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

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 776,409, filed December 6, 2018, and U.S. Provisional Application No. 62 / 778,062, filed December 11, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to polypeptides comprising at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine ​​protease (CP); to activatable antibodies and other larger molecules comprising these polypeptides comprising at least CM1 that is a substrate for at least one MMP protease and at least CM2 that is a substrate for at least one SP protease and / or at least one CP protease; and to methods of making and using these polypeptides comprising at least CM1 that is a substrate for at least one MMP protease and at least CM2 that is a substrate for at least one SP protease and / or at least one CP protease in a variety of therapeutic, diagnostic, and prophylactic indications.

[0004] Reference to a sequence listing

[0005] Pursuant to Title 37 of the United States Code of Federal Regulations (CFR) Section 1.821, the Sequence Listing filed electronically herewith in computer-readable form (CFR) via EFS-Web as the file name "CYTX-058-PCT_ST25" is incorporated herein by reference. The electronic copy of the Sequence Listing was created on November 26, 2019, and has a disk capacity of 159 kilobytes. Background Art

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

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

[0008] In one aspect of the present invention, provided herein is an isolated polypeptide comprising a tandem substrate, wherein the tandem substrate comprises at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) or cysteine ​​protease (CP), wherein CM1 comprises the amino acid sequence AHGL or PRQV, and wherein the N-terminus to C-terminus arrangement of the tandem substrate is CM1-CM2 or CM2-CM1. 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 connected by a connecting peptide. In some embodiments, the CM1 and CM2 of the isolated polypeptide are directly connected to each other. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 containing a substrate of a CP enzyme, and wherein the CP enzyme is legumain. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises a substrate of an SP enzyme selected from the group consisting of: urokinase, matriptase, and neutrophil elastase. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2, which comprises a substrate of an SP enzyme selected from the group consisting of: urokinase, matriptase, and neutrophil elastase; and a substrate of a CP enzyme, and wherein the CP enzyme is legumain. In some embodiments, the isolated polypeptide of the present disclosure comprises CM1, which comprises a substrate of an MMP enzyme selected from the group consisting of: MMP2, MMP9, or MMP14.

[0009] In some embodiments, the isolated polypeptide of the present 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, the isolated polypeptide of the present 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), LSGRSANI (SEQ ID NO: 108), LSGRSANP (SEQ ID NO: 114), LSGRSDYH (SEQ ID NO:86), LSGRSDTH (SEQ ID NO:92), LSGRSDQH (SEQ ID NO:96), LSGRSDIH (SEQ ID NO: 100), and LSGRSDDH (SEQ ID NO: 104). In some embodiments, the isolated polypeptide of the present disclosure comprises 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, the isolated polypeptide of the present disclosure comprises 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, the isolated polypeptide of the present disclosure comprises 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, the isolated polypeptide of the present 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 present 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), LSGRSANI (SEQ ID NO: 108), LSGRSANP (SEQ ID NO: 114), LSGRSDYH (SEQ ID NO: 86), LSGRSDTH (SEQ ID NO: 92), LSGRSDQH (SEQ ID NO: 96), LSGRSDIH (SEQ ID NO: 117). NO: 100) and LSGRSDDH (SEQ ID NO: 104).

[0010] In some embodiments, the N-terminus to C-terminus of the tandem substrates of the isolated polypeptides of the present disclosure are arranged as CM1-CM2. In some embodiments, the N-terminus to C-terminus of the tandem substrates of the isolated polypeptides of the present disclosure are arranged as CM2-CM1.

[0011] In some embodiments, the cleavage rates of MMP9 and MMP14 of the isolated polypeptides of the present disclosure towards the first cleavable moiety of CM1 are each at least 80%. In some embodiments, the cleavage rates of MMP9 and MMP14 of the isolated polypeptides of the present disclosure towards the first cleavable moiety of CM1 are each at least 85%. In some embodiments, the cleavage rates of MMP9 and MMP14 of the isolated polypeptides of the present disclosure towards the first cleavable moiety of CM1 are each at least 90%.

[0012] In some embodiments, the in vivo stability of the CM1 first cleavable portion of the isolated polypeptide of the present disclosure is less than 30% activation. In some embodiments, the in vivo stability of the CM1 first cleavable portion of the isolated polypeptide of the present disclosure is less than 25% activation.

[0013] In some embodiments, the cleavage rate of the tandem substrate by MMP9 and MMP14 of the isolated polypeptides of the present disclosure is each at least 30%. In some embodiments, the cleavage rate of the tandem substrate by MMP9 and MMP14 of the isolated polypeptides of the present disclosure is each at least 50%. In some embodiments, the cleavage rate of the tandem substrate by MMP9 and MMP14 of the isolated polypeptides of the present disclosure is each at least 70%.

[0014] In some embodiments, the cleavage rates of the MMP9 and MMP14 of the isolated polypeptides of the present invention for the tandem substrate are each at least 15%, and the cleavage rates of the proteases for the tandem substrate are at least 30%. In some embodiments, the cleavage rates of the MMP9 and MMP14 of the isolated polypeptides of the present invention for the tandem substrate are each at least 30%, and the cleavage rates of the proteases for the tandem substrate are at least 30%. In some embodiments, the cleavage rates of the MMP9 and MMP14 of the isolated polypeptides of the present invention for the tandem substrate are each at least 50%, and the cleavage rates of the proteases for the tandem substrate are at least 50%. In some embodiments, the cleavage rates of the MMP9 and MMP14 of the isolated polypeptides of the present invention for the tandem substrate are each at least 70%, and the cleavage rates of the proteases for the tandem substrate are at least 70%.

[0015] In some embodiments, the isolated polypeptides of the present disclosure have tandem substrates with an in vivo stability lower than 40% activation. In some embodiments, the isolated polypeptides of the present disclosure have tandem substrates with an in vivo stability lower than 30% activation. In some embodiments, the isolated polypeptides of the present disclosure have tandem substrates with an in vivo stability lower than 25% activation. In some embodiments, the isolated polypeptides of the present disclosure have tandem substrates with an in vivo stability lower than 20% activation.

[0016] 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: 25-43. 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: 25, 26, 29, 31, 32, 34, 36, and 37. 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: 27, 28, 30, 33, and 35. 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: 25, 29, 31, 36, and 37. 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: 26, 32, and 34. 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: 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.

[0017] 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 to a target; at least a first cleavable portion (CM1) that is a substrate for at least one matrix metalloproteinase (MMP); and at least a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP) or cysteine ​​protease (CP), wherein CM1 comprises the amino acid sequence AHGL or PRQV, and wherein the N-terminus to C-terminus of the tandem substrates are arranged as CM1-CM2 or CM2-CM1. In some embodiments, at least one of the MMP, CP, and SP is co-localized with the target in the 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, a scFv, a 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, the isolated polypeptide of the present disclosure comprises an AB directly linked to CM1. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB connected to CM1 via a connecting peptide. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB connected to CM2. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB directly connected to CM2. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB connected to CM2 via a connecting peptide. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB comprising a light chain variable region of an antibody or antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the light chain variable region of the AB. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB comprising a heavy chain variable region of an antibody or antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the heavy chain variable region of the AB.

[0018] 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 an MM, the dissociation constant of which binds to AB is greater than the dissociation constant of AB binding to the target. In some embodiments, the isolated polypeptide of the present disclosure comprises an MM, which is a polypeptide of no more than 40 amino acids in length. In some embodiments, the isolated polypeptide of the present disclosure comprises an MM, which is linked to CM1, such that the isolated polypeptide comprises the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, the isolated polypeptide of the present disclosure comprises a connecting peptide between MM and CM1. In some embodiments, the isolated polypeptide of the present disclosure comprises a connecting peptide between CM2 and AB. In some embodiments, the isolated polypeptide of the present disclosure comprises a connecting peptide between MM and CM1 and a connecting peptide between CM2 and AB. In some embodiments, the isolated polypeptide of the present disclosure comprises MM linked to CM1, such that, in the uncleaved state, the isolated polypeptide comprises the following structural arrangement from N-terminus to C-terminus: MM-CM2-CM1-AB or AB-CM1-CM2-MM. In some embodiments, the isolated polypeptide of the present disclosure comprises a connecting peptide between MM and CM2. In some embodiments, the isolated polypeptide of the present disclosure comprises a connecting peptide between CM1 and AB. In some embodiments, the isolated polypeptide of the present disclosure comprises a connecting peptide between MM and CM2 and a connecting peptide between CM1 and AB.

[0019] In some embodiments, the isolated polypeptide of the present disclosure comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and wherein the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus in the 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 different from each other. In some embodiments, each of LP1 and LP2 is a peptide having a length of about 1 to 20 amino acids. In some embodiments, the isolated polypeptide comprises a third connecting peptide (LP') between CM1 and CM2. In some embodiments, the isolated polypeptide of the present disclosure comprises AB, which comprises a light chain variable region of an antibody or antigen-binding fragment thereof, and wherein LP2 is connected to the N-terminus of the light chain variable region of AB. In some embodiments, the isolated polypeptide of the present disclosure comprises an AB comprising a heavy chain variable region of an antibody or antigen-binding fragment thereof, and wherein LP2 is linked to the N-terminus of the heavy chain variable region of the AB.

[0020] In some embodiments, the isolated polypeptides of the present disclosure comprise an MM, wherein the amino acid sequence of the MM differs from the amino acid sequence of the target and is no more than 10% identical to the amino acid sequence of the natural binding partner of the AB. In some embodiments, the isolated polypeptides of the present disclosure comprise an MM that does not interfere with or compete with the binding of the AB to the target in a cleaved state.

[0021] In some embodiments, the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450-462. In some embodiments, the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450-462 and a heavy chain amino acid sequence comprising SEQ ID NO: 400.

[0022] In another aspect of the present invention, an activatable antibody that specifically binds to a target in an activated state is provided herein, the activatable antibody comprising an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target; a masking portion (MM) coupled to AB, wherein when the activatable antibody is in an uncleaved state, MM inhibits the binding of AB to the target; and a cleavable portion (CM) comprising a tandem substrate according to any one of the tandem substrates provided herein. In some embodiments, the activatable antibody comprises an MM that binds to AB with a dissociation constant greater than the dissociation constant for binding of AB to the target. In some embodiments, the activatable antibody comprises an MM that is a polypeptide of no more than 40 amino acids 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, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the activatable antibody comprises an MM that binds to AB with a dissociation constant greater than the dissociation constant for binding of AB to the target.

[0023] In some embodiments, the activatable antibody comprises MM linked to CM1, such that, in the uncleaved state, the activatable antibody 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 connecting peptide between MM and CM1. In some embodiments, the activatable antibody comprises a connecting peptide between CM2 and AB. In some embodiments, the activatable antibody comprises a connecting peptide between MM and CM1 and a connecting peptide between CM2 and AB.

[0024] In some embodiments, the activatable antibody comprises MM linked to CM1, such that, in the uncleaved state, the activatable antibody 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 connecting peptide between MM and CM2. In some embodiments, the activatable antibody comprises a connecting peptide between CM1 and AB. In some embodiments, the activatable antibody comprises a connecting peptide between MM and CM2 and a connecting peptide between CM1 and AB.

[0025] In some embodiments, the activatable antibody comprises an AB comprising a light chain variable region of an antibody or antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the light chain variable region of the AB. In some embodiments, the activatable antibody comprises an AB comprising a heavy chain variable region of an antibody or antigen-binding fragment thereof, and wherein CM1 or CM2 is linked to the N-terminus of the heavy chain variable region of the AB.

[0026] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and wherein the activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM. In some embodiments, the two connecting peptides are different from each other. In some embodiments, each of LP1 and LP2 is a peptide of approximately 1 to 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 an AB comprising a light chain variable region of an antibody or antigen-binding fragment thereof, and wherein LP2 is linked to the N-terminus of the light chain variable region of the AB. In some embodiments, the activatable antibody comprises an AB comprising a heavy chain variable region of an antibody or antigen-binding fragment thereof, and wherein LP2 is linked to the N-terminus of the heavy chain variable region of the AB.

[0027] In some embodiments, the activatable antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450-462. In some embodiments, the activatable antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 450-462 and a heavy chain amino acid sequence comprising SEQ ID NO: 400.

