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

Abstract

To provide an isolated polypeptide comprising a tandem substrate, an activatable antibody that specifically binds to a target and a production method therefor, and methods for using these in a variety of therapeutic, diagnostic and prophylactic indications.SOLUTION: The invention provides polypeptides that include at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) or at least one cysteine protease (CP).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 776,409, filed December 6, 2018, and U.S. Provisional Patent Application No. 62 / 778,062, filed December 11, 2018, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to polypeptides comprising at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine ​​protease (CP), to activatable antibodies and other larger molecules that comprise these polypeptides that comprise at least CM1 that is a substrate for at least one MMP protease and CM2 that is a substrate for at least one SP protease and / or at least one CP protease, and to methods for making these polypeptides that comprise at least CM1 that is a substrate for at least one MMP protease and CM2 that is a substrate for at least one SP protease and / or at least one CP protease and to methods for using them in various therapeutic, diagnostic and prophylactic applications.

[0003] Array list reference The "Sequence Listing," filed electronically via EFS-Web under file name "CYTX-058-PCT_ST25" in computer-readable format contemporaneously with the present specification pursuant to 37 CFR § 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 technology]

[0004] Proteases are enzymes that degrade proteins by cleaving peptide bonds between amino acid residues. Proteases are naturally present in all living organisms and are involved in a variety of physiological reactions, from simple degradation to highly regulated pathways. Some proteases are known to cleave specific peptide bonds based on the presence of specific amino acid sequences within the protein. Thus, there is a need to identify novel substrates for proteases and to use these substrates for a variety of therapeutic, diagnostic and prophylactic applications. Summary of the Invention

[0005] In some embodiments, the 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, the 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 via a linking peptide. In some embodiments, the 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 comprising a substrate for a CP enzyme, wherein the CP enzyme is legumain. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 comprising a substrate for 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 comprising a substrate for an SP enzyme selected from the group consisting of urokinase, matriptase, and neutrophil elastase, and a substrate for a CP enzyme. In some embodiments, the isolated polypeptide of the present disclosure comprises CM1 comprising a substrate for an MMP enzyme selected from the group consisting of MMP2, MMP9, or MMP14.

[0006] In some embodiments, the isolated polypeptide of the disclosure comprises a CM2 comprising 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, an isolated polypeptide of the disclosure comprises a CM2 comprising 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), LSGRSNI (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). In some embodiments, an isolated polypeptide of the disclosure comprises a CM2 comprising 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, an isolated polypeptide of the disclosure comprises a CM2 comprising 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, an isolated polypeptide of the disclosure comprises a CM2 comprising an amino acid sequence selected from the group consisting of 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). In some embodiments, an isolated polypeptide of the disclosure comprises a CM2 comprising 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 disclosure comprises a CM2 comprising 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), LSGRSNI (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).

[0007] In some embodiments, the isolated polypeptide of the disclosure has an N-terminal to C-terminal arrangement of tandem substrates CM1-CM2. In some embodiments, the isolated polypeptide of the disclosure has an N-terminal to C-terminal arrangement of tandem substrates CM2-CM1.

[0008] In some embodiments, the isolated polypeptide of the present disclosure has a cleavability of the first cleavable portion of CM1 by MMP9 and MMP14, respectively, that is at least 80%. In some embodiments, the isolated polypeptide of the present disclosure has a cleavability of the first cleavable portion of CM1 by MMP9 and MMP14, respectively, that is at least 85%. In some embodiments, the isolated polypeptide of the present disclosure has a cleavability of the first cleavable portion of CM1 by MMP9 and MMP14, respectively, that is at least 90%.

[0009] In some embodiments, the isolated polypeptide of the present disclosure has an in vivo stability of the first cleavable portion of CM1 that is less than 30% active. In some embodiments, the isolated polypeptide of the present disclosure has an in vivo stability of the first cleavable portion of CM1 that is less than 25% active.

[0010] In some embodiments, the isolated polypeptide of the present disclosure has a tandem substrate cleavability by MMP9 and MMP14, respectively, that is at least 30%. In some embodiments, the isolated polypeptide of the present disclosure has a tandem substrate cleavability by MMP9 and MMP14, respectively, that is at least 50%. In some embodiments, the isolated polypeptide of the present disclosure has a tandem substrate cleavability by MMP9 and MMP14, respectively, that is at least 70%.

[0011] In some embodiments, the isolated polypeptide of the present disclosure has a tandem substrate cleavability by MMP9 and MMP14, respectively, that is at least 15%, and by matriptase, that is at least 30%. In some embodiments, the isolated polypeptide of the present disclosure has a tandem substrate cleavability by MMP9 and MMP14, respectively, that is at least 30%, and by matriptase, that is at least 30%. In some embodiments, the isolated polypeptide of the present disclosure has a tandem substrate cleavability by MMP9 and MMP14, respectively, that is at least 50%, and by matriptase, that is at least 50%. In some embodiments, the isolated polypeptide of the present disclosure has a tandem substrate cleavability by MMP9 and MMP14, respectively, that is at least 70%, and by matriptase, that is at least 70%.

[0012] In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability of activation of less than 40%. In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability of activation of less than 30%. In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability of activation of less than 25%. In some embodiments, the isolated polypeptides of the present disclosure have a tandem substrate with an in vivo stability of activation of less than 20%.

[0013] In some embodiments, an isolated polypeptide of the present disclosure comprises a tandem substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43. In some embodiments, an isolated polypeptide of the present disclosure comprises a tandem substrate comprising 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, an isolated polypeptide of the present disclosure comprises a tandem substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35. In some embodiments, an isolated polypeptide of the present disclosure comprises a tandem substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 29, 31, 36, and 37. In some embodiments, an isolated polypeptide of the present disclosure comprises a tandem substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, and 34. In some embodiments, an isolated polypeptide of the present disclosure comprises a tandem substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 33. In some embodiments, the isolated polypeptide of the present disclosure comprises a tandem substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35.

[0014] In some embodiments, provided herein is an isolated polypeptide of the present disclosure comprising an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP), and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) or cysteine ​​protease (CP), wherein CM1 comprises the amino acid sequence AHGL or PRQV, and the arrangement of the tandem substrate from N- to C-terminus is CM1-CM2 or CM2-CM1. In some embodiments, at least one of the MMP, CP, and SP co-localizes with the target in a tissue. In some embodiments, the isolated polypeptide of the present disclosure comprises an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB linked to CM1. In some embodiments, an isolated polypeptide of the present disclosure comprises an AB directly linked to CM1. In some embodiments, an isolated polypeptide of the present disclosure comprises an AB linked to CM1 via a connecting peptide. In some embodiments, an isolated polypeptide of the present disclosure comprises an AB linked to CM2. In some embodiments, an isolated polypeptide of the present disclosure comprises an AB directly linked to CM2. In some embodiments, an isolated polypeptide of the present disclosure comprises an AB linked to CM2 via a connecting peptide. In some embodiments, an isolated polypeptide of the present disclosure comprises an AB comprising an antibody light chain variable region, or an antigen-binding fragment thereof, wherein CM1 or CM2 is linked to the N-terminus of the light chain variable region of AB. In some embodiments, an isolated polypeptide of the present disclosure comprises an AB comprising an antibody heavy chain variable region, or an antigen-binding fragment thereof, wherein CM1 or CM2 is linked to the N-terminus of the heavy chain variable region of AB.

[0015] In some embodiments, the isolated polypeptide of the present disclosure comprises a masking moiety (MM). In some embodiments, the isolated polypeptide of the present disclosure comprises a MM that has a higher dissociation constant for binding to AB than the dissociation constant for binding of AB to its target. In some embodiments, the isolated polypeptide of the present disclosure comprises a MM that is a polypeptide 40 amino acids in length or less. In some embodiments, the isolated polypeptide of the present disclosure comprises a MM linked to CM1 such that in its uncleaved state, the isolated polypeptide comprises the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, the isolated polypeptide of the present disclosure comprises a linking peptide between MM and CM1. In some embodiments, the isolated polypeptide of the present disclosure comprises a linking peptide between CM2 and AB. In some embodiments, the isolated polypeptide of the present disclosure comprises a linking peptide between MM and CM1 and a linking peptide between CM2 and AB. In some embodiments, an isolated polypeptide of the disclosure comprises MM linked to CM1, such that the isolated polypeptide in its uncleaved state comprises the following structural arrangement from N-terminus to C-terminus: MM-CM2-CM1-AB or AB-CM1-CM2-MM. In some embodiments, an isolated polypeptide of the disclosure comprises a linking peptide between MM and CM2. In some embodiments, an isolated polypeptide of the disclosure comprises a linking peptide between CM1 and AB. In some embodiments, an isolated polypeptide of the disclosure comprises a linking peptide between MM and CM2 and a linking peptide between CM1 and AB.

[0016] In some embodiments, an isolated polypeptide of the present disclosure comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the isolated polypeptide has the following structural configuration from N-terminus to C-terminus in its uncleaved state: 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 connecting peptides are not identical to each other. In some embodiments, each of LP1 and LP2 is a peptide about 1-20 amino acids in length. In some embodiments, the isolated polypeptide comprises a third connecting peptide (LP') between CM1 and CM2. In some embodiments, the isolated polypeptide of the present disclosure comprises AB comprising an antibody light chain variable region, or an antigen-binding fragment thereof, and LP2 is linked to the N-terminus of the light chain variable region of AB. In some embodiments, an isolated polypeptide of the disclosure comprises an AB comprising a heavy chain variable region of an antibody or an antigen-binding fragment thereof, wherein LP2 is linked to the N-terminus of the heavy chain variable region of the AB.

[0017] In some embodiments, the isolated polypeptide of the disclosure comprises a MM whose amino acid sequence differs from that of the target and is 10% or less identical to the amino acid sequence of the natural binding partner of the AB. In some embodiments, the isolated polypeptide of the disclosure comprises a MM that, in its cleaved state, does not interfere with or compete with the AB for binding to the target.

[0018] In some embodiments, the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450 to 462. In some embodiments, the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450 to 462, and a heavy chain amino acid sequence comprising SEQ ID NO: 400.

[0019] In another aspect of the present invention, provided herein is an activatable antibody that, in an activated state, specifically binds to a target, the activatable antibody comprising: an antibody or antigen-binding fragment (AB) that specifically binds to the target; a masking moiety (MM) attached to the AB, the MM inhibiting binding of the AB to the target when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) comprising a tandem substrate according to any one of the tandem substrates provided herein. In some embodiments, the activatable antibody comprises a MM that has a dissociation constant for binding to the AB that is higher than the dissociation constant for binding of the AB to the target. In some embodiments, the activatable antibody comprises a MM that is a polypeptide 40 amino acids or less in length. In some embodiments, the activatable antibody comprises an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. In some embodiments, the activatable antibody comprises a MM that has a dissociation constant for binding to the AB that is higher than the dissociation constant for binding of the AB to the target.

[0020] In some embodiments, the activatable antibody comprises MM linked to CM1, such that the activatable antibody in its uncleaved state comprises the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, the activatable antibody comprises a linking peptide between MM and CM1. In some embodiments, the activatable antibody comprises a linking peptide between CM2 and AB. In some embodiments, the activatable antibody comprises a linking peptide between MM and CM1 and a linking peptide between CM2 and AB.

[0021] In some embodiments, the activatable antibody comprises MM linked to CM1, such that the activatable antibody in its uncleaved state comprises the following structural arrangement from N-terminus to C-terminus: MM-CM2-CM1-AB or AB-CM1-CM2-MM. In some embodiments, the activatable antibody comprises a linking peptide between MM and CM2. In some embodiments, the activatable antibody comprises a linking peptide between CM1 and AB. In some embodiments, the activatable antibody comprises a linking peptide between MM and CM2 and a linking peptide between CM1 and AB.

[0022] In some embodiments, the activatable antibody comprises an AB comprising a light chain variable region of an antibody or an antigen-binding fragment thereof, and CM1 or CM2 is linked to the N-terminus of the light chain variable region of AB. In some embodiments, the activatable antibody comprises an AB comprising a heavy chain variable region of an antibody or an antigen-binding fragment thereof, and CM1 or CM2 is linked to the N-terminus of the heavy chain variable region of AB.

[0023] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody has the following structural configuration from N-terminus to C-terminus in its uncleaved state: 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 connecting peptides are not identical to each other. In some embodiments, each of LP1 and LP2 is a peptide approximately 1-20 amino acids in length. In some embodiments, the activatable antibody comprises a third connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises AB comprising an antibody light chain variable region, or an antigen-binding fragment thereof, and LP2 is linked to the N-terminus of the light chain variable region of AB. In some embodiments, the activatable antibody comprises an AB comprising a heavy chain variable region of an antibody or an antigen-binding fragment thereof, and LP2 is linked to the N-terminus of the heavy chain variable region of the AB.

[0024] In some embodiments, the activatable antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450 to 462. In some embodiments, the activatable antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450 to 462, and a heavy chain amino acid sequence comprising SEQ ID NO: 400.

[0025] In some embodiments, the activatable antibody comprises a MM that differs from the amino acid sequence of the target and is 10% or less identical to the amino acid sequence of the natural binding partner of the AB, hi some embodiments, the activatable antibody comprises a MM that, in the cleaved state, does not interfere with or compete with the AB for binding to the target.

[0026] In another aspect of the invention, provided herein is a conjugated activatable antibody comprising an activatable antibody conjugated to a drug. In some embodiments, the drug is conjugated to AB via a linker. In some embodiments, the drug is conjugated to AB via a cleavable linker. In some embodiments, the drug is conjugated to AB via a non-cleavable linker.

[0027] 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 a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, and a calicheamicin or a derivative thereof, auristatin E or a derivative thereof, monomethylauristatin E (MMAE), monomethylauristatin 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.

[0028] In another aspect of the invention, provided herein is a pharmaceutical composition comprising an isolated polypeptide of this disclosure, or an activatable antibody of this disclosure, or a conjugated activatable antibody of this disclosure, and a carrier. In some embodiments, the pharmaceutical composition comprises an additional agent. In some embodiments, the pharmaceutical composition comprises an additional agent that is a therapeutic agent.

[0029] In another aspect of the invention, provided herein is an isolated nucleic acid molecule encoding an isolated polypeptide of this disclosure or an activatable antibody of this disclosure. In some embodiments, a vector comprises the isolated nucleic acid molecule of this disclosure.

[0030] In another aspect of the invention, provided herein is a method of producing an antibody or activatable antibody by culturing cells under conditions that result in expression of an isolated polypeptide of this disclosure or an activatable antibody of this 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 containing a nucleic acid construct encoding an activatable antibody of this disclosure and recovering the activatable antibody.

[0031] In another aspect of the present invention, provided herein are methods of treating, alleviating the symptoms of, or slowing the progression of a disorder or disease, comprising administering a therapeutically effective amount of an isolated polypeptide of this disclosure, or an activatable antibody of this disclosure, or a conjugated activatable antibody of this disclosure, or a pharmaceutical composition of this disclosure to a subject in need thereof. 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 explanation of the drawings]

[0032] [Figure 1]Graphs showing exemplary results of the percent of the indicated activatable anti-EGFR antibodies of the present disclosure observed to be cleaved in vivo following their administration to nu / nu mice. These exemplary results showed that some of the activatable antibodies tested exhibited greater stability than other substrates that are cleavable by multiple enzymes. [Figure 2] 2A and 2B are graphs showing exemplary results of in vitro binding of the indicated anti-EGFR activatable antibodies of the present disclosure to EGFR. These exemplary results demonstrate that MMP substrates affect the masking efficiency of the prodomain of the activatable antibody. [Figure 3] 3A and 3B are graphs showing exemplary results of in vivo efficacy of the indicated anti-EGFR activatable antibodies of the present disclosure using a mouse H292 xenograft model. These exemplary results demonstrate that activatable antibodies with specific MMP substrates of the present disclosure demonstrated efficacy in this xenograft model that was comparable to unmasked anti-EGFR cetuximab. [Figure 4] 1 is a graph showing exemplary results of the percent of the indicated activatable anti-EGFR antibodies of the disclosure that were activated in intratumoral tissue following administration to a mouse H292 xenograft model. [Figure 5] Graphs showing exemplary results of the percentage of the indicated activatable anti-EGFR antibodies of the present disclosure with single MMP or tandem substrates of the present disclosure that were observed to be cleaved in vivo after their administration to nu / nu mice. These exemplary results showed that some of the activatable antibodies with tandem substrates tested exhibited greater in vivo stability than other substrates that are cleavable by multiple enzymes. [Figure 6] Graphs showing exemplary results of in vivo efficacy of the indicated anti-EGFR activatable antibodies of the present disclosure using a mouse H292 xenograft model. These exemplary results showed that activatable antibodies with specific MMP substrates of the present disclosure demonstrated efficacy in this xenograft model that was comparable to that of unmasked anti-EGFR cetuximab antibodies. [Figure 7]1 is a graph showing exemplary results of the percent of the indicated activatable anti-EGFR antibodies of the disclosure that were activated in intratumoral tissue following administration to a mouse H292 xenograft model. [Figure 8] 1 is a graph showing exemplary results of calculated masking efficiencies calculated from in vitro binding of the indicated anti-EGFR activatable antibodies of the present disclosure to EGFR. These exemplary results showed that tandem substrates affected the masking efficiency of the pro domain of the activatable antibody. [Figure 9] 1 is a graph showing exemplary results of the percent of the indicated activatable anti-EGFR antibodies of the disclosure that were activated after incubation with human tumor tissue. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure provides amino acid sequences comprising at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) 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 imply any requirement regarding the orientation or other structural arrangement of the first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and the at least second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine ​​protease (CP). Thus, the term "tandem substrate" encompasses CM1-CM2 substrates having an N-terminal to C-terminal structural arrangement, such as CM1-CM2 or CM2-CM1. The term "tandem substrate" also encompasses substrates in which at least a portion of the CM1 sequence overlaps at least a portion of the CM2 sequence.

