comprise the amino acid sequence NYGVH (SEQ ID NO: 220); VH CDR2 sequences that at least comprise the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); VH CDR3 sequences that at least comprise the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); and combinations thereof.

[0133] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains a combination of VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence is selected from VL CDR1 sequences that at least comprise the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); VL CDR2 sequences that at least comprise the amino acid sequence KYASESIS (SEQ ID NO: 224); and VL CDR3 sequences that at least comprise the amino acid sequence QQNNNWPTT (SEQ ID NO: 225); and combinations thereof.

[0134] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, wherein at least one of the VH CDR1, VH CDR2, and VH CDR3 sequences is selected from VH CDR1 sequences comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 220); VH CDR2 sequences comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); VH CDR3 sequences comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); and combinations thereof.

[0135] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, wherein at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences is selected from VL CDR1 sequences comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); VL CDR2 sequences comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO: 224); and VL CDR3 sequences comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 225); and combinations thereof.

[0136] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains VH CDR1 sequence, VH CDR2 sequence, VH CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, wherein the VH CDR1 sequence comprises at least the amino acid sequence NYGVH (SEQ ID NO: 220); the VH CDR2 sequence comprises at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); the VH CDR3 sequence comprises at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); the VL CDR1 sequence comprises at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); the VL CDR2 sequence comprises at least the amino acid sequence KYASESIS (SEQ ID NO: 224); and the VL CDR3 sequence comprises at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 225).

[0137] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 220); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO: 224); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 225).

[0138] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43; and an anti-Jagged antibody comprising the amino acid sequence of the anti-Jagged antibody disclosed herein. In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43; and an antibody having a light chain comprising the amino acid sequence SEQ ID NO: 401 and a heavy chain comprising the amino acid sequence SEQ ID NO: 400.

[0139] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least the heavy chain amino acid sequence of SEQ ID NO: 400 and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 403-423 and 450-462.

[0140] In some embodiments, the activatable antibody further comprises an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agents. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is rhizoxin or a derivative thereof. In some embodiments, the agent is rhizoxin E or a derivative thereof. In some embodiments, the agent is monomethyl rhizoxin E (MMAE). In some embodiments, the agent is monomethyl rhizoxin D (MMAD). In some embodiments, the agent is maytansine or a maytansine derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamycin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine 。In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0141] In some embodiments, the agent is an anti-inflammatory agent.

[0142] In some embodiments, the activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0143] In some embodiments, the conjugated antibody comprises a detectable label. In some embodiments, the detectable label includes an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent includes a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the contrast agent includes iodine, gadolinium, or iron oxide. In some embodiments, the enzyme includes horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label includes yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or an europium derivative. In some embodiments, the luminescent label includes an N-methyl acridine derivative. In some embodiments, the label includes Alexa label, such as Alex 680 or Alexa 750. In some embodiments, the ligand-based label includes biotin, avidin, streptavidin, or one or more haptens.

[0144] In some embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of the signal peptide. In some embodiments, the spacer is directly linked to the MM of the activatable antibody. In some embodiments, the spacer is arranged in a structure from the N-terminus to the C-terminus as spacer-MM-CM1-CM2 substrate-AB and is directly linked to the MM of the activatable antibody. Examples of spacers directly linked to the N-terminus of the MM of the activatable antibody are amino acid sequences selected from the group consisting of: QGQSGQ (SEQ ID NO:153), GQSGQ (SEQ ID NO:154), QSGQ (SEQ ID NO:155), SGQ, GQ, and Q. In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO:153). In some embodiments, the spacer comprises at least the amino acid sequence GQSGQ (SEQ ID NO:154). In some embodiments, the spacer comprises at least the amino acid sequence QSGQ (SEQ ID NO:155). In some embodiments, the spacer comprises at least the amino acid sequence SGQ. In some embodiments, the spacer comprises at least the amino acid sequence GQ. In some embodiments, the spacer comprises at least the amino acid sequence Q.

[0145] In some embodiments, the AB of the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0146] In some embodiments, the serum half-life of the activatable antibody is longer than the serum half-life of the corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 15 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 11 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 9 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 7 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 6 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 5 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 4 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 3 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 2 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 24 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 20 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 18 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 16 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 14 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 6 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 4 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 3 hours.

[0147] The present disclosure also provides compositions and methods comprising activatable antibodies, which comprise an antibody or antibody fragment (AB) that specifically binds to a given target, wherein the AB is conjugated to a masking moiety (MM) that reduces the ability of the AB to bind to its target. In some embodiments, the activatable antibody further comprises a CM1-CM2 substrate that is a substrate for at least one MMP and at least one SP. The compositions and methods provided herein enable attachment of one or more agents to one or more cysteine residues in the AB without compromising the activity of the activatable antibody (e.g., masking, activation, or binding activity). In some embodiments, the compositions and methods provided herein enable attachment of one or more agents to one or more cysteine residues in the AB without reducing or otherwise interfering with one or more disulfide bonds in the MM. The compositions and methods provided herein produce an activatable antibody conjugated to one or more agents (e.g., any of a variety of therapeutic, diagnostic, and / or prophylactic agents), e.g., in some embodiments, without any of the one or more agents being conjugated to the MM of the activatable antibody. The compositions and methods provided herein produce a conjugated activatable antibody in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in the uncleaved state. The compositions and methods provided herein produce a conjugated activatable antibody in which the activatable antibody is still activated, i.e., cleaved, in the presence of an MMP that cleaves the CM1-CM2 substrate.

[0148] The activatable antibody has at least one conjugation point for an agent, but in the methods and compositions provided herein, fewer than all possible conjugation points are available for conjugation to the agent. In some embodiments, one or more conjugation points are sulfur atoms involved in disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain thio bonds rather than interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms of cysteine or other amino acid residues containing sulfur atoms. Such residues may be naturally present in the antibody structure or may be incorporated into the antibody by site-directed mutagenesis, chemical conversion, or misincorporation of unnatural amino acids.

[0149] Also provided are methods of preparing conjugates of activatable antibodies having one or more interchain disulfide bonds in AB and one or more intrachain disulfide bonds in MM, and a drug that reacts with free thiols. The methods generally include partially reducing the interchain disulfide bonds in the activatable antibody with a reducing agent such as TCEP; and conjugating the drug that reacts with free thiols to the partially reduced activatable antibody. As used herein, the term partial reduction refers to a situation in which the activatable antibody is contacted with a reducing agent and less than all of the disulfide bonds (e.g., less than all possible conjugation sites) are reduced. In some embodiments, less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5% of all possible conjugation sites are reduced.

[0150] In some embodiments, a method is provided for reducing and conjugating an agent (e.g., a drug) to an activatable antibody such that the agent has a selective disposition. The method generally includes partially reducing the activatable antibody with a reducing agent such that any conjugation sites in the masking portion or other non-AB portion of the activatable antibody are not reduced, and conjugating the agent to the interchain thiols in the AB. One or more conjugation sites are selected to allow for the desired agent disposition to permit conjugation to occur at the desired site. The reducing agent is, for example, TCEP. The reduction reaction conditions (e.g., such as the ratio of the reducing agent to the activatable antibody, incubation time, temperature during incubation, pH of the reduction reaction solution, etc.) are determined by identifying the conditions that result in the conjugated activatable antibody, wherein the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in the uncleaved state. The ratio of the reducing agent to the activatable antibody will vary depending on the activatable antibody. In some embodiments, the ratio of the reducing agent to the activatable antibody will range from about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the ratio ranges from about 5:1 to 1:1. In some embodiments, the ratio ranges from about 5:1 to 1.5:1. In some embodiments, the ratio ranges from about 4:1 to 1:1. In some embodiments, the ratio ranges from about 4:1 to 1.5:1. In some embodiments, the ratio ranges from about 8:1 to about 1:1. In some embodiments, the ratio ranges from about 2.5:1 to 1:1.

[0151] In some embodiments, provided is a method of reducing an interchain disulfide bond in an AB of a reducible activatable antibody and conjugating an agent (e.g., a thiol-containing agent such as a drug) to the resulting interchain thiol to selectively localize one or more agents on the AB. The method generally includes partially reducing the AB with a reducing agent to form at least two interchain thiols without forming all possible interchain thiols in the activatable antibody; and conjugating the agent to the interchain thiols of the partially reduced AB. For example, the AB of the activatable antibody is partially reduced at about 37 °C for about 1 hour at a desired reducing agent:activatable antibody ratio. In some embodiments, the range of the ratio of the reducing agent to the activatable antibody will be from about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments, the range of the ratio is from about 5:1 to 1:1. In some embodiments, the range of the ratio is from about 5:1 to 1.5:1. In some embodiments, the range of the ratio is from about 4:1 to 1:1. In some embodiments, the range of the ratio is from about 4:1 to 1.5:1. In some embodiments, the range of the ratio is from about 8:1 to about 1:1. In some embodiments, the range of the ratio is from about 2.5:1 to 1:1.

[0152] The thiol-containing reagent can be, for example, cysteine or N-acetylcysteine. The reducing agent can be, for example, TCEP. In some embodiments, the reduced activatable antibody can be purified using, for example, column chromatography, dialysis, or diafiltration before conjugation. In some embodiments, the reduced antibody is not purified after partial reduction and before conjugation.

[0153] The present disclosure also provides a partially reduced activatable antibody, wherein at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody comprises an antibody or an antigen-binding fragment (AB) thereof that specifically binds to a target, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in the uncleaved state to the target, and a CM1-CM2 substrate coupled to the AB, wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for at least one MMP and at least one SP. In some embodiments, the MM is coupled to the AB via the CM1-CM2 substrate. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM within the activatable antibody are not disturbed by the reducing agent. In some embodiments, the activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0154] The present disclosure also provides a partially reduced activatable antibody, including but not limited to the multispecific activatable antibodies of the present disclosure, wherein at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without disturbing or otherwise impairing the activity and / or efficacy of the activatable antibody, wherein the activatable antibody comprises an antibody or an antigen-binding fragment (AB) thereof that specifically binds to a target, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in the uncleaved state to the target, and a CM1-CM2 substrate coupled to the AB, and the CM1-CM2 substrate is a polypeptide that serves as a substrate for a protease. By way of non-limiting example, the activity and / or efficacy of the activatable antibody is masking activity, activation of the activatable antibody, and / or binding activity of the activated activatable antibody. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM within the activatable antibody are not disturbed by the reducing agent. In some embodiments, the activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0155] The present disclosure also provides conjugated activatable antibodies that comprise an activatable antibody linked to a monomethyl auristatin D (MMAD) payload, wherein the activatable antibody comprises an antibody or an antigen-binding fragment (AB) thereof that specifically binds to a target, a masking moiety (MM) that inhibits binding of the AB of the activatable antibody in the uncleaved state to the target, and a CM1-CM2 substrate conjugated to the AB, and the CM1-CM2 substrate is a polypeptide that serves as a substrate for at least one MMP protease and at least one SP protease.

[0156] In some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any one of several methods for attaching an agent to an AB: (a) attaching to a carbohydrate moiety of the AB, or (b) attaching to a thiol group of the AB, or (c) attaching to an amino group of the AB, or (d) attaching to a carboxyl group of the AB.

[0157] In some embodiments, the MMAD payload is conjugated to the AB via a linker. In some embodiments, the MMAD payload is conjugated to a cysteine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to a lysine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to another residue of the AB, such as those residues disclosed herein, via a linker. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is selected from the group consisting of the linkers shown in Tables 5 and 6. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide-PEG-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide-PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker. In some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation techniques disclosed herein.

[0158] The present disclosure also provides polypeptides and other larger molecules that comprise one or more of the CM1-CM2 substrate sequences presented herein. By way of non-limiting example, the CM1-CM2 substrate sequences presented herein can be used in prodrug compositions and methods of using the same. These CM1-CM2 substrate sequences presented herein can also be used in probes and other detection agents and methods of using the same. For example, the CM1-CM2 substrate sequences presented herein can be used in combination with fluor and other quenchers to produce detection agents such as imaging agents and / or other diagnostic agents. Those of ordinary skill in the art will understand that the CM1-CM2 substrate sequences presented herein can be used in any composition and / or method in the art that uses a substrate cleavable by at least one MMP and at least one SP.

[0159] The present disclosure also provides an isolated nucleic acid molecule encoding an antibody and / or an activatable antibody described herein, and a vector comprising such isolated nucleic acid sequences. The present disclosure provides methods of producing an antibody and / or an activatable antibody by culturing a cell comprising such a vector under conditions that result in the expression of the antibody and / or the activatable antibody.

[0160] The present disclosure provides a method of making a conjugated antibody of the present disclosure that binds a given target, the method comprising (a) culturing a cell comprising a nucleic acid construct encoding an antibody under conditions that result in the expression of the antibody, (i) wherein the antibody comprises a CM1-CM2 substrate, and (ii) wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for a matrix metalloproteinase and a serine protease; (b) recovering the antibody; and (c) conjugating the recovered antibody with one or more additional agents.

[0161] The present disclosure also provides a method of making an activatable antibody of the present disclosure that binds a given target in an activated state, the method comprising: (a) culturing a cell comprising a nucleic acid construct encoding an activatable antibody under conditions that result in the expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a CM1-CM2 substrate, and an antibody or an antigen-binding fragment thereof (AB) that specifically binds the target, (i) wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for an MMP and an SP; and (ii) wherein the CM1-CM2 substrate is positioned in the activatable antibody such that in the uncleaved state, the MM interferes with the specific binding of the AB to the target, and in the cleaved state, the MM does not interfere with or compete with the specific binding of the AB to the target; and (b) recovering the activatable antibody.

[0162] The present disclosure also provides a method of making a conjugated activatable antibody of the present disclosure that binds a given target in an activated state, the method comprising: (a) culturing a cell comprising a nucleic acid construct encoding the activatable antibody under conditions that result in the expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a CM1-CM2 substrate, and an antibody or antigen-binding fragment thereof (AB) that specifically binds the target, (i) wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for MMP and SP and / or CP; and (ii) wherein the CM1-CM2 substrate is positioned in the activatable antibody such that in the uncleaved state, MM interferes with the specific binding of AB to the target, and in the cleaved state, MM does not interfere with or compete with the specific binding of AB to the target; (b) recovering the activatable antibody; and (c) conjugating the recovered antibody with one or more additional agents.

[0163] The present disclosure provides a method of preventing a target-related disease in a subject, delaying the progression of the disease, treating the disease, alleviating the symptoms of the disease, or otherwise improving the disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody as described herein.