[0028] In some embodiments, the activatable antibody comprises an MM that is different from the target and is no more than 10% identical in amino acid sequence to the natural binding partner of the AB. In some embodiments, the activatable antibody comprises an MM that does not interfere with or compete with the binding of the AB to the target in the cleaved state.

[0029] In another aspect of the present invention, provided herein is a conjugated activatable antibody comprising an activatable antibody conjugated to an agent. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker.

[0030] In some embodiments, the activatable antibody of conjugation includes an agent as a toxin or a fragment thereof. In some embodiments, the activatable antibody of conjugation includes an agent as a microtubule inhibitor. In some embodiments, the activatable antibody of conjugation includes an agent as a nucleic acid damaging agent. In some embodiments, the activatable antibody of conjugation includes an agent, and the agent is dolastatin or its derivatives, auristatin or its derivatives, maytansinoid or its derivatives, duocarmycin or its derivatives, calicheamicin or its derivatives, auristatin E or its derivatives, monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD) or a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the activatable antibody of conjugation includes an agent as a detectable portion or a diagnostic agent.

[0031] In another aspect of the present invention, a pharmaceutical composition is provided herein, comprising an isolated polypeptide of the present invention, an activatable antibody of the present invention, or a conjugated activatable antibody of the present invention and a carrier. In some embodiments, the pharmaceutical composition comprises an additional agent. In some embodiments, the pharmaceutical composition comprises an additional agent as a therapeutic agent.

[0032] In another aspect of the present invention, provided herein is an isolated nucleic acid molecule encoding an isolated polypeptide of the present disclosure or an activatable antibody of the present disclosure. In some embodiments, a vector comprises an isolated nucleic acid molecule of the present disclosure.

[0033] In another aspect of the present invention, provided herein is a method for producing an antibody or an activatable antibody by culturing cells under conditions that result in expression of an isolated polypeptide of the present invention or an activatable antibody of the present invention. In another aspect of the present invention, provided herein is a method for producing an activatable antibody that binds to a target in an activated state, the method comprising: culturing cells comprising a nucleic acid construct encoding an activatable antibody of the present invention, and recovering the activatable antibody.

[0034] In another aspect of the present invention, provided herein is a method for treating a condition or disease, alleviating the symptoms of the condition or disease, or delaying the progression of the condition or disease, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide of the present disclosure, an activatable antibody of the present disclosure, a conjugated activatable antibody of the present disclosure, or a pharmaceutical composition of the present disclosure. In some embodiments, the condition or disease is cancer. In some embodiments, the method comprises administering an additional agent to the subject. In some embodiments, the method comprises administering an additional therapeutic agent to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Graphs showing exemplary results of the percentage of in vivo cleavage observed for the indicated activatable anti-EGFR antibodies of the present disclosure after administration to nu / nu mice. These exemplary results demonstrate that several tested activatable antibodies exhibit a higher degree of stability than other substrates cleavable by various enzymes.

[0036] Figure 2A and Figure 2B is a graph 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 antibodies.

[0037] Figure 3A and Figure 3B Graphs showing exemplary results of the in vivo efficacy of indicated activatable anti-EGFR antibodies of the present disclosure obtained using a mouse H292 xenograft model. These exemplary results demonstrate that the activatable antibodies of the present disclosure with certain MMP substrates exhibit comparable efficacy to unmasked anti-EGFR cetuximab in this xenograft model.

[0038] Figure 4 is a graph showing exemplary results of the percentage of activation of the indicated activatable anti-EGFR antibodies of the present disclosure within intratumoral tissue after administration to a mouse H292 xenograft model.

[0039] Figure 5 Graphs showing exemplary results of the percentage of in vivo cleavage observed for the indicated activatable anti-EGFR antibodies of the present disclosure with either a single MMP or tandem substrate following administration to nu / nu mice. These exemplary results demonstrate that several tested activatable antibodies with tandem substrates exhibited a higher degree of in vivo stability than other substrates cleavable by multiple enzymes.

[0040] Figure 6Graphs showing exemplary results of in vivo efficacy of indicated activatable anti-EGFR antibodies of the present disclosure obtained using a mouse H292 xenograft model. These exemplary results demonstrate that activatable antibodies of the present disclosure with certain MMP substrates exhibit comparable efficacy to unmasked anti-EGFR cetuximab antibody in this xenograft model.

[0041] Figure 7 is a graph showing exemplary results of the percentage of activation of the indicated activatable anti-EGFR antibodies of the present disclosure within intratumoral tissue after administration to a mouse H292 xenograft model.

[0042] Figure 8 is a graph showing exemplary results of calculated masking efficiencies calculated from in vitro binding of indicated anti-EGFR activatable antibodies of the present disclosure to EGFR. These exemplary results demonstrate that the tandem substrate affects the masking efficiency of the prodomain of the activatable antibody.

[0043] Figure 9 is a graph showing exemplary results of the percentage of activation of the indicated activatable anti-EGFR antibodies of the present disclosure after incubation with human tumor tissue. DETAILED DESCRIPTION

[0044] The present disclosure provides amino acid sequences comprising at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine ​​protease (CP). These amino acid sequences are collectively referred to herein as "tandem substrates." This term is not intended to convey any requirements regarding the orientation or other structural arrangement of the first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and the at least second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP) and / or at least one cysteine ​​protease (CP). Thus, the term "tandem substrate" encompasses CM1-CM2 substrates having the following structural arrangement from N-terminus to C-terminus: CM1-CM2 or CM2-CM1. The term "tandem substrate" also encompasses substrates in which at least a portion of the CM1 sequence overlaps with at least a portion of the CM2 sequence.

[0045] 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.

[0046] The CM1-CM2 substrates described herein can be used in a variety of therapeutic, diagnostic, and prophylactic indications. For example, these CM1-CM2 substrates can be used in activatable antibodies comprising an antibody or antigen-binding fragment thereof (AB) comprising a prodomain. The prodomain comprises at least one masking moiety (MM) that is linked to at least one antigen- or epitope-binding domain of the AB such that coupling of the MM reduces the ability of the AB to bind to its target.

[0047] In some embodiments, the activatable antibody comprises at least a first CM (CM1) and at least a second CM (CM2). In some embodiments, at least a portion of the CM1 substrate sequence overlaps with at least a portion of the CM2 sequence. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least one common amino acid residue. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least two common amino acid residues. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share at least three common amino acid residues. In some embodiments, the CM1 substrate sequence and the CM2 substrate sequence share three or more common amino acid residues.

[0048] In some embodiments, CM1 and CM2 are separate polypeptides that are operably linked together.

[0049] In some embodiments, CM1 and CM2 are separate polypeptides that are directly linked together, i.e., the N-terminus of one substrate is directly linked to the C-terminus of the other substrate polypeptide. In some embodiments, the N-terminus of CM1 is directly linked to the C-terminus of CM2. In some embodiments, the N-terminus of CM2 is directly linked to the C-terminus of CM1.

[0050] In some embodiments, CM1 and CM2 are separate polypeptides operably linked together by at least one linking moiety.

[0051] In some embodiments, the first cleavable moiety CM1 and the second cleavable moiety CM2 in the activatable antibody have the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM.

[0052] 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.

[0053] 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 a 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.

[0054] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0055] 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.

[0056] 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 of the same MMP. In some embodiments, CM1 comprises at least two or more substrates for MMP2. In some embodiments, CM1 comprises at least two or more MMP9 substrates. In some embodiments, CM1 comprises at least two or more MMP14 substrates.

[0057] In some embodiments, CM1 is a substrate of a MMP and comprises at least the sequences ALAHGLF (SEQ ID NO: 1); DLAHPLL (SEQ ID NO: 2); AFRHLR (SEQ ID NO: 3); PHGFFQ (SEQ ID NO: 4); SVHHLI (SEQ ID NO: 5); RGPKLYW (SEQ ID NO: 6); RFPYGVW (SEQ ID NO: 7); HVPRQV (SEQ ID NO: 8); SNPFKY (SEQ ID NO: 9); RFPLKV (SEQ ID NO: 10); PFHLSR (SEQ ID NO: 11); STVFHM (SEQ ID NO: 12); MGPWFM (SEQ ID NO: 13); RHLAKL (SEQ ID NO: 14); PLGVRGK (SEQ ID NO: 15); and QNQALRIA (SEQ ID NO: 16).

[0058] 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).

[0059] 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.

[0060] 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.

[0061] 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. By way of non-limiting example, examples of other SPs that cleave CM2 described herein include activated protein C; cathepsin A; cathepsin G; chymotrypsin; coagulation factor proteases, such as, for example, FVIIa, FIXa, FXa, FXIa, FXIIa; elastase; granzyme B; guanidinobenzoatase; HtrA1; lactoferrin; marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine protease (TTSP), such as, for example, DESC1, DPP-4, FAP, Hepsin, matriptase-2, TMPRSS2, TMPRSS3 and / or TMPRSS4.

[0062] For example, a suitable CM2 is cleaved by at least one serine protease and can comprise at least one sequence selected from the group consisting of the 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), NO:88), SGRSDY (SEQ ID NO:89), LSGRSDNP (SEQ ID NO:90), SGRSDNP (SEQ ID NO:91), LSGRSDTH (SEQ ID NO:92), LSGRSDT (SEQ ID NO:93), SGRSDTH (SEQ ID NO:94), SGRSDT (SEQ ID NO:95), LSGRSDQH (SEQ ID NO:96), LSGRSDQ (SEQ ID NO:97), SGRSDQH (SEQ ID NO:98), SGRSDQ (SEQ ID NO:99), LSGRSDIH (SEQ ID NO:100), LSGRSDI (SEQ ID NO:101), SGRSDIH (SEQ ID NO:102), SGRSDI (SEQ ID NO:103), LSGRSDDH (SEQ ID NO:104), LSGRSDD (SEQ ID NO:105), SGRSDDH (SEQ ID NO:106), SGRSDD (SEQ ID NO:107), LSGRSANI (SEQ ID NO:108), LSGRSAN (SEQ ID NO:109), LSGRSA (SEQ ID NO:110), SGRSANI (SEQ ID NO:111), SGRSAN (SEQ ID NO:112), SGRSA (SEQID NO:113), LSGRSANP (SEQ ID NO:114) and SGRSANP (SEQ ID NO:115).

[0063] In some embodiments, suitable CM2 is cleaved by at least one serine protease and may comprise at least one sequence selected from the group consisting of 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.

[0064] In some embodiments, suitable CM2 is cleaved by at least one serine protease and may comprise at least one sequence selected from the group consisting of APRSF (SEQ ID NO: 130), APRS (SEQ ID NO: 131), and PRSF (SEQ ID NO: 132).

[0065] In some embodiments, suitable CM2 is cleaved by at least one serine protease and may comprise at least one sequence selected from the group consisting of 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).

[0066] In some embodiments, CM2 is a substrate for at least one cysteine ​​protease (CP). In some embodiments, the CP is a leguminase. In some embodiments, suitable CM2 is cleaved by at least one cysteine ​​protease and may comprise at least one amino acid sequence selected from the group consisting of AAN, SAN, and GPTN (SEQ ID NO: 152).

[0067] In some embodiments, the CM1-CM2 substrate comprises an amino acid sequence selected from the group consisting of LSGRSALAHGLF (SEQ ID NO:25), ALAHGLFSGRSAN (SEQ ID NO:26), HVPRQVLSGRS (SEQ ID NO:27), HVPRQVLSGRSAN (SEQ ID NO:28), TARGPALAHGLF (SEQ ID NO:29), TARGPVPRQV (SEQ ID NO:30), APRSALAHGLF (SEQ ID NO:31), ALAHGLFAPRSF (SEQ ID NO:32), HVPRQVAPRSF (SEQ ID NO:33), ALAHGLPTFVHL (SEQ ID NO:34), GLPTFVHLPRQV (SEQ ID NO:35), AANALAHGLF (SEQ ID NO:36), GPTNALAHGLF (SEQ ID NO:37), ISSGLLSGRSNI (SEQ ID NO:38), AVGLLAPPGGLSGRSNI (SEQ ID NO:39), NO:39), ISSGLLSGRSNIGS (SEQ ID NO:40), AVGLLAPPGGLSGRSNIGS (SEQ ID NO:41), ISSGLLSGRSNIG (SEQ ID NO:42) and AVGLLAPPGGLSGRSNIG (SEQ ID NO:43).