[0034] In some embodiments, CM2 comprises at least two substrates. In some embodiments, CM2 comprises a substrate for a first serine protease and a substrate for a second serine protease. In some embodiments, CM2 comprises a substrate for a serine protease and a substrate for a cysteine ​​protease.

[0035] The CM1-CM2 substrates described herein are useful for a variety of therapeutic, diagnostic, and prophylactic applications. For example, these CM1-CM2 substrates are useful for activatable antibodies, including antibodies or antigen-binding fragments thereof (AB), that include a prodomain. The prodomain includes at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the AB, such that binding of the MM reduces the ability of the AB to bind its target.

[0036] In some embodiments, the activatable antibody comprises at least a first CM (CM1) and a second CM (CM2). In some embodiments, at least a portion of the CM1 substrate sequence overlaps with at least a portion of the CM2 sequence. In some embodiments, the CM1 and CM2 substrate sequences share at least one amino acid residue. In some embodiments, the CM1 and CM2 substrate sequences share at least two amino acid residues. In some embodiments, the CM1 and CM2 substrate sequences share at least three amino acid residues. In some embodiments, the CM1 and CM2 substrate sequences share three or more amino acid residues.

[0037] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together.

[0038] In some embodiments, CM1 and CM2 are separate polypeptides that are directly linked together, i.e., the N-terminus of one substrate polypeptide is directly linked to the C-terminus of the other substrate polypeptide. In some embodiments, the N-terminus of CM1 is directly linked to the C-terminus of CM2. In some embodiments, the N-terminus of CM2 is directly linked to the C-terminus of CM1.

[0039] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together via at least one linking moiety.

[0040] In some embodiments, the first cleavable moiety CM1 and the second cleavable moiety CM2 of the uncleaved activatable antibody have the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM.

[0041] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between the masking moiety (MM) and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1 and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between MM and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and AB.

[0042] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP'") between CM2 and the masking moiety (MM). In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP'") between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP") between AB and CM1, a connecting peptide (LP") between CM1 and CM2, and a connecting peptide (LP'") between CM2 and MM.

[0043] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0044] 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.

[0045] In some embodiments, CM1 is a substrate for MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and / or MMP19. In some embodiments, CM1 is a substrate for MMP2. In some embodiments, CM1 is a substrate for MMP9. In some embodiments, CM1 is a substrate for MMP14. In some embodiments, CM1 is a substrate for two or more MMPs. In some embodiments, CM1 is a substrate for at least MMP9 and MMP14. In some embodiments, CM1 is a substrate for at least MMP2 and MMP9. In some embodiments, CM1 is a substrate for at least MMP2 and MMP14. In some embodiments, CM1 is a substrate for three or more MMPs. In some embodiments, CM1 is a substrate for at least MMP2, MMP9, and MMP14. In some embodiments, CM1 comprises two or more substrates for the same MMP. In some embodiments, CM1 comprises at least two or more MMP2 substrates. In some embodiments, CM1 comprises at least two or more MMP9 substrates. In some embodiments, CM1 comprises at least two or more MMP14 substrates.

[0046] In some embodiments, CM1 is a substrate for MMPs and includes 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).

[0047] 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 (SEQ ID 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 (SEQ ID 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).

[0048] 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.

[0049] 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.

[0050] In some embodiments, CM2 is a substrate for at least one serine protease (SP), in some embodiments, the SP is selected from u-type plasminogen activator (uPA, also known as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1), neutrophil elastase (e.g., human neutrophil elastase), and combinations thereof. Non-limiting examples of other SPs that cleave CM2 described herein include activated protein C; cathepsin G; chymase; coagulation factor proteases, such as FVIIa, FIXa, FXa, FXIa, FXIIa, etc.; elastase; granzyme B; guanidinobenzoatase; HtrA1; lactoferrin; marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, TMPRSS2, TMPRSS3, and / or TMPRSS4.

[0051] For example, a suitable CM2 may be 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). 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), L SGRSDDH (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 (SEQ ID NO: 113), LSGRSANP (SEQ ID NO: 114), and SGRSANP (SEQ ID NO: 115).

[0052] In some embodiments, a suitable CM2 may be cleaved by at least one serine protease and 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.

[0053] In some embodiments, a suitable CM2 may be 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).

[0054] In some embodiments, a suitable CM2 may be cleaved by at least one serine protease and 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).

[0055] In some embodiments, CM2 is a substrate for at least one cysteine ​​protease (CP). In some embodiments, the CP is legumain. In some embodiments, a suitable CM2 is cleaved by at least one cysteine ​​protease and may include at least one sequence selected from the following amino acid sequences: AAN, SAN, and GPTN (SEQ ID NO: 152).

[0056] In some embodiments, the CM1-CM2 substrate is 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), The amino acid sequence comprises an amino acid sequence selected from the group consisting of 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).

[0057] In some embodiments, the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM.

[0058] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and in its uncleaved state, the antibody has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM. In some embodiments, LP1 and LP2 are each peptides about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other.

[0059] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between the masking moiety (MM) and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP2) between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between MM and CM1 and a connecting peptide (LP2) between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between MM and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP2) between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between MM and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP2) between CM2 and AB.

[0060] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between AB and CM1. In some embodiments, the activatable antibody comprises a connecting peptide (LP2) between CM2 and the masking moiety (MM). In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between AB and CM1 and a connecting peptide (LP2) between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between AB and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP2) between CM2 and MM. In some embodiments, the activatable antibody comprises a connecting peptide (LP1) between AB and CM1, a connecting peptide (LP') between CM1 and CM2, ...

[0061] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0062] In some embodiments, at least one of LP1 or LP2 is (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.

[0063] 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).

[0064] 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), GSSGGSGGSGS (SEQ ID NO: 213), GGGSSGGS (SEQ ID NO: 214), and GSSGGSGGSGGSG (SEQ ID NO: 215).

[0065] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 216), GSSGT (SEQ ID NO: 217), or GSSG (SEQ ID NO: 218). In some embodiments, LP2 comprises the amino acid sequence GGS. In some embodiments, LP2 comprises the amino acid sequence GGGS (SEQ ID NO: 216).

[0066] In some embodiments, CM1 is a substrate for MMPs and includes 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).

[0067] In some embodiments, an isolated polypeptide of this disclosure or an activatable antibody of this disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 is 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), The LP2 linker comprises an amino acid sequence selected from the group consisting of 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), ISSGLLLSGRSNI (SEQ ID NO: 38), and AVGLLAPPGGLSGRSNI (SEQ ID NO: 39), and the LP2 linker comprises GGGS (SEQ ID NO: 216).

[0068] In some embodiments, an isolated polypeptide of this disclosure or an activatable antibody of this disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 comprise 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), ISSGLLLSGRSNI (SEQ ID NO:38), and AVGLLAPPGGLSGRSNI (SEQ ID NO:39), and wherein the LP2 linker comprises GGS.

[0069] In some embodiments, an isolated polypeptide of this disclosure or an activatable antibody of this disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 and LP2 are 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), APRSALAHGLFG GGS (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), ISSGLLSLSGRSNIGGGS (SEQ ID NO: 239), and AVGLLAPPGGLSGRSNIGGGS (SEQ ID NO: 240).

[0070] In some embodiments, an isolated polypeptide of this disclosure or an activatable antibody of this disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 and LP2 are 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), APRSALAHGLF GGS (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).

[0071] In some embodiments, the AB has a dissociation constant for binding to the target of about 100 nM or less.

[0072] In some embodiments, the activatable antibody comprises an antibody or antigen-binding fragment (AB) thereof that specifically binds 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, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, scab, dAb, single domain heavy chain antibody, or single domain light chain antibody. In some embodiments, such AB is a murine, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0073] In some embodiments, the MMP protease co-localizes with the target in a tissue, and the MMP protease cleaves the CM1 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the SP protease co-localizes with the target in a tissue, and the SP protease cleaves the CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the CP protease co-localizes with the target in a tissue, and the CP protease cleaves the CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and / or SP protease co-localizes with the target in a tissue, and the MMP protease and / or SP protease cleaves the CM1-CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and the SP protease co-localize with the target in a tissue, and at least one of the MMP protease and the SP protease cleaves the CM1-CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and / or the CP protease co-localize with the target in a tissue, and the MMP protease and / or the CP protease cleaves the CM1-CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and the CP protease co-localize with the target in a tissue, and at least one of the MMP protease and the CP protease cleaves the CM1-CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and / or CP protease and / or SP protease co-localize with the target in a tissue, and the MMP protease and / or CP protease and / or SP protease cleaves a CM1-CM2 substrate in the antibody when the antibody is exposed to the protease. In some embodiments, the MMP protease and CP protease and SP protease co-localize with the target in a tissue, and at least one of the MMP protease and SP protease and CP protease cleaves a CM1-CM2 substrate in the antibody when the antibody is exposed to the protease.

[0074] In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide up to 15 amino acids in length.

[0075] 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 substantially identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of the CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence of a CM1-CM2 substrate is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease.

[0076] 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 substantially 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 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 substantially 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 substantially identical to 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 at most 90% or more identical to any polypeptide sequence that is naturally cleaved by the same SP and / or CP protease, e.g., any animal polypeptide sequence. 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 at most 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 at most 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same SP and / or CP protease.

[0077] In some embodiments, a CM1-CM2 substrate of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43. In some embodiments, a CM1-CM2 substrate of the present disclosure 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, a CM1-CM2 substrate of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35. In some embodiments, a CM1-CM2 substrate of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 29, 31, 36, and 37. In some embodiments, a CM1-CM2 substrate of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, and 34. In some embodiments, a CM1-CM2 substrate of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 33. In some embodiments, a CM1-CM2 substrate of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35.

[0078] In some embodiments, an activatable antibody of the present disclosure comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43, an antibody or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind the target. In some embodiments, an activatable antibody of the present disclosure comprises 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, an antibody or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind the target. In some embodiments, an activatable antibody of the present disclosure comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35, an antibody or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind the target. In some embodiments, an activatable antibody of the disclosure comprises 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 or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind the target. In some embodiments, an activatable antibody of the disclosure comprises 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 or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind the target. In some embodiments, an activatable antibody of the disclosure comprises 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 or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind the target.In some embodiments, an activatable antibody of the present disclosure comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35, and an antibody or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target.

[0079] 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 an anti-EGFR antibody disclosed herein. In some embodiments, the activatable antibody comprises a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43, and an antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 401 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 400.

[0080] In some embodiments, CM1-CM2 is comprised 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.

[0081] In some embodiments, the isolated polypeptide of the present disclosure comprises a CM1 substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to 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 is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any mammalian polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% identical to 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 is a substrate for at least one MMP and comprises a polypeptide sequence that is at most 90% 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 at most 90% identical to any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence that is a substrate for at least one MMP 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).

[0082] In some embodiments, an activatable antibody of the present disclosure comprises 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 antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target.

[0083] In some embodiments, an 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 not substantially identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence comprises a polypeptide sequence that is a substrate for at least one SP and is not substantially identical to any mammalian polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence comprises a polypeptide sequence that is a substrate for at least one SP and is not substantially identical to any human polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence comprises a polypeptide sequence that is at most 90% or more identical to any polypeptide sequence, e.g., any animal polypeptide sequence, that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence comprises a polypeptide sequence that is at most 90% or more identical to any mammalian polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence is a substrate for at least one SP and comprises a polypeptide sequence that is at most 90% or more identical to any human polypeptide sequence that is naturally cleaved by the same SP protease. In some embodiments, the CM2 substrate sequence that is a substrate for at least one SP comprises a polypeptide sequence selected from the group consisting of APRSF (SEQ ID NO: 130) and GLPTFVHL (SEQ ID NO: 135).

[0084] In some embodiments, an activatable antibody of the present disclosure comprises 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 antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target.

[0085] In some embodiments, an activatable antibody of the present disclosure comprises a substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16, and an antibody or antigen-binding fragment thereof (AB) that binds a target, and a masking moiety (MM) that reduces the ability of the antigen or epitope-binding domain of the AB to bind its target.

[0086] In some embodiments, the CM1-CM2, CM1, or CM2 substrate is also a substrate for at least one additional protease.

[0087] In some embodiments, the at least one additional protease is an MMP protease that is different from the MMP protease that cleaves CM1. In some embodiments, the at least one additional protease is an MMP protease selected from the group consisting of MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.

[0088] In some embodiments, the at least one additional protease is a SP protease that is 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, matriptase, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor proteases such as FVIIa, FIXa, FXa, FXIa, and FXIIa, elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, and type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, TMPRSS2, TMPRSS3, and TMPRSS4.

[0089] In some embodiments, the at least one additional protease is selected from the group consisting of those shown in Table 6. [Table 1]

[0090] The present disclosure also provides an antibody conjugated to a drug, comprising at least a first CM1 and a second CM2. In some embodiments, the first CM1 and the second CM2 are each polypeptides 15 amino acids or less in length. In some embodiments, the activatable antibody is a conjugated activatable antibody, which in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB-drug, drug-AB-CM2-CM1-MM, MM-CM2-CM1-AB-drug, or drug-AB-CM1-CM2-MM. In some embodiments, the activatable antibody is a conjugated activatable antibody, which in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: drug-MM-CM1-CM2-AB, AB-CM2-CM1-MM-drug, drug-MM-CM2-CM1-AB, or AB-CM1-CM2-MM-drug. In some embodiments, the activatable antibody is a conjugated activatable antibody, which in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: drug-MM-CM1-CM2-AB-drug, drug-AB-CM2-CM1-MM-drug, drug-MM-CM2-CM1-AB-drug, or drug-AB-CM1-CM2-MM-drug.

[0091] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first connecting peptide (LP1), and a second connecting peptide (LP2), wherein the antibody in its uncleaved state has the following structural arrangement from N- to C-terminus: MM1-LP1-CM1-CM2-LP2-AB-drug, drug-AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB-drug, or drug-AB-LP2-CM1-CM2-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other.

[0092] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first connecting peptide (LP1), and a second connecting peptide (LP2), wherein the antibody in its uncleaved state has the following structural arrangement from N- to C-terminus: drug-MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM-drug, drug-MM1-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM-drug. In some embodiments, each of LP1 and LP2 is a peptide about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other.

[0093] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first connecting peptide (LP1), and a second connecting peptide (LP2), wherein the antibody in its uncleaved state has the following structural arrangement from N- to C-terminus: drug-MM1-LP1-CM1-CM2-LP2-AB-drug, drug-AB-LP2-CM2-CM1-LP1-MM-drug, drug-MM1-LP1-CM2-CM1-LP2-AB-drug, or drug-AB-LP2-CM1-CM2-LP1-MM-drug. In some embodiments, each of LP1 and LP2 is a peptide about 1-20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other.

[0094] In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between the masking moiety (MM) and CM1. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP2) between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between MM and CM1 and a connecting peptide (LP2) between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between MM and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP2) between CM2 and AB. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between MM and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP2) between CM2 and AB.

[0095] In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between AB and CM1. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP2) between CM2 and the masking moiety (MM). In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between AB and CM1 and a connecting peptide (LP2) between CM2 and MM. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between AB and CM1 and a connecting peptide (LP') between CM1 and CM2. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP') between CM1 and CM2 and a connecting peptide (LP2) between CM2 and MM. In some embodiments, the conjugated activatable antibody comprises a connecting peptide (LP1) between AB and CM1, a connecting peptide (LP') between CM1 and CM2, and a connecting peptide (LP2) between CM2 and MM.

[0096] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0097] In some embodiments, at least one of LP1 or LP2 is (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.

[0098] 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).

[0099] 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), GSSGGSGGSGS (SEQ ID NO: 213), and GGGSSGGS (SEQ ID NO: 214).

[0100] 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).

[0101] In some embodiments, the CM1-CM2 substrate is linked or otherwise attached to an antibody. For example, CM1-CM2 is used to link one or more agents to an antibody or antigen-binding fragment (AB) that binds a predetermined target, such that upon exposure to MMPs and / or SPs and / or CPs, CM1-CM2 is cleaved and the agent is released from the AB. Exemplary targets include, but are not limited to, those listed in Table 1. Exemplary ABs include, but are not limited to, those listed in Table 2.

[0102] In some embodiments, the AB has a dissociation constant for binding to the target of about 100 nM or less.

[0103] In some embodiments, the antibody includes an antibody or antigen-binding fragment thereof that specifically binds a target. In some embodiments, the target-binding antibody or immunologically active fragment thereof is a monoclonal antibody, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, scab, dAb, single-domain heavy chain antibody, or single-domain light chain antibody. In some embodiments, such target-binding antibody or immunologically active fragment thereof is a murine, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0104] In some embodiments, the MM has a dissociation constant for binding to the AB that is less than or equal to the dissociation constant of the AB to the target.