[0164] The present disclosure provides a method of preventing inflammation and / or an inflammatory disorder in a subject, delaying the progression of the disorder, treating the disorder, alleviating the symptoms of the disorder, or otherwise improving the disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody as described herein. The present disclosure also provides a method of preventing cancer in a subject, delaying the progression of the cancer, treating the cancer, alleviating the symptoms of the cancer, or otherwise improving the cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody as described herein. The present disclosure also provides a method of preventing an autoimmune disease in a subject, delaying the progression of the disease, treating the disease, alleviating the symptoms of the disease, or otherwise improving the disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, an activatable antibody, and / or a conjugated activatable antibody as described herein.

[0165] The conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies used in any embodiment of these methods and uses can be administered at any stage of the disease. For example, such conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be administered to a patient with cancer at any stage from early to metastatic. The terms subject and patient are used interchangeably herein.

[0166] In some embodiments, the subject is a mammal, such as a human, non-human primate, companion animal (e.g., cat, dog, horse), farm animal, working animal, or zoo animal. In some embodiments, the subject is a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal under veterinary care.

[0167] The conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies and their therapeutic formulations are administered to a subject having or susceptible to a disease or disorder associated with abnormal target expression and / or activity. A subject having or susceptible to a disease or disorder associated with abnormal target expression and / or activity is identified using any of a variety of methods known in the art. For example, a subject having cancer or other neoplastic conditions is identified using any of a variety of clinical and / or laboratory tests (such as physical examination and blood, urine, and / or fecal analysis) to assess health status. For example, a subject having an inflammation and / or inflammatory disorder is identified using any of a variety of clinical and / or laboratory tests (such as physical examination and / or body fluid analysis, e.g., blood, urine, and / or fecal analysis) to assess health status.

[0168] Administration of the conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies to a patient having a disease or disorder associated with abnormal target expression and / or activity is considered successful if any of a variety of laboratory or clinical goals are achieved. For example, administration of the conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies to a patient having a disease or disorder associated with abnormal target expression and / or activity is considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, suppressed, or do not progress to a further (i.e., worse) state. Administration of the conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies to a patient having a disease or disorder associated with abnormal target expression and / or activity is considered successful if the disease or disorder enters a remission or does not progress to a further (i.e., worse) state.

[0169] In some embodiments, the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies described herein are used in combination with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended application such as cancer. For example, the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be used in combination with additional chemotherapeutic or anti-tumor agents.

[0170] In some embodiments, one or more additional agents are chemotherapeutic agents, such as chemotherapeutic agents selected from the group consisting of: docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0171] In some embodiments, one or more additional agents are checkpoint inhibitors, kinase inhibitors, agents that inhibit inhibitors in the tumor microenvironment, and / or T cell or NK agonists. In some embodiments, one or more additional agents are radiotherapy, either alone or in combination with one or more other additional agents such as chemotherapeutic agents or anti-tumor agents. In some embodiments, one or more additional agents are vaccines, oncolytic viruses, and / or DC activators, by way of non-limiting examples, such as toll-like receptor (TLR) agonists and / or α-CD40. In some embodiments, one or more additional agents are tumor-targeting antibodies designed to kill tumors by ADCC or by direct conjugation with toxins (e.g., antibody-drug conjugates (ADCs)).

[0172] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of: CTLA-4, LAG-3, PD-1, PD-L1, TIGIT, TIM-3, B7H4, BTLA, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors (such as ibrutinib). In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of: IDO inhibitors, α-CSF1R inhibitors, α-CCR4 inhibitors, TGF-β, myeloid-derived suppressor cells, or T regulatory cells. In some embodiments, the agonist is selected from the group consisting of: Ox40, GITR, CD137, ICOS, CD27, and HVEM.

[0173] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is TGF-β. In some embodiments, the inhibitor is myeloid-derived suppressor cells. In some embodiments, the inhibitor is regulatory T cells.

[0174] In some embodiments, the agonist is Ox40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.

[0175] In some embodiments, an antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody is administered in combination with one or more additional agents (such as a chemotherapeutic agent, anti-inflammatory agent, and / or immunosuppressive agent) during and / or after treatment. In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent are formulated into a single therapeutic composition, and the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent are administered simultaneously. Alternatively, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent are separate from each other, for example, they are each formulated into separate therapeutic compositions, and the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent are administered simultaneously, or the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent are administered at different times in a treatment regimen. For example, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody is administered before the additional agent, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody is administered after the additional agent, or the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent are administered in an alternating manner. As described herein, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent are administered in a single dose or multiple doses.

[0176] In some embodiments, an antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and one or more additional agents are administered simultaneously. For example, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and one or more additional agents can be formulated into a single composition or administered as two or more separate compositions. In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and one or more additional agents are administered sequentially, or the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and one or more additional agents are administered at different times in a treatment regimen.

[0177] In some embodiments, a conjugated antibody, activatable antibody, and / or conjugated activatable antibody is administered in combination with one or more additional agents during and / or after treatment, the one or more additional agents being, by way of non-limiting example, an anti-inflammatory agent, immunosuppressive agent, chemotherapeutic agent (such as an alkylating agent), antimetabolite, antimicrotubule agent, topoisomerase inhibitor, cytotoxic antibiotic, and / or any other nucleic acid damaging agent. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g. )。In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapeutic agent, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeting agent, such as a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a targeting agent, such as another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteasome inhibitor, such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulator, such as lenalidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent regarded as standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art.

[0178] In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody. In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody that targets the same target as the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody. In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody that targets a different target than the target of the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody.

[0179] In some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody and one or more additional agents are administered simultaneously. For example, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody and one or more additional agents can be formulated as a single composition or administered as two or more separate compositions. In some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody and one or more additional agents are administered sequentially, or the antibody and / or conjugated antibody and the additional agent are administered at different times in a treatment regimen. For example, the antibody and / or conjugated antibody is administered before the additional agent, the antibody and / or conjugated antibody is administered after the additional agent, or the antibody and / or conjugated antibody and the additional agent are administered in an alternating manner. As described herein, the antibody and / or conjugated antibody and the additional agent are single-dose or multi-dose.

[0180] The present disclosure also provides methods and kits for using conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies in a variety of diagnostic and / or prophylactic indications.

[0181] The pharmaceutical compositions according to the present disclosure may comprise the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies of the present disclosure and a carrier. These pharmaceutical compositions may be included in kits, such as diagnostic kits.

[0182] The conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies comprise an antibody or an antigen-binding fragment thereof (AB) that specifically binds to a target. Exemplary classes of targets of the AB include, but are not necessarily limited to, cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), etc. In some embodiments, the conjugated antibody and / or activatable antibody has an AB that binds to an extracellular target (usually an extracellular protein target). In some embodiments, the conjugated antibody and / or activatable antibody is designed for cellular uptake and can be converted intracellularly.

[0183] As a non-limiting example, the AB is a binding partner of any of the targets listed in Table 2.

[0184] Table 2: Exemplary Targets

[0185]

[0186]

[0187] As a non-limiting example, the AB is or is derived from an antibody listed in Table 3.

[0188] Table 3: Exemplary Sources of Ab

[0189]

[0190]

[0191] The activatable antibodies and activatable antibody compositions provided herein comprise at least an antibody or an antibody fragment thereof (collectively referred to as AB throughout the present disclosure) that specifically binds to a target, such as a human target, wherein the AB is modified with a masking moiety (MM).

[0192] In some embodiments, a masking moiety is selected for use with a specific antibody or antibody fragment. For example, suitable masking moieties for use with an antibody that binds EGFR include MM containing the sequence CISPRG (SEQ ID NO:165). By way of non-limiting example, MM can include sequences such as the following: CISPRGC (SEQ ID NO:166); CISPRGCG (SEQ ID NO:167); CISPRGCPDGPYVMY (SEQ ID NO:168); CISPRGCPDGPYVM (SEQ ID NO:169); CISPRGCEP GTYVPT (SEQ ID NO:170); and CISPRGCPGQIWHPP (SEQ ID NO:171). Other suitable masking moieties include any of the EGFR-specific masks disclosed in PCT Publication No. WO 2010 / 081173, by way of non-limiting example, such as GSHCLIPINMGAPSC (SEQ ID NO:172); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO:173); CNHHYFYTCGCISPRG CPG (SEQ ID NO:174); ADHVFWGSYGCISPRGCPG (SEQ ID NO:175); CHHVYWGHCGCISPRGCPG (SEQ ID NO:176); CPHFTTTSCGCISPRGCPG (SEQ ID NO:177); CNHHYHYYCGCISPRG CPG (SEQ ID NO:178); CPHVSFGSCGCISPRGCPG (SEQ ID NO:179); CPYYTLSYCGCISPRGCPG (SEQ ID NO:180); CNHVYFGTCGCISPRGCPG (SEQ ID NO:181); CNHFTLTTCGCISPRGCP G (SEQ ID NO:182); CHHFTLTTCGCISPRGCPG (SEQ ID NO:183); YNPCATPMCCISPRGCPG (SEQ ID NO:184); CNHHYFYTCGCISPRGCG (SEQ ID NO:185); CNHHYHYYCGCISPRGCG (SEQ ID NO:186); CNHVYFGTCGCISPRGCG (SEQ ID NO:187); CHHVYWGHCGCISPRGCG (SEQ ID NO:188); CPHFTTTSCGCI SPRGCG (SEQ ID NO:189);CNHFTLTTCGCISPRGCG (SEQ ID NO:190); CHHFTLTTCGCISPRGCG (SEQ ID NO:191); CPYYT LSYCGCISPRGCG (SEQ ID NO:192); CPHVSFGSCGCISPRGCG (SEQ ID NO:193); ADHVFWGSYGCISPRGCG (SEQ ID NO:194); YNPCATPMCCISPRGCG (SEQ ID NO:195); CHHVYWGHC GCISPRGCG (SEQ ID NO:196); C(N / P)H(H / V / F)(Y / T)(F / W / T / L)(Y / G / T / S)(T / S / Y / H)CGCISPRGCG (SEQ ID NO:197); CISPRGCGQPIPSVK (SEQ ID NO:198); CISPRGCTQPYHVSR (SEQ ID NO:199); and / or CISPRGCNAVSGLGS (SEQID NO:164).;

[0193] Compared with the specific binding of AB without MM modification or the specific binding of the parental AB to the target, when AB is modified with MM and in the presence of the target, the specific binding of AB to its target is reduced or inhibited.

[0194] The K of AB modified with MM for the target d Compared with the K of AB without MM modification or the parental AB for the target dat least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000-fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1,000,000 fold, 10-10,000,000 fold, 100-1,000 fold, 100-10,000 fold, 100-100,000 fold, 100-1,000,000 fold, 100-10,000,000 fold, 1,000-10,000 fold, 1,000-100,000 fold, 1,000-1,000,000 fold, 1000-10,000,000 fold, 10,000-100,000 fold, 10,000-1,000,000 fold, 10,000-10,000,000 fold, 100,000-1,000,000 fold or 100,000-10,000,000 fold. Conversely, the binding affinity of AB modified with MM for the target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000-fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1,000,000 fold, 10-10,000,000 fold, 100-1,000 fold, 100-10,000 fold, 100-100,000 fold, 100-1,000,000 fold, 100-10,000,000 fold, 1,000-10,000 fold, 1,000-100,000 fold, 1,000-1,000,000 fold, 1000-10,000,000 fold, 10,000-100,000 fold, 10,000-1,000,000 fold, 10,000-10,000,000 fold, 100,000-1,000,000 fold or 100,000-10,000,000 fold lower than the binding affinity of unmodified AB or parental AB for the target.

[0195] The dissociation constant (K d ) of MM for AB is generally greater than the K d of AB for the target. The K of MM for ABd The K of Comparable AB against the target d is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times greater. In contrast, the binding affinity of MM for AB is generally lower than the binding affinity of AB for the target. The binding affinity of MM for AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lower than the binding affinity of AB for the target.

[0196] Compared with the specific binding of AB not modified with MM or the specific binding of parental AB to the target, when AB is modified with MM and in the presence of the target, the specific binding of AB to its target is reduced or inhibited. When measured in in vivo or in vitro assays, compared with the binding of AB not modified with MM to the target or the binding of parental AB to the target, when modified with MM, the ability of AB to bind to the target can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100%, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer.

[0197] MM inhibits the binding of AB to the target. MM binds to the antigen-binding domain of AB and inhibits the binding of AB to the target. MM can sterically inhibit the binding of AB to the target. MM can allosterically inhibit the binding of AB to its target. In these embodiments, when measured in in vivo or in vitro assays, compared with the binding of AB not modified with MM, parental AB or AB not conjugated to MM to the target, when AB is modified or conjugated to MM and in the presence of the target, AB does not bind or substantially does not bind to the target, or the binding of AB to the target does not exceed 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50%, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer.

[0198] When AB is conjugated to MM or modified by MM, MM "masks" or reduces or otherwise inhibits the specific binding of AB to the target. When AB is conjugated to MM or modified by MM, such conjugation or modification can cause structural changes that reduce or inhibit the ability of AB to specifically bind its target.

[0199] An AB conjugated to MM or modified with MM can be represented by the following formula (in the order from the amino (N)-terminal region to the carboxyl (C)-terminal region):

[0200] (MM)-(AB)

[0201] (AB)-(MM)

[0202] (MM)-L-(AB)

[0203] (AB)-L-(MM)

[0204] wherein MM is a masking moiety, AB is an antibody or an antibody fragment thereof, and L is a linker. In many embodiments, it may be desirable to insert one or more linkers (such as flexible linkers) into the composition to provide flexibility.

[0205] In certain embodiments, MM is not a natural binding partner of AB. In some embodiments, MM contains no or substantially no homology to any natural binding partner of AB. In some embodiments, MM is not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% similar to any natural binding partner of AB. In some embodiments, MM is not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% identical to any natural binding partner of AB. In some embodiments, MM is not more than 25% identical to any natural binding partner of AB. In some embodiments, MM is not more than 50% identical to any natural binding partner of AB. In some embodiments, MM is not more than 20% identical to any natural binding partner of AB. In some embodiments, MM is not more than 10% identical to any natural binding partner of AB.

[0206] In some embodiments, the activatable antibody comprises an AB modified by an MM, and further comprises at least one cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). Such activatable antibodies exhibit activatable / convertible binding to the target of the AB. An activatable antibody typically comprises an antibody or antibody fragment (AB) modified or conjugated with a masking moiety (MM) and CM1-CM2 substrates.