[0068] In some embodiments, the activatable antibody has the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM.

[0069] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other.

[0070] 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.

[0071] 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 a 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, and a connecting peptide (LP2) between CM2 and MM.

[0072] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0073] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: (GS) n 、(GGS) n 、(GSGGS) n (SEQ ID NO: 200) and (GGGS) n (SEQ ID NO: 201), wherein n is an integer of at least 1.

[0074] 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).

[0075] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 208), GSSGGSGGSGG (SEQ ID NO: 209), GSSGGSGGSGGS (SEQ ID NO: 210), GSSGGSGGSGGSGGGS (SEQ ID NO: 211), GSSGGSGGSG (SEQ ID NO: 212), or GSSGGSGGSGS (SEQ ID NO: 213), GGGSSGGS (SEQ ID NO: 214), and GGGS (SEQ ID NO: 215).

[0076] 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). In some embodiments, LP2 comprises the amino acid sequence GGS. In some embodiments, LP2 comprises the amino acid sequence GGGS (SEQ ID NO: 215).

[0077] In some embodiments, CM1 is a substrate of a MMP and comprises at least the sequences ALAHGLF (SEQ ID NO: 1); DLAHPLL (SEQ ID NO: 2); AFRHLR (SEQ ID NO: 3); PHGFFQ (SEQ ID NO: 4); SVHHLI (SEQ ID NO: 5); RGPKLYW (SEQ ID NO: 6); RFPYGVW (SEQ ID NO: 7); HVPRQV (SEQ ID NO: 8); SNPFKY (SEQ ID NO: 9); RFPLKV (SEQ ID NO: 10); PFHLSR (SEQ ID NO: 11); STVFHM (SEQ ID NO: 12); MGPWFM (SEQ ID NO: 13); RHLAKL (SEQ ID NO: 14); PLGVRGK (SEQ ID NO: 15); and QNQALRIA (SEQ ID NO: 16).

[0078] In some embodiments, the isolated polypeptide of the present disclosure or the activatable antibody of the present disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 comprises an amino acid sequence selected from the group consisting of LSGRSALAHGLF (SEQ ID NO: 25), ALAHGLFSGRSAN (SEQ ID NO: 26), HVPRQVLSGRS (SEQ ID NO: 27), HVPRQVLSGRSAN (SEQ ID NO: 28), TARGPALAHGLF (SEQ ID NO: 29), TARGPVPRQV (SEQ ID NO: 30), APRSALAHGLF (SEQ ID NO: 31), ALAHGLFAPRSF (SEQ ID NO: 32), HVPRQVAPRSF (SEQ ID NO: 33), ALAHGLPTFVHL (SEQ ID NO: 34), GLPTFVHLPRQV (SEQ ID NO: 35), AANALAHGLF (SEQ ID NO: 36), GPTNALAHGLF (SEQ ID NO: 37), NO:37), ISSGLLSGRSNI (SEQ ID NO:38), and AVGLLAPPGGLSGRSNI (SEQ ID NO:39), and the LP2 linker comprises GGGS (SEQ ID NO:215).

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

[0080] In some embodiments, the isolated polypeptide of the present disclosure or the activatable antibody of the present disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 and LP2 comprise an amino acid sequence selected from the group consisting of LSGRSALAHGLFGGGS (SEQ ID NO: 226), ALAHGLFSGRSANGGGS (SEQ ID NO: 227), HVPRQVLSGRSGGGS (SEQ ID NO: 228), HVPRQVLSGRSANGGGS (SEQ ID NO: 229), TARGPALAHGLFGGGS (SEQ ID NO: 230), TARGPVPRQVGGGS (SEQ ID NO: 231), APRSALAHGLFGGGS (SEQ ID NO: 232), ALAHGLFAPRSFGGGS (SEQ ID NO: 233), HVPRQVAPRSFGGGS (SEQ ID NO: 234), ALAHGLPTFVHLGGGS (SEQ ID NO: 235). NO:235), GLPTFVHLPRQVGGGS (SEQ ID NO:236), AANALAHGLFGGGS (SEQ ID NO:237), GPTNALAHGLFGGGS (SEQ ID NO:238), ISSGLLSGRSNIGGGS (SEQ ID NO:239) and AVGLLAPPGGLSGRSNIGGGS (SEQ ID NO:240).

[0081] In some embodiments, the isolated polypeptide of the present disclosure or the activatable antibody of the present disclosure comprises a CM1-CM2 substrate and an LP2 linker, wherein CM1-CM2 and LP2 comprise an amino acid sequence selected from the group consisting of LSGRSALAHGLFGGS (SEQ ID NO: 241), ALAHGLFSGRSANGGS (SEQ ID NO: 242), HVPRQVLSGRSGGS (SEQ ID NO: 243), HVPRQVLSGRSANGGS (SEQ ID NO: 244), TARGPALAHGLFGGS (SEQ ID NO: 245), TARGPVPRQVGGS (SEQ ID NO: 246), APRSALAHGLFGGS (SEQ ID NO: 247), ALAHGLFAPRSFGGS (SEQ ID NO: 248), HVPRQVAPRSFGGS (SEQ ID NO: 249), ALAHGLPTFVHLGGS (SEQ ID NO: 250), GLPTFVHLPRQVGGS (SEQ ID NO: 251). NO:251), AANALAHGLFGGS (SEQ ID NO:252), GPTNALAHGLFGGS (SEQ ID NO:253), ISSGLLSGRSNIGGS (SEQ ID NO:254) and AVGLLAPPGGLSGRSNIGGS (SEQ ID NO:255).

[0082] In some embodiments, the AB binds to the target with a dissociation constant of about 100 nM or less.

[0083] In some embodiments, the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target. In some embodiments, the AB is a full-length antibody. In some embodiments, the AB is an immunologically active fragment. In some embodiments, the AB is an antigen-binding fragment. In some embodiments, the AB is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an AB is a mouse antibody, other rodent antibodies, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0084] In some embodiments, the MMP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease cleaves CM1 in the antibody. In some embodiments, the SP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the SP protease cleaves a CM2 substrate in the antibody. In some embodiments, the CP protease is co-localized with the target in the tissue, and when the antibody is exposed to the protease, the CP protease cleaves a CM2 substrate in the antibody. In some embodiments, the MMP protease and / or SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease and / or SP protease cleave a CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, at least one of the MMP protease and SP protease cleaves a CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and / or CP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease and / or CP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and CP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, at least one of the MMP protease and CP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and / or CP protease and / or SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, the MMP protease and / or CP protease and / or SP protease cleaves the CM1-CM2 substrate in the antibody. In some embodiments, the MMP protease and CP protease and SP protease are co-localized with the target in the tissue, and when the antibody is exposed to the protease, at least one of the MMP protease, SP protease and CP protease cleaves the CM1-CM2 substrate in the antibody.

[0085] In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide of up to 15 amino acids in length.

[0086] 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 not substantially identical to any polypeptide sequence naturally cleaved by the same MMP protease (e.g., any animal polypeptide sequence). 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 not substantially identical to any mammalian polypeptide sequence 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 not substantially identical to any human polypeptide sequence 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 no more than 90% identical or more to any polypeptide sequence naturally cleaved by the same MMP protease (e.g., any animal polypeptide sequence). 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 no more than 90% identical or more to any mammalian polypeptide sequence 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 no more than 90% identical, or more, to any human polypeptide sequence that is naturally cleaved by the same MMP protease.

[0087] In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP and comprises a polypeptide sequence that is substantially non-identical to any polypeptide sequence 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 substantially non-identical to any mammalian polypeptide sequence naturally cleaved by the same SP and / or CP protease. In some embodiments, the CM2 substrate sequence of the CM1-CM2 substrate is a substrate for at least one SP and / or at least one CP and comprises a polypeptide sequence that is substantially non-identical to any human polypeptide sequence 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 no more than 90% identical, or more, to any polypeptide sequence 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 no more than 90% identical, or more, 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 no more than 90% identical, or more, to any human polypeptide sequence that is naturally cleaved by the same SP and / or CP protease.

[0088] In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 29, 31, 32, 34, 36, and 37. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 29, 31, 36, and 37. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, and 34. In some embodiments, the CM1-CM2 substrates of the present disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 33. In some embodiments, the CM1-CM2 substrate of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35.

[0089] In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43; and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking portion (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 29, 31, 32, 34, 36, and 37; and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking portion (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 30, 33, and 35; and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking portion (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 29, 31, 36, and 37; and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, and 34; and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 33; and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target. In some embodiments, the activatable antibodies of the present disclosure comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 and 35; and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the isolated polypeptides of the present disclosure comprise a CM1 substrate that is a substrate for at least one MMP and comprises a polypeptide sequence that is not substantially identical to any polypeptide sequence 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 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 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 no more than 90% identical or more to any polypeptide sequence 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 no more than 90% identical or more to any mammalian polypeptide sequence 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 no more than 90% identical, or more, to any human polypeptide sequence that is naturally cleaved by the same MMP protease. In some embodiments, the CM1 substrate sequence is a substrate for at least one MMP and comprises a polypeptide sequence selected from the group consisting of: ALAHGLF (SEQ ID NO: 1), DLAHPLL (SEQ ID NO: 2), RGPKLYW (SEQ ID NO: 6), RFPYGVW (SEQ ID NO: 7), and QNQALRIA (SEQ ID NO: 16).

[0093] In some embodiments, the activatable antibodies of the present disclosure comprise a CM1 substrate comprising an amino acid sequence selected from the group consisting of: ALAHGLF (SEQ ID NO: 1), DLAHPLL (SEQ ID NO: 2), RGPKLYW (SEQ ID NO: 6), RFPYGVW (SEQ ID NO: 7), and QNQALRIA (SEQ ID NO: 16); and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target.

[0094] In some embodiments, the isolated polypeptides of the present disclosure comprise 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 naturally cleaved by the same SP protease (e.g., any animal polypeptide sequence). In some embodiments, the CM2 substrate sequence is a substrate for at least one SP and comprises a polypeptide sequence that is not substantially identical to any mammalian polypeptide sequence 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 not substantially identical to any human polypeptide sequence 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 no more than 90% identical or more to any polypeptide sequence naturally cleaved by the same SP protease (e.g., any animal polypeptide sequence). In some embodiments, the CM2 substrate sequence is a substrate for at least one SP and comprises a polypeptide sequence that is no more than 90% identical or more to any mammalian polypeptide sequence 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 no more than 90% identical or more to any human polypeptide sequence 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 selected from the group consisting of: APRSF (SEQ ID NO: 130) and GLPTFVHL (SEQ ID NO: 135).

[0095] In some embodiments, the activatable antibodies of the present disclosure comprise a CM2 substrate comprising an amino acid sequence selected from the group consisting of APRSF (SEQ ID NO: 130) and GLPTFVHL (SEQ ID NO: 135); and an antibody or antigen-binding fragment thereof (AB) that binds to a target and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target.

[0096] In some embodiments, the activatable antibodies of the present disclosure comprise 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 to a target and a masking moiety (MM) that reduces the ability of the antigen- or epitope-binding domain of the AB to bind to its target.

[0097] In some embodiments, a CM1-CM2, CM1, or CM2 substrate is also a substrate for at least one additional protease.

[0098] In some embodiments, the at least one additional protease is a different MMP protease than the MMP protease that cleaves CM1. In some embodiments, the at least one additional protease is a MMP protease selected from the group consisting of: MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.

[0099] In some embodiments, the at least one additional protease is a SP protease different from the SP protease that cleaves CM2. In some embodiments, the at least one additional SP protease is selected from the group consisting of: uPA; matriptase; activated protein C; cathepsin A; cathepsin G; chymotrypsin; coagulation factor proteases, such as, for example, FVIIa, FIXa, FXa, FXIa, FXIIa; elastase; granzyme B; guanidinobenzoylase; HtrA1; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; plasmin; PSA; tPA; thrombin; tryptase; type II transmembrane serine protease (TTSP), such as, for example, DESC1, DPP-4, FAP, Hepsin, matriptase-2, TMPRSS2, TMPRSS3, and TMPRSS4.

[0100] In some embodiments, the at least one additional protease is selected from the group consisting of those proteases shown in Table 1.