[0105] In some embodiments, the MM does not interfere with or compete with the AB for binding to the target in the cleaved state.

[0106] In some embodiments, the MM is a polypeptide between 2 and 40 amino acids in length, for example, the MM is a polypeptide up to about 40 amino acids in length.

[0107] In some embodiments, the MM polypeptide sequence differs from the sequence of any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is 50% or less identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is 40%, 30%, 25%, 20%, 15%, or 10% or less identical to any natural binding partner of AB.

[0108] In some embodiments, the agent conjugated to the AB or the AB of the activatable antibody is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. As used herein, a toxin fragment is a fragment that retains toxic activity. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalator, or other DNA damaging agent. In some embodiments, the agent is an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0109] In some embodiments, the agent is an anti-inflammatory agent.

[0110] In some embodiments, the antibody and / or activatable antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0111] In some embodiments, the conjugated antibody and / or conjugated activatable antibody comprises a detectable label. In some embodiments, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent comprises a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the imaging agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label comprises an N-methylacridium derivative. In some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0112] In some embodiments, the antibody and / or AB of the activatable antibody naturally contain one or more disulfide bonds. In some embodiments, the AB can be modified to contain one or more disulfide bonds.

[0113] In some embodiments, the antibodies and / or conjugated antibodies are monospecific. In some embodiments, the antibodies and / or conjugated antibodies are multispecific, also referred to herein as multispecific antibodies and / or conjugated multispecific antibodies. In some embodiments, the multispecific antibodies and / or conjugated multispecific antibodies are bispecific or trispecific. In some embodiments, the antibodies and / or conjugated antibodies are formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule. In some embodiments, the antibodies and / or conjugated antibodies are formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other genetically modified receptor or other immune effector cell, such as a CAR-modified NK cell. In some embodiments, the activatable antibodies and / or conjugated activatable antibodies are formulated as part of a pro-chimeric antigen receptor (CAR) modified T cell. In some embodiments, the activatable antibodies and / or conjugated activatable antibodies are formulated as part of a pro-chimeric antigen receptor (CAR) modified NK cell.

[0114] In some embodiments, the activatable antibody and / or conjugated activatable antibody is monospecific. In some embodiments, the activatable antibody and / or conjugated activatable antibody is multispecific and is also referred to herein as a multispecific activatable antibody and / or a conjugated multispecific activatable antibody. As used herein, the term "activatable antibody" and all grammatical variations thereof, unless otherwise specified, are intended to encompass, but are not limited to, embodiments in which the activatable antibody is a multispecific activatable antibody of the present disclosure. As used herein, the term "conjugated activatable antibody" and all grammatical variations thereof, unless otherwise specified, are intended to encompass, but are not limited to, embodiments in which the conjugated activatable antibody is a conjugated multispecific activatable antibody of the present disclosure. In some embodiments, the multispecific activatable antibody and / or conjugated multispecific activatable antibody is bispecific or trispecific. In some embodiments, the activatable antibody and / or conjugated activatable antibody 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 genetically modified receptor.

[0115] In some embodiments, the antibodies, antibody conjugates, activatable antibodies, conjugated activatable antibodies, multispecific activatable antibodies, and / or conjugated multispecific activatable antibodies described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include, for example, current pharmaceutical and / or surgical therapies for an intended use, such as cancer. For example, the activatable antibodies, conjugated activatable antibodies, multispecific activatable antibodies, and / or conjugated multispecific activatable antibodies can be used in conjunction with an additional chemotherapeutic or anti-neoplastic agent.

[0116] In some embodiments, the activatable antibody is a multispecific activatable antibody. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize multiple antigens or epitopes and include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody such that binding of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. In some embodiments, the MM is attached to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM1-CM2 substrate) that functions as a substrate for at least one MMP protease and at least one SP. The activatable multispecific antibodies provided herein are stable in circulation, are activated at the intended therapeutic and / or diagnostic site but not in normal, i.e., healthy tissue, and, when activated, exhibit target binding at least comparable to that of the corresponding unmodified multispecific antibody.

[0117] 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 antigen-binding fragment thereof (AB1) that specifically binds epidermal growth factor receptor (EGFR) and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence NYGVH (SEQ ID NO: 220); a VH CDR2 sequence comprising at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); a VH CDR3 sequence comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); and combinations thereof.

[0118] 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 antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); a VL CDR2 sequence comprising at least the amino acid sequence KYASESIS (SEQ ID NO: 224); a VL CDR3 sequence comprising the amino acid sequence QQNNNWPTT (SEQ ID NO: 225); and combinations thereof.

[0119] 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 antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 220). a CDR1 sequence; a VH CDR2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); and combinations thereof.

[0120] 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 antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a VL CDR1 sequence that comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); a VL CDR1 sequence that comprises a sequence 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); a CDR2 sequence; a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 225); and combinations thereof.

[0121] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence at least comprises the amino acid sequence NYGVH (SEQ ID NO: 220); the VH CDR2 sequence at least comprises the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); the VH CDR3 sequence comprises the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); the VL CDR1 sequence at least comprises the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); the VL CDR2 sequence at least comprises the amino acid sequence KYASESIS (SEQ ID NO: 224); The CDR3 sequence comprises the amino acid sequence QQNNNWPTT (SEQ ID NO: 225).

[0122] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure, specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 220); The CDR2 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); the VH CDR3 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); the VL CDR1 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); The CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO: 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).

[0123] In some embodiments, activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, 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 an anti-Jagged antibody disclosed herein. In some embodiments, activatable antibodies and / or conjugated activatable antibodies provided herein, including but not limited to, 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 of SEQ ID NO: 401 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 400.

[0124] 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.

[0125] In some embodiments, the activatable antibody also includes a drug conjugated to AB. In some embodiments, the drug is a therapeutic agent. In some embodiments, the drug is an anti-tumor drug. In some embodiments, the drug is a toxin or a fragment thereof. In some embodiments, the drug is conjugated to AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the drug is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the drug is conjugated to AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the drug is an agent selected from the group listed in Table 3. In some embodiments, the drug is a dolastatin. In some embodiments, the drug is an auristatin or a derivative thereof. In some embodiments, the drug is auristatin E or a derivative thereof. In some embodiments, the drug is monomethyl auristatin E (MMAE). In some embodiments, the drug is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0126] In some embodiments, the agent is an anti-inflammatory agent.

[0127] In some embodiments, the activatable antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0128] In some embodiments, the conjugated antibody comprises a detectable label. In some embodiments, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent comprises a radioisotope. In some embodiments, the radioisotope is indium or technetium. In some embodiments, the imaging agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label comprises an N-methylacridium derivative. In some embodiments, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0129] In some embodiments, the activatable antibody also comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is directly connected to the MM of the activatable antibody. In some embodiments, the spacer is directly connected to the MM of the activatable antibody in the N- to C-terminal structural arrangement of spacer-MM-CM1-CM2substrate-AB. An example of a spacer directly connected to the N-terminus of the MM of an activatable antibody is an amino acid sequence 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 includes at least the amino acid sequence GQ. In some embodiments, the spacer includes at least the amino acid sequence Q.

[0130] In some embodiments, the AB in the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be modified to contain one or more disulfide bonds.

[0131] In some embodiments, the serum half-life of an activatable antibody is longer than the serum half-life of its corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of an activatable antibody is similar to the half-life of its corresponding multi-antibody. In some embodiments, the serum half-life of an activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism.In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism.

[0132] The present disclosure also provides compositions and methods comprising activatable antibodies, including antibodies or antibody fragments (AB) that specifically bind a predetermined target, wherein the AB is conjugated to a masking moiety (MM) that reduces the ability of the AB to bind its target. In some embodiments, the activatable antibody further comprises a CM1-CM2 substrate that is a substrate for at least one MMP and at least one SP. The compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues in the AB without impairing the activity (e.g., masking, activation, or binding activity) of the activatable antibody. In some embodiments, the compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues in the AB without reducing or interfering with one or more disulfide bonds in the MM. The compositions and methods provided herein produce activatable antibodies conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic, and / or prophylactic agents; for example, in some embodiments, no agents are conjugated to any MM of the activatable antibody. The compositions and methods provided herein generate conjugated activatable antibodies in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in its uncleaved state. The compositions and methods provided herein generate conjugated activatable antibodies in which the activatable antibody is still activated, i.e., cleaved, in the presence of an MMP capable of cleaving the CM1-CM2 substrate.

[0133] Although an activatable antibody has at least one conjugation point for an agent, all possible conjugation points are available for conjugation to an agent in the methods and compositions provided herein. In some embodiments, one or more conjugation points are sulfur atoms involved in disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain sulfide bonds, but not intrachain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. Such residues may occur naturally in antibody structures or may be incorporated into antibodies by site-directed mutagenesis, chemical conversion, or misincorporation of unnatural amino acids.

[0134] Also provided is a method for preparing a conjugate of an activatable antibody having one or more interchain disulfide bonds in AB and one or more intrachain disulfide bonds in MM, wherein an agent that reacts with free thiols is provided. The method generally involves partially reducing the interchain disulfide bonds in AA with a reducing agent, such as TCEP, and conjugating a 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 a multispecific activatable antibody is contacted with a reducing agent to reduce fewer than all disulfide bonds, e.g., fewer than all possible binding sites. In some embodiments, less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of all possible conjugation sites are reduced.

[0135] In some embodiments, a method is provided for reducing a drug, e.g., a drug, and conjugating it to an activatable antibody, allowing selectivity in drug placement. The method generally involves partially reducing the activatable antibody with a reducing agent, such that any conjugation sites in the masking portion or other non-AB portions of the activatable antibody are not reduced, and then conjugating the drug to the interchain thiol in the AB. The conjugation site is selected to allow for the desired placement of the drug so that conjugation can occur at the desired site. An example of a reducing agent is TCEP. The reduction reaction conditions, such as the ratio of reducing agent to activatable antibody, the length of incubation, the temperature during incubation, the pH of the reduction reaction solution, etc., are determined by identifying conditions that produce a conjugated activatable antibody in which the MM retains its ability to effectively and efficiently mask the AB of the activatable antibody in its uncleaved state. The ratio of reducing agent to activatable antibody will vary depending on the activatable antibody. In some embodiments, the ratio of reducing agent to activatable antibody will be in the range of about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0136] In some embodiments, methods are provided for reducing the interchain disulfide bond in the AB of an activatable antibody and conjugating a thiol-containing agent, such as a drug, to the resulting interchain thiol to selectively place the agent on the AB. The methods generally involve 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 an activatable antibody is partially reduced at a desired ratio of reducing agent:activatable antibody at about 37°C for about 1 hour. In some embodiments, the ratio of reducing agent to activatable antibody will be in the range of about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0137] The thiol-containing reagent can be, for example, cysteine ​​or N-acetylcysteine. The reducing agent can be, for example, TCEP. In some embodiments, the reduced activatable antibody can be purified prior to conjugation, for example, using column chromatography, dialysis, or diafiltration. In some embodiments, the reduced antibody is not purified after partial reduction and prior to conjugation.

[0138] The present disclosure also provides a partially reduced activatable antibody in which at least one interchain disulfide bond in the activatable antibody has been reduced by a reducing agent without disrupting any intrachain disulfide bonds in the activatable antibody, the activatable antibody comprising an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that inhibits binding of the activatable antibody AB to the target in its uncleaved state, and a CM1-CM2 substrate (a polypeptide that functions as a substrate for at least one MMP and one SP) bound to the AB. In some embodiments, the MM is bound to the AB via the CM1-CM2 substrate. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disrupted by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM in the activatable antibody are not disrupted by the reducing agent. In some embodiments, the activatable antibody in its uncleaved state has the following structural configuration from N-terminus to C-terminus: MM-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0139] The present disclosure also provides partially reduced activatable antibodies, including but not limited to the multispecific activatable antibodies of the present disclosure, in which at least one intrachain disulfide bond in the activatable antibody has been reduced by a reducing agent without interfering with or impairing the activity and / or efficacy of the activatable antibody, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that, in its uncleaved state, inhibits binding of the activatable antibody to the target, and a CM1-CM2 substrate attached to the AB, wherein the CM1-CM2 substrate is a polypeptide that functions as a substrate for a protease. The activity and / or efficacy of the activatable antibody, by non-limiting example, is the masking activity, activation of the activatable antibody, and / or the binding activity of the activated activatable antibody. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disrupted by a reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM in the activatable antibody are not disrupted by a reducing agent. In some embodiments, the activatable antibody in its uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0140] The present disclosure also provides a conjugated activatable antibody comprising an activatable antibody linked to a monomethyl auristatin D (MMAD) payload, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that inhibits binding of the AB of the activatable antibody in its uncleaved state to the target, and a CM1-CM2 substrate conjugated to the AB, wherein the CM1-CM2 substrate is a polypeptide that functions as a substrate for at least one MMP protease and at least one SP protease.

[0141] In some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any of several methods for attaching an agent to the AB, such as: (a) attachment to a carbohydrate moiety of the AB, or (b) attachment to a sulfhydryl group of the AB, or (c) attachment to an amino group of the AB, or (d) attachment to a carboxylate group of the AB.

[0142] In some embodiments, the MMAD payload is conjugated to AB via a linker. In some embodiments, the MMAD payload is conjugated to a cysteine ​​in AB via a linker. In some embodiments, the MMAD payload is conjugated to a lysine in AB via a linker. In some embodiments, the MMAD payload is conjugated to another residue of AB, such as a residue 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 linkers shown in Tables 5 and 6. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidoPEG-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation techniques disclosed herein.

[0143] The present disclosure also provides polypeptides and other larger molecules comprising one or more CM1-CM2 substrate sequences provided herein. As a non-limiting example, the CM1-CM2 substrate sequences provided herein are useful in prodrug compositions and methods of use thereof. These CM1-CM2 substrate sequences provided herein are also useful in probes and other detection agents and methods of use thereof. For example, the CM1-CM2 substrate sequences provided herein can be used in combination with fluorescent quenchers and other quenchers to generate detection agents, such as imaging agents and / or other diagnostic agents. Those skilled in the art will understand that the CM1-CM2 substrate sequences provided herein are useful in any composition and / or method in the art that may employ a substrate cleavable by at least one MMP and at least one SP.

[0144] The present disclosure also provides isolated nucleic acid molecules encoding the antibodies and / or activatable antibodies described herein, and vectors comprising these isolated nucleic acid sequences. The present disclosure provides methods of producing the antibodies and / or activatable antibodies by culturing cells comprising such vectors under conditions that result in expression of the antibodies and / or activatable antibodies.

[0145] The present disclosure provides methods for producing a conjugated antibody of the present disclosure that binds a predetermined target by (a) culturing cells containing a nucleic acid construct encoding the antibody under conditions that result in expression of the antibody, where (i) the antibody comprises a CM1-CM2 substrate, and (ii) the CM1-CM2 substrate is a polypeptide that functions as a substrate for matrix metalloproteases and serine proteases; (b) recovering the antibody; and (c) conjugating the recovered antibody to one or more additional agents.

[0146] The present disclosure also provides a method for producing an activatable antibody of the present disclosure that binds a predetermined target in an activated state by (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions that result in expression of the activatable antibody, the activatable antibody comprising a masking moiety (MM), a CM1-CM2 substrate, and an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, where (i) the CM1-CM2 substrate is a polypeptide that functions as a substrate for MMPs and SPs; and (ii) the CM1-CM2 substrate is positioned in the activatable antibody such that, in an uncleaved state, the MM interferes with specific binding of the AB to the target, and in a cleaved state, the MM does not interfere with or compete with specific binding of the AB to the target; and (b) recovering the activatable antibody.

[0147] The present disclosure also provides a method for producing a conjugated activatable antibody of the present disclosure that binds a predetermined target in an activated state by: (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions that result in expression of the activatable antibody, the activatable antibody comprising a masking moiety (MM), a CM1-CM2 substrate, and an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, where (i) the CM1-CM2 substrate is a polypeptide that functions as a substrate for an MMP and an SP and / or CP; (ii) the CM1-CM2 substrate is positioned in the activatable antibody such that, in an uncleaved state, the MM interferes with specific binding of the AB to the target, and in a cleaved state, the MM does not interfere with or compete with specific binding of the AB to the target; (b) recovering the activatable antibody; and (c) conjugating the recovered antibody to one or more additional agents.

[0148] The present disclosure provides methods of preventing, delaying the progression of, treating, alleviating symptoms of, or ameliorating a target-related disease in a subject by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody and / or conjugated activatable antibody described herein.

[0149] The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating the symptoms of, or ameliorating inflammation and / or inflammatory diseases in a subject by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody described herein. The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating the symptoms of, or ameliorating cancer in a subject by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody described herein. The present disclosure provides methods of preventing, slowing the progression of, treating, alleviating the symptoms of, or ameliorating autoimmune diseases in a subject by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody described herein.

[0150] The conjugated activating antibodies, activatable antibodies, and / or conjugated activatable antibodies used in any of these method and use embodiments can be administered at any stage of disease. For example, such conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be administered to patients with cancer at any stage, from early to metastatic. The terms "subject" and "patient" are used interchangeably.

[0151] In some embodiments, the subject is a mammal, such as a human, a non-human primate, a companion animal (e.g., a cat, dog, horse), a livestock animal, a work animal, or a zoo animal. In some embodiments, the subject is a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal under veterinarian care.