[0207] Arrange the elements of the activatable antibody such that the MM and CM1-CM2 substrates are positioned such that, in the cleaved (or relatively active) state and in the presence of the target, the AB binds the target, while in the uncleaved (or relatively inactive) state and in the presence of the target, the specific binding of the AB to its target is reduced or inhibited. Due to the inhibition or masking of the ability of the MM to specifically bind its target by the AB, the specific binding of the AB to its target can be reduced.

[0208] The K of the AB modified with MM and CM1-CM2 substrates for the target d is lower than the K of the AB not modified with MM and CM1-CM2 substrates or the parental AB for the target dat least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000-fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1,000,000 fold, 10-10,000,000 fold, 100-1,000 fold, 100-10,000 fold, 100-100,000 fold, 100-1,000,000 fold, 100-10,000,000 fold, 1,000-10,000 fold, 1,000-100,000 fold, 1,000-1,000,000 fold, 1,000-10,000,000 fold, 10,000-100,000 fold, 10,000-1,000,000 fold, 10,000-10,000,000 fold, 100,000-1,000,000 fold or 100,000-10,000,000 fold. Conversely, the binding affinity of the AB modified with MM and CM1-CM2 substrates for the target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000-fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1,000,000 fold, 10-10,000,000 fold, 100-1,000 fold, 100-10,000 fold, 100-100,000 fold, 100-1,000,000 fold, 100-10,000,000 fold, 1,000-10,000 fold, 1,000-100,000 fold, 1,000-1,000,000 fold, 1,000-10,000,000 fold, 10,000-100,000 fold, 10,000-1,000,000 fold, 10,000-10,000,000 fold, 100,000-1,000,000 fold or 100,000-10,000,000 fold lower than the binding affinity of the AB not modified with MM and CM1-CM2 substrates or the parental AB for the target.

[0209] Compared with the specific binding of AB or the parental AB without modification with MM and CM1-CM2 substrates to the target, when AB is modified with MM and CM1-CM2 substrates and in the presence of the target but in the absence of modifiers (such as MMP and SP), the specific binding of AB to its target is reduced or inhibited. When measured in in vivo or in vitro assays, compared with the binding of the parental AB to its target or the binding of AB without modification with MM and CM1-CM2 substrates to its target, when modified with MM and CM1-CM2 substrates, the ability of AB to bind to the target can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer.

[0210] As used herein, the term cleavage state refers to the condition of an activatable antibody after the CM1-CM2 substrate has been modified (i.e., cleaved) by at least one matrix metalloproteinase and / or at least one serine protease. As used herein, the term uncleaved state or fully uncleaved refers to the condition of an activatable antibody in the absence of cleavage of the CM1-CM2 substrate by MMP and / or SP. As described above, the term "activatable antibody" is used herein to refer to an activatable antibody in its uncleaved (native) state as well as in its cleavage state. An activatable antibody in its cleavage state is also referred to herein as an activated antibody and / or an activated activatable antibody. It will be clear to those of ordinary skill in the art that in some embodiments, due to cleavage of the CM1-CM2 substrate by a protease, resulting in the release of at least MM, the cleaved activatable antibody may lack MM.

[0211] Activatable or convertible means that when in an inhibited, masked or uncleaved state (i.e., the first conformation), the activatable antibody exhibits a first binding level to the target, and when in an uninhibited, unmasked and / or cleaved state (i.e., the second conformation), the activatable antibody exhibits a second binding level to the target, wherein the second target binding level is greater than the first binding level. Generally, the access of the target to the AB of the activatable antibody is greater in the presence of a cleavage agent capable of cleaving the CM1-CM2 substrate than in the absence of such a cleavage agent. Thus, when the activatable antibody is in the uncleaved state, the binding of AB to the target is inhibited and its target binding may be masked (i.e., the first conformation prevents AB from binding to the target), and in the cleavage state, AB is not inhibited or unmasked for target binding.

[0212] Select CM1-CM2 substrates and ABs of activatable antibodies such that the AB represents the binding portion of a given target and the CM1-CM2 substrate represents the substrate of MMP and SP, where MMP and / or SP are co-localized with the target at a therapeutic or diagnostic site in a subject. Particular uses of the activatable antibodies disclosed herein are found, where, for example, MMP and SP, each capable of cleaving a site in the CM1-CM2 substrate, are present at relatively higher levels in target-containing tissue at a therapeutic or diagnostic site than in tissue at a non-therapeutic site (e.g., in healthy tissue).

[0213] In some embodiments, the activatable antibody provides reduced toxicity and / or adverse side effects that may otherwise be caused by the binding of the AB at non-therapeutic sites if the binding of the AB to the target is not masked or otherwise inhibited.

[0214] Generally, an activatable antibody can be designed by selecting an AB of interest and constructing the remainder of the activatable antibody such that, when conformationally restricted, the MM provides masking of the AB or reduced binding of the AB to its target. Structural design criteria can be considered to provide this functional feature.

[0215] Provided are activatable antibodies with a convertible phenotype that exhibit a desired dynamic range of target binding in a conformationally inhibited versus an uninhibited conformation. The dynamic range generally refers to the ratio of (a) the maximum detected level of a parameter under a first set of conditions to (b) the minimum detected value of the parameter under a second set of conditions. For example, in the context of an activatable antibody, the dynamic range refers to the ratio of (a) the maximum detected level of a target protein bound by the activatable antibody in the presence of MMP and SP capable of cleaving the CM1-CM2 substrate of the activatable antibody to (b) the minimum detected level of the target protein bound by the activatable antibody in the absence of protease. The dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of the activatable antibody with a lysing agent (e.g., an enzyme) treatment to the dissociation constant of the activatable antibody without lysing agent treatment. The greater the dynamic range of the activatable antibody, the better the convertible phenotype of the activatable antibody. An activatable antibody with a relatively high dynamic range value (e.g., greater than 1) exhibits a more desirable conversion phenotype such that the extent of target protein binding by the activatable antibody occurs to a greater degree (e.g., predominantly) in the presence of a lysing agent (e.g., an enzyme) capable of cleaving the CM1-CM2 substrate of the activatable antibody compared to in the absence of the lysing agent.

[0216] Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulas of activatable antibodies are provided below. It is specifically contemplated that the N-terminal to C-terminal order of the AB, MM, and CM1-CM2 substrate can be reversed in the activatable antibody. It is also specifically contemplated that the CM and MM can overlap in the amino acid sequence, such that, for example, the CM1-CM2 substrate is at least partially contained within the MM.

[0217] For example, an activatable antibody can be represented by the following formula (in the order from the amino (N)-terminal region to the carboxyl (C)-terminal region):

[0218] (MM)-(CM1-CM2 substrate)-(AB)

[0219] (AB)-(CM1-CM2 substrate)-(MM)

[0220] wherein MM is a masking moiety, CM1-CM2 substrate is a cleavable moiety, and AB is an antibody or a fragment thereof. As described above, the term "CM1-CM2 substrate" is not intended to convey any requirement regarding the orientation or other structural arrangement of the first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and the at least second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). Thus, the term "CM1-CM2 substrate" encompasses CM1-CM2 substrates having the following structural arrangement from the N-terminus to the C-terminus: CM1-CM2 or CM2-CM1. The term "CM1-CM2 substrate" also encompasses substrates in which at least a portion of the CM1 sequence overlaps with at least a portion of the CM2 sequence. It should also be noted that although MM and CM1-CM2 substrate are indicated as different components in the above formula, in all exemplary embodiments (including the formula) disclosed herein, it is contemplated that the amino acid sequences of MM and CM1-CM2 substrate may overlap, for example, such that the CM1-CM2 substrate is fully or partially contained within MM. Additionally, the above formula provides additional amino acid sequences that can be located at the N-terminus or C-terminus of the activatable antibody element.

[0221] In certain embodiments, MM is not a natural binding partner of AB. In some embodiments, MM does not contain or is substantially free of homology to any natural binding partner of AB. In some embodiments, MM is not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% similar to any natural binding partner of AB. In some embodiments, MM is not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% identical to any natural binding partner of AB. In some embodiments, MM is not more than 50% identical to any natural binding partner of AB. In some embodiments, MM is not more than 25% identical to any natural binding partner of AB. In some embodiments, MM is not more than 20% identical to any natural binding partner of AB. In some embodiments, MM is not more than 10% identical to any natural binding partner of AB.

[0222] In many embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) into the activatable antibody construct to provide flexibility at one or more of the MM-CM1-CM2 substrate junction, the CM1-CM2 substrate-AB junction, or both. For example, the AB, MM, and / or CM1-CM2 substrate may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly Gly) to provide the desired flexibility. Thus, the convertible phenotype of such activatable antibody constructs may benefit from the introduction of one or more amino acids to provide flexible linkers. Additionally, as described below, when the activatable antibody is provided as a conformationally restricted construct, a flexible linker may be operably inserted to facilitate the formation and maintenance of a cyclic structure in the uncleaved activatable antibody.

[0223] For example, in certain embodiments, the activatable antibody comprises one of the following formulas (wherein the following formulas represent amino acid sequences in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction):

[0224] (MM)-LP1-(CM1-CM2 substrate)-(AB)

[0225] (MM)-(CM1-CM2 substrate)-LP2-(AB)

[0226] (MM)-LP1-(CM1-CM2 substrate)-LP2-(AB)

[0227] wherein MM, CM1-CM2 substrate, and AB are as defined above; wherein each of LP1 and LP2 is independently and optionally present or absent and is the same or different flexible linker comprising at least 1 flexible amino acid (e.g., Gly). Additionally, the above formulas provide additional amino acid sequences that can be positioned at the N-terminus or C-terminus of the activatable antibody elements. Examples include, but are not limited to, targeting moieties (e.g., ligands of cell receptors present in target tissues) and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HAS)).

[0228] The rate of cleavage of the CM1-CM2 substrate by at least one MMP is about 0.001 - 1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M -1 S-1 and is cleaved by at least one SP at a rate of about 0.001 - 1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250 or 1500 x 10 4 M -1 S -1 。

[0229] For enzymatic specific cleavage, contact between the enzyme and the CM1 - CM2 substrate is made. When the activatable antibody comprising AB conjugated to MM and the CM1 - CM2 substrate is in the presence of the target and sufficient enzyme activity, the CM1 - CM2 substrate can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to contact the CM1 - CM2 substrate and effect cleavage. It can be readily envisioned that the enzyme may be near the CM1 - CM2 substrate but unable to cleave due to protein modification by other cytokines or enzymes.

[0230] Linkers suitable for use in the compositions described herein are generally linkers that provide flexibility to the modified AB or activatable antibody to facilitate inhibition of the binding of the inhibitory AB to the target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can have any suitable different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and the length can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0231] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:381), and (GGGS)n (SEQ ID NO:382), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and may thus be able to act as neutral tethers between components. Glycine is closer to a significantly larger phi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly (SEQ ID NO:202), Gly-Gly-Ser-Gly-Gly (SEQ ID NO:203), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:204), Gly-Ser-Gly-Gly-Gly (SEQ ID NO:205), Gly-Gly-Gly-Ser-Gly (SEQ ID NO:206), Gly-Ser-Ser-Ser-Gly (SEQ ID NO:207), etc. One of ordinary skill in the art will recognize that the design of an activatable antibody may include a linker that is fully or partially flexible such that the linker may include a flexible linker and one or more portions that impart a less flexible structure to provide the desired activatable antibody structure.

[0232] In some embodiments, the activatable antibodies described herein further comprise an agent conjugated to the activatable antibody. In some embodiments, the conjugated agent is a therapeutic agent, such as an anti-inflammatory agent and / or an anti-tumor agent. In such embodiments, the agent is conjugated to a carbohydrate moiety of the activatable antibody, e.g., in some embodiments, where the carbohydrate moiety is outside the antigen-binding region of the antibody or antigen-binding fragment of the activatable antibody. In some embodiments, the agent is conjugated to a sulfhydryl group in the antibody or antigen-binding fragment of the activatable antibody.

[0233] In some embodiments, the agent is a cytotoxic agent (such as a toxin (e.g., an enzymatically active toxin or fragment thereof from a bacterial, fungal, plant, or animal source)) or a radioisotope (i.e., a radioactive conjugate).

[0234] In some embodiments, the agent is a detectable moiety, such as a label or other marker. For example, the agent is or comprises a radiolabeled amino acid, one or more biotin moieties detectable by streptavidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optics or calorimetry), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety is attached via a spacer molecule.

[0235] The present disclosure also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent (such as a toxin (e.g., an enzymatically active toxin or fragment thereof from bacterial, fungal, plant, or animal origin)) or a radioisotope (i.e., a radio-conjugate). Suitable cytotoxic agents include, for example, dolastatin and its derivatives (e.g., auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD). In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansine alkaloid or a maytansine alkaloid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine . In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0236] In some embodiments, an agent is linked to AB using a maleimidocaproyl-valine-citrulline linker or a maleimidomethyl-PEG-valine-citrulline linker. In some embodiments, an agent is linked to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, an agent is linked to AB using a maleimidomethyl-PEG-valine-citrulline linker. In some embodiments, the agent is monomethyl auristatin D (MMAD) linked to AB using a maleimidomethyl-PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as "vc-MMAD". In some embodiments, the agent is monomethyl auristatin E (MMAE) linked to AB using a maleimidomethyl-PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as "vc-MMAE". The structures of vc-MMAD and vc-MMAE are shown below:

[0237] vc-MMAD:

[0238]

[0239] vc-MMAE:

[0240]

[0241] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecene. A variety of radionuclides can be used to generate radiolabeled antibodies. Examples include 212 Bi, 131 I, 131 In,90 Y and 186 Re.

[0242] Conjugates of antibodies and cytotoxic agents are made using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propionic acid N-succinimidyl ester (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters such as dimethyl adipimidate HCL, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bisazido compounds such as bis(p-azidobenzoyl)hexanediamine, diazo derivatives such as bis(p-diazobenzoyl)-ethylenediamine, diisocyanates such as toluene 2,6-diisocyanate, and bifunctional fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene. For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-benzylisothiocyanato-3-methyl-diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radio nucleotides to antibodies. (See WO94 / 11026).

[0243] Table 4 lists some exemplary agents that can be employed in the disclosures described herein, but is in no way meant to be an exhaustive list.