[0101] Table 1: Exemplary proteases and / or enzymes

[0102]

[0103]

[0104] The present disclosure also provides an antibody comprising at least a first CM1 and at least a second CM2, and conjugated to an agent. In some embodiments, the first CM1 and the second CM2 are each polypeptides of no more than 15 amino acids in length. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM-CM1-CM2-AB-agent, agent-AB-CM2-CM1-MM, MM-CM2-CM1-AB-agent, or agent-AB-CM1-CM2-MM. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from N-terminus to C-terminus in the uncleaved state: agent-MM-CM1-CM2-AB, AB-CM2-CM1-MM-agent, agent-MM-CM2-CM1-AB, or AB-CM1-CM2-MM-agent. In some embodiments, the activatable antibody is a conjugated activatable antibody having the following structural arrangement from N-terminus to C-terminus in the uncleaved state: Agent-MM-CM1-CM2-AB-Agent, Agent-AB-CM2-CM1-MM-Agent, Agent-MM-CM2-CM1-AB-Agent, or Agent-AB-CM1-CM2-MM-Agent.

[0105] In some embodiments, the activatable antibody is a conjugated activatable antibody comprising a masking moiety (MM), a first connecting peptide (LP1), and a second connecting peptide (LP2), and the antibody, in the uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-CM2-LP2-AB-agent, agent-AB-LP2-CM2-CM1-LP1-MM, MM1-LP1-CM2-CM1-LP2-AB-agent, or agent-AB-LP2-CM1-CM2-LP1-MM. In some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. In some embodiments, the two connecting peptides need not be identical to each other.

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

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

[0108] 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.

[0109] 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 a 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.

[0110] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0111] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: (GS) n 、(GGS) n 、(GSGGS) n (SEQ ID NO: 381) and (GGGS) n (SEQ ID NO:382), wherein n is an integer of at least 1.

[0112] 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).

[0113] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 208), GSSGGSGGSGG (SEQ ID NO: 209), GSSGGSGGSGGS (SEQ ID NO: 210), GSSGGSGGSGGSGGGS (SEQ ID NO: 211), GSSGGSGGSG (SEQ ID NO: 212), or GSSGGSGGSGS (SEQ ID NO: 213), and GGGSSGGS (SEQ ID NO: 214).

[0114] 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).

[0115] In some embodiments, the CM1-CM2 substrate is linked to or otherwise attached to an antibody. For example, CM1-CM2 is used to link one or more agents to an antibody or antigen-binding fragment thereof (AB) that binds to a given target, such that the CM1-CM2 is cleaved upon exposure to an MMP and / or SP and / or CP, and the agent is released from the AB. Exemplary targets include, but are not limited to, those listed in Table 2. Exemplary ABs include, but are not limited to, the antibodies listed in Table 3.

[0116] In some embodiments, the AB binds to the target with a dissociation constant of about 100 nM or less.

[0117] In some embodiments, the antibody comprises an antibody or antigen-binding fragment thereof that specifically binds to a target. In some embodiments, the antibody or immunologically active fragment thereof that binds to a target is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds to a target is a mouse antibody, other rodent antibodies, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0118] In some embodiments, the MM binds to the AB with a dissociation constant that is no greater than the dissociation constant of the AB to the target.

[0119] In some embodiments, the MM does not interfere with or compete for binding of the AB to the target in the cleaved state.

[0120] In some embodiments, the MM is a polypeptide of about 2 to 40 amino acids in length. For example, the MM is a polypeptide of up to about 40 amino acids in length.

[0121] In some embodiments, the MM polypeptide sequence is not identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of AB.

[0122] In some embodiments, the agent conjugated to the AB or the AB of an 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 fragment of a toxin is a fragment that retains toxin 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 intercalating agent, or other DNA damaging agent. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is riocetin or a derivative thereof. In some embodiments, the agent is riocetin E or a derivative thereof. In some embodiments, the agent is monomethylriocetin E (MMAE). In some embodiments, the agent is monomethylriocetin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0123] In some embodiments, the agent is an anti-inflammatory agent.

[0124] In some embodiments, the antibody and / or activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0125] In some embodiments, the conjugated antibody and / or the 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 radioactive isotope. In some embodiments, the radioactive isotope is indium or technetium. In some embodiments, the contrast agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or beta-galactosidase. In some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label comprises an N-methylacrydinium derivative. In some embodiments, the label comprises Alexa Fluor Tags, such as Alex 680 or Alexa 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0126] In some embodiments, the AB of an antibody and / or activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0127] In some embodiments, antibody and / or conjugated antibody is monospecific. In some embodiments, antibody and / or conjugated antibody is multispecific, referred to herein as multispecific antibody and / or conjugated multispecific antibody. In some embodiments, multispecific antibody and / or conjugated multispecific antibody is bispecific or trifunctional. In some embodiments, antibody and / or conjugated antibody is formulated as a part of pro-bispecific T cell adapter protein (pro-BITE) molecule. In some embodiments, antibody and / or conjugated antibody is formulated as a part of pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor or other immune effector cell (such as CAR modified NK cell). In some embodiments, activatable antibody and / or conjugated activatable antibody is formulated as a part of pro-chimeric antigen receptor (CAR) modified T cell. In some embodiments, activatable antibody and / or conjugated activatable antibody is formulated as a part of pro-chimeric antigen receptor (CAR) modified NK cell.

[0128] In some embodiments, the activatable antibody and / or the conjugated activatable antibody is monospecific. In some embodiments, the activatable antibody and / or the conjugated activatable antibody is multispecific, referred to herein as a multispecific activatable antibody and / or a conjugated multispecific activatable antibody. Unless otherwise indicated, as used herein, terms such as "activatable antibody" and all grammatical variations thereof are intended to encompass, but are not limited to, embodiments in which the activatable antibody is a multispecific activatable antibody of the present disclosure. Unless otherwise indicated, as used herein, terms such as "conjugated activatable antibody" and all grammatical variations thereof are intended to encompass, but are not limited to, embodiments in which the activatable antibody is a conjugated multispecific activatable antibody of the present disclosure. In some embodiments, the multispecific activatable antibody and / or the conjugated multispecific activatable antibody is bispecific or trifunctional. In some embodiments, the activatable antibody and / or the conjugated activatable antibody is formulated as part of a pro-bispecific T cell adaptor protein (pro-BITE) molecule. In some embodiments, the activatable antibody and / or conjugated activatable antibody is formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell or other engineered receptor.

[0129] In some embodiments, the antibodies, antibody conjugates, activatable antibodies, conjugated activatable antibodies, multispecific activatable antibodies, and / or conjugated multispecific activatable antibodies described herein are used in combination with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended application, such as cancer. For example, an activatable antibody, conjugated activatable antibody, multispecific activatable antibody, and / or conjugated multispecific activatable antibody can be used in combination with another chemotherapeutic agent or anti-tumor agent.

[0130] In some embodiments, the activatable antibody is a multispecific activatable antibody. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize two or more different antigens or epitopes and comprise at least one masking moiety (MM) that is linked to at least one antigen or epitope binding domain of the multispecific antibody such that coupling of the MM reduces the ability of the antigen or epitope binding domain to bind to its target. In some embodiments, the MM is coupled to the antigen or epitope binding domain of the multispecific antibody via a CM1-CM2 substrate that acts as a substrate for at least one MMP protease and at least one SP protease. The activatable multispecific antibodies provided herein are stable in circulation, activated at the intended therapeutic and / or diagnostic site, but not activated in normal tissue (i.e., healthy tissue), and upon activation, exhibit at least comparable binding to the target as the corresponding unmodified multispecific antibody.

[0131] 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 to epidermal growth factor receptor (EGFR) and contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising 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 at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); and combinations thereof.

[0132] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising 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); and a VLCDR3 sequence comprising at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 225); and combinations thereof.

[0133] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 220); a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); a VH CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 222); NO: 221); a VH CDR2 sequence comprising 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); and combinations thereof.

[0134] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); a VL CDR1 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO: 224); NO: 224); and a VL CDR3 sequence comprising a sequence 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.

[0135] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains 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 at least the amino acid sequence NYGVH (SEQ ID NO: 220); the VH CDR2 sequence comprises at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); the VH CDR3 sequence comprises at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 222); the VL CDR1 sequence comprises at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 223); the VL CDR2 sequence comprises at least the amino acid sequence KYASESIS (SEQ ID NO: 224); and the VL The CDR3 sequence comprises at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 225).

[0136] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to EGFR and contains 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 VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 221); and the VH The 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 of ALTYYDYEFAY (SEQ ID NO: 222); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of RASQSIGTNIH (SEQ ID NO: 223); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of 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 of QQNNNWPTT (SEQ ID NO: 225). NO:225) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence.

[0137] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise 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, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise a CM1-CM2 substrate comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-43; and an antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 401 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 400.

[0138] In some embodiments, the activatable antibodies and / or conjugated activatable antibodies provided herein (including but not limited to the multispecific activatable antibodies and / or conjugated multispecific activatable antibodies of the present disclosure) comprise 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.

[0139] In some embodiments, the activatable antibody further comprises an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is riocetin or a derivative thereof. In some embodiments, the agent is riocetin E or a derivative thereof. In some embodiments, the agent is monomethylriocetin E (MMAE). In some embodiments, the agent is monomethylriocetin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0140] In some embodiments, the agent is an anti-inflammatory agent.

[0141] In some embodiments, the activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0142] 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 radioactive isotope. In some embodiments, the radioactive isotope is indium or technetium. In some embodiments, the contrast agent comprises iodine, gadolinium, or iron oxide. In some embodiments, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or beta-galactosidase. In some embodiments, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label comprises an N-methylacridine derivative. In some embodiments, the label comprises Alexa Fluor Tags, such as Alex 680 or Alexa 750. In some embodiments, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0143] In some embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is directly attached to the MM of the activatable antibody. In some embodiments, the spacer is directly attached to the MM of the activatable antibody in a structure of spacer-MM-CM1-CM2 substrate-AB from N-terminus to C-terminus. Examples of spacers directly attached to the N-terminus of the MM of the activatable antibody include amino acid sequences selected from the group consisting of: QGQSGQ (SEQ ID NO: 153), GQSGQ (SEQ ID NO: 154), QSGQ (SEQ ID NO: 155), SGQ, GQ, and Q. In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 153). In some embodiments, the spacer comprises at least the amino acid sequence GQSGQ (SEQ ID NO: 154). In some embodiments, the spacer comprises at least the amino acid sequence QSGQ (SEQ ID NO: 155). In some embodiments, the spacer comprises at least the amino acid sequence SGQ. In some embodiments, the spacer comprises at least the amino acid sequence GQ. In some embodiments, the spacer comprises at least the amino acid sequence Q.

[0144] In some embodiments, the AB of the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to contain one or more disulfide bonds.

[0145] In some embodiments, the serum half-life of the activatable antibody is longer than the serum half-life of the corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 15 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 11 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 9 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 7 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 6 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 5 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 4 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 3 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 2 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 24 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 20 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 18 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 16 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 14 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 12 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 10 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 8 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 6 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 4 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 3 hours.

[0146] The present disclosure also provides compositions and methods comprising activatable antibodies comprising an antibody or antibody fragment (AB) that specifically binds to a given target, wherein the AB is coupled to a masking moiety (MM) that reduces the ability of the AB to bind to its target. In some embodiments, the activatable antibody further comprises a CM1-CM2 substrate that is a substrate for at least one MMP and at least one SP. The compositions and methods provided herein enable one or more agents to be attached 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 enable one or more agents to be attached to one or more cysteine ​​residues in the AB without reducing or otherwise interfering with one or more disulfide bonds in the MM. The compositions and methods provided herein produce activatable antibodies conjugated to one or more agents (e.g., any one of a variety of therapeutic agents, diagnostic agents, and / or prophylactic agents), for example, in some embodiments, no one or more agents are conjugated to the MM of the activatable antibody. The compositions and methods provided herein produce conjugated activatable antibodies, wherein the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in its uncleaved state. The compositions and methods provided herein produce conjugated activatable antibodies, wherein the activatable antibodies are still activated, ie, cleaved, in the presence of an MMP that can cleave a CM1-CM2 substrate.