[0152] The conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and their therapeutic formulations, are administered to a subject suffering from or susceptible to a disease or disorder associated with abnormal target expression and / or activity. A subject suffering from or susceptible to a disease or disorder associated with abnormal target expression and / or activity can be identified using any of a variety of methods known in the art. For example, a subject suffering from cancer or other tumor conditions can be identified using any of a variety of clinical and / or laboratory tests, such as physical examination and blood, urine, and / or stool analysis to assess health status. For example, a subject suffering from inflammation and / or an inflammatory disorder can be identified using any of a variety of clinical and / or laboratory tests, such as physical examination and / or body fluid analysis to assess health status, such as blood, urine, and / or stool analysis.

[0153] Administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody to a patient having a disease or disorder associated with aberrant target expression and / or activity is considered successful if any of a variety of laboratory or clinical objectives are achieved. For example, administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody to a patient having a disease or disorder associated with aberrant target expression and / or activity is considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, or inhibited, or do not progress to a further worsening state. Administration of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody to a patient having a disease or disorder associated with aberrant target expression and / or activity is considered successful if the disease or disorder goes into remission or does not progress to a further worsening state.

[0154] In some embodiments, the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies described herein are used in conjunction with or in combination with one or more additional agents. Suitable additional agents include, for example, current pharmaceutical and / or surgical therapies for a desired application, such as cancer. For example, the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be used in conjunction with an additional chemotherapeutic or anti-neoplastic agent.

[0155] In some embodiments, the additional agent is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-bound paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0156] In some embodiments, the additional agent is a checkpoint inhibitor, a kinase inhibitor, an agent that targets inhibitors in the tumor microenvironment, and / or a T cell or NK agonist. In some embodiments, the additional agent is radiation therapy, alone or in combination with another additional agent, such as a chemotherapeutic or anti-neoplastic agent. In some embodiments, the additional agent is a vaccine, an oncovirus, and / or a DC activator, such as, by way of non-limiting example, a Toll-like receptor (TLR) agonist and / or α-CD40. In some embodiments, the additional agent is a tumor-targeting antibody (e.g., an antibody-drug conjugate (ADC)) designed to kill tumors via ADCC or via direct conjugation to a toxin.

[0157] In some embodiments, the checkpoint inhibitor is selected from the group consisting of CTLA-4, LAG-3, PD-1, PD-1, TIGIT, TIM-3, B7H4, BTLA, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and a Btk inhibitor such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an α-CSF1R inhibitor, an α-CCR4 inhibitor, TGF-β, a myeloid-derived suppressor cell, or a T-regulatory cell. In some embodiments, the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM.

[0158] 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 TIM-3. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is TGF-β. In some embodiments, the inhibitor is a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T regulatory cell.

[0159] 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.

[0160] In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody is administered in combination with one or more additional agents, e.g., chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressive agents, during and / or after treatment. In some embodiments, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody, and the additional agent 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 one another, e.g., each formulated into a separate therapeutic composition, 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 during the therapeutic regimen. For example, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody may be administered prior to administration of the additional agent, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody may be administered subsequent to administration of the additional agent, or the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent may be administered in alternation. As described herein, the antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody and the additional agent may be administered in a single dose or multiple doses.

[0161] In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent are administered simultaneously. For example, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent may be formulated in a single composition or administered as multiple separate compositions. In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent are administered sequentially, or the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent are administered at different times during the treatment regimen.

[0162] In some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody are administered during and / or after treatment in combination with one or more additional agents, including, but not limited to, anti-inflammatory agents, immunosuppressants, chemotherapeutic agents, such as alkylating agents, antimetabolites, anti-microtubule inhibitors, topoisomerase inhibitors, cytotoxic antibiotics, and / or any other nucleic acid damaging agents. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapy agent, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, such as a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a targeted agent, such as another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteasome inhibitor, such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulatory agent, such as lenalidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered standard of care by those of skill in the art. In some embodiments, the additional agent is a chemotherapeutic agent known to those of skill in the art.

[0163] In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody. In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody directed against the same target as the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody. In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody directed against a target different from that of the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody.

[0164] In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent are administered simultaneously. For example, the conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent may be formulated into a single composition or administered as multiple separate compositions. In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody, and additional agent are administered sequentially, or the antibody and / or conjugated antibody and additional agent are administered at different times during the treatment regimen. For example, the antibody and / or conjugated antibody is administered before administration of the additional agent, the antibody and / or conjugated antibody is administered following administration of the additional agent, or the antibody and / or conjugated antibody and additional agent are administered alternately. As described herein, the antibody and / or conjugated antibody and additional agent may be administered in a single dose or multiple doses.

[0165] The present disclosure also provides methods and kits for the use of conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies in various diagnostic and / or prophylactic applications.

[0166] Pharmaceutical compositions according to the present disclosure can include an antibody, conjugated antibody, activatable antibody, and / or conjugated activatable antibody of the present disclosure and a carrier. These pharmaceutical compositions can be included in a kit, such as, for example, a diagnostic kit.

[0167] Conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies comprise an antibody or antigen-binding fragment (AB) that specifically binds a target. Exemplary types of targets for ABs include, but are not limited to, cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), and the like. In some embodiments, the conjugated antibodies and / or activatable antibodies have an AB that binds an extracellular target, usually an extracellular protein target. In some embodiments, the conjugated antibodies and / or activatable antibodies are designed for cellular uptake and are switchable intracellularly.

[0168] As a non-limiting example, AB is a binding partner for any of the targets listed in Table 1. [Table 2] TIFF0007781254000003.tif110162

[0169] As a non-limiting example, AB is or is derived from an antibody listed in Table 2. [Table 3] TIFF0007781254000005.tif93161

[0170] The activatable antibodies and activatable antibody compositions provided herein include at least an antibody or antibody fragment thereof (collectively referred to throughout this disclosure as AB) that specifically binds a target, e.g., a human target, wherein the AB is modified with a masking moiety (MM).

[0171] In some embodiments, the masking moiety is selected for use with a specific antibody or antibody fragment. For example, a suitable masking moiety for use with an antibody that binds EGFR includes a MM comprising the sequence CISPRG (SEQ ID NO: 165). By way of non-limiting example, the MM may comprise a sequence such as CISPRGC (SEQ ID NO: 166); CISPRGCG (SEQ ID NO: 167); CISPRGCPDGPYVMY (SEQ ID NO: 168); CISPRGCPDGPYVM (SEQ ID NO: 169), CISPRGCEPGTYVPT (SEQ ID NO: 170) and CISPRGCPGQIWHPP (SEQ ID NO: 171).Other suitable masking moieties include, but are not limited to, GSHCLIPINMGAPSC (SEQ ID NO: 172); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 173); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 174); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 175); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 176); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 177); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 178). ;CPHVSFGSCGCISPRGCPG (SEQ ID NO: 179);CPYYTLSYCGCISPRGCPG (SEQ ID NO: 180);CNHVYFGTCGCISPRGCPG (SEQ ID NO: 181);CNHFTLTTCGCISPRGCPG (SEQ ID NO: 182);CHHFTLTTCGCISPRGCPG (SEQ ID NO: 183);YNPCATPMCCISPRGCPG (SEQ ID NO: 184);CNHHYFYTCGCISPRGCG (SEQ ID NO: 185);CNHHYHYYCGCISPRGCG (SEQ ID NO: 186);CNHV YFGTCGCISPRGCG (SEQ ID NO: 187); CHHVYWGHCGCISPRGCG (SEQ ID NO: 188); CPHFTTTSCGCISPRGCG (SEQ ID NO: 189); CNHFTLTTCGCISPRGCG (SEQ ID NO: 190); CHHFTLTTCGCISPRGCG (SEQ ID NO: 191); CPYYTLSYCGCISPRGCG (SEQ ID NO: 192); CPHVSFGSCGCISPRGCG (SEQ ID NO: 193); ADHVFWGSYGCISPRGCG (SEQ ID NO: 194); YNPCATPMCCISPR GCG (SEQ ID NO: 195); CHHVYWGHCGCISPRGCG (SEQ ID NO: 196); C(N / P)H(H / V / F)(Y / T)(F / W / T / L)(Y / G / T / S)(T / S / Y / H)CGCISPRGCG (SEQ ID NO: 197); CISPRGCGQPIPSVK ​​(SEQ ID NO: 198); CISPRGCTQPYHVSR (SEQ ID NO: 199); and / or any of the EGFR-specific masks disclosed in International Publication No. WO2010 / 081173, such as CISPRGCNAVSGLGS (SEQ ID NO: 164).

[0172] When the AB is modified with a MM and in the presence of a target, the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM to its target or the specific binding of the parent AB to its target.

[0173] K of MM-modified AB against target d is the K of the AB unmodified by MM or the parent AB against 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 times or more than or equal to 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 10-10,000,000, 10-10 ,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times larger. Conversely, the binding affinity of an AB modified with a MM to a target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100 times or more, greater than the binding affinity of an AB not modified with a MM or the parent AB to a target. ,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times smaller.

[0174] The dissociation constant of MM with AB (K d ) is generally the K of AB against the targetd K of MM against AB is larger than d is the K of AB against the target d The binding affinity of the MM to the AB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the binding affinity of the AB to the target. Conversely, the binding affinity of the MM to the AB is generally less than the binding affinity of the AB to the target. The binding affinity of the MM to the AB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times less than the binding affinity of the AB to the target.

[0175] When the AB is modified with a MM and in the presence of a target, the specific binding of the AB to the target is reduced or inhibited compared to the specific binding of the AB not modified with a MM or the specific binding of the parent AB to the target. The ability of the AB to bind the target when modified with a MM is reduced or inhibited for at least 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 28 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, or 96 hours, 5 days, 10 days, 15 days, 30 days, or 48 hours, as measured in an in vivo or in vitro assay, compared to the binding of the AB not modified with a MM or the binding of the parent AB to the target. , 45, 60, 90, 120, 150, or 180 days, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months or longer, the risk of urinary tract infection may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.

[0176] The MM inhibits the binding of the AB to its target. The MM binds the antigen-binding domain of the AB and inhibits the binding of the AB to its target. The MM can sterically inhibit the binding of the AB to its target. The MM can allosterically inhibit the binding of the AB to its target. In these embodiments, when the AB is modified with or bound to the MM and in the presence of a target, the binding of the AB to its target is inhibited for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for at least 5, 10, 15, 30, 45, 60, 90, 12, 16, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, as measured in an in vivo or in vitro assay, compared to the binding of the AB not modified with the MM, the parent AB, or the AB not bound to the MM to the target. Over a period of 0, 150 or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer, there is no, or substantially no, or only 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% binding of AB to the target.

[0177] When an AB is bound to or modified by an MM, the MM "masks" or reduces or inhibits the specific binding of the AB to its target. When an AB is bound to or modified by an MM, such binding or modification may cause a conformational change that reduces or inhibits the ability of the AB to specifically bind to its target.

[0178] AB bound to or modified with MM can be represented by the following formula (from the amino (N)-terminal region to the carboxyl (C)-terminal region): (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) wherein MM is a masking moiety, AB is an antibody or antibody fragment thereof, and L is a linker. In many embodiments, it may be desirable to insert one or more linkers, e.g., flexible linkers, into the composition to provide flexibility.

[0179] In certain embodiments, the MM is not a natural binding partner of AB. In some embodiments, the MM does not include or is substantially free of homology to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0180] In some embodiments, an activatable antibody comprises an AB that is modified with a MM and also comprises at least one cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). Such an activatable antibody exhibits activatable / switchable binding of the AB to its target. An activatable antibody generally comprises an antibody or antibody fragment (AB) that is modified with or conjugated to a masking moiety (MM) and a CM1-CM2 substrate.

[0181] The elements in the activatable antibody are aligned such that the MM and CM1-CM2 substrate are appropriately 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, specific binding of the AB to its target is reduced or inhibited. Specific binding of the AB to its target is inhibited or masked by the MM's ability to specifically bind its target. can be reduced by

[0182] K of AB modified by MM and CM1-CM2 substrates against the target d is the K of the AB unmodified by the MM and CM1-CM2 substrates, or the parent AB, against the target. dat least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or more than or between 5 and 10, 10 and 100, 10 and 1,000, 10 and 10,000, 10 and 100,000, 10 and 1,000,000, 10 and 10,000,000, 10 0-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times larger. Conversely, the binding affinity of an AB modified with MM and CM1-CM2 substrates to a target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or more, or 5-10, 10-100, 10-1,000, 10-10 times, or 10-10 times, greater than the binding affinity of an AB not modified with MM and CM1-CM2 substrates or a parent AB to a target. 0,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-10,000, 1,000-100, 000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times smaller.

[0183] When an AB is modified with MM and CM1-CM2 substrates and in the presence of its target but not in the presence of a modifying agent (e.g., MMP and SP), the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with MM and CM1-CM2 substrates or the specific binding of the parent AB to its target. The ability of the AB to bind a target when modified with the MM and CM1-CM2 substrates may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% over a period of at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more as measured in an in vivo or in vitro assay when compared to the binding of the parent AB or the binding of an AB not modified with the MM and CM1-CM2 substrate to a target.

[0184] As used herein, the term cleaved state refers to the state of an activatable antibody after modification, i.e., cleavage, of the CM1-CM2 substrate by at least one matrix metalloprotease. The terms uncleaved state or completely uncleaved state, as used herein, refer to the state of an activatable antibody in the absence of cleavage of the CM1-CM2 substrate by MMPs and / or SPs. As discussed above, the term "activatable antibody" is used herein to refer to an activatable antibody in both its uncleaved (native) state and its cleaved state. An activatable antibody in its cleaved state is also referred to herein as an activated antibody and / or an activated activatable antibody. It will be apparent to those skilled in the art that in some embodiments, a cleaved activatable antibody lacks MM due to cleavage of the CM1-CM2 substrate by a protease, resulting in the release of at least MM.

[0185] By activatable or switchable, we mean that the activatable antibody exhibits a first level of target binding (first conformation) in an inhibited, masked, or uncleaved state, and a second level of target binding (i.e., second conformation) in an uninhibited, unmasked, and / or cleaved state, where the second level of target binding is greater than the first level of binding. Generally, target access to the AB of an activatable antibody is higher in the presence of a cleaving agent capable of cleaving the CM1-CM2 substrate than in the absence of such a cleaving agent. Thus, when the activatable antibody is in the uncleaved state, the AB can be inhibited or masked from target binding (i.e., the first conformation is such that the AB cannot bind the target), and in the cleaved state, the AB is not inhibited or masked from target binding.

[0186] The CM1-CM2 substrate and AB of the activatable antibody are selected such that AB is a binding moiety for a predetermined target and the CM1-CM2 substrate is a substrate for an MMP and SP, where the MMP and / or SP co-localize with the target at a therapeutic or diagnostic site of a subject. The activatable antibodies disclosed herein are particularly useful, for example, when the MMP and SP capable of cleaving the site in the CM1-CM2 substrate, respectively, are present at relatively higher levels in target-containing tissue at the therapeutic or diagnostic site than in tissue at a non-therapeutic site (e.g., healthy tissue).

[0187] In some embodiments, the activatable antibody results in a reduction of toxic and / or adverse side effects that might otherwise result from binding of the AB at a non-therapeutic site if the AB were unmasked or otherwise inhibited from binding to its target.

[0188] In general, an activatable antibody can be designed by selecting an AB of interest and constructing the remainder of the activatable antibody so that, if conformationally constrained, the MM provides for masking of the AB or reducing binding of the AB to its target. Structural design criteria can be considered to confer this functional characteristic.

[0189] Activatable antibodies are provided that exhibit a desired dynamic range of switchable phenotypes for target binding in inhibited versus uninhibited conformations. Dynamic range typically refers to the ratio of (a) the maximum detectable level of a parameter under a first set of conditions to (b) the minimum detectable value of that parameter under a second set of conditions. For example, in the case of an activatable antibody, the dynamic range refers to the ratio of (a) the maximum detectable level of target protein binding to 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 detectable level of target protein binding to 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 treatment with an activatable antibody cleaving agent (e.g., an enzyme) to the dissociation constant of treatment with multiple activatable antibody cleaving agents. The greater the dynamic range of an activatable antibody, the better the switchable phenotype of the activatable antibody. Activatable antibodies with relatively high dynamic range values ​​(e.g., values ​​higher than 1) exhibit a more desirable switching phenotype, such that target protein binding by the activatable antibody occurs to a greater extent (e.g., occurs primarily) in the presence of a cleaving agent (e.g., an enzyme) that can cleave the CM1-CM2 substrate of the activatable antibody than in the absence of the cleaving agent.

[0190] Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulas for activatable antibodies are provided below. It is specifically contemplated that the N- to C-terminal order of the AB, MM, and CM1-CM2 substrate can be reversed within an activatable antibody. It is also specifically contemplated that the CM and MM can overlap in amino acid sequence, such that, for example, the CM1-CM2 substrate is at least partially contained within the MM.