[0244] Table 4: Exemplary Agents for Conjugation

[0245]

[0246]

[0247]

[0248] One of ordinary skill in the art will recognize that a variety of possible moieties can be conjugated to the resulting antibodies of the present disclosure. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0249] Coupling can be accomplished by any chemical reaction that binds the two molecules together, so long as the antibody and the other moiety retain their respective activities. This linkage can include a number of chemical mechanisms such as covalent binding, affinity binding, intercalation, coordination binding, and complexation. However, in some embodiments, the binding is covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of an external bridging molecule. Many divalent or multivalent linkers can be used to couple protein molecules such as the antibodies of the present disclosure to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimidyl esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamine. This list is not intended to be exhaustive of the types of coupling agents known in the art, but is exemplary of more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0250] In some embodiments, in addition to the compositions and methods provided herein, the conjugated activatable antibody can be modified for site-specific conjugation by insertion or otherwise incorporating a modified amino acid sequence into the activatable antibody sequence. These modified amino acid sequences are designed to allow control of the placement and / or dosage of the conjugating agent within the conjugated activatable antibody. For example, an activatable antibody can be engineered to contain cysteine substitutions at positions on the light and heavy chains that provide reactive thiol groups and do not adversely affect protein folding and assembly, nor alter antigen binding. In some embodiments, an activatable antibody can be engineered to contain or otherwise introduce one or more unnatural amino acid residues within the activatable antibody to provide suitable conjugation sites. In some embodiments, an activatable antibody can be engineered to contain or otherwise introduce an enzyme-activatable peptide sequence within the activatable antibody sequence.

[0251] Suitable linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of haloacetyl hydrazide derivatives coupled to antibodies via oligopeptide linkers. In some embodiments, suitable linkers include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene) (Pierce Chem. Co., catalog (21558G)); (iii) SPDP (succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate) (Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate) (Pierce Chem. Co. catalog number 2165-G); and (v) sulfo-NHS conjugated to EDC (N-hydroxysulfosuccinimide: Pierce Chem. Co., catalog number 24510). Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB).

[0252] The linkers described above contain components with different properties, and thus result in conjugates with different physicochemical properties. For example, the sulfo-NHS ester of a carboxylic acid alkyl ester is more stable than the sulfo-NHS ester of a carboxylic acid aryl ester. The solubility of linkers containing NHS esters is lower than that of sulfo-NHS esters. In addition, the linker SMPT contains a sterically hindered disulfide bond and can form conjugates with increased stability. Generally, the stability of disulfide linkages is lower than that of other linkages because disulfide linkages are cleaved in vitro, resulting in fewer available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. The esters formed by carbodiimide coupling (such as EDC) when used in combination with sulfo-NHS are more resistant to hydrolysis than the esters formed by the carbodiimide coupling reaction alone.

[0253] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are two or more linkers. The two or more linkers are the same, i.e., cleavable or non-cleavable, or the two or more linkers are different, i.e., at least one is cleavable and at least one is non-cleavable.

[0254] The present disclosure utilizes several methods for attaching an agent to AB: (a) attaching to the carbohydrate moiety of AB, or (b) attaching to the thiol group of AB, or (c) attaching to the amino group of AB, or (d) attaching to the carboxyl group of AB. According to the present disclosure, AB can be covalently attached to the agent through an intermediate linker having at least two reactive groups, one of which reacts with AB and one of which reacts with the agent. A linker can be selected that can include any compatible organic compound such that the reaction with AB (or the agent) does not adversely affect the reactivity and selectivity of AB. In addition, the attachment of the linker to the agent may not disrupt the activity of the agent. Suitable linkers for reacting with oxidized antibodies or oxidized antibody fragments include those containing amines selected from the group consisting of: primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and thiosemicarbazide groups. Such reactive functional groups can be present as part of the linker structure or can be introduced by appropriate chemical modification of a linker that does not contain such groups.

[0255] According to the present disclosure, suitable linkers for attaching to reduced AB include those having certain reactive groups capable of reacting with the thiol groups of reduced antibodies or fragments. Such reactive groups include, but are not limited to: reactive haloalkyls (including, for example, haloacetyls), p-mercuribenzoate groups, and groups capable of undergoing Michael-type addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101:3097-3110).

[0256] According to the present disclosure, suitable linkers for attaching to Ab that is neither oxidized nor reduced include those having certain functional groups capable of reacting with the primary amino groups present in unmodified lysine residues in Ab. Such reactive groups include, but are not limited to, NHS carboxylates or carbonates, sulfo-NHS carboxylates or carbonates, 4-nitrophenyl carboxylates or carbonates, pentafluorophenyl carboxylates or carbonates, acyl imidazoles, isocyanates, and isothiocyanates.

[0257] According to the present disclosure, suitable linkers for attaching to Ab that is neither oxidized nor reduced include those having certain functional groups capable of reacting with the carboxyl groups present in aspartic acid residues or glutamic acid residues in Ab, which have been activated with a suitable reagent. Suitable activating reagents include EDC with or without added NHS or sulfo-NHS, and other dehydrating agents for carboxamide formation. In these cases, the functional groups present in the suitable linker will include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0258] The agent can be attached to the linker either before or after attaching the linker to AB. In some applications, it may be desirable to first prepare an AB-linker intermediate, where the linker is free of the associating agent. Depending on the particular application, a specific agent can then be covalently attached to the linker. In some embodiments, for conjugation purposes, AB is first attached to MM, CM1-CM2 substrate, and the associating linker, and then attached to the linker.

[0259] Branched linker: In certain embodiments, a branched linker having multiple sites for attaching agents is utilized. For a multi-site linker, a single covalent attachment to AB will result in an AB-linker intermediate capable of binding agents at multiple sites. The sites can be any chemical site that can be attached to an aldehyde or a thiol or an agent.

[0260] In some embodiments, higher specific activity (or a higher ratio of agent to AB) can be achieved by attaching single-site linkers at multiple sites on AB. The multiple sites can be introduced into AB by either of two methods. First, multiple aldehyde groups and / or thiol groups can be generated in the same AB. Second, a "branched linker" having multiple functional sites for subsequent attachment to the linker can be attached to the aldehyde or thiol of AB. The functional sites of the branched linker or multi-site linker can be aldehyde or thiol, or can be any chemical site that can be attached to the linker. Higher specific activity can be obtained by combining these two methods (i.e., attaching a multi-site linker at multiple sites on AB).

[0261] Cleavable linker: A peptide linker that is readily cleaved by enzymes of the complement system can be used in one embodiment of the present disclosure, such enzymes including but not limited to urokinase, tissue plasminogen activator, trypsin, plasmin, or another enzyme having proteolytic activity. According to one method of the present disclosure, the agent is attached via a linker that is readily cleaved by complement. The antibody is selected from the classes that can activate complement. Thus, the antibody-agent conjugate activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, the agent is attached via a linker that is readily cleaved by an enzyme having proteolytic activity, such as urokinase, tissue plasminogen activator, plasmin, or trypsin. These cleavable linkers can be used in activatable antibodies including conjugation of extracellular toxins, which extracellular toxins are, by way of non-limiting example, any of the extracellular toxins shown in Table 4.

[0262] Table 5 provides non-limiting examples of cleavable linker sequences.

[0263] Table 5: Exemplary linker sequences for conjugation

[0264]

[0265]

[0266] Alternatively, the agent can be attached to the AB via a disulfide bond (e.g., a disulfide bond on a cysteine molecule). Since many tumors naturally release high levels of glutathione (a reducing agent), it can reduce the disulfide bond and subsequently release the agent at the delivery site. In certain specific embodiments, the reducing agent that modifies the CM1-CM2 substrate will also modify the linker of the conjugated activatable antibody.

[0267] Spacer and cleavable element: In some embodiments, it may be necessary to construct the linker in a way that optimizes the spacing between the agent and the AB of the activatable antibody. This can be done by using a linker with the following general structure:

[0268] W–(CH2)n–Q

[0269] where

[0270] W is --NH--CH2-- or --CH2--;

[0271] Q is an amino acid, a peptide; and

[0272] n is an integer from 0 to 20.

[0273] In some embodiments, the linker can include a spacer element and a cleavable element. The spacer element is used to position the cleavable element away from the core of the AB so that the cleavable element is more accessible to the enzyme responsible for cleavage. Some of the branched linkers described above can act as spacer elements.

[0274] Throughout the discussion, it should be understood that the attachment of the linker to the agent (or the attachment of the spacer element to the cleavable element, or the attachment of the cleavable element to the agent) does not need to be a specific attachment or reaction mode. Any reaction that provides a product with appropriate stability and biocompatibility is acceptable.

[0275] Serum complement and linker selection: According to one method of the present disclosure, when it is necessary to release the agent, an AB that is a class of antibodies that can activate complement is used. The resulting conjugate retains the ability to bind both the antigen complement and activate the complement cascade. Thus, according to this embodiment of the present disclosure, the agent is linked to one end of a cleavable linker or a cleavable element, and the other end of the linker group is attached to a specific site on the AB. For example, if the agent has a hydroxyl or amino group, it can be attached to the carboxyl terminus of a peptide, an amino acid, or other suitably selected linker via an ester or amide bond, respectively. For example, such an agent can be attached to the linker peptide via a carbodiimide reaction. If the agent contains functional groups that will interfere with the attachment to the linker, these interfering functional groups can be blocked before attachment and deblocked after the preparation of the product conjugate or intermediate. Then, the opposite end or the amino terminus of the linker is used, either directly or after further modification, to bind to an AB that can activate complement.

[0276] The linker (or spacer element of the linker) can have any desired length, and one end thereof can be covalently attached to a specific site on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker.

[0277] Thus, when these conjugates bind to an antigen in the presence of complement, the amide or ester bond of the linker attaching the agent will be cleaved, resulting in the release of the agent in its active form. When administered to a subject, these conjugates will accomplish the delivery and release of the agent at the target site and are particularly effective in delivering in vivo agents such as those presented in but not limited to Table 4, antibiotics, antimetabolites, anti-proliferative agents, and the like.

[0278] Linkers that release in the absence of complement activation: In another application of targeted delivery, since the activation of the complement cascade will ultimately lyse the target cell, it is necessary to release the agent in the absence of complement activation. Thus, this method is useful when the delivery and release of the agent should be accomplished without killing the target cell. This is the goal when it is desired to deliver a cell mediator such as a hormone, an enzyme, a corticosteroid, a neurotransmitter, a gene, or an enzyme to a target cell. These conjugates can be prepared by attaching the agent to an AB that cannot activate complement via a linker that is moderately susceptible to cleavage by serum proteases. When such a conjugate is administered to an individual, the antigen-antibody complex will form rapidly, while the cleavage of the agent will occur slowly, resulting in the release of the compound at the target site.

[0279] Biochemical crosslinking agents: In some embodiments, certain biochemical crosslinking agents can be used to conjugate an activatable antibody to one or more therapeutic agents. The crosslinking reagent forms a molecular bridge that links the functional groups of two different molecules. To link two different proteins in a stepwise manner, a heterobifunctional crosslinking agent that eliminates the formation of unwanted homopolymers can be used.

[0280] Peptide-based linkers that can be cleaved by lysosomal proteases are also useful, such as Val-Cit, Val-Ala, or other dipeptides. Additionally, acid-labile linkers that can be cleaved in the low pH environment of the lysosome can be used, such as: disialyl ethers. Other suitable linkers include cathepsin-labile substrates, particularly those that exhibit optimal function at acidic pH.

[0281] Exemplary heterobifunctional crosslinking agents are cited in Table 6.

[0282] Table 6: Exemplary heterobifunctional crosslinking agents

[0283]

[0284]

[0285] Non-cleavable linker or direct attachment: In some embodiments of the present disclosure, the conjugate can be designed such that the agent is delivered to the target but not released. This can be accomplished by attaching the agent directly or via a non-cleavable linker to the AB.

[0286] These non-cleavable linkers can include amino acids, peptides, D-amino acids, or other organic compounds that can be modified to contain functional groups that can subsequently be used to attach to the AB by the methods described herein. The general formula for such organic linkers can be

[0287] W–(CH2)n–Q

[0288] where

[0289] W is --NH--CH2-- or --CH2--;

[0290] Q is an amino acid, peptide; and

[0291] n is an integer from 0 to 20.

[0292] Non-cleavable conjugates: In some embodiments, the compound can be attached to an AB that does not activate complement. When using an AB that cannot effect complement activation, this attachment can be accomplished using a linker that is susceptible to cleavage by activated complement or a linker that is not susceptible to cleavage by activated complement.

[0293] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those described below: Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation times are disclosed in U.S. Patent No. 5,013,556.

[0294] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to produce liposomes of the desired diameter. The Fab' fragments of the antibodies of the present disclosure can be conjugated to the liposomes by a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0295] Definition :

[0296] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. The term "a / an" entity refers to one or more of said entities. For example, a compound refers to one or more compounds. Thus, the terms "a / an", "one or more", and "at least one" are used interchangeably herein. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and the techniques used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those that are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly done in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclature and the laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those that are well known and commonly used in these chemical fields. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of subjects.

[0297] As used in accordance with the present disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0298] As used herein, the term "antibody" refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin (Ig) molecule that contains a molecule having an antigen-binding site that specifically binds an antigen (immunoreacts with the antigen). "Specifically binds" or "immunoreacts with" or "immunologically specifically binds" means that an antibody reacts with one or more antigenic determinants of a desired antigen and does not react with or binds with much lower affinity to other polypeptides (K d > 10 -6 ). Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, domain antibodies, single-chain antibodies, Fab and F(ab')2 fragments, scFv, and Fab expression libraries.

[0299] Known basic antibody structural units comprise tetramers. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector functions. Generally, antibody molecules obtained from humans are related to any one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another by the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, etc. In addition, in humans, the light chain can be a κ chain or a λ chain.

[0300] As used herein, the term "monoclonal antibody" (mAb) or "monoclonal antibody composition" refers to a population of antibody molecules containing only one molecular species of antibody molecules consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are the same in all molecules of the population. The MAb contains an antigen-binding site capable of immunoreacting with a specific epitope of an antigen, the epitope being characterized by a unique binding affinity for the antigen-binding site.

[0301] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent segments (termed "hypervariable regions") in the V regions of the heavy and light chains are interposed between more conserved flanking segments termed "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences that naturally occur between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to one another in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are termed "complementarity determining regions" or "CDRs". The amino acid assignments for each domain are made according to the definitions of the Kabat sequences of proteins of immunological interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia and Lesk J. Mol. Biol. 196:901 - 917 (1987), Chothia et al. Nature 342:878 - 883 (1989).