[0147] The activatable antibody has at least one conjugation point for the agent, but in the methods and compositions provided herein, fewer than all possible conjugation points can be used for conjugation to the agent. In some embodiments, one or more conjugation points are sulfur atoms involved in disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms involved in interchain sulfide bonds, rather than sulfur atoms involved in interchain disulfide bonds. In some embodiments, one or more conjugation points are sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. Such residues may be naturally present in the antibody structure or may be incorporated into the antibody by site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0148] Also provided are methods for preparing conjugates of activatable antibodies having one or more interchain disulfide bonds in the AB and one or more intrachain disulfide bonds in the MM, and a drug that reacts with free thiols. The methods generally comprise partially reducing the interchain disulfide bonds in the activatable antibody with a reducing agent, such as TCEP; and conjugating the drug that reacts with free thiols to the partially reduced activatable antibody. As used herein, the term partially reduced refers to a situation in which the activatable antibody is contacted with a reducing agent and less than all disulfide bonds (e.g., less than all possible conjugation sites) are reduced. In some embodiments, less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5% of all possible conjugation sites are reduced.

[0149] In some embodiments, a method is provided in which an agent (e.g., a drug) is reduced and conjugated to an activatable antibody, thereby causing the agent to have a selective arrangement. The method generally includes partially reducing the activatable antibody with a reducing agent so that any conjugation site in the masking portion or other non-AB portion of the activatable antibody is not reduced, and conjugating the agent to the interchain thiol in the AB. One or more conjugation sites are selected to allow the desired agent to be arranged to allow conjugation to occur at the desired site. The reducing agent is, for example, TCEP. The reduction reaction conditions (e.g., the ratio of reducing agent to activatable antibody, incubation time, temperature during incubation, pH of the reduction reaction solution, etc.) are determined by identifying the conditions for producing conjugated activatable antibodies, wherein MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in the uncleaved state. The ratio of reducing agent to activatable antibody will vary according to the activatable antibody. In some embodiments, the ratio of reducing agent to activatable antibody will range from about 20: 1 to 1 : 1, about 10: 1 to 1 : 1, about 9: 1 to 1 : 1, about 8: 1 to 1 : 1, about 7: 1 to 1 : 1, about 6: 1 to 1 : 1, about 5: 1 to 1 : 1, about 4: 1 to 1 : 1, about 3: 1 to 1 : 1, about 2: 1 to 1 : 1, about 20: 1 to 1 : 1.5, about 10: 1 to 1 : 1.5, about 9: 1 to 1 : 1.5, about 8: 1 to 1 : 1.5, about 7: 1 to 1 : 1.5, about 6: 1 to 1 : 1.5, about 5: 1 to 1 : 1.5, about 4: 1 to 1 : 1.5, about 3: 1 to 1 : 1.5, about 2: 1 to 1 : 1.5, about 1.5: 1 to 1 : 1.5, or about 1 : 1 to 1 : 1.5. In some embodiments, the ratio ranges from about 5:1 to 1:1. In some embodiments, the ratio ranges from about 5:1 to 1.5:1. In some embodiments, the ratio ranges from about 4:1 to 1:1. In some embodiments, the ratio ranges from about 4:1 to 1.5:1. In some embodiments, the ratio ranges from about 8:1 to about 1:1. In some embodiments, the ratio ranges from about 2.5:1 to 1:1.

[0150] In some embodiments, a method is provided for reducing interchain disulfide bonds in the AB of an activatable antibody and conjugating an agent (e.g., a thiol-containing agent such as a drug) to the resulting interchain thiol to selectively position one or more agents on the AB. The method generally comprises partially reducing the AB with a reducing agent to form at least two interchain thiols without forming all possible interchain thiols in the activatable antibody; and conjugating the agent to the interchain thiol of the partially reduced AB. For example, the AB of the activatable antibody is partially reduced at a desired reducing agent:activatable antibody ratio for about 1 hour at about 37°C. In some embodiments, the ratio of reducing agent to activatable antibody will range from about 20: 1 to 1 : 1, about 10: 1 to 1 : 1, about 9: 1 to 1 : 1, about 8: 1 to 1 : 1, about 7: 1 to 1 : 1, about 6: 1 to 1 : 1, about 5: 1 to 1 : 1, about 4: 1 to 1 : 1, about 3: 1 to 1 : 1, about 2: 1 to 1 : 1, about 20: 1 to 1 : 1.5, about 10: 1 to 1 : 1.5, about 9: 1 to 1 : 1.5, about 8: 1 to 1 : 1.5, about 7: 1 to 1 : 1.5, about 6: 1 to 1 : 1.5, about 5: 1 to 1 : 1.5, about 4: 1 to 1 : 1.5, about 3: 1 to 1 : 1.5, about 2: 1 to 1 : 1.5, about 1.5: 1 to 1 : 1.5, or about 1 : 1 to 1 : 1.5. In some embodiments, the ratio ranges from about 5:1 to 1:1. In some embodiments, the ratio ranges from about 5:1 to 1.5:1. In some embodiments, the ratio ranges from about 4:1 to 1:1. In some embodiments, the ratio ranges from about 4:1 to 1.5:1. In some embodiments, the ratio ranges from about 8:1 to about 1:1. In some embodiments, the ratio ranges from about 2.5:1 to 1:1.

[0151] The thiol-containing reagent can be, for example, cysteine ​​or N-acetylcysteine. The reducing agent can be, for example, TCEP. In some embodiments, the reduced activatable antibody can be purified prior to conjugation using, for example, column chromatography, dialysis, or diafiltration. In some embodiments, the reduced antibody is not purified after partial reduction and prior to conjugation.

[0152] The present disclosure also provides partially reduced, activatable antibodies, wherein at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody comprises an antibody or 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 to the target in its uncleaved state, and a CM1-CM2 substrate coupled to the AB, wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for at least one MMP and at least one SP. In some embodiments, the MM is coupled to the AB via the CM1-CM2 substrate. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM within the activatable antibody are not disturbed by the reducing agent. In some embodiments, the activatable antibody has the following structural arrangement from N-terminus to C-terminus in its uncleaved state: MM-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0153] The present disclosure also provides partially reduced activatable antibodies, including but not limited to the multispecific activatable antibodies of the present disclosure, wherein at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without interfering with or otherwise impairing the activity and / or efficacy of the activatable antibody, wherein the activatable antibody comprises an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, a masking portion (MM) that inhibits binding of the AB of the activatable antibody in an uncleaved state to the target, and a CM1-CM2 substrate coupled to the AB, and the CM1-CM2 substrate is a polypeptide that serves as a substrate for a protease. By way of non-limiting example, the activity and / or efficacy of the activatable antibody is masking activity, activation of the activatable antibody, and / or binding activity of the activated activatable antibody. In some embodiments, one or more intrachain disulfide bonds of the activatable antibody are not interfered with by the reducing agent. In some embodiments, one or more intrachain disulfide bonds of the MM within the activatable antibody are not interfered with by the reducing agent. In some embodiments, the activatable antibody has the following structural arrangement from N-terminus to C-terminus in the uncleaved state: MM-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-MM. In some embodiments, the reducing agent is TCEP.

[0154] The present disclosure also provides a conjugated activatable antibody comprising an activatable antibody linked to a monomethylrioxetine 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 an uncleaved state to the target, and a CM1-CM2 substrate coupled to the AB, wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for at least one MMP protease and at least one SP protease.

[0155] In some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any of the following methods for attaching an agent to the AB: (a) to the carbohydrate portion of the AB, or (b) to the sulfhydryl group of the AB, or (c) to the amino group of the AB, or (d) to the carboxyl group of the AB.

[0156] In some embodiments, the MMAD payload is conjugated to the AB via a linker. In some embodiments, the MMAD payload is conjugated to a cysteine ​​in the AB via a linker. In some embodiments, the MMAD payload is conjugated to a lysine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to another residue of the AB, such as those disclosed herein, via a linker. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is selected from the group consisting of the linkers shown in Table 5 and Table 6. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are connected via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker. In some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation techniques disclosed herein.

[0157] The present disclosure also provides polypeptides and other larger molecules comprising one or more CM1-CM2 substrate sequences set forth herein. By way of non-limiting example, the CM1-CM2 substrate sequences set forth herein can be used in prodrug compositions and methods of use thereof. These CM1-CM2 substrate sequences set forth herein can also be used in probes and other detection agents and methods of use thereof. For example, the CM1-CM2 substrate sequences set forth herein can be used in combination with fluor and other quenchers to produce detection agents, such as imaging agents and / or other diagnostic agents. One of ordinary skill in the art will appreciate that the CM1-CM2 substrate sequences set forth herein can be used in the art in any composition and / or method using a substrate cleavable by at least one MMP and at least one SP.

[0158] The present disclosure also provides an isolated nucleic acid molecule encoding an antibody and / or an activatable antibody as described herein, and a vector comprising such isolated nucleic acid sequences. The present disclosure provides a method for producing an antibody and / or an activatable antibody by culturing a cell under conditions that result in expression of the antibody and / or the activatable antibody, wherein the cell comprises such a vector.

[0159] The present disclosure provides a method of making a conjugated antibody of the present disclosure that binds a given target, the method comprising (a) culturing a cell comprising a nucleic acid construct encoding the antibody, (i) wherein the antibody comprises a CM1-CM2 substrate, and (ii) wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for matrix metalloproteinases and serine proteases, under conditions that result in expression of the antibody; (b) recovering the antibody; and (c) conjugating the recovered antibody to one or more additional agents.

[0160] The present disclosure also provides a method for producing an activatable antibody of the present disclosure that binds to a given target in an activated state, the method being performed by: (a) culturing a cell containing a nucleic acid construct encoding the activatable antibody under conditions that result in expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a CM1-CM2 substrate, and an antibody or antigen-binding fragment thereof (AB) that specifically binds to the target, (i) wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for MMPs and SPs; and (ii) wherein the CM1-CM2 substrate is positioned in the activatable antibody such that, in the uncleaved state, the MM interferes with specific binding of the AB to the target, and in the 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.

[0161] The present disclosure also provides a method for producing a conjugated, activatable antibody of the present disclosure that binds to a given target in an activated state, the method being performed by: (a) culturing a cell containing a nucleic acid construct encoding the activatable antibody under conditions that result in expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a CM1-CM2 substrate, and an antibody or antigen-binding fragment thereof (AB) that specifically binds to the target, (i) wherein the CM1-CM2 substrate is a polypeptide that serves as a substrate for MMPs and SPs and / or CPs; and (ii) wherein the CM1-CM2 substrate is positioned in the activatable antibody such that, in the uncleaved state, the MM interferes with specific binding of the AB to the target, and in the 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.

[0162] The present disclosure provides methods for preventing, delaying progression of, treating, alleviating symptoms of, or otherwise ameliorating a target-associated 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.

[0163] The present disclosure provides a method for preventing inflammation and / or inflammatory conditions in a subject, delaying the progression of the condition, treating the condition, alleviating the symptoms of the condition, or otherwise improving the condition, by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody as described herein. The present disclosure also provides a method for preventing cancer in a subject, delaying the progression of the cancer, treating the cancer, alleviating the symptoms of the cancer, or otherwise improving the cancer, by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody as described herein. The present disclosure also provides a method for preventing an autoimmune disease in a subject, delaying the progression of the disease, treating the disease, alleviating the symptoms of the disease, or otherwise improving the disease, by administering to a subject in need thereof a therapeutically effective amount of a conjugated antibody, activatable antibody, and / or conjugated activatable antibody as described herein.

[0164] The conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies used in any embodiment of these methods and uses can be administered at any stage of the disease. For example, such conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be administered to a patient with cancer at any stage from early stage to metastasis. The terms subject and patient are used interchangeably herein.

[0165] In some embodiments, the subject is a mammal, such as a human, a non-human primate, a companion animal (e.g., a cat, a dog, a horse), a farm animal, a working animal, or a zoo animal. In some embodiments, the subject is a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal under veterinary care.