[0191] For example, an activatable antibody may have the following formula (from amino (N) terminal region to carboxyl (C) terminal region): (MM)-(CM1-CM2 substrate)-(AB) (AB)-(CM1-CM2 substrate)-(MM) where MM is a masking moiety, the CM1-CM2 substrate is a cleavable moiety, and AB is an antibody or fragment thereof. As previously noted, the term "CM1-CM2 substrate" is not intended to imply any requirement regarding the orientation or other structural arrangement of the first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and the 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 arrangements from N-terminus to C-terminus: CM1-CM2 or CM2-CM1. The term "CM1-CM2 substrate" also encompasses substrates in which at least a portion of the CM1 sequence overlaps with at least a portion of the CM2 sequence. It should also be noted that although the MM and CM1-CM2 substrate are shown as separate components in the above formula, in all exemplary embodiments (including formulas) disclosed herein, it is contemplated that the amino acid sequences of the MM and CM1-CM2 substrate may overlap such that the CM1-CM2 substrate is fully or partially contained within the MM. In addition, the above formulas provide for additional amino acid sequences that may be located N- or C-terminal to the activatable antibody element.

[0192] In certain embodiments, the MM is not a natural binding partner of AB. In some embodiments, the MM does not include or is substantially free of homology to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0193] In many embodiments, it may be desirable to insert one or more linkers, e.g., flexible linkers, into an activatable antibody construct to provide flexibility at one or more of the MM-CM1-CM2 substrate bonds, the CM1-CM2 substrate-AB bonds, or both. For example, the AB, MM, and / or CM1-CM2 substrates do not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, particularly Gly) to provide the desired flexibility. Thus, the switchable phenotype of such an activatable antibody construct may benefit from the introduction of one or more amino acids to provide a flexible linker. Furthermore, as described below, when an activatable antibody is provided as a conformationally constrained construct, a flexible linker may be operatively introduced to promote the formation and maintenance of a cyclic structure in the uncleaved activatable antibody.

[0194] For example, in certain embodiments, the activatable antibody has the following formula (the formula below represents the amino acid sequence from N-terminal to C-terminal or C-terminal to N-terminal): (MM)-LP1-(CM1-CM2 substrate)-(AB) (MM)-(CM1-CM2 substrate)-LP2-(AB) (MM)-LP1-(CM1-CM2 substrate)-LP2-(AB) wherein MM, CM1-CM2 substrate, and AB are as defined above; LP1 and LP2 are each independently the same or different flexible linkers, optionally present or absent, comprising at least one flexible amino acid (e.g., Gly). In addition, the above formula provides additional amino acid sequences that can be placed N-terminally or C-terminally to the activatable antibody element. Examples include, but are not limited to, targeting moieties (e.g., ligands for receptors on cells present in target tissues) and moieties that extend serum half-life (e.g., polypeptides that bind serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HAS))).

[0195] The CM1-CM2 substrate is approximately 0.001 to 1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250 or 1500x10 4 M -1 S -1 It is specifically cleaved by at least one MMP at a rate of approximately 0.001 to 1500x10 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 1500x10 4 M-1 S -1 is specifically cleaved by at least one SP at a rate of

[0196] For specific cleavage by the enzyme, contact between the enzyme and the CM1-CM2 substrate is achieved. When an activatable antibody, including MM and AB bound to the CM1-CM2 substrate, is in the presence of a target and sufficient enzymatic activity, the CM1-CM2 substrate can be cleaved. Sufficient enzymatic activity may refer to the ability of the enzyme to contact the CM1-CM2 substrate and effect cleavage. It is readily envisioned that the enzyme may be near the CM1-CM2 substrate but unable to cleave it due to other cellular factors or protein modifications of the enzyme.

[0197] Linkers suitable for use in the compositions described herein are generally those that provide flexibility to the modified AB or activatable antibody to facilitate inhibition of AB binding to a target. Such linkers are commonly referred to as flexible linkers. Suitable linkers can be readily selected and can be any of a variety of suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids in length, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0198] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO:381), and (GGGS)n (SEQ ID NO:382), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can function as neutral tethers between components. Glycine has access to much more Φ-Ψ space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly (SEQ ID NO: 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. Those skilled in the art will recognize that the design of an activatable antibody may include a linker that is fully or partially flexible, and consequently, the linker may include a flexible linker and one or more moieties that confer a less flexible structure to provide the desired multispecific activatable antibody structure.

[0199] In some embodiments, the activatable antibodies described herein also encompass drugs conjugated to the activatable antibody. In some embodiments, the conjugated drug is a therapeutic agent, such as an anti-inflammatory agent and / or an anti-neoplastic agent. In such embodiments, the drug is conjugated to a carbohydrate moiety of the activatable antibody; for example, in some embodiments, the carbohydrate moiety is located outside the antigen-binding region of the antibody or antigen-binding fragment within the activatable antibody. In some embodiments, the drug is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment within the activatable antibody.

[0200] In some embodiments, the agent is a cytotoxic drug such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

[0201] In some embodiments, the agent is a detectable moiety, such as, for example, a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotinyl moieties detectable by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity detectable by optical or calorimetric methods), 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 by a spacer molecule.

[0202] The present disclosure also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragment thereof), or a radioisotope (i.e., a radioconjugate). Suitable cytotoxic agents include, for example, dolastatin and its derivatives (e.g., auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethylauristatin E (MMAE) or monomethylauristatin D (MMAD). In some embodiments, the agent is an agent selected from the group listed in Table 3. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0203] In some embodiments, the drug is linked to AB using a maleimidocaproyl-valine-citrulline linker or a maleimidoPEG-valine-citrulline linker. In some embodiments, the drug is linked to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, the drug is linked to AB using a maleimidoPEG-valine-citrulline linker. In some embodiments, the drug is linked to AB using a maleimidoPEG-valine-citrulline linker. In some embodiments, the drug is monomethyl auristatin D (MMAD) linked to AB using a maleimidoPEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, this linker-payload construct is referred to herein as "vc-MMAD." In some embodiments, the drug is monomethyl auristatin E (MMAE) linked to AB using a maleimidoPEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, this linker-payload construct is referred to herein as "vc-MMAE." The structures of vc-MMAD and vc-MMAE are shown below: vc-MMAD: [ka] vc-MMAE: [ka]

[0204] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Jatropha forsi protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, soapwort (saponaria officinalis) inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecene. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is an example.

[0205] Conjugates of antibodies and cytotoxic agents have been prepared using a variety of bifunctional protein coupling agents, including N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate hydrochloride), activated esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). 14 C-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies (see International Publication No. WO 94 / 11026).

[0206] Table 3 lists some exemplary pharmaceutical agents that may be used in the disclosure described herein, but is by no means meant to be an exhaustive list. [Table 4] TIFF0007781254000009.tif167161

[0207] Those skilled in the art will appreciate that a wide variety of suitable moieties may be conjugated to the antibodies provided by the present disclosure (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0208] Conjugation can be achieved by any chemical reaction that links two molecules, so long as the antibody and other moiety retain their respective activities. This linkage can involve many chemical mechanisms, including covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. However, in some embodiments, the linkage is covalent. Covalent bonding can be achieved either by direct condensation of existing side chains or by the incorporation of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful for linking protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various coupling agents known in the art, but rather is illustrative of some of the more commonly used representative 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)).

[0209] In some embodiments, in addition to the compositions and methods provided herein, conjugated activatable antibodies can be modified for site-specific conjugation via modified amino acid sequences inserted or otherwise incorporated into the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled placement and / or dosage of the conjugated agent within the conjugated activatable antibody. For example, activatable antibodies can be modified 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 or alter antigen binding. In some embodiments, activatable antibodies can be modified to include or incorporate one or more non-naturally occurring amino acid residues within the activatable antibody to provide suitable sites for conjugation. In some embodiments, activatable antibodies can be modified to include or incorporate enzymatically activatable peptide sequences within their activatable antibody sequence.

[0210] Suitable linkers are described in the literature (see, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester)). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives attached to antibodies 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., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. No. 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co. Cat. No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide: Pierce Chem. Co. Cat. No. 2165-G) conjugated to EDC. Chem. Co., catalog number 24510. Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

[0211] The above linkers contain components with different properties, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, which allows for the formation of conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. Sulfo-NHS, in particular, can enhance the stability of carbodiimide bonds. Carbodiimide coupling (e.g., EDC) when used in combination with sulfo-NHS forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0212] 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 can all be the same, i.e., cleavable or non-cleavable, or the two or more linkers can be different, i.e., at least one cleavable and at least one non-cleavable.

[0213] The present disclosure utilizes several methods for attaching a drug to an AB: (a) attachment to the carbohydrate moiety of the AB, or (b) attachment to a sulfhydryl group of the AB, or (c) attachment to an amino group of the AB, or (d) attachment to a carboxylate group of the AB. In the present disclosure, the AB may be covalently attached to the drug via an intermediate linker having at least two reactive groups, one that reacts with the AB and one that reacts with the drug. The linker, which may comprise any compatible organic compound, can be selected so that reaction with the AB (or drug) does not adversely affect the reactivity and selectivity of the AB. Furthermore, attachment of the linker to the drug does not destroy the activity of the drug. Linkers suitable for reaction with oxidized antibodies or oxidized antibody fragments include those containing an amine selected from the group consisting of primary amine groups, secondary amine groups, hydrazine groups, hydrazide groups, hydroxylamine groups, phenylhydrazine groups, semicarbazide groups, and thiosemicarbazide groups. Such reactive functional groups may be present as part of the structure of the linker or may be introduced by appropriate chemical modification of a linker that does not contain such groups.

[0214] In the present disclosure, linkers suitable for attachment to reduced AB include those having specific reactive groups capable of reacting with sulfhydryl groups on the reduced antibody or fragment, including, but not limited to, reactive haloalkyl groups (e.g., haloacetyl groups), p-mercuribenzoate groups, and groups capable of Michael-type addition reactions (e.g., maleimides and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101:3097-3110).

[0215] In the present disclosure, suitable linkers that are not attached to either the oxidized or reduced Ab include those that have specific functional groups that can react with primary amino groups present in unmodified lysine residues in the Ab. Such reactive groups include, but are not limited to, NHS carboxylic acid or carbonate, sulfo-NHS carboxylic acid or carbonate, 4-nitrophenyl carboxylic acid or carbonate, pentafluorophenyl carboxylic acid or carbonate, acylimidazole, isocyanate, and isothiocyanate.

[0216] In the present disclosure, suitable linkers that are not attached to either oxidized or reduced Abs include linkers with specific functional groups that can react with carboxylic acid groups present in aspartic acid or glutamic acid residues in Abs when activated with a suitable reagent. Suitable activation reagents include EDC, with or without added NHS or sulfo-NHS, and other dehydrating agents that utilize carboxamide formation. In these cases, suitable functional groups present on the linker include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0217] The drug can be attached to the linker before or after the linker is attached to AB. In certain applications, it may be desirable to first generate an AB-linker intermediate in which the linker does not contain the associated drug. Depending on the particular application, a particular drug can be covalently attached to the linker. In some embodiments, AB is first attached to the MM, CM1-CM2 substrate, and associated linker, and then attached to the linker for conjugation purposes.

[0218] Branched Linkers: In certain embodiments, branched linkers are utilized that have multiple sites for drug attachment. In the case of multi-site linkers, a single covalent bond to AB results in an AB linker intermediate that can attach drugs at several sites. These sites can be aldehyde or sulfhydryl groups, or any chemical site to which a drug can be attached.

[0219] In some embodiments, higher specific activity (or higher drug to AB ratio) can be achieved by attaching a single-site linker at multiple sites on the AB. These multiple sites can be introduced into the AB by either of two methods. First, multiple aldehyde and / or sulfhydryl groups can be generated in the same AB. Second, a "branched linker" with multiple functional sites for subsequent attachment to a linker can be attached to the aldehydes or sulfhydryls of the AB. The functional sites of the branched linker or multi-site linker can be aldehyde or sulfhydryl groups, or any chemical site to which a linker can be attached. Even higher specific activity can be obtained by combining these two approaches, i.e., attaching a multi-site linker at multiple sites on the AB.

[0220] Cleavable linker: A peptide linker susceptible to cleavage by an enzyme of the complement system, such as, but not limited to, urokinase, tissue plasminogen activator, trypsin, plasmin, or another enzyme with proteolytic activity, can be used in one embodiment of the present disclosure. In one method of the present disclosure, a drug is attached via a linker susceptible to cleavage by complement. The antibody is selected from a class that can activate complement. The antibody-drug conjugate thus activates the complement cascade and releases the drug at the target site. In another method of the present disclosure, a drug is attached via a linker susceptible to cleavage by an enzyme with proteolytic activity, such as urokinase, tissue plasminogen activator, plasmin, or trypsin. These cleavable linkers are useful in conjugated activatable antibodies, including, by non-limiting example, any of the extracellular toxins listed in Table 3.

[0221] Non-limiting examples of cleavable linker sequences are provided in Table 4. [Table 5]

[0222] Additionally, drugs can be attached to AB via disulfide bonds (e.g., disulfide bonds on cysteine ​​molecules). Many tumors naturally release high levels of glutathione (a reducing agent), which can reduce the disulfide bond, resulting in the subsequent release of the drug at the delivery site. In certain embodiments, the reducing agent that modifies the CM1-CM2 substrate will also modify the linker of the conjugated activatable antibody.

[0223] Spacer and cleavable elements: In some embodiments, it may be necessary to construct the linker in such a way as to optimize the spacing between the drug and the AB of the activatable antibody. This can be achieved by using a linker with the following general structure: W-(CH2)nQ This can be achieved by the use of a linker of the formula: W is either -NH-CH2- or -CH2-; Q is an amino acid, a peptide, and n is an integer of 0 to 20.

[0224] In some embodiments, the linker may comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the AB core so that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain branched linkers described above may function as spacer elements.

[0225] It should be understood throughout this discussion that the attachment of a linker to a drug (or a spacer element to a cleavable element, or a cleavable element to a drug) does not require a particular mode of attachment or reaction: any reaction resulting in a product of suitable stability and biological compatibility is acceptable.

[0226] Selection of serum complement and linker: In one method of the present disclosure, if release of a drug is desired, an AB, a class of antibody capable of activating complement, is used. The resulting conjugate retains both the ability to bind antigen and the ability to activate the complement cascade. Thus, in this embodiment of the present disclosure, the drug is connected to one end of a cleavable linker or cleavable element, and the other end of the linker group is attached to a specific site on the AB. For example, if the drug has a hydroxy or amino group, it can be attached to the carboxy terminus of a peptide, amino acid, or other appropriately selected linker via an ester or amide bond, respectively. For example, such drugs can be attached to a linker peptide via a carbodiimide reaction. If the drug contains functional groups that interfere with attachment to the linker, these interfering functional groups can be blocked prior to attachment and then unblocked once the product conjugate or intermediate is produced. The opposite or amino terminus of the linker can then be used directly or after further modification for conjugation to an AB capable of activating complement.

[0227] The linker (or spacer element of the linker) can be of any desired length, and one end can be covalently attached to a specific site on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker.

[0228] Thus, when these conjugates bind to an antigen in the presence of complement, the amide or ester bond attaching the drug to the linker is cleaved, resulting in the release of the drug in its active form. These conjugates, when administered to a subject, achieve delivery or release of the drug at the target site and are particularly effective for the in vivo delivery of pharmaceuticals, antibiotics, antimetabolites, antiproliferative agents, and the like, including but not limited to, those presented in Table 3.

[0229] Linkers for release without complement activation: In yet another application of targeted delivery, drug release without complement activation is desirable because activation of the complement cascade ultimately lyses target cells. This approach is therefore useful when drug delivery and release must be achieved without killing the target cells. This is the goal when delivery of cellular mediators, such as hormones, enzymes, corticosteroids, neurotransmitters, genes, or enzymes, to target cells is desired. These conjugates can be prepared by attaching a drug to an AB that cannot activate complement via a linker that is somewhat susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complexes form rapidly, while drug cleavage occurs slowly, resulting in the release of the compound at the target site.

[0230] Biochemical crosslinkers: In some embodiments, activatable antibodies can be conjugated to one or more therapeutic agents using specific biochemical crosslinkers. Crosslinkers form molecular bridges that link functional groups of two different molecules. To link two different proteins in a stepwise manner, heterobifunctional crosslinkers can be used, eliminating undesired homopolymer formation.

[0231] Peptidyl linkers that are cleavable by lysosomal proteases, such as Val-Cit, Val-Ala, or other dipeptides, are also useful. Additionally, acid-labile linkers that are cleavable in the low pH environment of the lysosome, such as bis-sialyl ether, can be used. Other suitable linkers include cathepsin-labile substrates, particularly those that function optimally at acidic pH.

[0232] Exemplary hetero-bifunctional cross-linkers are shown in Table 5. [Table 6]

[0233] Non-cleavable linker or direct attachment: In some embodiments of the present disclosure, conjugates can be designed so that the drug is delivered to the target but not released. This can be achieved by attaching the drug to the AB directly or via a non-cleavable linker.

[0234] These non-cleavable linkers may include amino acids, peptides, D-amino acids, or other organic compounds that can be modified by the methods described herein to contain a functional group that can then be used for attachment to AB. A general formula for such organic linkers is: W-(CH2)nQ wherein W is either -NH-CH2- or -CH2-; Q is an amino acid, a peptide, and n is an integer of 0 to 20.

[0235] Non-cleavable conjugates: In some embodiments, a compound can be attached to an AB that does not activate complement. When an AB that cannot activate complement is used, this attachment can be achieved using a linker that is susceptible to cleavage by activated complement, or using a linker that is not susceptible to cleavage by activated complement.