[0302] As used herein, the term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin, scFv or T cell receptor. The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. Epitope determinants are generally composed of the chemically active surface groups of a molecule (such as amino acids or sugar side chains), and generally have specific three-dimensional structural features as well as specific charge features. For example, antibodies can be generated against the N-terminal or C-terminal peptides of a polypeptide. An antibody is considered to specifically bind an antigen when the dissociation constant ≤ 1 μM; in some embodiments ≤ 100 nM and in some embodiments ≤ 10 nM.

[0303] As used herein, the terms "specifically bind", "immunobind" and "immunobinding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunobinding interaction can be expressed according to the dissociation constant (K d ) of the interaction, where a smaller K d represents a greater affinity. Methods well known in the art can be used to quantify the immunobinding properties of a selected polypeptide. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, the "association rate constant" (K on ) and the "dissociation rate constant" (K off ) can be determined by calculating the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of K off / K on can eliminate all parameters unrelated to affinity and is equal to the dissociation constant K d . (Generally see Davies et al. (1990) Annual Rev Biochem 59:439-473). As measured by an assay such as a radioligand binding assay or a similar assay known to those of skill in the art, an antibody of the present disclosure is considered to specifically bind to a target when the binding constant (K d ) is ≤ 1 μM, in some embodiments ≤ 100 nM, in some embodiments ≤ 10 nM, and in some embodiments ≤ 100 pM to about 1 pM.

[0304] As used herein, the term "isolated polynucleotide" shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that (1) is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not exist in nature as part of a larger sequence. Polynucleotides according to the present disclosure include nucleic acid molecules encoding heavy chain immunoglobulin molecules as shown herein and nucleic acid molecules encoding light chain immunoglobulin molecules as shown herein.

[0305] The term "isolated protein" as used herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; the "isolated protein" is not associated with a protein found in nature according to its origin or derived origin, (2) does not contain other proteins from the same source, e.g., does not contain murine proteins, (3) is expressed by cells from different species, or (4) does not exist in nature.

[0306] The term "polypeptide" is used herein as a general term to refer to natural proteins, fragments, or analogs having a polypeptide sequence. Thus, natural protein fragments and analogs are species of the genus polypeptide. Polypeptides according to the present disclosure include heavy chain immunoglobulin molecules as shown herein and light chain immunoglobulin molecules as shown herein, as well as antibody molecules formed by the combination of a heavy chain immunoglobulin molecule and a light chain immunoglobulin molecule (such as a κ light chain immunoglobulin molecule, and vice versa), and fragments and analogs thereof.

[0307] As used herein, the term "naturally occurring" when applied to an object means the fact that the object is found in nature. For example, a polypeptide or polynucleotide sequence that is isolated from a source in nature and not intentionally modified by a person in the laboratory and that exists in a biological (including a virus) or otherwise naturally is naturally occurring.

[0308] As used herein, the term "operably linked" means that the described components are in a relationship that permits them to function in the intended manner. A control sequence that is "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

[0309] As used herein, the term "control sequence" refers to polynucleotide sequences that are necessary to effect the expression and processing of the coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosome binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is necessary for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "polynucleotide" as referred to herein means a nucleotide of at least 10 bases in length, which is a ribonucleotide or deoxyribonucleotide or a modified form of either type of nucleotide. The term includes single-stranded and double-stranded forms of DNA.

[0310] The term oligonucleotide as referred to herein includes naturally occurring and modified nucleotides joined together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides that generally include 200 bases or fewer in length. In some embodiments, the oligonucleotide is 10 to 60 bases in length, and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 bases in length. Oligonucleotides are generally single-stranded, such as for probes, but oligonucleotides can be double-stranded, such as for constructing gene mutants. The oligonucleotides of the present disclosure are sense or antisense oligonucleotides.

[0311] As used herein, the term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" as used herein includes nucleotides having modified or substituted sugar moieties, etc. The term "oligonucleotide linkage" as used herein includes oligonucleotide linkages such as phosphorothioate, dithiophosphate, selenophosphate, diselenophosphate, phosphoroanilothioate, phoshoraniladate, phosphoramidate, etc. See, e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et al., Nucl. Acids Res. 16:3209 (1988), Zon et al., Anti Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). If desired, the oligonucleotide may include a label for detection.

[0312] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd ed., E.S. Golub and D.R. Gren eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers of the twenty conventional amino acids (e.g., D - amino acids), unnatural amino acids (such as α,α - disubstituted amino acids), N - alkyl amino acids, lactic acid, and other non - conventional amino acids may also be suitable components of the polypeptides of the present disclosure. Examples of non - conventional amino acids include: 4 - hydroxyproline, γ - carboxyglutamic acid, ε - N,N,N - trimethyllysine, ε - N - acetyllysine, O - phosphoserine, N - acetylserine, N - formylmethionine, 3 - methylhistidine, 5 - hydroxylysine, σ - N - methylarginine, and other similar amino acids and imino acids (e.g., 4 - hydroxyproline). In the polypeptide notation used herein, according to standard usage and convention, the left - hand direction is the amino - terminal direction, and the right - hand direction is the carboxyl - terminal direction.

[0313] Similarly, unless otherwise stated, the left end of a single-stranded polynucleotide sequence is the 5' end, and the leftward direction of a double-stranded polynucleotide sequence is called the 5' direction. The 5' to 3' addition direction of a primary RNA transcript is called the transcription direction, and the sequence region on the DNA strand having the same sequence as the RNA and being 5' to the 5' end of the RNA transcript is called the "upstream sequence", and the sequence region on the DNA strand having the same sequence as the RNA and being 3' to the 3' end of the RNA transcript is called the "downstream sequence".

[0314] When applied to polypeptides, the term "substantial identity" means that two peptide sequences share at least 80% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, at least 90% sequence identity in some embodiments, at least 95% sequence identity in some embodiments, and at least 99% sequence identity in some embodiments.

[0315] In some embodiments, the non-identical residue positions differ due to conservative amino acid substitutions.

[0316] As discussed herein, it is contemplated that the present disclosure encompasses minor changes in the amino acid sequence of an antibody or immunoglobulin molecule, provided that the changes in the amino acid sequence are maintained at at least 75%, in some embodiments at least 80%, 90%, 95%, and in some embodiments 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those substitutions that occur within an amino acid family and are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid, glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include (i) serine and threonine, which are the aliphatic-hydroxyl family; (ii) asparagine and glutamine, which are the amide-containing family; (iii) alanine, valine, leucine, and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonably expected that the substitution of leucine with isoleucine or valine, the substitution of aspartic acid with glutamic acid, the substitution of threonine with serine, or the substitution of an amino acid with a structurally related amino acid analog will not have a significant impact on the binding or properties of the resulting molecule, especially where the substitution does not involve an amino acid within the framework site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of an antibody or immunoglobulin molecule can be readily prepared by one of ordinary skill in the art. Suitable amino and carboxyl termini of the fragment or analog occur near the boundaries of the functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with publicly available or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformational domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that, in accordance with the present disclosure, one of ordinary skill in the art can identify sequence motifs and structural conformations that can be used to define structural and functional domains.

[0317] Suitable amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinities for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins having sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (such as conservative amino acid substitutions) can be made in the naturally occurring sequence (e.g., in the polypeptide portion outside of one or more domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parental sequence (e.g., the amino acid replacement should not tend to disrupt a helix present in the parental sequence or disrupt other types of secondary structure that characterize the parental sequence). Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles (ed. Creighton, W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (eds. C. Branden and J. Tooze, Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0318] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or one or more internal deletions, but wherein the remaining amino acid sequence is the same as the corresponding positions in a naturally occurring sequence, e.g., deduced from a full-length cDNA sequence. The fragment length is typically at least 5, 6, 8, or 10 amino acids, at least 14 amino acids in some embodiments, at least 20 amino acids in some embodiments, typically at least 50 amino acids, and at least 70 amino acids in some embodiments. As used herein, the term "analog" refers to a polypeptide consisting of a segment of at least 25 amino acids that has substantial identity to a portion of the deduced amino acid sequence and specifically binds to a target under suitable binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) relative to the naturally occurring sequence. The analog length is typically at least 20 amino acids, at least 50 amino acids or longer in some embodiments, and can often be as long as the full-length naturally occurring polypeptide.

[0319] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from a biological material.

[0320] As used herein, the term "labeled" or "label" refers to the incorporation of a detectable moiety, such as by incorporation of a radiolabeled amino acid or attachment of a polypeptide to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent moiety or enzymatic activity that can be detected by optical or colorimetric methods). In some cases, the label or marker can also be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the label is attached via a spacer arm of varying lengths to reduce potential steric hindrance. As used herein, the term "agent or drug" refers to a compound or composition that is capable of inducing a desired therapeutic effect when appropriately administered to a patient.

[0321] Other chemical terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., McGraw-Hill, San Francisco (1985)).

[0322] As used herein, "substantially pure" means that the target species is the major species present (i.e., it is more abundant on a molar basis than any other individual species in the composition), and in some embodiments, a substantially purified fraction is a composition in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present.

[0323] Generally, a substantially pure composition will comprise greater than about 80% of all macromolecular species present in the composition, and in some embodiments greater than about 85%, 90%, 95%, and 99%. In some embodiments, the target species is purified to the necessary homogeneity (contaminant species in the composition cannot be detected by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0324] The term patient includes human and veterinary subjects.

[0325] The activatable antibodies of the present disclosure specifically bind to a given target, such as a human target protein. Also included in the present disclosure are activatable antibodies that bind to the same epitope as the activatable antibodies described herein.

[0326] One of ordinary skill in the art will recognize that, without undue experimentation, it can be determined whether a monoclonal antibody (e.g., a murine monoclonal antibody or a humanized antibody) has the same specificity as a monoclonal antibody used in the methods described herein by determining whether the former prevents the monoclonal antibody used in the methods described herein from binding to the target. If the monoclonal antibody being tested competes with the monoclonal antibody of the present disclosure, as shown by a decrease in the binding of the monoclonal antibody of the present disclosure, then the two monoclonal antibodies bind to the same or closely related epitopes. One method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody of the present disclosure is to pre-incubate the monoclonal antibody of the present disclosure with the target and then add the monoclonal antibody being tested to determine whether the ability of the monoclonal antibody being tested to bind to the target is inhibited. If the monoclonal antibody being tested is inhibited, then it is highly likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present disclosure.

[0327] Multispecific activatable antibody

[0328] The present disclosure also provides multispecific activatable antibodies. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize two or more different antigens or epitopes and comprise at least one masking moiety (MM) that is linked to at least one antigen or epitope binding domain of the multispecific antibody such that the coupling of the MM reduces the ability of the antigen or epitope binding domain to bind its target. In some embodiments, the MM is coupled to the antigen or epitope binding domain of the multispecific antibody via a CM1-CM2 substrate that serves as a substrate for at least one MMP protease and at least one SP. The activatable multispecific antibodies provided herein are stable in circulation, are activated at the intended therapeutic and / or diagnostic site, but are not activated in normal tissue (i.e., healthy tissue), and upon activation exhibit binding to the target that is at least comparable to that of the corresponding unmodified multispecific antibody.

[0329] In some embodiments, the multispecific activatable antibody is designed to engage an immune effector cell, also referred to herein as an immune effector cell-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody is designed to engage a leukocyte, also referred to herein as a leukocyte-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody is designed to engage a T cell, also referred to herein as a T cell-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody engages a surface antigen on a leukocyte, such as a T cell, a natural killer (NK) cell, a myeloid monocyte, a macrophage, and / or a surface antigen on another immune effector cell. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell is a monocyte, such as a myeloid monocyte. In some embodiments, the multispecific activatable antibody is designed to bind to or otherwise interact with more than one target and / or more than one epitope, also referred to herein as a multi-antigen targeting activatable antibody. As used herein, the terms "target" and "antigen" are used interchangeably.

[0330] In some embodiments, the immune effector cell - conjugated multispecific activatable antibody of the present disclosure comprises a targeting antibody or an antigen - binding fragment thereof and an immune effector cell - conjugating antibody or an antigen - binding portion thereof, wherein at least one of the targeting antibody or an antigen - binding fragment thereof and / or the immune effector cell - conjugating antibody or an antigen - binding portion thereof is masked. In some embodiments, the immune effector cell - conjugating antibody or an antigen - binding fragment thereof comprises a first antibody or an antigen - binding fragment thereof (AB1) that binds to a first immune effector cell - conjugating target, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the targeting antibody or an antigen - binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen - binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the antibody or an antigen - binding fragment thereof that engages the immune effector cell comprises a first antibody or an antigen - binding fragment thereof (AB1) that binds to a first immune effector cell - conjugating target, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind to the first target, and the targeting antibody or an antigen - binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen - binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the non - immune effector cell - conjugating antibody is a cancer - targeting antibody. In some embodiments, the non - immune cell effector antibody is IgG. In some embodiments, the immune effector cell - conjugating antibody is scFv. In some embodiments, the targeting antibody (e.g., non - immune cell effector antibody) is IgG and the immune effector cell - conjugating antibody is scFv. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell is a myeloid monocyte.

[0331] In some embodiments, the T cell engaging multispecific activatable antibodies of the present disclosure comprise a targeting antibody or an antigen-binding fragment thereof and a T cell engaging antibody or an antigen-binding portion thereof, wherein at least one of the targeting antibody or an antigen-binding fragment thereof and / or the T cell engaging antibody or an antigen-binding portion thereof is masked. In some embodiments, the T cell engaging antibody or an antigen-binding fragment thereof comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the targeting antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof that comprises a second antibody or an antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the T cell engaging antibody or an antigen-binding fragment thereof comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind to the first target, and the targeting antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof that comprises a second antibody or an antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind to the second target.

[0332] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-cancer antibody or an antigen-binding fragment thereof and a T cell engaging antibody or an antigen-binding portion thereof, wherein at least one of the anti-cancer antibody or an antigen-binding fragment thereof and / or the T cell engaging antibody or an antigen-binding portion thereof is masked. In some embodiments, the T cell engaging antibody or an antigen-binding fragment thereof comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the anti-cancer antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds to a second cancer-related target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind to the second cancer-related target. In some embodiments, the T cell engaging antibody or an antigen-binding fragment thereof comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind to the first target, and the anti-cancer antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds to a second cancer-related target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind to the second cancer-related target.