[0166] The conjugated antibody, activatable antibody and / or conjugated activatable antibody and therapeutic preparation thereof are applied to a subject suffering from or susceptible to a disease or condition related to abnormal target expression and / or activity. Any of a variety of methods known in the art is used to identify a subject suffering from or susceptible to a disease or condition related to abnormal target expression and / or activity. For example, any of a variety of clinical and / or laboratory tests (such as physical examination and blood, urine and / or fecal analysis) is used to identify a subject suffering from cancer or other tumor conditions to assess health status. For example, any of a variety of clinical and / or laboratory tests (such as physical examination and / or body fluid analysis, such as blood, urine and / or fecal analysis) is used to identify a subject suffering from inflammation and / or inflammatory conditions to assess health status.

[0167] If any of a variety of laboratory or clinical goals is achieved, it is considered that the application of conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies to patients suffering from diseases or conditions related to abnormal target expression and / or activity is successful. For example, if one or more symptoms associated with a disease or condition are alleviated, alleviated, suppressed or do not progress to a further (i.e., worse) state, it is considered that the application of conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies to patients suffering from diseases or conditions related to abnormal target expression and / or activity is successful. If a disease or condition enters remission or does not progress to a further (i.e., worse) state, it is considered that the application of conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies to patients suffering from diseases or conditions related to abnormal target expression and / or activity is successful.

[0168] In some embodiments, the antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies described herein are used in combination with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended application, such as cancer. For example, antibodies, conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies can be used in combination with additional chemotherapeutic agents or anti-tumor agents.

[0169] In some embodiments, the one or more additional agents is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0170] In some embodiments, one or more additional agents are checkpoint inhibitors, kinase inhibitors, agents targeting inhibitors in the tumor microenvironment and / or T cells or NK agonists. In some embodiments, one or more additional agents are radiotherapy alone or in combination with one or more additional agents such as chemotherapeutic agents or anti-tumor agents. In some embodiments, one or more additional agents are vaccines, tumor viruses and / or DC activators, by way of non-limiting examples, such as toll-like receptor (TLR) agonists and / or α-CD40. In some embodiments, one or more additional agents are tumor-targeting antibodies designed to kill tumors by ADCC or by being directly conjugated to toxins (e.g., antibody-drug conjugates (ADC)).

[0171] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of: CTLA-4, LAG-3, PD-1, PD-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 Btk inhibitors (such as ibrutinib). In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of: IDO inhibitors, α-CSF1R inhibitors, α-CCR4 inhibitors, TGF-β, myeloid-derived suppressor cells, or T regulatory cells. In some embodiments, the agonist is selected from the group consisting of: Ox40, GITR, CD137, ICOS, CD27, and HVEM.

[0172] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is TGF-β. In some embodiments, the inhibitor is myeloid-derived suppressor cells. In some embodiments, the inhibitor is a T regulatory cell.

[0173] 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.

[0174] In some embodiments, antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies are administered in combination with one or more other agents (e.g., chemotherapeutic agents, anti-inflammatory agents and / or immunosuppressants) during and / or after treatment. In some embodiments, antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies and other agents are formulated into a single therapeutic composition, and antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies and other agents are administered simultaneously. Alternatively, antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies and other agents are separated from each other, for example, each of them is formulated into a separate therapeutic composition, and antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies and other agents are administered simultaneously, or antibodies, conjugated antibodies, activatable antibodies and / or conjugated activatable antibodies and other agents are administered at different times in a treatment regimen. For example, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody is administered before the administration of the additional agent, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody is administered after the administration of the additional agent, or the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and the additional agent are administered in an alternating manner. As described herein, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and the additional agent are administered in a single dose or multiple doses.

[0175] In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and one or more additional agents are administered simultaneously. For example, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and one or more additional agents can be formulated into a single composition or administered as two or more separate compositions. In some embodiments, the antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody and one or more additional agents are administered sequentially or at different times in the treatment regimen.

[0176] In some embodiments, the conjugated antibody, activatable antibody, and / or conjugated activatable antibody is administered during and / or after treatment in combination with one or more additional agents, such as, by way of non-limiting example, an anti-inflammatory agent, an immunosuppressant, a chemotherapeutic agent (such as an alkylating agent), an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and / or any other nucleic acid damaging agent. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., ). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as a platinum-based chemotherapeutic agent, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeting agent, such as a kinase inhibitor, for example, sorafenib or erlotinib. In some embodiments, the additional agent is a targeting agent, such as another antibody, for example, a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteasome inhibitor, such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulator, such as lenalidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent that is considered standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art.

[0177] In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody. In some embodiments, the additional agent is an antibody, another conjugated antibody, another activatable antibody, and / or another conjugated activatable antibody that is 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 that is directed against a target different from the target of the first conjugated antibody, activatable antibody, and / or conjugated activatable antibody.

[0178] In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody and one or more additional agents are administered simultaneously. For example, the conjugated antibody, activatable antibody and / or conjugated activatable antibody and one or more additional agents can be formulated into a single composition or administered as two or more separate compositions. In some embodiments, the conjugated antibody, activatable antibody and / or conjugated activatable antibody and one or more additional agents are administered sequentially, or the antibody and / or conjugated antibody and additional agent are administered at different times in the treatment regimen. For example, the antibody and / or conjugated antibody is administered before administering the additional agent, the antibody and / or conjugated antibody is administered after administering the additional agent, or the antibody and / or conjugated antibody and additional agent are administered in an alternating manner. As described herein, the antibody and / or conjugated antibody and additional agent are single doses or multiple doses.

[0179] The present disclosure also provides methods and kits for using the conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies in a variety of diagnostic and / or prophylactic indications.

[0180] Pharmaceutical compositions according to the present disclosure may comprise an antibody, conjugated antibody, activatable antibody and / or conjugated activatable antibody of the present disclosure and a carrier. These pharmaceutical compositions may be included in a kit, such as, for example, a diagnostic kit.

[0181] Conjugated antibodies, activatable antibodies, and / or conjugated activatable antibodies comprise an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target. Exemplary categories of targets for AB include, but are not necessarily limited to, cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), and the like. In some embodiments, conjugated antibodies and / or activatable antibodies have ABs that bind to extracellular targets (typically extracellular protein targets). In some embodiments, conjugated antibodies and / or activatable antibodies are designed for cellular uptake and can be converted intracellularly.

[0182] As a non-limiting example, AB is a binding partner for any of the targets listed in Table 2.

[0183] Table 2: Exemplary targets

[0184]

[0185]

[0186] As non-limiting examples, AB is or is derived from an antibody listed in Table 3.

[0187] Table 3: Exemplary sources of Abs

[0188]

[0189]

[0190] The activatable antibodies and activatable antibody compositions provided herein contain at least an antibody or antibody fragment thereof (collectively referred to throughout this disclosure as AB) that specifically binds to a target, eg, a human target, wherein the AB is modified with a masking moiety (MM).

[0191] In some embodiments, a 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 to EGFR includes an MM comprising the sequence CISPRG (SEQ ID NO: 165). By way of non-limiting example, an MM may comprise sequences such as: CISPRGC (SEQ ID NO: 166); CISPRGCG (SEQ ID NO: 167); CISPRGCPDGPYVMY (SEQ ID NO: 168); CISPRGCPDGPYVM (SEQ ID NO: 169); CISPRGCEPGTYVPT (SEQ ID NO: 170); and CISPRGCPGQIWHPP (SEQ ID NO: 171). Other suitable masking moieties include any of the EGFR-specific masks disclosed in PCT Publication No. WO 2010 / 081173, such as, by way of non-limiting example, GSHCLIPINMGAPSC (SEQ ID NO: 172); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 173); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 174); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 175); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 176); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 177); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 178); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 179); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 180); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 181). 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); CNHVYFGTCGCISPRGCG (SEQ ID NO:187); CHHVYWGHCGCISPRGCG (SEQ ID NO:188); CPHFTTTSCGCISPRGCG (SEQ ID NO:189);CNHFTLTTCGCISPRGCG (SEQ ID NO:190); CHHFTLTTCGCISPRGCG (SEQ ID NO:191); CPYYTLSYCGCISPRGCG (SEQ ID NO:192); CPHVSFGSCGCISPRGCG (SEQ ID NO:193); ADHVFWGSYGCISPRGCG (SEQ ID NO:194); YNPCATPMCCISPRGCG (SEQ ID NO:194) 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 CISPRGCNAVSGLGS (SEQ IDNO: 164).

[0192] When the AB is modified with an MM and in the presence of the target, the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB not modified with the MM or the specific binding of the parent AB to the target.

[0193] K of MM-modified AB against target d Compared with the K of AB without MM modification or parental AB against target dat least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or more, or between 5-10 times, 10-100 times, 10-1,000 times, 10-10,000 times, 10-100,000 times, 10-1,000,000 times, 10-10,000,000 times, 100-1,000 times, 1 00-10,000 times, 100-100,000 times, 100-1,000,000 times, 100-10,000,000 times, 1,000-10,000 times, 1,000-10,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 10,000-10,000,000 times, 100,000-1,000,000 times or between 100,000-10,000,000 times. In contrast, the binding affinity of the AB modified with MM for the target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1,000,000 fold, 10-1,000,000 fold or more, than the binding affinity of the AB not modified with MM or the parental AB for the target. times, 10-10,000,000 times, 100-1,000 times, 100-10,000 times, 100-1,000,000 times, 100-10,000,000 times, 1,000-10,000,000 times, 1,000-10,000,000 times, 1,000-10,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 10,000-10,000,000 times, 100,000-1,000,000 times or between 100,000-10,000,000 times.

[0194] The dissociation constant (K) of MM against AB d ) is usually greater than the K of AB against the target d MM against AB's Kd Comparable AB K against target d AB. MMs can have a binding affinity that is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the binding affinity of AB for the target. Conversely, the binding affinity of MMs for AB is typically lower than the binding affinity of AB for the target. The binding affinity of MMs for AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times less than the binding affinity of AB for the target.

[0195] When the AB is modified with an MM and in the presence of the target, the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB not modified with the MM or the specific binding of the parent AB to the target. When modified with a MM, the ability of the AB to bind to a target may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% compared to the binding of the AB not modified with the MM or the binding of the parental AB to the target when measured in an in vivo or in vitro assay for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or longer.

[0196] The MM inhibits the binding of the AB to the target. The MM binds to the antigen binding domain of the AB and inhibits the binding of the AB to the target. The MM can sterically inhibit the binding of the AB to the target. The MM can allosterically inhibit the binding of the AB to its target. In these embodiments, when the AB is modified or coupled to the MM and in the presence of the target, the AB does not bind or does not substantially bind to the target, or the AB binds to the target no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% when compared to the binding of the AB not modified with the MM, the parent AB, or the AB not coupled to the MM to the target when measured in an in vivo or in vitro assay. , 9, 10, 15, 20, 25, 30, 35, 40, or 50% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer.

[0197] When AB is coupled to or modified by MM, MM "masks" or reduces or otherwise inhibits the specific binding of AB to its target. When AB is coupled to or modified by MM, such coupling or modification may cause a structural change that reduces or inhibits the ability of AB to specifically bind to its target.

[0198] AB coupled to or modified with MM can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region):

[0199] (MM)-(AB)

[0200] (AB)-(MM)

[0201] (MM)-L-(AB)

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

[0203] 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 (eg, flexible linkers) into the composition to provide flexibility.

[0204] In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM has no or substantially no homology to any natural binding partner of the 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 the 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 the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB.

[0205] In some embodiments, the activatable antibody comprises an AB modified with a MM and further comprises at least one cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). Such activatable antibodies exhibit activatable / switchable binding to the target of the AB. The activatable antibody typically comprises an antibody or antibody fragment (AB) modified with or coupled to a masking moiety (MM) and a CM1-CM2 substrate.

[0206] The elements of the activatable antibody are arranged such that the MM and the CM1-CM2 substrate are positioned such that in the cleaved (or relatively active) state and in the presence of the target, the AB binds to the target, while in the uncleaved (or relatively inactive) state, in the presence of the target, the specific binding of the AB to its target is reduced or inhibited. The specific binding of the AB to its target may be reduced due to the inhibition or masking of the MM on the ability of the AB to specifically bind to its target.