[0236] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556.

[0237] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0238] Definition: Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meaning commonly understood by one of ordinary skill in the art. The term "a" or "an" entity refers to one or more of that entity. For example, a compound refers to one or more compounds. Thus, the terms "a," "an," "one or more," and "at least one" may be used interchangeably. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with and in connection with the cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization techniques described herein is that which is well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection, etc.). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the nuclear art or as described herein. The aforementioned techniques and procedures are generally carried out according to conventional methods well known in the art, 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, NY (1989)).The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as these laboratory procedures and techniques, are well known and commonly used in the art.Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

[0239] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0240] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with, or with a much lower affinity (K d >10 -6 ) Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibodies, single chain, Fab and F(ab')2 fragments, scFv, and a Fab expression library.

[0241] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Antibody molecules obtained from humans generally belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Specific classes also have subclasses, such as IgG1 and IgG2. Furthermore, in humans, the light chains can be kappa or lambda chains.

[0242] The term "monoclonal antibody" (mAb) or "monoclonal antibody composition," as used herein, refers to a population of antibody molecules containing only one molecular species of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0243] 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 from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, termed "hypervariable regions," are interposed between more conserved adjacent stretches known as "framework regions" or "FRs." Thus, the term "FR" refers to the naturally occurring amino acid sequences between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to be bound, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." The assignment of amino acids to each domain follows the definitions of the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989).

[0244] As used herein, the term "epitope" encompasses any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" encompasses any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific conformational and charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. An antibody is said to specifically bind an antigen if the dissociation constant is 1 μm or less, in some embodiments, 100 nM or less, and in some embodiments, 10 nM or less.

[0245] As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ) and smaller K d indicates a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and "off rate constant" (K off Both the K and K can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The ratio of α to β allows us to eliminate all parameters unrelated to affinity and to determine the dissociation constant K d (For a review, see Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present disclosure have a binding constant (K d ) is said to specifically bind to a target when the ion concentration is 1 μM or less, in some embodiments 100 nM or less, in some embodiments 10 nM or less, and in some embodiments 100 pM or less to about 1 pM.

[0246] The term "isolated polynucleotide," as used herein, means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an "isolated polynucleotide" can mean (1) that it is not associated with all or part of a polynucleotide in which it is found in nature, (2) that it is operably linked to a polynucleotide with which it is not linked in nature, or (3) that it is not naturally occurring as part of a larger sequence. Polynucleotides of the present disclosure include nucleic acid molecules that encode heavy chain immunoglobulin molecules as set forth herein, and nucleic acid molecules that encode light chain immunoglobulin molecules as set forth herein.

[0247] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, and depending on its origin or source of derivation, an "isolated protein" is (1) not associated with proteins in which it is found in nature, (2) free from other proteins of the same source, e.g., free from mouse proteins, (3) expressed by cells from a different species, or (4) not occurring in nature.

[0248] The term "polypeptide" is used herein as a generic term to refer to naturally occurring proteins, fragments, or analogs of a polypeptide sequence. Naturally occurring protein fragments and analogs are therefore species of the polypeptide genus. Polypeptides of the present disclosure include heavy chain immunoglobulin molecules as set forth herein, and light chain immunoglobulin molecules as set forth herein, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules together with light chain immunoglobulin molecules, e.g., kappa light chain immunoglobulins, and vice versa, as well as fragments and analogs thereof.

[0249] The term "naturally occurring" as applied to an object herein refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (such as a virus) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory or otherwise is naturally occurring.

[0250] As used herein, the term "operably linked" refers to the positioning of the components so described, and is in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0251] The term "control sequence" as used herein refers to a polynucleotide sequence required to affect the expression and processing of the coding sequence to which it is linked. The nature of such control sequences depends on the host organism in prokaryotes, and generally includes a promoter, a ribosomal binding site, and a transcription termination sequence in eukaryotes; generally, such control sequences include a promoter and a transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences. The term "polynucleotide" referred to herein means a nucleotide of at least 10 bases in length, ribonucleotides or deoxynucleotides, or modified forms of any type of nucleotide. This term includes single-stranded and double-stranded forms of DNA.

[0252] The term oligonucleotide, as used herein, includes naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset typically containing 200 or fewer bases in length. In some embodiments, oligonucleotides are 10-60 bases in length, and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length. Oligonucleotides are typically single-stranded, e.g., for probes, but oligonucleotides can be double-stranded, e.g., for use in constructing gene mutants. Oligonucleotides of the present disclosure can be either sense or antisense oligonucleotides.

[0253] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkage" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroseleroleate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoronmidate, etc. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anticancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp.87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. US Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). Oligonucleotides may optionally contain a label for detection.

[0254] As used herein, the 20 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 20 conventional amino acids, α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unnatural amino acids (e.g., D-amino acids) may also be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0255] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition of the nascent RNA transcript is referred to as the transcription direction sequence, and the region of the sequence on the DNA strand that has the same sequence as the RNA and is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the sequence on the DNA strand that has the same sequence as the RNA and is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0256] As applied to polypeptides, the term "substantially identical" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weighting, share at least 80% sequence identity, in some embodiments at least 90% sequence identity, in some embodiments at least 95% sequence identity, and in some embodiments at least 99% sequence identity.

[0257] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0258] As discussed herein, minor changes in the amino acid sequence in an antibody or immunoglobulin molecule are considered to be encompassed by the present disclosure, provided that the changes in amino acid sequence maintain at least 75%, in some embodiments at least 80%, 90%, 95%, and in some embodiments, 99% integrity. Conservative amino acid substitutions are particularly contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are typically classified into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic-hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, it is reasonable to predict that the single substitution of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar substitution of amino acids with structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule, especially when the substitution does not involve an amino acid within a framework region. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative, assays which are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Suitable amino and carboxy termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequences to public or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformation 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 those skilled in the art can recognize sequence motifs and structural conformations that can be used to determine structural and functional domains in accordance with the present disclosure.

[0259] Suitable amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties of such analogs. Analogs can include various muteins of sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in naturally occurring sequences (e.g., portions of polypeptides outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt helices occurring in the parent sequence or other types of secondary structure that characterize the parent sequence). Art-recognized examples of polypeptide secondary and tertiary structure are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0260] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino- and / or carboxy-terminal deletion and / or one or more internal deletions, but with the remaining amino acid sequence identical to the corresponding positions in a naturally occurring sequence, e.g., derived from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, e.g., in some embodiments, at least 14 amino acids in length, in some embodiments, at least 20 amino acids in length, usually at least 50 amino acids in length, and in some embodiments, 70 amino acids in length. As used herein, the term "analog" refers to a polypeptide composed of a segment of at least 25 amino acids that has substantial identity to a portion of the predicted amino acid sequence and has specific binding to a target under suitable binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) with respect to the naturally occurring sequence. Analogs are usually at least 20 amino acids in length, e.g., in some embodiments, at least 50 or more amino acids in length, and can often be as long as a full-length naturally occurring polypeptide.

[0261] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract from biological materials.

[0262] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation or attachment of a radiolabeled amino acid to a polypeptide of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain circumstances, the label or marker can also be a therapeutic agent. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H,14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, and predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. As used herein, the term "pharmaceutical agent or drug" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0263] Other chemical terms herein are used in accordance with conventional usage in the art as exemplified in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0264] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition); in some embodiments, a substantially purified fraction is a composition in which the target species constitutes at least about 50 percent (on a molar basis) of all macromolecular species present.

[0265] Generally, a substantially pure composition will comprise greater than about 80%, and in some embodiments, greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity (contaminating species in the composition cannot be detected by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0266] The term "patient" includes human and veterinary subjects.

[0267] The activatable antibodies of the present disclosure specifically bind a predetermined target, e.g., a human target protein. Activatable antibodies that bind to the same epitope as the activatable antibodies described herein are also encompassed by the present disclosure.

[0268] Those skilled in the art will understand that it is possible to determine, without undue experimentation, whether a monoclonal antibody (e.g., a murine monoclonal or a humanized antibody) has the same specificity as the monoclonal antibody used in the methods described herein by determining whether the former interferes with the latter's binding to its target. If the monoclonal antibody being tested competes with the monoclonal antibody of the present disclosure, as indicated by reduced binding by the monoclonal antibody of the present disclosure, then the two monoclonal antibodies bind to the same or closely related epitopes. A method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody of the present disclosure is to preincubate the monoclonal antibody of the present disclosure with the target, 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 almost certainly has the same or a functionally equivalent epitope specificity as the monoclonal antibody of the present disclosure.

[0269] Multispecific activatable antibodies The present disclosure also provides multispecific activatable antibodies. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize multiple antigens or epitopes and include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody such that binding of the MM reduces the ability of the antigen- or epitope-binding domain to bind to its target. In some embodiments, the MM is attached to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM1-CM2 substrate) that functions as a substrate for at least one MMP protease and at least one SP. The activatable multispecific antibodies provided herein are stable in circulation, are activated at the intended therapeutic and / or diagnostic site but not in normal, i.e., healthy tissue, and, when activated, exhibit target binding at least comparable to that of the corresponding unmodified multispecific antibody.

[0270] In some embodiments, the multispecific activatable antibody is designed to engage immune effector cells, and is referred to herein as an immune effector-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody is designed to engage leukocytes, and is referred to herein as a leukemia-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody is designed to engage T cells, and is 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, e.g., on a T cell, on a natural killer (NK) cell, on a bone marrow mononuclear cell, on a macrophage, and / or 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 mononuclear cell, such as a bone marrow mononuclear cell. In some embodiments, multispecific activatable antibodies are designed to bind to or react with multiple targets and / or multiple epitopes, and are referred to herein as multi-antigen-targeting activatable antibodies. As used herein, the terms "target" and "antigen" are used interchangeably.

[0271] In some embodiments, the immune effector cell engaging multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and an immune effector cell engaging antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the immune effector cell engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the immune effector cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising 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 binding of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the immune effector cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell engaging target, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof (AB2) that binds a second target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the non-immune effector cell engaging antibody is a cancer-targeting antibody. In some embodiments, the non-immune cell effector antibody is an IgG. In some embodiments, the immune effector cell engaging antibody is an scFv. In some embodiments, the targeting antibody (e.g., non-immune cell effector antibody) is an IgG and the immune effector cell engaging antibody is an scFv.In some embodiments, the immune effector cells are white blood cells. In some embodiments, the immune effector cells are T cells. In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are bone marrow mononuclear cells.

[0272] In some embodiments, the T cell engaging multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and a T cell engaging antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the T cell engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the T cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second target. In some embodiments, the T cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell engaging target, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment (AB2) that binds a second target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second target.

[0273] In some embodiments, the T cell engaging multispecific activatable antibody comprises a cancer-targeting antibody or antigen-binding fragment thereof and a T cell engaging antibody or antigen-binding portion thereof, wherein at least one of the cancer-targeting antibody or antigen-binding fragment thereof and / or the T cell engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the T cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the T cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell engaging target, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment (AB2) that binds a second cancer-associated target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0274] In some embodiments, the T cell engaging multispecific activatable antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof and a T cell engaging scFv, wherein at least one of the cancer-targeting IgG antibody or antigen-binding fragment thereof and / or the T cell engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the T cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the T cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T cell engaging target, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind the first target, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment (AB2) that binds a second cancer-associated target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0275] In some embodiments of immune effector-engaging multispecific activatable antibodies, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, e.g., any target listed in Table 1, such as, but not limited to, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and another antigen is typically a stimulatory or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of T cells or NK cells. Examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also 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 NK-expressed KIR. The antibody domain that provides specificity for a T cell surface antigen can also be replaced with a ligand or ligand domain that binds to a T cell receptor, an NK cell receptor, a macrophage receptor, and / or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9.

[0276] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the cancer microenvironment and includes antibodies, e.g., IgG or scFv, against tumor-targeting and agonist antibodies, e.g., IgG or scFv against costimulatory receptors expressed on the surface of activated T cells or NK cells, where at least one of the cancer-targeting and / or agonist antibodies is masked. Examples of costimulatory receptors include, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, and OX40. In this embodiment, upon activation by tumor-associated proteases, the multispecific activatable antibody effectively crosslinks and activates T cell- or NK cell-expressed costimulatory receptors in a tumor-dependent manner, enhancing the activity of T cells responding to any tumor antigen via their endogenous T cell antigen or NK-activating receptor. The activation-dependent nature of these T cell or NK cell costimulatory receptors focuses the activity of the activated multispecific activatable antibody toward tumor-specific T cells without activating all T cells regardless of their antigen specificity. In one embodiment, at least the costimulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of autoreactive T cells that may be present in tissues that also express the antigen recognized by the tumor target-specific antibody in the multispecific activatable antibody, but whose activity is limited by the lack of co-receptor engagement.

[0277] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the microenvironment of a disease characterized by T cell hyperstimulation, such as, but not limited to, an autoimmune disease or inflammatory disease. Such a multispecific activatable antibody includes an antibody, e.g., an IgG or scFv, directed against a target comprising a surface antigen expressed in tissues targeted by T cells in autoimmune or inflammatory diseases, and an antibody, e.g., an IgG or scFv, directed against an inhibitory receptor expressed on the surface of T cells or NK cells, wherein at least one of the disease tissue-targeting 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 NK-expressed KIR. Examples of tissue antigens targeted by T cells in autoimmune diseases include, but are not limited to, surface antigens expressed on myelin or nerve cells in multiple sclerosis, or surface antigens expressed on pancreatic islet cells in type 1 diabetes. In this embodiment, the multispecific activatable antibodies, upon localization in tissues under autoimmune attack or inflammation, become activated and co-engage T cell or NK cell inhibitory receptors to suppress the activity of autoreactive T cells responding to any diseased tissue-targeted antigens through their endogenous TCRs or activating receptors. In one embodiment, at least one or more antibodies are masked to prevent the suppression of desired T cell responses in non-diseased tissues where the target antigen may also be expressed.

[0278] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-CD3 epsilon (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the targeting antibody or antigen-binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that binding 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, where AB1 is attached to a masking moiety (MM1) such that binding 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 comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second target.

[0279] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the anti-anti-anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0280] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0281] In some embodiments, the T cell engaging multispecific activatable antibody comprises an OKT3-derived anti-CD3 epsilon (CD3ε) scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the OKT3 scFv or OKT3-derived scFv is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that binding 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 antigen-binding fragment thereof (AB1) that binds to CD3ε, where AB1 is attached to a masking moiety (MM1) such that binding 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 comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second target.

[0282] In some embodiments, the T cell engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0283] In some embodiments, the T cell-engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0284] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv, and at least one of the targeting antibody or antigen-binding fragment thereof and / or the anti-CTLA-4 scFv is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CTLA-4. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that binding 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 antigen-binding fragment thereof (AB1) that binds CTLA-4, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second target.

[0285] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv and a targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CTLA-4 scFv and / or the targeting IgG antibody or antigen-binding portion thereof is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CTLA-4, wherein AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CTLA-4. In some embodiments, the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that binding 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 antigen-binding fragment thereof (AB1) that binds CTLA-4, where AB1 is attached to a masking moiety (MM1) such that binding of MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, where AB2 is attached to a masking moiety (MM2) such that binding of MM2 reduces the ability of AB2 to bind the second target.

[0286] In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody comprises at least a first antibody or antigen-binding fragment thereof that binds a first target and / or a first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and / or a second epitope. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different targets. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different epitopes on the same target. In some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds a combination of multiple different targets and multiple different epitopes on the same target.

[0287] In some embodiments, the multispecific activatable antibody comprising an IgG has a masked IgG variable domain. In some embodiments, the multispecific activatable antibody comprising an scFv has a masked scFv domain. In some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, and at least one of the IgG variable domains is attached to a masking moiety. In some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, and at least one of the scFv domains is attached to a masking moiety. In some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, and at least one of the IgG variable domains is attached to a masking moiety and at least one of the scFv domains is attached to a masking moiety. In some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, and each of the IgG variable domain and the scFv domain is attached to its own masking moiety. In some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T cell surface antigen. In some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a different target antigen, hi some embodiments, one antibody domain of the multispecific activatable antibody has specificity for an epitope of the target antigen and another antibody domain has specificity for a different epitope of the target antigen.

[0288] In multispecific activatable antibodies, scFvs may be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, to the carboxyl terminus of the light chain of an IgG activatable antibody, or to the carboxyl terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs may be fused to the amino terminus of the heavy chain of an IgG activatable antibody, to the amino terminus of the light chain of an IgG activatable antibody, or to the amino terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs may be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. In some embodiments, a masking moiety (MM) linked to the CM1-CM2 substrate is attached to the antigen-binding domain of the IgG, masking that domain. In some embodiments, a masking moiety (MM) linked to the CM1-CM2 substrate is attached to the antigen-binding domain of at least one scFv, masking that domain. In some embodiments, the masking moiety (MM) linked to the CM1-CM2 substrate is attached to the antigen-binding domain of an IgG, masking that domain, and the masking moiety (MM) linked to the CM1-CM2 substrate is attached to the antigen-binding domain of at least one scFv, masking that domain.