[0333] In some embodiments, the T cell engaging multispecific activatable antibody comprises a cancer-targeting IgG antibody or an antigen-binding fragment thereof and a T cell engaging scFv, wherein at least one of the cancer-targeting IgG antibody or an antigen-binding fragment thereof and / or the T cell engaging antibody or an antigen-binding portion thereof is masked. In some embodiments, the T cell engaging antibody or an antigen-binding fragment thereof comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the cancer-targeting IgG antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind to the second cancer-associated target. In some embodiments, the T cell engaging antibody or an antigen-binding fragment thereof comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind to the first target, and the cancer-targeting IgG antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0334] In some embodiments of the immune effector cell engaging multispecific activatable antibodies, one antigen is typically an antigen present on tumor cells or other cell types associated with a disease, such as but not limited to any of the targets listed in Table 2, such as but not limited to EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and the other antigen is typically a stimulatory or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myeloid monocytes, macrophages, and / or other immune effector cells, such as but not limited to B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of T cells or NK cells; examples of such stimulatory receptors include but are not limited to CD3, CD27, CD28, CD137 (also known as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments, the antigen is an inhibitory receptor present on the surface of T cells; examples of such inhibitory receptors include but are not limited to BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and KIR expressed by NK. The antibody domain conferring antigen specificity on the surface of T cells can also be replaced by a ligand or ligand domain that binds to a T cell receptor, NK cell receptor, macrophage receptor, and / or other immune effector cell receptor (such as but not limited to B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9).

[0335] One embodiment of the present disclosure is a multispecific activatable antibody that is activatable in the cancer microenvironment and includes an antibody against a tumor target (e.g., IgG or scFv) and an agonist antibody against a costimulatory receptor expressed on the surface of activated T cells or NK cells (e.g., IgG or scFv), wherein at least one of the cancer target antibody and / or the agonist antibody is masked. Examples of costimulatory receptors include, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, and OX40. In this embodiment, once activated by a tumor-associated protease, the multispecific activatable antibody will effectively crosslink and activate the costimulatory receptors expressed on T cells or NK cells in a tumor-dependent manner to enhance the activity of T cells that respond to any tumor antigen through their endogenous T cell antigen or NK activation receptors. The activation-dependent nature of these T cell or NK cell costimulatory receptors will focus the activity of the activated multispecific activatable antibody on tumor-specific T cells without activating all T cells independent of their antigen specificity. In one embodiment, at least the costimulatory receptor antibody of the multispecific activatable antibody is masked to prevent the activation of autoreactive T cells that may be present in tissues that also express the antigen recognized by the antibody against the tumor target in the multispecific activatable antibody, but whose activity is restricted due to the lack of coreceptor engagement.

[0336] One embodiment of the present disclosure is a multispecific activatable antibody that is activatable in diseases characterized by T cell overstimulation, such as, but not limited to, autoimmune diseases or inflammatory disease microenvironments. Such a multispecific activatable antibody comprises an antibody against a target that includes a surface antigen expressed in a tissue targeted by T cells in an autoimmune or inflammatory disease, e.g., IgG or scFv; and an antibody against an inhibitory receptor expressed on the surface of T cells or NK cells, e.g., IgG or scFv, wherein at least one of the disease tissue target antibody and / or the T cell inhibitory receptor antibody is masked. Examples of inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and KIR expressed by NK. Examples of tissue antigens targeted by T cells in autoimmune diseases include, but are not limited to, surface antigens expressed on myelin or nerve cells in multiple sclerosis or surface antigens expressed on pancreatic islet cells in type 1 diabetes. In this embodiment, when localized in a tissue under autoimmune attack or inflammation, the multispecific activatable antibody is activated and co-engages with the T cell or NK cell inhibitory receptor to inhibit the activity of autoreactive T cells that respond to any disease tissue-targeted antigen through their endogenous TCR or activation receptors. In one embodiment, at least one or more antibodies are masked to prevent the inhibition of T cell responses in non-disease tissues that may also express the target antigen.

[0337] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-CD3ε (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the targeting antibody or antigen-binding portion thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind the second target.

[0338] In some embodiments, the T cell engaging multi-specific activatable antibody comprises an anti-CD3ε scFv and an antibody targeting cancer or an antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the antibody targeting cancer or its antigen-binding portion is masked. In some embodiments, the CD3ε scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the antibody targeting cancer or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the antibody targeting cancer or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0339] In some embodiments, the T cell engaging multi-specific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting IgG antibody or an antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting IgG antibody or its antigen-binding portion is masked. In some embodiments, the CD3ε scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting IgG antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody IgG or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0340] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-CD3ε (CD3ε) scFv derived from OKT3, wherein at least one of the targeting antibody or its antigen-binding fragment and / or the OKT3 scFv or OKT3-derived scFv is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the targeting antibody or its antigen-binding fragment comprises a second antibody or its fragment, the second antibody or its fragment comprising a second antibody or its antigen-binding fragment (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or its antigen-binding fragment comprises a second antibody or its fragment, the second antibody or its fragment comprising a second antibody or its antigen-binding fragment (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second target.

[0341] In some embodiments, the T cell engaging multi-specific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and an anti-cancer antibody or an antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or the OKT3-derived scFv and / or the anti-cancer antibody or its antigen-binding portion is masked. In some embodiments, the OKT3 scFv or the OKT3-derived scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the anti-cancer antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the OKT3 scFv or the OKT3-derived scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CD3ε, and the anti-cancer antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0342] In some embodiments, the T cell engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting IgG antibody or an antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or the OKT3-derived scFv and / or the cancer-targeting IgG antibody or its antigen-binding portion is masked. In some embodiments, the OKT3 scFv or the OKT3-derived scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting IgG antibody or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the OKT3 scFv or the OKT3-derived scFv comprises a first antibody or an antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody IgG or an antigen-binding fragment thereof comprises a second antibody or a fragment thereof, the second antibody or a fragment thereof comprising a second antibody or an antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0343] In some embodiments, the T cell engaging multi-specific activatable antibody comprises an anti-CTLA-4 scFv, wherein at least one of the targeting antibody or its antigen-binding fragment and / or the anti-CTLA-4 scFv is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds CTLA-4, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CTLA-4. In some embodiments, the targeting antibody or its antigen-binding fragment comprises a second antibody or its fragment, and the second antibody or its fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds CTLA-4, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting antibody or its antigen-binding fragment comprises a second antibody or its fragment, and the second antibody or its fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second target.

[0344] In some embodiments, the T cell engaging multi-specific activatable antibody comprises an anti-CTLA-4 scFv and a targeting IgG antibody or its antigen-binding fragment, wherein at least one of the anti-CTLA-4 scFv and / or the targeting IgG antibody or its antigen-binding portion is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds CTLA-4, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CTLA-4. In some embodiments, the targeting IgG antibody or its antigen-binding fragment comprises a second antibody or its fragment, and the second antibody or its fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds CTLA-4, wherein AB1 is attached to a masking moiety (MM1) such that the conjugation of MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting antibody IgG or its antigen-binding fragment comprises a second antibody or its fragment, and the second antibody or its fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that the conjugation of MM2 reduces the ability of AB2 to bind the second target.

[0345] In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody comprises at least a first antibody or an antigen-binding fragment thereof that binds to a first target and / or a first epitope and a second antibody or an antigen-binding fragment thereof that binds to a second target and / or a second epitope. In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different targets. In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different epitopes on the same target. In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to a combination of two or more different targets and two or more different epitopes on the same target.

[0346] In some embodiments, the multi-specific activatable antibody comprising IgG has a masked IgG variable domain. In some embodiments, the multi-specific activatable antibody comprising scFv has a masked scFv domain. In some embodiments, the multi-specific activatable antibody has an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. In some embodiments, the multi-specific activatable antibody has an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking moiety. In some embodiments, the multi-specific activatable antibody has an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. In some embodiments, the multi-specific activatable antibody has an IgG variable domain and an scFv domain, wherein each of the IgG variable domain and the scFv domain is coupled to its own masking moiety. In some embodiments, one antibody domain of the multi-specific activatable antibody is specific for a target antigen and the other antibody domain is specific for a T cell surface antigen. In some embodiments, one antibody domain of the multi-specific activatable antibody is specific for a target antigen and the other antibody domain is specific for another target antigen. In some embodiments, one antibody domain of the multi-specific activatable antibody is specific for an epitope of a target antigen and the other antibody domain is specific for another epitope of the target antigen.

[0347] In a multi-specific activatable antibody, the scFv can be fused to the carboxyl terminus of the heavy chain of the IgG activatable antibody, the carboxyl terminus of the light chain of the IgG activatable antibody, or the carboxyl termini of the heavy and light chains of the IgG activatable antibody. In a multi-specific activatable antibody, the scFv can be fused to the amino terminus of the heavy chain of the IgG activatable antibody, the amino terminus of the light chain of the IgG activatable antibody, or the amino termini of the heavy and light chains of the IgG activatable antibody. In a multi-specific activatable antibody, the scFv can be fused to any combination of one or more carboxyl termini and one or more amino termini of the IgG activatable antibody. In some embodiments, a masking moiety (MM) linked to a CM1-CM2 substrate is attached to and masks the antigen-binding domain of the IgG. In some embodiments, a masking moiety (MM) linked to a CM1-CM2 substrate is attached to and masks the antigen-binding domain of at least one scFv. In some embodiments, a masking moiety (MM) linked to a CM1-CM2 substrate is attached to and masks the antigen-binding domain of the IgG, and a masking moiety (MM) linked to a CM1-CM2 substrate is attached to and masks the antigen-binding domain of at least one scFv.

[0348] The present disclosure provides examples of multispecific activatable antibody constructs, including but not limited to the following: (VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2; (VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2; (MM-LP1-CM1-CM2 substrate-LP2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*)2; (MM-LP1-CM1-CM2 substrate-LP2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*)2; (VL-CL)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (MM-LP1-CM1-CM2 substrate-LP2-VL-CL)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (MM-LP1-CM1-CM2 substrate-LP2-VL-CL)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(VH-CH1-CH2-CH3)2; (MM-LP1-CM 1-CM2 substrate-LP2-VL*-L3-VH*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; or (VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2, wherein: VL and VH represent the light and heavy variable domains with the first specificity contained in IgG; VL* and VH* represent the variable domains with the second specificity contained in scFv; LP1 is a linker peptide connecting the masking moiety (MM) and the CM1-CM2 substrate; LP2 is a linker peptide connecting the CM1-CM2 substrate and the antibody; L3 is a linker peptide connecting the variable domains of scFv; L4 is a linker peptide connecting the antibody with the first specificity to the antibody with the second specificity; CL is the light chain constant domain;And CH1, CH2, and CH3 are heavy chain constant domains. The first specificity and the second specificity can be directed against any antigen or epitope.;

[0349] In some embodiments of the T cell engaging multispecific activatable antibody, one antigen is typically an antigen present on the surface of tumor cells or other cell types associated with a disease, such as but not limited to any of the targets listed in Table 2, such as but not limited to EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and the other antigen is typically a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myeloid monocytes, macrophages, and / or other immune effector cells, such as but not limited to B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also known as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain conferring surface antigen specificity to T cells can also be replaced by a ligand or ligand domain that binds to a T cell receptor, NK cell receptor, macrophage receptor, and / or other immune effector cell receptor (such as but not limited to B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9). In some embodiments of the multi-antigen targeting activatable antibody, one antigen is selected from the group of targets listed in Table 2, and the other antigen is selected from the group of targets listed in Table 2.

[0350] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v4, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v6, which is specific for binding to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which is specific for binding to human and mouse Jagged1 and Jagged2. In some embodiments, the targeting antibody is 4D11v2, which is specific for binding to human and mouse Jagged1 and Jagged2.

[0351] In some embodiments, the targeting antibody can be in the form of an activatable antibody. In some embodiments, one or more scFvs can be in the form of Pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0352] In some embodiments, the scFv pair is specific for binding CD3ε and is or is derived from an antibody or fragment thereof that binds CD3ε, such as CH2527, FN18, H2C, OKT3, 2C11, UCHT1 or V9. In some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).

[0353] In some embodiments, the anti-CTLA-4 scFv comprises the following amino acid sequence:

[0354] GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVTVSSAS (SEQ ID NO:347)

[0355] In some embodiments, the anti-CTLA-4 scFv comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:347.

[0356] In some embodiments, the anti-CD3ε scFv comprises the following amino acid sequence:

[0357] GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR(SEQ ID NO:349)

[0358] In some embodiments, the anti-CD3ε scFv comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:349.

[0359] In some embodiments, the scFv is specific for binding to one or more T cells, one or more NK cells, and / or one or more macrophages. In some embodiments, the scFv is specific for binding to a target selected from the group consisting of: B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3 or VISTA.

[0360] In some embodiments, the multispecific activatable antibody further comprises an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the multispecific activatable antibody via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agents. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is a raucousine or a derivative thereof. In some embodiments, the agent is raucousine E or a derivative thereof. In some embodiments, the agent is monomethyl raucousine E (MMAE). In some embodiments, the agent is monomethyl raucousine D (MMAD). In some embodiments, the agent is a maytansine alkaloid or a maytansine alkaloid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0361] In some embodiments, the multispecific activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0362] In some embodiments, the multispecific activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the multispecific activatable antibody can be engineered to contain one or more disulfide bonds.

[0363] The present disclosure also provides an isolated nucleic acid molecule encoding the multispecific activatable antibody described herein, and a vector comprising these isolated nucleic acid sequences. The present disclosure provides a method for producing a multispecific activatable antibody, which is carried out by culturing cells under conditions that result in the expression of the activatable antibody, wherein the cells comprise such a nucleic acid molecule. In some embodiments, the cells comprise such a vector.

[0364] The present disclosure also provides a method of making the multispecific activatable antibodies of the present disclosure, which proceeds by the following steps: (a) culturing cells comprising a nucleic acid construct encoding a multispecific activatable antibody under conditions that result in the expression of the multispecific activatable antibody, and (b) recovering the multispecific activatable antibody.

[0365] The present disclosure also provides multispecific activatable antibodies and / or multispecific activatable antibody compositions that comprise at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds a first target or first epitope and a second antibody or an antigen-binding fragment thereof (AB2) that binds a second target or second epitope, wherein at least AB1 is coupled to or otherwise attached to a masking moiety (MM1) such that the coupling of MM1 reduces the ability of AB1 to bind its target. In some embodiments, MM1 is coupled to AB1 via the CM1-CM2 substrate of MMP and SP, wherein at least one of MMP and SP is co-localized with the target of AB1 at a therapeutic or diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, are activated at a desired therapeutic and / or diagnostic site, but are not activated in normal tissue (i.e., healthy tissue), and upon activation exhibit binding to the target of AB1 that is at least comparable to that of the corresponding unmodified multispecific antibody.