[0207] K of AB modified with MM and CM1-CM2 substrates against targets d Compared with the K of AB not modified with MM and CM1-CM2 substrates or parental 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, or between 5-10 times, 10-100 times, 10-1,000 times, 10-10,000 times, 10-100,000 times, 10-1,000,000 times, 10-10,000,000 times, 100-1,000 times, times, 100-10,000 times, 100-100,000 times, 100-1,000,000 times, 100-10,000,000 times, 1,000-10,000 times, 1,000-10,000 times, 1,000-1,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 100,000-1,000,000 times, or between 100,000-10,000,000 times. In contrast, the binding affinity of the AB modified with MM and CM1-CM2 substrates for the target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 fold or more, or between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-100,000 fold, 10-100,000 fold or more, than the binding affinity of the AB not modified with MM and CM1-CM2 substrates or the parental AB for the target. -1,000,000 times, 10-10,000,000 times, 100-1,000 times, 100-10,000 times, 100-100,000 times, 100-1,000,000 times, 100-10,000,000 times, 1,000-10,000 times, 1,000-100,000 times, 1, 000-1,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 10,000-10,000,000 times, 100,000-1,000,000 times or between 100,000-10,000,000 times.

[0208] When the AB is modified with MM and CM1-CM2 substrates and in the presence of the target but not the modifying agents (e.g., MMPs and SPs), the specific binding of the AB to its target is reduced or inhibited, compared to the specific binding of the AB not modified with MM and CM1-CM2 substrates or the parental AB to the target. When modified with MM and CM1-CM2 substrates, the ability of the AB to bind 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 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or longer, compared to the binding of the parent AB to its target or the binding of the AB not modified with MM and CM1-CM2 substrates when measured in an in vivo or in vitro assay.

[0209] As used herein, the term cleavage state refers to the state of an activatable antibody after the CM1-CM2 substrate is modified (i.e., cleaved) by at least one matrix metalloproteinase and / or at least one serine protease. As used herein, the term uncleaved state or completely uncleaved refers to the state of an activatable antibody in the absence of cleavage of the CM1-CM2 substrate by MMP and / or SP. As described above, the term "activatable antibody" is used herein to refer to an activatable antibody in its uncleaved (native) state and in its cleaved state. The activatable antibody in its cleaved state is also referred to herein as an activated antibody and / or an activated activatable antibody. It is clear to those of ordinary skill that in some embodiments, the cleaved activatable antibody may lack MM due to the cleavage of the CM1-CM2 substrate by a protease, thereby resulting in the release of at least MM.

[0210] Activatable or convertible means that when in an inhibited, masked or uncleaved state (i.e., a first conformation), the activatable antibody exhibits a first binding level to the target, and when in an uninhibited, unmasked and / or cleaved state (i.e., a second conformation), the activatable antibody exhibits a second binding level to the target, wherein the second target binding level is greater than the first binding level. In general, the target is closer to the AB of the activatable antibody in the presence of a cleavage agent capable of cleaving the CM1-CM2 substrate than in the absence of such a cleavage agent. Thus, when the activatable antibody is in an uncleaved state, the binding of the AB to the target is inhibited and its target binding may be masked (i.e., the first conformation renders the AB unable to bind to the target), and in the cleaved state, the AB is not inhibited or unmasked for target binding.

[0211] The CM1-CM2 substrate and AB of the activatable antibody are selected such that the AB represents the binding portion of a given target and the CM1-CM2 substrate represents a substrate for an MMP and SP, wherein the MMP and / or SP are co-localized with the target at a therapeutic site or diagnostic site in a subject. Particular uses of the activatable antibodies disclosed herein are found, for example, where the MMP and SP, each capable of cleaving a site in the CM1-CM2 substrate, are present at relatively higher levels in target-containing tissue at the therapeutic site or diagnostic site than in tissue at a non-therapeutic site (e.g., in healthy tissue).

[0212] In some embodiments, the activatable antibody provides reduced toxicity and / or adverse side effects that would otherwise result from binding of the AB at non-therapeutic sites if binding of the AB to the target was not masked or otherwise inhibited.

[0213] In general, an activatable antibody can be designed by selecting an AB of interest and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides masking of the AB or reduced binding of the AB to its target. Structural design criteria can be considered to provide these functional characteristics.

[0214] Provided are activatable antibodies that exhibit a convertible phenotype of the desired dynamic range of target binding in an inhibited conformation versus an uninhibited conformation. Dynamic range generally refers to the ratio of the maximum detection level of a parameter (a) under a first set of conditions to the minimum detection value of a parameter (b) under a second set of conditions. For example, in the context of activatable antibodies, dynamic range refers to the ratio of the maximum detection level of a target protein bound to an activatable antibody (a) in the presence of MMPs and SPs that can cleave the CM1-CM2 substrates of an activatable antibody to the minimum detection level of a target protein bound to an activatable antibody (b) in the absence of a protease. The dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of an activatable antibody cleavage agent (e.g., enzyme) to the dissociation constant of an activatable antibody cleavage agent. The larger the dynamic range of an activatable antibody, the better the convertible phenotype of the activatable antibody. Activatable antibodies having relatively high dynamic range values ​​(e.g., greater than 1) exhibit a more desirable switching phenotype, such that target protein binding by the activatable antibody occurs to a greater extent (e.g., predominantly) in the presence of a cleavage agent (e.g., an enzyme) capable of cleaving the CM1-CM2 substrate of the activatable antibody than in the absence of the cleavage agent.

[0215] Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulas of activatable antibodies are provided below. It is specifically contemplated that the N-terminus to C-terminus order of the AB, MM, and CM1-CM2 substrate can be reversed in the activatable antibody. It is also specifically contemplated that the CM and MM can overlap in amino acid sequence, for example, such that the CM1-CM2 substrate is at least partially contained within the MM.

[0216] For example, an activatable antibody can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region):

[0217] (MM)-(CM1-CM2 substrate)-(AB)

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

[0219] wherein MM is a masking moiety, CM1-CM2 substrate is a cleavable moiety, and AB is an antibody or fragment thereof. As described above, the term "CM1-CM2 substrate" is not intended to convey any requirements regarding the orientation or other structural arrangement of the first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and the at least second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). Thus, the term "CM1-CM2 substrate" encompasses CM1-CM2 substrates having the following structural arrangement from 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 MM and CM1-CM2 substrate are indicated as distinct components in the above formula, in all exemplary embodiments disclosed herein (including the formula), it is contemplated that the amino acid sequences of MM and CM1-CM2 substrate may overlap, e.g., such that the CM1-CM2 substrate is fully or partially contained within the MM. Additionally, the above formula provides additional amino acid sequences that can be positioned at the N-terminus or C-terminus of the activatable antibody element.

[0220] In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM has no or substantially no homology to any natural binding partner of the 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 the 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 the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB.

[0221] In many embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) into the activatable antibody construct to provide flexibility at one or more of the MM-CM1-CM2 substrate junction, the CM1-CM2 substrate-AB junction, or both. For example, the AB, MM, and / or CM1-CM2 substrates may not contain residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly Gly) sufficient to provide the desired flexibility. Thus, the convertible phenotype of such activatable antibody constructs may benefit from the introduction of one or more amino acids to provide a flexible linker. Additionally, as described below, when the activatable antibody is provided as a conformationally constrained construct, a flexible linker may be operably inserted to promote the formation and maintenance of a ring structure in the uncleaved activatable antibody.

[0222] For example, in certain embodiments, the activatable antibody comprises one of the following formulae (wherein the following formulae represent an amino acid sequence in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction):

[0223] (MM)-LP1-(CM1-CM2 substrate)-(AB)

[0224] (MM)-(CM1-CM2 substrate)-LP2-(AB)

[0225] (MM)-LP1-(CM1-CM2 substrate)-LP2-(AB)

[0226] Wherein MM, CM1-CM2 substrate and AB are as defined above; wherein each of LP1 and LP2 is independently and optionally present or absent, the same or different flexible linker comprising at least one flexible amino acid (e.g., Gly). In addition, the above formula provides additional amino acid sequences that can be positioned at the N-terminus or C-terminus of the activatable antibody element. Examples include, but are not limited to, targeting moieties (e.g., ligands for cell receptors present in target tissues) and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HAS)).

[0227] The rate of specific cleavage of the CM1-CM2 substrate by at least one MMP is about 0.001-1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M -1 S-1 , and the rate of specific cleavage by at least one SP is about 0.001-1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M -1 S -1 .

[0228] To achieve specific enzymatic cleavage, the enzyme is brought into contact with the CM1-CM2 substrate. When an activatable antibody comprising an AB coupled to a MM and the CM1-CM2 substrate is present in the presence of a target and sufficient enzymatic activity, the CM1-CM2 substrate can be cleaved. Sufficient enzymatic activity can refer to the ability of the enzyme to contact and cleave the CM1-CM2 substrate. It is readily conceivable that an enzyme may be in the vicinity of the CM1-CM2 substrate but unable to cleave it due to protein modification by other cytokines or enzymes.

[0229] The linker suitable for use in the compositions described herein is generally a linker that provides flexibility of the modified AB or activatable antibody to promote inhibition of the combination of AB and target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be easily selected and can have any suitable different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and the length can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0230] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 381), and (GGGS)n (SEQ ID NO: 382), where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to act as neutral tethers between components. Glycine accesses a significantly larger phi-psi space than even alanine and is much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly (SEQ ID NO: 202), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 203), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 204), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 205), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 206), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 207), etc. One of ordinary skill will recognize that the design of an activatable antibody can include a fully or partially flexible linker, such that a linker can include a flexible linker and one or more portions that impart a less flexible structure to provide the desired activatable antibody structure.

[0231] In some embodiments, the activatable antibodies described herein further comprise an agent conjugated to the activatable antibody. In some embodiments, the conjugated agent is a therapeutic agent, such as an anti-inflammatory agent and / or an anti-tumor agent. In such embodiments, the agent is conjugated to a carbohydrate moiety of the activatable antibody, for example, in some embodiments, wherein the carbohydrate moiety is located outside the antigen-binding region of the antibody or antigen-binding fragment of the activatable antibody. In some embodiments, the agent is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment of the activatable antibody.

[0232] In some embodiments, the agent is a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) or a radioactive isotope (ie, a radioconjugate).

[0233] In some embodiments, the agent is a detectable moiety, such as a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotin moieties detectable by a marker avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetry), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety is attached via a spacer molecule.

[0234] The present disclosure also relates to immunoconjugates comprising antibodies conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) or radioactive isotopes (i.e., radioconjugates). Suitable cytotoxic agents include, for example, dolastatin and its derivatives (e.g., riocetin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethyl riocetin E (MMAE) or monomethyl riocetin D (MMAD). In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is riocetin or its derivatives. In some embodiments, the agent is riocetin E or its derivatives. In some embodiments, the agent is monomethyl riocetin E (MMAE). In some embodiments, the agent is monomethyl riocetin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0235] In some embodiments, the agent is linked to AB using a maleimidocaproyl-valine-citrulline linker or a maleimidocaproyl PEG-valine-citrulline linker. In some embodiments, the agent is linked to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, the agent is linked to AB using a maleimidocaproyl-valine-citrulline linker. In some embodiments, the agent is linked to AB using a maleimidocaproyl PEG-valine-citrulline linker. In some embodiments, the agent is monomethylreoxetine D (MMAD) linked to AB using a maleimidocaproyl PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as "vc-MMAD". In some embodiments, the agent is monomethylreoxetine E (MMAE) linked to AB using a maleimidocaproyl PEG-valine-citrulline-p-aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as "vc-MMAE". The structures of vc-MMAD and vc-MMAE are shown below:

[0236] vc-MMAD:

[0237]

[0238] vc-MMAE:

[0239]

[0240] 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, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. 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.

[0241] Conjugates of antibodies and cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see WO94 / 11026).

[0242] Table 4 lists some exemplary agents that may be employed in the disclosure described herein, but is by no means intended to be an exhaustive list.

[0243] Table 4: Exemplary Agents for Conjugation

[0244]

[0245]

[0246]

[0247] One of ordinary skill in the art will recognize that a wide variety of possible moieties can be coupled to the resulting antibodies of the present disclosure (see, for example, "Conjugate Vaccines," in 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).

[0248] As long as the antibody and other parts maintain their respective activity, coupling can be accomplished by any chemical reaction that binds the two molecules. This linkage can include many chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding, and complexation. However, in some embodiments, the binding is covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linkers can be used to couple protein molecules (such as 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 an exhaustive description of various coupling agents known in the art, but rather to illustrate more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987)).