[0289] The present disclosure provides examples of multispecific activatable antibody structures, including, but not limited to: (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 matrix-LP2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL-CL)2:(MM-LP1-CM1-CM2 matrix-LP2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(MM-LP1-CM1-CM2 matrix-LP2-VL-CL)2:(VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(MM-LP1-CM1-CM2 matrix-LP2-VL-CL)2:(VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH * -L3-VL * -LP2-CM1-CM2 matrix-LP1-MM)2:(VH-CH1-CH2-CH3)2;(VL-CL-L4-VL) * -L3-VH * -LP2-CM1-CM2 matrix-LP1-MM)2:(VH-CH1-CH2-CH3)2;(MM-LP1-CM1-CM2 matrix-LP2-VL * -L3-VH * -L4-VL-CL)2:(VH-CH1-CH2-CH3)2;(MM-LP1-CM1-CM2 matrix-LP2-VH) * -L3-VL * -L4-VL-CL)2:(VH-CH1-CH2-CH3)2;(VL-CL-L4-VH * -L3-VL * -LP2-CM1-CM2 matrix-LP1-MM)2:(MM-LP1-CM1-CM2 matrix-LP2-VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH * -L3-VL * -LP2-CM1-CM2 matrix-LP1-MM)2:(MM-LP1-CM1-CM2 matrix-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); or (VL-CL-L4-VL * -L3-VH * -LP2-CM1-CM2 substrate-LP1-MM)2:(VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2, where VL and VH are the light and heavy chain variable domains of the first specificity contained in IgG; * and V.H. * is the variable domain of the second specificity contained in the scFv; LP1 is the linker peptide connecting the masking moiety (MM) and the CM1-CM2 substrate; LP2 is the linker peptide connecting the CM1-CM2 substrate and the antibody; L3 is the linker peptide connecting the variable domains of the scFv; L4 is the linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is the light chain constant domain; and CH1, CH2, and CH3 are heavy chain constant domains. The first and second specificities can be for any antigen or epitope.

[0290] In some embodiments of the T cell engaging multispecific activatable antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, such as, but not limited to, any of the targets listed in Table 1, including, but not limited to, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and another antigen is typically an antigen present on the surface of a T cell, natural killer (NK) cell, bone marrow mononuclear cell, macrophage, or other target. Stimulatory (also referred to herein as activating) or inhibitory receptors present on the surface of T cell and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. Antibody domains that provide specificity for T cell surface antigens can also be replaced with ligands or ligand domains that bind to T cell receptors, NK cell receptors, macrophage receptors, and / or other immune effector cell receptors, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9. In some embodiments of the multi-antigen targeted activatable antigen, one antigen is selected from the group of targets listed in Table 1 and another antigen is selected from the group of targets listed in Table 1.

[0291] 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 Jagged 1 and Jagged 2. In some embodiments, the targeting antibody is 4D11v2, which is specific for binding to human and mouse Jagged 1 and Jagged 2.

[0292] In some embodiments, the targeting antibody may be in the form of an activatable antibody. In some embodiments, the scFv may be in the form of a Pro-scFv (see, e.g., WO2009 / 025846, WO2010 / 081173).

[0293] In some embodiments, the scFv is specific for binding to CD3ε and is or is derived from an antibody or fragment thereof that binds CD3ε, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).

[0294] In some embodiments, the anti-CTLA-4 scFv comprises the following amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVTVSSAS (SEQ ID NO: 347)

[0295] 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.

[0296] In some embodiments, the anti-CD3ε scFv comprises the following amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 349)

[0297] 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.

[0298] In some embodiments, the scFv is specific for binding one or more T cells, one or more NK cells, and / or one or more macrophages. In some embodiments, the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.

[0299] In some embodiments, the multispecific activatable antibody also includes a drug conjugated to AB. In some embodiments, the drug is a therapeutic agent. In some embodiments, the drug is an anti-tumor drug. In some embodiments, the drug is a toxin or a fragment thereof. In some embodiments, the drug is conjugated to the multispecific activatable antibody via a linker. In some embodiments, the drug is conjugated to AB via a cleavable linker. In some embodiments, the drug is conjugated to AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the drug is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the drug is an agent selected from the group listed in Table 4. In some embodiments, the drug is a dolastatin. In some embodiments, the drug is an auristatin or a derivative thereof. In some embodiments, the drug is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0300] In some embodiments, the multispecific activatable antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0301] In some embodiments, the multispecific activatable antibody naturally comprises one or more disulfide bonds. In some embodiments, the multispecific activatable antibody can be engineered to comprise one or more disulfide bonds.

[0302] The present disclosure also provides isolated nucleic acid molecules encoding the multispecific activatable antibodies described herein, and vectors comprising these isolated nucleic acid sequences. The present disclosure provides methods of producing multispecific antibodies by culturing cells under conditions that result in expression of the activatable antibodies, wherein the cells comprise such nucleic acid molecules. In some embodiments, the cells comprise such vectors.

[0303] The present disclosure also provides methods for producing a multispecific activatable antibody of the present disclosure by (a) culturing a cell containing a nucleic acid construct encoding the multispecific activatable antibody under conditions that result in expression of the multispecific activatable antibody, and (b) recovering the multispecific activatable antibody.

[0304] The present disclosure also provides multispecific antibody and / or multispecific activatable antibody compositions comprising at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that binds a second target or a second epitope, wherein at least AB1 is conjugated or otherwise attached to a masking moiety (MM1) such that the binding of MM1 reduces the ability of AB1 to bind its target. In some embodiments, MM1 is conjugated to AB1 via a CM1-CM2 substrate for MMPs and SPs, and at least one of the MMPs and SPs co-localizes with the target of AB1 at a therapeutic or diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, are activated at the intended site of treatment and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the target of AB1 at least comparable to that of the corresponding unmodified multispecific antibody.

[0305] In some embodiments, the multispecific activatable antibody comprises a linking peptide between the MM1 and CM1-CM2 substrates.

[0306] In some embodiments, the multispecific activatable antibody comprises a linking peptide between the CM1-CM2 substrate and AB1.

[0307] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and at least a portion of the multispecific activatable antibody has the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM1-LP1-CM1-CM2 substrate-LP2-AB1 or AB1-LP2-CM1-CM2 substrate-LP1-MM1. In some embodiments, the two connecting peptides need not be identical to each other.

[0308] In some embodiments, at least one of LP1 or LP2 is (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).

[0309] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2.

[0310] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1), a second connecting peptide (LP2), and a connecting peptide between CM1 and CM2 (LP'), and at least a portion of the multispecific activatable antibody has the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM1-LP1-CM1-CM2 substrate-LP2-AB1 or AB1-LP2-CM1-CM2 substrate-LP1-MM1. In some embodiments, the connecting peptides need not be identical to each other.

[0311] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0312] In some embodiments, the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds a second target or a second epitope. In some embodiments, each AB in the multispecific activatable antibody is independently selected from the group consisting of monoclonal antibodies, domains, antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single-domain heavy chain antibodies, and single-domain light chain antibodies. In some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., murine or rat), chimeric, humanized, or fully human, monoclonal antibody.

[0313] In some embodiments, each AB in the multispecific activatable antibody has a dissociation constant of about 100 nM or less for binding to its corresponding target or epitope.

[0314] In some embodiments, MM1 has a dissociation constant for binding to its corresponding AB that is higher than the dissociation constant for binding of AB to its corresponding target or epitope.

[0315] In some embodiments, MM1 has a dissociation constant for binding to its corresponding AB that is less than or equal to the dissociation constant for binding of AB to its corresponding target or epitope.

[0316] In some embodiments, MM1 does not interfere with or compete with its corresponding AB for binding to its corresponding target or epitope when the multispecific activatable antibody is in its cleaved state.

[0317] In some embodiments, MM1 is a polypeptide between about 2 and 40 amino acids in length. In some embodiments, each MM in the multispecific activatable antibody is a polypeptide less than or equal to 40 amino acids in length.

[0318] In some embodiments, MM1 has a polypeptide sequence that differs from the sequence of the target of its corresponding AB.

[0319] In some embodiments, MM1 has a polypeptide sequence that is 50% or less identical to any natural binding partner of its corresponding AB. In some embodiments, MM1 has a polypeptide sequence that is 25% or less identical to any natural binding partner of its corresponding AB. In some embodiments, MM1 has a polypeptide sequence that is 10% or less identical to any natural binding partner of its corresponding AB.

[0320] In some embodiments, the binding of MM1 is determined by the dissociation constant (K d ) is not bound to MM1 for its corresponding target or epitope. d reduces the ability of its corresponding AB to bind its target or epitope so that it is at least 20-fold greater than

[0321] In some embodiments, the binding of MM1 is determined by the dissociation constant (K d) is not bound to MM1 for its corresponding target or epitope. d reduces the ability of its corresponding AB to bind its target or epitope so that it is at least 40-fold greater than its corresponding AB.

[0322] In some embodiments, the binding of MM1 is determined by the dissociation constant (K d ) is not bound to MM1 for its corresponding target or epitope. d reduces the ability of its corresponding AB to bind its target or epitope so that it is at least 100-fold greater than

[0323] In some embodiments, the binding of MM1 is determined by the dissociation constant (K d ) is not bound to MM1 for its corresponding target or epitope. d The target or epitope binding ability of the corresponding AB is reduced so that the target or epitope binding ability of the corresponding AB is at least 1000 times greater than the target or epitope binding ability of the corresponding AB.

[0324] In some embodiments, the binding of MM1 is determined by the dissociation constant (K d ) is not bound to MM1 for its corresponding target or epitope. d The ability of its corresponding AB to bind its target or epitope is reduced so that it is at least 10,000 times greater than the corresponding AB.

[0325] In some embodiments, MM1 is an amino acid sequence selected from the MMs disclosed herein.

[0326] In some embodiments, the multispecific activatable antibody comprises at least a second masking moiety (MM2) that inhibits binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state, and an additional cleavable moiety (CM') attached to AB2, where CM' is either a CM1-CM2 substrate or a polypeptide that functions as a substrate for a second protease. In some embodiments, CM' is a polypeptide 15 amino acids or less in length. In some embodiments, CM' is a CM1-CM2 substrate, where CM1 and CM2 in the CM1-CM2 substrate are each independently a polypeptide 15 amino acids or less in length.

[0327] In some embodiments, the MMP protease, SP protease, and / or second protease co-localize with a second target or epitope in a tissue, and the MMP protease, SP protease, and / or second protease cleaves the CM' in the multispecific activatable antibody upon exposure of the multispecific activatable antibody to the MMP protease, SP protease, and / or second protease. In some embodiments, the MMP protease, SP protease, and / or second protease co-localize with a first target or epitope and a second target or epitope in a tissue. In some embodiments, the MMP protease, SP protease, and / or 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 a protease selected from the group consisting of those shown in Table 6.

[0328] In some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has a dissociation constant for binding to its corresponding AB that is greater than the dissociation constant of AB for its corresponding target or epitope.

[0329] In some embodiments, each MM in the multispecific activatable antibody has a dissociation constant for binding to its corresponding AB that is less than or equal to the dissociation constant of the AB for its corresponding target or epitope.

[0330] In some embodiments, each MM in a multispecific activatable antibody does not interfere with or compete with its corresponding AB for binding to its corresponding target or epitope when the multispecific activatable antibody is in a cleaved state.

[0331] In some embodiments, each MM in the multispecific activatable antibody is a polypeptide between about 2 and 40 amino acids in length, hi some embodiments, each MM in the multispecific activatable antibody is a polypeptide less than or equal to 40 amino acids in length.

[0332] In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that differs from the sequence of the target of the corresponding AB.

[0333] In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is 50% or less identical to any natural binding partner of its corresponding AB. In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is 25% or less identical to any natural binding partner of its corresponding AB. In some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is 10% or less identical to any natural binding partner of its corresponding AB.

[0334] In some embodiments, the binding of each MM is determined by the dissociation constant (Kd ) is not bound to MM for its corresponding target or epitope d reduces the ability of its corresponding AB to bind its target or epitope so that it is at least 20-fold greater than

[0335] In some embodiments, the binding of each MM is determined by the dissociation constant (K d ) is not bound to MM for its corresponding target or epitope d reduces the ability of its corresponding AB to bind its target or epitope so that it is at least 40-fold greater than its corresponding AB.

[0336] In some embodiments, the binding of each MM is determined by the dissociation constant (K d ) is not bound to MM for its corresponding target or epitope d reduces the ability of its corresponding AB to bind its target or epitope so that it is at least 100-fold greater than

[0337] In some embodiments, the binding of each MM is determined by the dissociation constant (K d ) is not bound to MM for its corresponding target or epitope d The target or epitope binding ability of the corresponding AB is reduced so that the target or epitope binding ability of the corresponding AB is at least 1000 times greater than the target or epitope binding ability of the corresponding AB.

[0338] In some embodiments, the binding of each MM is determined by the dissociation constant (K d ) is not bound to MM for its corresponding target or epitope d The ability of its corresponding AB to bind its target or epitope is reduced so that it is at least 10,000 times greater than the corresponding AB.

[0339] In some embodiments, each MM is an amino acid sequence selected from the MMs disclosed herein.

[0340] In some embodiments, the protease that cleaves the CM1-CM2 substrate sequence co-localizes in tissues with the target of AB1 in the multispecific activatable antibody, and the MMP protease and / or SP protease, i.e., at least one of the MMP protease and the SP protease, cleaves the CM1-CM2 substrate in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0341] In some embodiments, the multispecific activatable antibody comprises multiple CM1-CM2 substrate sequences, and an MMP protease and / or SP protease that cleaves at least one CM1-CM2 substrate sequence co-localizes in tissue with a target of at least one region of the AB region in the multispecific activatable antibody, and the MMP protease and / or SP protease cleaves the CM1-CM2 substrate in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0342] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least two-fold greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0343] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least three times greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0344] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least four times greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0345] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least 5-fold greater than the dissociation constant of unmodified AB binding to that target, whereas in the cleaved state, AB binds its target.

[0346] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least 10-fold greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0347] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least 20-fold greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0348] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least 40-fold greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0349] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least 50-fold greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0350] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least 100-fold greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0351] In some embodiments, the CM1-CM2 substrate is positioned in the multispecific activatable antibody such that, in the uncleaved state, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced and occurs with a dissociation constant that is at least 200-fold greater than the dissociation constant of unmodified AB binding to that target, while in the cleaved state, AB binds its target.

[0352] The present disclosure also provides compositions and methods encompassing multispecific activatable antibodies comprising 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 conjugated to a masking moiety (MM1) that reduces the ability of AB1 to bind its target. In some embodiments, each AB is conjugated to a MM that reduces the ability of its corresponding AB for each target. For example, in embodiments of a multispecific activatable antibody, AB1 is conjugated to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, and AB2 is conjugated to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target. In some embodiments, a multispecific activatable antibody comprises multiple AB regions, and in such embodiments, for each AB of the activatable antibody, AB1 is linked to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, AB2 is linked to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target, AB3 is linked to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target, and so on.

[0353] In some embodiments, the multispecific activatable antibody further comprises at least one CM1-CM2 substrate that is a substrate for MMP proteases and SP proteases, where the CM1-CM2 substrate links the MM to the AB. For example, in some embodiments, the multispecific activatable antibody comprises at least a first antibody or antibody fragment (AB1) and a second antibody or antibody fragment (AB2) that specifically bind a target, where at least the first AB in the multispecific activatable antibody is conjugated to a masking moiety (MM1) that reduces the ability of AB1 to bind its target via a first CM1-CM2 substrate. In some multispecific activatable antibody embodiments, AB1 is conjugated to MM1 via a first CM1-CM2 substrate, and AB2 is conjugated to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target via a second CM1-CM2 substrate. In some embodiments, the multispecific activatable antibody comprises two or more AB regions; in some of these embodiments, AB1 is conjugated to MM1 via a first CM1-CM2 substrate, AB2 is conjugated to MM2 via a second CM1-CM2 substrate, AB3 is conjugated 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.

[0354] Activatable antibodies having a non-binding steric moiety or a binding partner of a non-binding steric moiety The present disclosure also provides activatable antibodies comprising a non-binding steric moiety (NB) or a binding partner (BP) for the non-binding steric moiety, where the BP recruits or attracts the NB to the activatable antibody. The activatable antibodies provided herein include, for example, activatable antibodies comprising a non-binding steric moiety (NB), a CM1-CM2 substrate, and an antibody or antibody fragment (AB) that binds to a target; activatable antibodies comprising a binding partner (BP) for the non-binding steric moiety, a CM1-CM2 substrate, and an AB; and activatable antibodies comprising a BP to which an NB has been recruited, a CM1-CM2 substrate, and an AB that binds a target. An activatable antibody in which the NB is covalently linked to the CM1-CM2 substrate and AB of the activatable antibody or is bound by interaction with a BP that is covalently linked to the CM1-CM2 substrate and AB of the activatable antibody is referred to herein as an "NB-containing activatable antibody." Activatable or switchable means that the activatable antibody exhibits a first level of target binding when in an inhibited, masked, or uncleaved state (a first conformation), and a second level of target binding when in an uninhibited, unmasked, and / or cleaved state (i.e., a second conformation, i.e., an activated antibody), where the second level of target binding is greater than the first level of binding. Activatable antibody compositions can exhibit enhanced bioavailability and better biodistribution compared to conventional antibody therapies.