[0366] In some embodiments, the multispecific activatable antibody comprises a linker peptide between MM1 and the CM1-CM2 substrate.

[0367] In some embodiments, the multispecific activatable antibody comprises a linker peptide between the CM1-CM2 substrate and AB1.

[0368] In some embodiments, the activatable antibody comprises a first linker peptide (LP1) and a second linker peptide (LP2), and at least a portion of the multispecific activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in an uncleaved state: MM1-LP1-CM1-CM2 substrate-LP2-AB1 or AB1-LP2-CM1-CM2 substrate-LP1-MM1. In some embodiments, the two linker peptides need not be identical to each other.

[0369] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO:381) and (GGGS) n(SEQ ID NO:382), where n is an integer of at least 1. In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: GGSG (SEQ ID NO:383), GGSGG (SEQ ID NO:384), GSGSG (SEQ ID NO:385), GSGGG (SEQ ID NO:386), GGGSG (SEQ ID NO:387), and GSSSG (SEQ ID NO:388).

[0370] In some embodiments, the activatable antibody comprises a linker peptide (LP') between CM1 and CM2.

[0371] In some embodiments, the activatable antibody comprises a first linker peptide (LP1), a second linker peptide (LP2), and a linker peptide (LP') between CM1 and CM2, and at least a portion of the multispecific activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: MM1-LP1-CM1-CM2 substrate-LP2-AB1 or AB1-LP2-CM1-CM2 substrate-LP1-MM1. In some embodiments, the linker peptides do not need to be identical to each other.

[0372] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO:218).

[0373] In some embodiments, the multispecific activatable antibody comprises at least a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to a first target or a first epitope and a second antibody or an antigen-binding fragment thereof (AB2) that specifically binds to a second target or a second epitope. In some embodiments, each AB in the multispecific activatable antibody is independently selected from the group consisting of: monoclonal antibody, domain antibody, single-chain, Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single-domain heavy-chain antibody, and single-domain light-chain antibody. In some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., mouse or rat) antibody, chimeric antibody, humanized antibody, or fully human monoclonal antibody.

[0374] In some embodiments, the dissociation constant of each AB in the multispecific activatable antibody binding to its corresponding target or epitope is about 100 nM or less.

[0375] In some embodiments, the dissociation constant of MM1 binding to its corresponding AB is greater than the dissociation constant of the AB binding to its corresponding target or epitope.

[0376] In some embodiments, the dissociation constant of MM1 binding to its corresponding AB does not exceed the dissociation constant of the AB binding to its corresponding target or epitope.

[0377] In some embodiments, when the multispecific activatable antibody is in a cleaved state, MM1 does not interfere with or compete for the binding of its corresponding AB to the corresponding target or epitope.

[0378] In some embodiments, MM1 is a polypeptide having a length of about 2 to 40 amino acids. In some embodiments, each MM in the multispecific activatable antibody is a polypeptide having a length not exceeding 40 amino acids.

[0379] In some embodiments, the polypeptide sequence of MM1 is different from the sequence of the target of the corresponding AB.

[0380] In some embodiments, the polypeptide sequence of MM1 is not more than 50% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of MM1 is not more than 25% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of MM1 is not more than 10% identical to any natural binding partner of the corresponding AB.

[0381] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K d ) of the AB coupled to MM1 for its corresponding target or epitope is at least 20 times greater than the K d of the AB not coupled to MM1 for its corresponding target or epitope.

[0382] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K d ) of the AB coupled to MM1 for its corresponding target or epitope is at least 40 times greater than the K d of the AB not coupled to MM1 for its corresponding target or epitope.

[0383] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K d ) of the AB coupled to MM1 for its corresponding target or epitope is at least 100 times greater than the K d of the AB not coupled to MM1 for its corresponding target or epitope.

[0384] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K d ) of the AB coupled to MM1 for its corresponding target or epitope is at least 100 times greater than the K dat least 1000-fold greater.

[0385] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (K d ) of the AB conjugated to MM1 for its corresponding target or epitope is d at least 10,000-fold greater than the K

[0386] In some embodiments, MM1 is an amino acid sequence selected from the MMs disclosed herein.

[0387] In some embodiments, the multispecific activatable antibody comprises at least a second masking moiety (MM2) that inhibits the binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state and an additional cleavable moiety (CM′) conjugated to AB2, wherein CM′ is a CM1-CM2 substrate or a polypeptide that serves as a substrate for a second protease. In some embodiments, CM′ is a polypeptide having a length of no more than 15 amino acids. In some embodiments, CM′ is a CM1-CM2 substrate, wherein the lengths of CM1 and CM2 in the CM1-CM2 substrate are each independently no more than 15 amino acids.

[0388] In some embodiments, the MMP protease, the SP protease, and / or the second protease are co-localized in the tissue with the second target or epitope, and wherein when the multispecific activatable antibody is exposed to the MMP protease, the SP protease, and / or the second protease, the MMP protease, the SP protease, and / or the second protease cleave the CM in the multispecific activatable antibody. ′In some embodiments, the MMP protease, the SP protease, and / or the second protease are co-localized in the tissue with the first target or epitope and the second target or epitope. In some embodiments, the MMP protease, the SP protease, and / or the second protease are the same MMP protease and the same SP protease. In some embodiments, the MMP protease, the SP protease, and / or the second protease are not the same MMP protease and are not the same SP protease. In some embodiments, the CM1-CM2 substrate and CM′ are different substrates for the same MMP protease and the same SP protease. In some embodiments, the protease that cleaves CM′ is selected from the group consisting of those shown in Table 1.

[0389] In some embodiments, the dissociation constant of each MM (e.g., MM1 and at least MM2) in the multispecific activatable antibody for binding to its corresponding AB is greater than the dissociation constant of the AB for its corresponding target or epitope.

[0390] In some embodiments, the dissociation constant of each MM in the multispecific activatable antibody binding to its corresponding AB does not exceed the dissociation constant of the AB binding to its corresponding target or epitope.

[0391] In some embodiments, when the multispecific activatable antibody is in the cleaved state, each MM in the multispecific activatable antibody does not interfere with or compete for the binding of its corresponding AB to the corresponding target or epitope.

[0392] In some embodiments, each MM in the multispecific activatable antibody is a polypeptide having a length of about 2 to 40 amino acids. In some embodiments, each MM in the multispecific activatable antibody is a polypeptide having a length not exceeding 40 amino acids.

[0393] In some embodiments, the polypeptide sequence of each MM in the multispecific activatable antibody is different from the sequence of the target of the corresponding AB.

[0394] In some embodiments, the polypeptide sequence of each MM in the multispecific activatable antibody is not more than 50% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of each MM in the multispecific activatable antibody is not more than 25% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of each MM in the multispecific activatable antibody is not more than 10% identical to any natural binding partner of the corresponding AB.

[0395] In some embodiments, the coupling of each MM reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K d ) of the AB coupled with MM to its corresponding target or epitope is at least 20 times greater than the K d of the AB not coupled with MM to its corresponding target or epitope.

[0396] In some embodiments, the coupling of each MM reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K d ) of the AB coupled with MM to its corresponding target or epitope is at least 40 times greater than the K d of the AB not coupled with MM to its corresponding target or epitope.

[0397] In some embodiments, the coupling of each MM reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K d ) of the AB coupled with MM to its corresponding target or epitope is at least 100 times greater than the K d of the AB not coupled with MM to its corresponding target or epitope.

[0398] In some embodiments, the coupling of each MM reduces the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (K d ) of the AB conjugated to MM for its corresponding target or epitope is at least 1000-fold greater than the K d of the AB not conjugated to MM for its corresponding target or epitope.

[0399] In some embodiments, the coupling of each MM reduces the ability of the corresponding AB to bind its target or epitope such that the dissociation constant (K d ) of the AB conjugated to MM for its corresponding target or epitope is at least 10,000-fold greater than the K d of the AB not conjugated to MM for its corresponding target or epitope.

[0400] In some embodiments, each MM is selected from the amino acid sequences of MMs disclosed herein.

[0401] In some embodiments, the protease that cleaves the CM1-CM2 substrate sequence co-localizes in the tissue with the target of AB1 in the multispecific activatable antibody, and when the multispecific activatable antibody is exposed to the protease, the MMP protease and / or the SP protease (i.e., at least one of the MMP protease and the SP protease) cleaves the CM1-CM2 substrate in the multispecific activatable antibody.

[0402] In some embodiments, the multispecific activatable antibody comprises more than one CM1-CM2 substrate sequence, and the MMP protease and / or the SP protease that cleaves at least one CM1-CM2 substrate sequence co-localizes in the tissue with the target of at least one AB region in the multispecific activatable antibody, and when the multispecific activatable antibody is exposed to the protease, the MMP protease and / or the SP protease cleaves the CM1-CM2 substrate in the multispecific activatable antibody.

[0403] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, the occurrence of binding of the multispecific activatable antibody to the target of one AB region is reduced, wherein the dissociation constant is at least two-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0404] In some embodiments, each CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, the occurrence of binding of the multispecific activatable antibody to the target of one AB region is reduced, wherein the dissociation constant is at least three-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0405] In some embodiments, each CM1-CM2 substrate is positioned within a multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of an AB region is reduced, where the dissociation constant is at least four-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0406] In some embodiments, each CM1-CM2 substrate is positioned within a multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of an AB region is reduced, where the dissociation constant is at least five-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0407] In some embodiments, each CM1-CM2 substrate is positioned within a multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of an AB region is reduced, where the dissociation constant is at least ten-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0408] In some embodiments, each CM1-CM2 substrate is positioned within a multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of an AB region is reduced, where the dissociation constant is at least 20-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0409] In some embodiments, each CM1-CM2 substrate is positioned within a multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of an AB region is reduced, where the dissociation constant is at least 40-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0410] In some embodiments, each CM1-CM2 substrate is positioned within a multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of an AB region is reduced, where the dissociation constant is at least 50-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0411] In some embodiments, each CM1-CM2 substrate is positioned within a multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of an AB region is reduced, where the dissociation constant is at least 100-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0412] In some embodiments, each CM1-CM2 substrate is positioned within the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to the target of one of the AB regions occurs less frequently, where the dissociation constant is at least 200-fold greater than the dissociation constant of the unmodified AB binding to its target, and in the cleaved state, the AB binds to its target.

[0413] The present disclosure also provides compositions and methods comprising a multispecific activatable antibody that comprises at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), wherein at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that reduces the ability of AB1 to bind its target. In some embodiments, each AB is coupled to an MM that reduces the ability of its respective AB to bind each target. For example, in an embodiment of a bispecific activatable antibody, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, and AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in such embodiments, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target, AB3 is coupled to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody.

[0414] In some embodiments, the multispecific activatable antibody further comprises a CM1-CM2 substrate that is a substrate for MMP protease and SP protease, wherein the CM1-CM2 substrate links MM to AB. For example, in some embodiments, the multispecific activatable antibody comprises at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), wherein at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that reduces the ability of AB1 to bind its target via a first CM1-CM2 substrate. In some embodiments of bispecific activatable antibodies, AB1 is coupled to MM1 via a first CM1-CM2 substrate, and AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target via a second CM1-CM2 substrate. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in some of these embodiments, AB1 is coupled to MM1 via a first CM1-CM2 substrate, AB2 is coupled to MM2 via a second CM1-CM2 substrate, and AB3 is coupled to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target via a third CM1-CM2 substrate, and so on for each AB in the multispecific activatable antibody.

[0415] Activatable antibody having a non-binding spacer moiety or a binding partner of a non-binding spacer moiety

[0416] The present disclosure also provides activatable antibodies that comprise a non-binding spacer moiety (NB) or a binding partner (BP) of the non-binding spacer moiety, wherein the BP recruits or otherwise attracts the NB to the activatable antibody. The activatable antibodies provided herein include, for example, activatable antibodies that comprise a non-binding spacer moiety (NB), a CM1-CM2 substrate, and an antibody or antibody fragment (AB) that binds a target; activatable antibodies that comprise a binding partner of the non-binding spacer moiety (NB), a CM1-CM2 substrate, and AB; and activatable antibodies that comprise a BP that has recruited the NB, a CM1-CM2 substrate, and AB that binds to the target. An activatable antibody in which the NB is covalently linked to the CM1-CM2 substrate and AB of the activatable antibody or associates by interacting with a BP that is covalently linked to the CM1-CM2 substrate and AB of the activatable antibody is referred to herein as a "NB-containing activatable antibody". Activatable or convertible means that when the activatable antibody is in an inhibited, masked, or uncleaved state (i.e., the first conformation), the activatable antibody exhibits a first level of binding to the target, and when the activatable antibody is in an uninhibited, unmasked, and / or cleaved state (i.e., the second conformation, i.e., the activated antibody), the activatable antibody exhibits a second level of binding to the target, wherein the second target binding level is greater than the first target binding level. Compared to conventional antibody therapies, activatable antibody compositions can exhibit increased bioavailability and more favorable biodistribution.

[0417] In some embodiments, if the AB is not masked or otherwise inhibited from binding to non-therapeutic and / or non-diagnostic sites, the activatable antibody provides reduced toxicity and / or adverse side effects that may otherwise be caused by binding to such sites.

[0418] In one embodiment, the activatable antibody comprises a non-binding spacer portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein the NB is a polypeptide that does not specifically bind to the AB; the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) of an enzyme; the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target; and the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. As used herein and throughout, the term polypeptide refers to any polypeptide comprising at least two amino acid residues, including larger polypeptides, full-length proteins, and fragments thereof, and the term polypeptide is not limited to single-chain polypeptides and may include multi-unit (e.g., multi-chain) polypeptides. In cases where the length of the polypeptide is shorter (e.g., less than 50 amino acids in total), the terms peptide and polypeptide may be used interchangeably herein, and in cases where the length of the polypeptide is longer (e.g., 50 amino acids or greater), the terms polypeptide and protein may be used interchangeably herein.

[0419] In one embodiment, the activatable antibody comprises a non-binding spacer portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide of up to 50 amino acids in length that comprises a substrate (S) of an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target; and (iv) the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. For example, the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate each independently have a length of up to 15 amino acids.

[0420] In one embodiment, an activatable antibody comprises a non-binding spacer portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target; (iv) the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme; and (v) the activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: NB-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-NB.

[0421] In one embodiment, an activatable antibody comprises a non-binding spacer portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target, and wherein the ability of the NB in the uncleaved activatable antibody to enable the AB to bind to the target is reduced by at least 50%, such as at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% as compared to the ability of the cleaved AB to bind to the target; and (iv) the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. For example, an assay as described herein or an in vitro target displacement assay (such as the assays described in PCT Publication Nos. WO 2009 / 025846 and WO2010 / 081173) is used to determine the reduction in the ability of the AB to bind to the target.

[0422] In one embodiment, an activatable antibody comprises a binding partner (BP) of a non-binding spacer moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein BP is a polypeptide that binds to NB upon exposure to NB; NB does not specifically bind to AB; the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) of an enzyme; the CM1-CM2 substrate is positioned such that in the uncleaved state in the presence of NB, NB interferes with the binding of AB to the target, and in the cleaved state, NB does not interfere with the binding of AB to the target and BP does not interfere with the binding of AB to the target; and NB and BP do not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. In some examples of this embodiment, the BP of the activatable antibody optionally binds to NB. In one embodiment, NB is recruited in vivo by the BP of the activatable antibody.

[0423] In some examples of any of these embodiments of activatable antibodies, the activatable antibody is formulated as a composition. In some embodiments of these embodiments, the composition further comprises NB, wherein NB is co-formulated with the activatable antibody comprising BP, CM1-CM2 substrate, and AB. In some examples of this embodiment, BP is selected from the group consisting of albumin-binding peptides, fibrinogen-binding peptides, fibronectin-binding peptides, hemoglobin-binding peptides, transferrin-binding peptides, immunoglobulin domain-binding peptides, and other serum protein-binding peptides.

[0424] In some examples of any of these embodiments of activatable antibodies, NB is a soluble globular protein. In some examples of any of these embodiments of activatable antibodies, NB is a protein that circulates in the bloodstream. In some examples of any of these embodiments of activatable antibodies, NB is selected from the group consisting of albumin, fibrinogen, fibronectin, hemoglobin, transferrin, immunoglobulin domains, and other serum proteins.

[0425] In some instances of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a protease-containing substrate (S). In some instances of any of these activatable antibody embodiments, the protease is co-localized with the tissue, and when the activatable antibody is exposed to the protease, the protease cleaves the CM1-CM2 substrate in the activatable antibody. In some instances of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide up to 50 amino acids in length. In some instances of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a substrate (S) up to 15 amino acids in length (e.g., 3 amino acids long, 4 amino acids long, 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, or 15 amino acids long).

[0426] In some instances of any of these activatable antibody embodiments, the activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: NB-CM1-CM2 substrate-AB, AB-CM1-CM2 substrate-NB, BP-CM1-CM2 substrate-AB, or AB-CM1-CM2 substrate-BP. In embodiments where the activatable antibody comprises BP and the corresponding NB is present in the activatable antibody, the activatable antibody has the following structural arrangement from the N-terminus to the C-terminus in the uncleaved state: NB:BP-CM1-CM2-AB, NB:BP-CM2-CM1-AB, AB-CM1-CM2-BP:NB, or AB-CM2-CM1-BP:NB, where ":" represents the interaction between NB and BP, such as binding.

[0427] In some instances of any of these activatable antibody embodiments, the activatable antibody comprises an antibody or an antigen-binding fragment thereof that specifically binds to a given target, and is a monoclonal antibody, a domain antibody, a single-chain, a Fab fragment, an F(ab')2 fragment, an scFv, an scab, a dAb, a single-domain heavy-chain antibody, or a single-domain light-chain antibody. In some embodiments, such an antibody or an immunologically active fragment thereof that binds to the target is a murine antibody, other rodent antibody, chimeric antibody, humanized antibody, or fully human monoclonal antibody.

[0428] In some instances of any of these activatable antibody embodiments, the activatable antibody comprises a combination of a variable heavy chain region having an amino acid sequence given herein and a variable light chain region having an amino acid sequence given herein. In some embodiments, the activatable antibody comprises a combination of a variable heavy chain region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence given herein and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence given herein.

[0429] In some instances of any of these activatable antibody embodiments, the activatable antibody further comprises an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the agent is dolastatin. In some embodiments, the agent is rhizoxin or a derivative thereof. In some embodiments, the agent is rhizoxin E or a derivative thereof. In some embodiments, the agent is monomethyl rhizoxin E (MMAE). In some embodiments, the agent is monomethyl rhizoxin D (MMAD). In some embodiments, the agent is a maytansine alkaloid or a maytansine alkaloid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine In some embodiments, the agent is pyrrolobenzodiazepine dimer.

[0430] In some instances of any of these activatable antibody embodiments, the activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0431] In some instances of any of these activatable antibody embodiments, the activatable antibody further comprises a spacer. In some instances of any of these activatable antibody embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some instances of any of these activatable antibody embodiments, the spacer is directly linked to the MM of the activatable antibody.

[0432] In some embodiments, the serum half-life of the activatable antibody is longer than the serum half-life of the corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 15 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 11 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 9 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 7 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 6 days. In some instances of any of these activatable antibody embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 5 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 4 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 3 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 2 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 24 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 20 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 18 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 16 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 14 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 6 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 4 hours. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 3 hours.

[0433] The present disclosure also provides an isolated nucleic acid molecule encoding any one of these activatable antibodies, as well as a vector comprising such isolated nucleic acid sequences. The present disclosure provides methods of generating an activatable antibody, which are carried out by culturing cells under conditions that result in the expression of the activatable antibody, wherein the cells comprise such nucleic acid sequences. In some embodiments, the cells comprise such a vector.

[0434] The dissociation constant (K d ) of the NB-containing activatable antibody for the target is greater than the K d of the AB that does not associate with NB or NB:BP for the target. The dissociation constant (K d ) of the NB-containing activatable antibody for the target is greater than the K d of the parental AB for the target. For example, the K d of the NB-containing activatable antibody for the target is greater than the K d of the AB that does not associate with NB or NB:BP for the target or the K dat least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000-fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1,000,000 fold, 10-10,000,000 fold, 100-1,000 fold, 100-10,000 fold, 100-100,000 fold, 100-1,000,000 fold, 100-10,000,000 fold, 1,000-10,000 fold, 1,000-100,000 fold, 1,000-1,000,000 fold, 1,000-10,000,000 fold, 10,000-100,000 fold, 10,000-1,000,000 fold, 10,000-10,000,000 fold, 100,000-1,000,000 fold or 100,000-10,000,000 fold. Conversely, the binding affinity of an activatable antibody containing an NB for a target is lower than the binding affinity of an AB that does not associate with an NB or NB:BP for the target or lower than the binding affinity of the parental AB for the target. For example, the binding affinity of an activatable antibody containing an NB for a target is lower than the binding affinity of an AB that does not associate with an NB or NB:BP for the target or lower than the binding affinity of the parental AB for the target by at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000-fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1,000,000 fold, 10-10,000,000 fold, 100-1,000 fold, 100-10,000 fold, 100-100,000 fold, 100-1,000,000 fold, 100-10,000,000 fold, 1,000-10,000 fold, 1,000-100,000 fold, 1,000-1,000,000 fold, 1,000-10,000,000 fold, 10,000-100,000 fold, 10,000-1,000,000 fold, 10,000-10,000,000 fold, 100,000-1,000,000 fold or 100,000-10,000,000 fold.

[0435] Compared with the specific binding of AB that does not associate with NB or NB:BP, when the activatable antibody containing NB is in the presence of the target, the specific binding of AB to the target is reduced or inhibited. Compared with the specific binding of the parental AB to the target, when the activatable antibody containing NB is in the presence of the target, the specific binding of AB to the target is reduced or inhibited. When measured in vitro and / or in vivo, compared with the binding of AB that does not associate with NB or NB:BP to the target or the binding of the parental AB to the target, the ability of the activatable antibody containing NB to bind the target is reduced by, for example, at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer.

[0436] Compared with the specific binding of AB that does not associate with NB or NB:BP, when the activatable antibody containing NB is in the presence of the target but in the absence of a modifier (such as a protease or other enzyme), the specific binding of AB to the target is reduced or inhibited. Compared with the specific binding of the parental AB to the target, when the activatable antibody containing NB is in the presence of the target but in the absence of a modifier (such as a protease, other enzyme, reducing agent or light), the specific binding of AB to the target is reduced or inhibited. When measured in vitro and / or in vivo, compared with the binding of AB that does not associate with NB or NB:BP to the target or the binding of the parental AB to the target, the ability of the activatable antibody containing NB to bind the target is reduced by, for example, at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer.

[0437] In some instances of any of these activatable antibody embodiments, the activatable antibody comprises an agent conjugated to the AB to produce an activatable antibody conjugate. In some embodiments of the activatable antibody conjugate, the agent is a therapeutic agent. In some embodiments, the agent is a diagnostic agent. In some embodiments, the agent is a detectable marker. In some embodiments of the activatable antibody conjugate, the agent is an anti-tumor agent. In some embodiments of the activatable antibody conjugate, the agent is a toxin or a fragment thereof. In some embodiments of the activatable antibody conjugate, the agent is conjugated to the AB via a linker. In some embodiments of the activatable antibody conjugate, the linker is a cleavable linker. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agents. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is rhizoxin or a derivative thereof. In some embodiments, the agent is rhizoxin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansine alkaloid or a maytansine alkaloid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is pyrrolobenzodiazepine . In some embodiments, the agent is pyrrolobenzodiazepine dimer.

[0438] In some instances of any of these activatable antibody embodiments, the activatable antibody is a dual-target binding activatable antibody. Such dual-target binding activatable antibodies contain two Abs that can bind the same or different targets. In certain embodiments, the dual-target activatable antibody contains a bispecific antibody or an antibody fragment.

[0439] Design a dual-target binding activatable antibody such that it has a CM1-CM2 substrate that can be cleaved by a cleaving agent, and the cleaving agent is co-localized with one or two targets of an AB that can bind to the activatable antibody in a target tissue. A dual-target binding activatable antibody having more than one AB against the same or different targets can be designed to have more than one CM1-CM2 substrate, where the first CM1-CM2 substrate can be cleaved by a cleaving agent in a first target tissue, and where the second CM1-CM2 substrate can be cleaved by a cleaving agent in a second target tissue, and one or more targets bind to the AB of the activatable antibody. In one embodiment, the first target tissue and the second target tissue are spatially separated, such as at different locations in an organism. In one embodiment, the first target tissue and the second target tissue are the same tissue separated in time, such as the same tissue at two different time points, such as the first time point is when the tissue is an early-stage tumor and the second time point is when the tissue is a late-stage tumor.

[0440] The present disclosure also provides nucleic acid molecules encoding the activatable antibodies described herein. The present disclosure also provides vectors comprising these nucleic acids. The activatable antibodies described herein are produced by culturing cells under conditions that result in the expression of the activatable antibody, where the cells comprise these nucleic acid molecules or vectors.

[0441] The present disclosure also provides a method for making an activatable antibody. In one embodiment, the method comprises the steps of: (a) culturing cells comprising a nucleic acid construct encoding an activatable antibody under conditions that result in the expression of the activatable antibody, where the activatable antibody comprises (i) a non-binding space portion (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or an antigen-binding fragment thereof (AB) that specifically binds to a target, where (1) NB does not specifically bind to AB; (2) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) of an enzyme; (3) the CM1-CM2 substrate is positioned such that in the uncleaved state, NB interferes with the binding of AB to the target, and in the cleaved state, NB does not interfere with the binding of AB to the target; and (4) NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody.

[0442] In some embodiments, the method comprises the steps of: (a) culturing a cell comprising a nucleic acid construct encoding an activatable antibody under conditions that result in the expression of the activatable antibody, wherein the activatable antibody comprises (i) a binding partner (BP) of a non-binding spacer portion (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or an antigen-binding fragment thereof (AB) that specifically binds to a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) of an enzyme; (3) the CM1-CM2 substrate is positioned such that in the uncleaved state of the NB, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target and the BP does not interfere with the binding of the AB to the target; and (4) the NB and the BP do not inhibit the cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody. In some examples of this embodiment, the BP of the activatable antibody binds to the NB.

[0443] Use of activatable antibodies and conjugated activatable antibodies

[0444] It should be understood that the administration of a therapeutic entity according to the present disclosure will be administered in conjunction with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th Edition, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour therein. These formulations include, for examp...

Claims

1. An isolated polypeptide comprising tandem substrates, wherein said tandem substrates comprise at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) or cysteine protease (CP). Wherein CM1 comprises the amino acid sequence AHGL (SEQ ID NO:54) or PRQV (SEQ ID NO:61), and wherein the N-terminus to C-terminus arrangement of said tandem substrates is CM1-CM2 or CM2-CM1.

2. The isolated polypeptide according to claim 1, wherein CM1 comprises an amino acid sequence selected from the group consisting of: ALAHGLF (SEQ ID NO:1), ALAHGL (SEQ ID NO:52), LAHGLF (SEQ ID NO:50), LAHGL (SEQ ID NO:53), and AHGLF (SEQ ID NO:51).

3. The isolated polypeptide according to claim 1, wherein CM1 comprises an amino acid sequence selected from the group consisting of: HVPRQV (SEQ ID NO:8) and VPRQV (SEQ ID NO:60).

4. The isolated polypeptide according to any one of claims 1 to 3, wherein CM1 and CM2 are linked by a linker peptide.

5. The isolated polypeptide according to any one of claims 1 to 3, wherein CM1 and CM2 are directly linked to each other.

6. The isolated polypeptide according to any one of claims 1 to 5, wherein said CM2 comprises a substrate for a CP enzyme, and wherein said CP enzyme is papain.

7. The isolated polypeptide according to any one of claims 1 to 6, wherein said CM2 comprises a substrate for an SP enzyme selected from the group consisting of: urokinase, proteolytic enzyme, and neutrophil elastase.

8. The isolated polypeptide according to any one of claims 1 to 7, wherein CM2 comprises a substrate for an SP enzyme selected from the group consisting of: urokinase, proteolytic enzyme, and neutrophil elastase; and a substrate for a CP enzyme, and wherein said CP enzyme is papain.

9. The isolated polypeptide according to any one of claims 1 to 8, wherein said MMP is MMP2, MMP9, or MMP14.

10. The isolated polypeptide according to any one of claims 1 to 9, wherein CM2 comprises an amino acid sequence selected from the group consisting of: SGR, LSGR (SEQ ID NO:73), ARG, PRS, TFVH (SEQ ID NO:141), AAN, SAN, LPTFV (SEQ ID NO:140), and GPTN (SEQ ID NO:152).

Images