[0355] In some embodiments, the activatable antibody results in a reduction of toxic and / or adverse side effects that would result from binding at non-therapeutic and / or non-diagnostic sites if the AB were not masked or otherwise inhibited from binding to such sites.

[0356] In one embodiment, the activatable antibody comprises a non-binding steric moiety (NB); a CM1-CM2 substrate; and at least an antibody or antibody fragment (AB) that specifically binds to a target, where NB is a polypeptide that does not specifically bind to AB; the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) for an enzyme; the CM1-CM2 substrate is configured such that, in its uncleaved state, NB prevents AB from binding to its target, and in its cleaved state, NB does not prevent AB from binding to its target; and NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. As used herein and throughout, the term polypeptide refers to any polypeptide comprising at least two amino acid residues, including larger polypeptides, full-length proteins, and fragments thereof, and the term polypeptide is not limited to single-chain polypeptides but may encompass multiple units, e.g., multi-chain polypeptides. When a polypeptide is of shorter length, e.g., less than 50 total amino acids in length, the terms peptide and polypeptide are used interchangeably herein, and when a polypeptide is of longer length, e.g., 50 amino acids in length or more, the terms polypeptide and protein are used interchangeably herein.

[0357] In one embodiment, the activatable antibody comprises a non-binding steric moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) NB comprises a polypeptide that does not specifically bind to AB; (ii) the CM1-CM2 substrate is a polypeptide up to 50 amino acids in length that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is configured such that, in its uncleaved state, NB interferes with binding of AB to its target, and in its cleaved state, NB does not interfere with binding of AB to its target; and (iv) NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. For example, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate independently has a length of up to 15 amino acids.

[0358] In one embodiment, the activatable antibody comprises a non-binding steric moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) NB is a polypeptide that does not specifically bind to AB; (ii) the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is configured such that, in the uncleaved state, NB interferes with binding of AB to its target and, in the cleaved state, NB does not interfere with binding of AB to its target; (iv) NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (v) the multispecific activatable antibody, in the uncleaved state, has the following structural configuration from N- to C-terminus: NB-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-NB.

[0359] In one embodiment, the activatable antibody comprises a non-binding steric moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) NB is a polypeptide that does not specifically bind to AB; (ii) the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is configured such that, in an uncleaved state, NB interferes with binding of AB to its target, and in a cleaved state, NB does not interfere with binding of AB to its target; the NB in ​​the uncleaved activatable antibody reduces the ability of AB to bind its target by at least 50%, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, compared to the ability of cleaved AB to bind its target; and (iv) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. The reduction in the ability of the AB to bind its target is determined, for example, using an assay as described herein or an in vitro target displacement assay, such as the assay described in International Publication Nos. WO2009 / 025846 and WO2010 / 081173.

[0360] In one embodiment, the activatable antibody comprises a binding partner (BP) for a non-binding steric moiety (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein the BP is a polypeptide that binds to the NB when exposed to the NB; the NB is a polypeptide that does not specifically bind to the AB; the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; the CM1-CM2 substrate is configured such that, in the presence of the NB, in an uncleaved state, the NB prevents the AB from binding to its target, and in a cleaved state, the NB does not prevent the AB from binding to its target and the BP does not prevent the AB from binding to its target; and the NB and BP do not inhibit cleavage of the CM1-CM2 substrate by the enzyme. In some examples of this embodiment, the BP of the activatable antibody is optionally bound to the NB. In one embodiment, the NB is recruited in vivo by the BP of the activatable antibody.

[0361] In some examples of these activatable antibody embodiments, the activatable antibody is formulated as a composition. In some of these embodiments, the composition also includes a NB, where the NB is co-formulated with the activatable antibody, including the BP, a CM1-CM2 substrate, and an AB. In some examples of this embodiment, the BP is selected from the group consisting of an albumin-binding peptide, a fibrinogen-binding peptide, a fibronectin-binding peptide, a hemoglobin-binding peptide, a transferrin-binding peptide, an immunoglobulin domain-binding peptide, and other serum protein-binding peptides.

[0362] In some examples of any of these activatable antibody embodiments, the NB is a soluble globular protein. In some examples of any of these activatable antibody embodiments, the NB is a protein that circulates in the bloodstream. In some examples of any of these activatable antibody embodiments, the NB is selected from the group consisting of albumin, fibrinogen, fibronectin, hemoglobin, transferrin, immunoglobulin domains, and other serum proteins.

[0363] In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a substrate (S) for a protease. In some examples of any of these activatable antibody embodiments, the protease co-localizes with its target in a tissue, and the protease cleaves the CM1-CM2 substrate in the activatable antibody upon exposure of the activatable antibody to the protease. In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide that is 50 amino acids in length. In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) that is up to 15 amino acids in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0364] In some examples of any of these activatable antibody embodiments, the activatable antibody has the following structural arrangement, N-terminal to C-terminal, in its uncleaved state: NB-CM1-CM2 substrate-AB, AB-CM1-CM2 substrate-NB, BP-CM1-CM2 substrate-AB, or AB-CM1-CM2 substrate-BP. In embodiments in which the activatable antibody comprises a BP and is in the presence of its corresponding NB, the activatable antibody has the following structural arrangement, N-terminal to C-terminal, in its uncleaved state: NB:BP-CM1-CM2-AB, NB:BP-CM2-CM1-AB, AB-CM1-CM2-BP:NB, or AB-CM2-CM1-BP:NB, where ":" represents an interaction, e.g., binding, between the NB and the BP.

[0365] In some examples of any of these activatable antibody embodiments, the activatable antibody specifically binds a predetermined target and includes antibodies or antigen-binding fragments thereof that are monoclonal antibodies, domain antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scabs, dAbs, single domain heavy chain antibodies, and single domain light chain antibodies. In some embodiments, such antibodies or immunologically active fragments thereof that bind their targets are murine, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0366] In some examples of any of these activatable antibody embodiments, the activatable antibody comprises a combination of a variable heavy chain region comprising an amino acid sequence provided herein and a variable light chain region comprising an amino acid sequence provided herein. In some embodiments, the activatable antibody comprises a combination of a variable heavy chain region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence provided herein and a variable light chain region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence provided herein.

[0367] In some examples of any of these activatable antibody embodiments, the activatable antibody also includes a drug conjugated to AB. In some embodiments, the drug is a therapeutic agent. In some embodiments, the drug is an anti-tumor drug. In some embodiments, the drug is a toxin or a fragment thereof. In some embodiments, the drug is conjugated to AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the drug is conjugated to AB via a non-cleavable linker. In some embodiments, the drug is a drug selected from the group listed in Table 3. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the drug is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalator, or other DNA damaging agent. In some embodiments, the drug is a dolastatin. In some embodiments, the drug is an auristatin or a derivative thereof. In some embodiments, the drug is auristatin E or a derivative thereof. In some embodiments, the drug is monomethylauristatin E (MMAE). In some embodiments, the drug is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0368] In some examples of any of these activatable antibody embodiments, the activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0369] In some examples of any of these activatable antibody embodiments, the activatable antibody also includes a spacer. In some examples of any of these activatable antibody embodiments, the activatable antibody also includes a signal peptide. In some embodiments, the signal peptide is joined to the activatable antibody via a spacer. In some examples of any of these activatable antibody embodiments, the spacer is connected directly to the MM of the activatable antibody.

[0370] In some embodiments, the serum half-life of an activatable antibody is longer than the serum half-life of its corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of an activatable antibody is similar to the half-life of its corresponding multi-antibody. In some embodiments, the serum half-life of an activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of an activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism.In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism.

[0371] The present disclosure also provides isolated nucleic acid molecules encoding any of these activatable antibodies, and vectors comprising these isolated nucleic acid sequences. The present disclosure provides methods of producing an activatable antibody by culturing cells under conditions that result in expression of the activatable antibody, wherein the cells comprise such nucleic acid sequences. In some embodiments, the cells comprise such vectors.

[0372] The number of dissociation events of NB-containing activatable antibodies against the target (K d ) is the K of AB against the target when AB is not bound to NB or NB:BP. d The dissociation constant (K d ) is the K of parent AB against the target d For example, the K of an NB-containing activatable antibody against a target d is the K of AB when AB is not combined with NB or NB:BP. d , or the K of parent AB relative to 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 or more times greater than, or 5 to 10, 10 to 100, 10 to 1,000, 10 to 10,000, 10 to 100,000, 10 to 1,000,000, 10 to 10,000,000, 10 to 10,000,000, 10 to 100,000, ,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times larger. Conversely, the binding affinity of the NB-containing activatable antibody for the target is less than the binding affinity of the AB when the AB is not conjugated with the NB or NB:BP, or is less than the binding affinity of the parent AB for the target.For example, the binding affinity of the NB-containing activatable antibody for a target may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more times greater, or at least 5-10, 10-100, 10-1,000, 10-10,000, 10- 100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times smaller.

[0373] When the NB-containing activatable antibody is in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB when the AB is not conjugated with the NB or NB:BP. When the NB-containing activatable antibody is in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the parent AB to its target. The ability of the NB-containing activatable antibody to bind its target when compared to the binding of an AB not conjugated to the NB or NB:BP, or the binding of the parent AB to its target, can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% over a period of at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, e.g., when measured in vitro and / or in vivo.

[0374] When the NB-containing activatable antibody is in the presence of its target but not in the presence of a modifying agent (e.g., a protease or other enzyme), specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB when the AB is not conjugated with the NB or NB:BP. When the NB-containing activatable antibody is in the presence of its target but not in the presence of a modifying agent (e.g., a protease, other enzyme, reducing agent, or light), specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the parent AB to its target. The ability of the NB-containing activatable antibody to bind its target when compared to the binding of an AB not conjugated with the NB or NB:BP, or the binding of the parent AB to its 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 more, e.g., when measured in vitro and / or in vivo.

[0375] In some examples of any of these activatable antibody embodiments, the activatable antibody also includes a drug that is conjugated to the AB to generate the activatable antibody conjugate. In some embodiments of the activatable antibody conjugate, the drug is a therapeutic agent. In some embodiments, the drug is a diagnostic agent. In some embodiments, the drug is a detectable marker. In some embodiments of the activatable antibody conjugate, the drug is an anti-tumor agent. In some embodiments of the activatable antibody conjugate, the drug is a toxin or a fragment thereof. In some embodiments of the activatable antibody conjugate, the drug 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 drug is conjugated to the AB via a non-cleavable linker. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the drug is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalator, or other DNA damaging agent. In some embodiments, the drug is an agent selected from the group listed in Table 3. In some embodiments, the drug is a dolastatin. In some embodiments, the drug is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0376] In some examples of any of these activatable antibody embodiments, the activatable antibody is a dual target binding activatable antibody. Such dual target binding activatable antibodies comprise two Abs capable of binding the same or different targets. In certain embodiments, the dual targeting activatable antibody comprises a bispecific antibody or fragment thereof.

[0377] A dual-target-binding activatable antibody is designed to have a CM1-CM2 substrate that can be cleaved by a cleaving agent co-localized in a target tissue, along with one or both targets that can bind to the AB of the activatable antibody. A dual-target-binding activatable antibody with multiple ABs for the same or different targets can be designed to have multiple CM1-CM2 substrates, where a first CM1-CM2 substrate can be cleaved by a cleaving agent in a first target tissue, and a second CM1-CM2 substrate can be cleaved by a cleaving agent in a second target tissue and has one or more targets that bind to the AB of the activatable antibody. In one embodiment, the first and second target tissues are spatially separated, for example, at different sites in an organism. In one embodiment, the first and second target tissues are the same tissue but temporally separated, for example, the same tissue at two different time points, for example, the first time point is when the tissue is an early-stage tumor and the second time point is when the tissue is a later-stage tumor.

[0378] The present disclosure also provides nucleic acid molecules encoding the activatable antibodies described herein. The present disclosure also provides vectors containing these nucleic acids. The activatable antibodies described herein are produced by culturing cells under conditions that result in expression of the activatable antibodies, where the cells contain these nucleic acid molecules or vectors.

[0379] The present disclosure also provides a method for producing an activatable antibody. In one embodiment, the method includes the steps of: (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions that result in expression of the activatable antibody, the activatable antibody comprising: (i) a non-binding steric moiety (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein: (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for an enzyme; (3) the CM1-CM2 substrate is configured such that, in an uncleaved state, the NB interferes with binding of the AB to the target and, in a cleaved state, the NB does not interfere with binding of the AB to the target; and (4) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody.

[0380] In another embodiment, the method includes the steps of: (a) culturing cells containing a nucleic acid construct encoding an activatable antibody under conditions that result in expression of the activatable antibody, the activatable antibody comprising (i) a binding partner (BP) to a non-binding steric moiety (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide that includes a substrate (S) for the enzyme; (3) the CM1-CM2 substrate is configured such that, in an uncleaved state and in the presence of the NB, the NB interferes with binding of the AB to the target, and in a cleaved state, the NB does not interfere with binding of the AB to the target and the BP does not interfere with binding of the AB to the target; (4) the NB and the BP do not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody. In some examples of this embodiment, the BP of the activatable antibody is bound to the NB.

[0381] Uses of activatable antibodies and conjugated activatable antibodies It will be understood that administration of therapeutic agents according to the present disclosure will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to every pharmacist, Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 therein (Blaug, Seymour). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA complexes, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. Any of the foregoing mixtures may be suitable for treatments and therapies according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and that the formulation is physiologically compatible and acceptable for the route of administration.See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998) and citations therein for additional information related to formulations, excipients, and carriers known to pharmacists.

[0382] Therapeutic formulations of the present disclosure comprising conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies are used to prevent, treat, or ameliorate diseases or disorders associated with aberrant target expression and / or activity. For example, therapeutic formulations of the present disclosure comprising conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies are used to treat or ameliorate inflammation, inflammatory disorders, autoimmune diseases, and / or cancer or other neoplastic conditions. In some embodiments, the cancer is a solid tumor or hematologic tumor in which the target is expressed. In some embodiments, the cancer is a solid tumor in which the target is expressed. In some embodiments, the cancer is a hematologic tumor in which the target is expressed. In some embodiments, the target is expressed in the parenchyma (e.g., the part of an organ or tissue that, in cancer, often performs the function of the organ or tissue). In some embodiments, the target is expressed in a cell, tissue, or organ. In some embodiments, the target is expressed in the stroma (i.e., the connective, supportive framework of a cell, tissue, or organ). In some embodiments, the target is expressed in osteoblasts. In some embodiments, the target is expressed in endothelium (vasculature). In some embodiments, the target is expressed in cancer stem cells. In some embodiments, the agent to which the activatable antibody is conjugated is a microtubule inhibitor. In some embodiments, the agent to which the activatable antibody is conjugated is a nucleic acid damaging agent.

[0383] Efficacy of prevention, amelioration, or treatment is determined in association with any known method for diagnosing or treating a disease or disorder associated with target expression and / or activity, e.g., aberrant target expression and / or activity. Prolonging the survival of the subject or slowing the progression of a disease or disorder associated with target expression and / or activity, e.g., aberrant target expression and / or activity, in the subject indicates that the conjugated antibody, activatable antibody, and / or conjugated activatable antibody confers a clinical benefit.

[0384] Conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be administered in the form of pharmaceutical compositions. Principles and considerations involved in the preparation of such compositions, as well as guidance in the selection of ingredients, are provided, for example, in Remington: The Science and Practice of Pharmacy, 19th ed. (Alfonso R. Gennaro, et al., editors), Mack Pub. Co., Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide and Protein Drug Delivery (Advances in Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0385] In some embodiments in which antibody fragments are used, the smallest fragment capable of specifically binding to the target protein binding domain is selected. For example, peptide molecules can be designed based on the variable region sequence of the antibody to retain the ability to bind the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain multiple active compounds necessary for the particular indication being treated, e.g., those compounds with complementary activities that, in some embodiments, do not adversely affect each other. In some embodiments, or in addition, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitor. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0386] The active ingredient may also be encapsulated in microcapsules, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions prepared, for example, by coacervation techniques or by interfacial polymerization.

[0387] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0388] Sustained-release prep...

Claims

1. 1. An isolated polypeptide comprising a tandem substrate, said tandem substrate comprising at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) or cysteine ​​protease (CP); CM1 comprises the amino acid sequence AHGL (SEQ ID NO:54); CM2 comprises the amino acid sequence SGR; An isolated polypeptide, wherein the N-terminal to C-terminal configuration of the tandem substrate is CM1-CM2 or CM2-CM1, and the tandem substrate comprises the amino acid sequence of SEQ ID NO: 25 or 26.

2. 2. The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises the amino acid sequence of LSGRSALAHGLF (SEQ ID NO: 25).

3. 2. The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises the amino acid sequence ALAHGLFSGRSAN (SEQ ID NO: 26).

4. A conjugated polypeptide comprising the isolated polypeptide of claim 1 conjugated to a drug.

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

6. The isolated polypeptide of claim 1 , wherein the CM2 comprises a substrate for a CP enzyme, and the CP enzyme is legumain.

7. 2. The isolated polypeptide of claim 1, wherein the CM2 comprises a substrate for an SP enzyme selected from the group consisting of urokinase, matriptase, and neutrophil elastase.

8. The isolated polypeptide described in claim 1, wherein the CM2 comprises a substrate for an SP enzyme selected from the group consisting of urokinase, matriptase, and neutrophil elastase, and a substrate for a CP enzyme, and the CP enzyme is legumain.

Images