[0249] In some embodiments, in addition to the compositions and methods provided herein, the activatable antibody of the conjugation can also be modified by inserting or otherwise comprising a modified amino acid sequence in the activatable antibody sequence to perform site-specific conjugation. These modified amino acid sequences are designed to allow the arrangement and / or dosage of the conjugate to be controlled within the activatable antibody of the conjugation. For example, the activatable antibody can be engineered to include cysteine ​​substitutions at positions on the light and heavy chains, which provide reactive thiol groups and do not adversely affect protein folding and assembly, nor change antigen binding. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce one or more non-natural amino acid residues within the activatable antibody to provide a suitable conjugation site. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce an enzyme-activatable peptide sequence within the activatable antibody sequence.

[0250] 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 haloacetic acid hydrazide derivatives coupled to antibodies via oligopeptide linkers. In some embodiments, suitable linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene) (Pierce Chem. Co., catalog (21558G)); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate) (Pierce Chem. Co., catalog (21558G)). Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6 [3- (2-pyridyldithio) -propionamido] hexanoate) (Pierce Chem. Co. catalog number 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 24510) conjugated to EDC. Additional linkers include, but are not limited to, SMCC, sulfoSMCC, SPDB, or sulfoSPDB.

[0251] The linkers described above 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 aryl carboxylates. Linkers containing NHS esters have lower solubility than sulfo-NHS esters. In addition, the linker SMPT contains sterically hindered disulfide bonds and can form conjugates with increased stability. In general, disulfide linkages are less stable than other linkages because they are cleaved in vitro, resulting in fewer usable conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. When used in combination with sulfo-NHS, the esters formed by carbodiimide coupling (such as EDC) are more resistant to hydrolysis than the esters formed by the carbodiimide coupling reaction alone.

[0252] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are two or more linkers. The two or more linkers are both identical, i.e., cleavable or non-cleavable, or the two or more linkers are different, i.e., at least one is cleavable and at least one is non-cleavable.

[0253] The present disclosure utilizes several methods for attaching the agent to the AB: (a) attachment to the carbohydrate portion of the AB, or (b) attachment to the sulfhydryl group of the AB, or (c) attachment to the amino group of the AB, or (d) attachment to the carboxyl group of the AB. According to the present disclosure, the AB can be covalently attached to the agent through an intermediate linker having at least two reactive groups, one of which reacts with the AB and one with the agent. The linker can be selected to include any compatible organic compound so that the reaction with the AB (or agent) does not adversely affect the reactivity and selectivity of the AB. In addition, the attachment of the linker to the agent may not destroy the activity of the agent. Suitable linkers for reacting with oxidized antibodies or oxidized antibody fragments include those containing an amine selected from the group consisting of: primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and thiosemicarbazides. Such reactive functional groups may be present as part of the linker structure or may be introduced by suitable chemical modification of a linker that does not contain such groups.

[0254] According to the present disclosure, suitable linkers for attachment to the reduced AB include those having certain reactive groups capable of reacting with the sulfhydryl groups of the reduced antibody or fragment. Such reactive groups include, but are not limited to, reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercurybenzoate groups, and groups capable of undergoing Michael-type addition reactions (including, for example, maleimide and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

[0255] According to the present disclosure, suitable linkers for attachment to Abs that are neither oxidized nor reduced include those having certain functional groups capable of reacting with primary amino groups present in unmodified lysine residues in the Ab. Such reactive groups include, but are not limited to, NHS carboxylates or carbonates, sulfo-NHS carboxylates or carbonates, 4-nitrophenyl carboxylates or carbonates, pentafluorophenyl carboxylates or carbonates, acyl imidazoles, isocyanates, and isothiocyanates.

[0256] According to the present disclosure, suitable linkers for attachment to Abs that are neither oxidized nor reduced include those with certain functional groups that react with the carboxylic acid groups present in the aspartic acid residues or glutamic acid residues in the Ab, which have been activated by suitable reagents. Suitable activating reagents include EDC with or without added NHS or sulfo-NHS, as well as other dehydrating agents for carboxamide formation. In these cases, the functional groups present in suitable linkers will include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0257] The agent can be attached to the linker before or after the linker is attached to the AB. In certain applications, it may be desirable to first prepare an AB-linker intermediate in which the linker does not contain an associating agent. Depending on the specific application, the specific agent can then be covalently attached to the linker. In some embodiments, for conjugation purposes, the AB is first attached to the MM, CM1-CM2 substrate, and the associated linker, and then attached to the linker.

[0258] Branched linkers: In certain embodiments, branched linkers with multiple sites for attachment of agents are utilized. For multi-site linkers, a single covalent attachment to the AB will produce an AB linker intermediate capable of binding agents at many sites. The site can be any chemical site that can be attached to an aldehyde or sulfhydryl group or an agent.

[0259] In some embodiments, higher specific activity (or higher agent to AB ratio) can be achieved by attaching a single site linker at multiple sites on the AB. The multiple sites can be introduced into the AB by either of two methods. First, multiple aldehyde groups and / or sulfhydryl groups can be generated in the same AB. Second, a "branched linker" having multiple functional sites for subsequent attachment to a linker can be attached to the aldehyde or sulfhydryl groups of the AB. The functional site of the branched linker or multi-site linker can be an aldehyde or sulfhydryl group, or can be any chemical site that can be attached to a linker. Higher specific activity can be obtained by combining these two methods (i.e., attaching a multi-site linker at multiple sites on the AB).

[0260] Cleavable linkers: Peptide linkers that are readily cleaved by enzymes 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. According to one method of the present disclosure, the agent is attached via a linker that is readily cleaved by complement. The antibody is selected from a class that can activate complement. Thus, the antibody-agent conjugate activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, the agent is attached via a linker that is readily cleaved by an enzyme with proteolytic activity, such as urokinase, tissue plasminogen activator, plasmin, or trypsin. These cleavable linkers can be used in conjugated activatable antibodies that include extracellular toxins, such as, by way of non-limiting example, any of the extracellular toxins shown in Table 4.

[0261] Table 5 provides non-limiting examples of cleavable linker sequences.

[0262] Table 5: Exemplary linker sequences for conjugation

[0263]

[0264]

[0265] In addition, the agent can be attached to the AB via a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Since many tumors naturally release high levels of glutathione (a reducing agent), it can reduce the disulfide bond and subsequently release the agent at the delivery site. In certain specific embodiments, the reducing agent that will modify the CM1-CM2 substrate will also modify the linker of the conjugated activatable antibody.

[0266] Spacer and cleavable element: In some embodiments, it may be necessary to construct the linker in a manner that optimizes the spacing between the agent and the AB of the activatable antibody. This can be accomplished by using a linker with the following general structure:

[0267] W–(CH2)n–Q

[0268] in

[0269] W is --NH--CH2-- or --CH2--;

[0270] Q is an amino acid or a peptide; and

[0271] n is an integer from 0 to 20.

[0272] In some embodiments, the linker may comprise a spacer element and a cleavable element. The spacer element is used to position the cleavable element away from the core of the AB so that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain branched linkers described above may serve as spacer elements.

[0273] Throughout this discussion, it will be understood that the attachment of the linker to the agent (or the attachment of the spacer element to the cleavable element, or the attachment of the cleavable element to the agent) does not require a specific mode of attachment or reaction. Any reaction that provides a product with suitable stability and biocompatibility is acceptable.

[0274] Selection of serum complement and linker: According to one method of the present disclosure, when a release agent is needed, an AB that is a class of antibodies that can activate complement is used. The resulting conjugate retains the ability to bind to antigen complement and activate both the complement cascade. Therefore, according to this embodiment of the present disclosure, the agent 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 agent has a hydroxyl or amino group, it can be attached to the carboxyl end of a peptide, amino acid, or other appropriately selected linker via an ester or amide bond, respectively. For example, such agents can be attached to a linker peptide via a carbodiimide reaction. If the agent contains functional groups that will interfere with attachment to the linker, these interfering functional groups can be blocked before attachment and unblocked after preparing the product conjugate or intermediate. The opposite end or amino end of the linker is then used directly or after further modification to bind to the AB that can activate complement.

[0275] The linker (or spacer element of the linker) can be of any desired length, one end of which 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.

[0276] Thus, when these conjugates are bound to the antigen in the presence of complement, the amide or ester bond attaching the agent to the linker will be cleaved, resulting in the release of the agent in its active form. When administered to a subject, these conjugates will achieve delivery and release of the agent at the target site and are particularly effective for in vivo delivery of agents such as, but not limited to, those presented in Table 4, antibiotics, antimetabolites, antiproliferative agents, and the like.

[0277] In the case of no complement activation, the joint released: in another application of targeted delivery, since the activation of the complement cascade will eventually dissolve the target cell, it is necessary to release the agent in the case of no complement activation. Therefore, when the delivery and release of the agent should be completed without killing the target cell, this method is useful. When it is necessary to deliver a cell mediator (such as a hormone, enzyme, corticosteroid, neurotransmitter, gene or enzyme) to the target cell, this is the goal. These conjugates can be prepared by attaching the agent to the AB that cannot activate complement via a joint that is easily cleaved by serum proteases. When this conjugate is applied to an individual, the antigen-antibody complex will form rapidly, and the cracking of the agent will occur slowly, thereby causing the compound to be released at the target site.

[0278] Biochemical cross-linkers: In some embodiments, certain biochemical cross-linkers can be used to conjugate the activatable antibody to one or more therapeutic agents. Cross-linking reagents form a molecular bridge that links the functional groups of two different molecules. To link two different proteins in a stepwise manner, heterobifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

[0279] Peptide-based linkers that are cleavable by lysosomal proteases are also useful, such as Val-Cit, Val-Ala, or other dipeptides. In addition, acid-labile linkers that are cleavable in the low pH environment of the lysosome, such as disialyl ethers, can be used. Other suitable linkers include substrates that are labile to cathepsins, particularly those that function best at acidic pH.

[0280] Exemplary heterobifunctional cross-linkers are cited in Table 6.

[0281] Table 6: Exemplary heterobifunctional cross-linkers

[0282]

[0283]

[0284] Non-cleavable linker or direct attachment: In some embodiments of the present disclosure, the conjugate can be designed so that the agent is delivered to the target but not released. This can be accomplished by attaching the agent directly or via a non-cleavable linker to the AB.

[0285] These non-cleavable linkers may include amino acids, peptides, D-amino acids, or other organic compounds that can be modified to contain functional groups that can then be used to attach to AB by the methods described herein. The general formula of such organic linkers may be

[0286] W–(CH2)n–Q

[0287] in

[0288] W is --NH--CH2-- or --CH2--;

[0289] Q is an amino acid or a peptide; and

[0290] n is an integer from 0 to 20.

[0291] Non-cleavable conjugates: In some embodiments, the 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 accomplished using a linker that is susceptible to cleavage by activated complement or a linker that is not susceptible to cleavage by activated complement.

[0292] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are 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 increased circulation time are disclosed in U.S. Patent No. 5,013,556.

[0293] Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to produce liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be conjugated to liposomes by a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982).

[0294] definition :

[0295] Unless otherwise defined, the scientific and technical terms used in conjunction with the present disclosure should have the meaning commonly understood by those of ordinary skill in the art. The term "a / an" entity refers to one or more entities. For example, a compound refers to one or more compounds. Therefore, the terms "a / an," "a / an or more," and "at least one" are used interchangeably herein. In addition, unless the context otherwise requires, singular terms should include plural and plural terms should include singular. In general, the nomenclature used in conjunction with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein and their techniques are well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as generally performed in the art or as described herein. The aforementioned techniques and procedures are typically performed according to conventional methods well-known in the art and as described in the 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 chemistry described herein, and the laboratory procedures and techniques of these chemical fields are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0296] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0297] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with or binds with much lower affinity (K) to other polypeptides. d >10 -6 ). Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, domain antibodies, single-chain antibodies, Fab and F(ab')2 fragments, scFv, and Fab expression libraries.

[0298] It is known that the basic antibody structural unit comprises a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector function. In general, antibody molecules obtained from humans are related to any one of the categories IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chains present in the molecules. Certain categories also have subclasses, such as IgG1, IgG2, etc. In addition, in humans, the light chain can be a kappa chain or a lambda chain.

[0299] As used herein, the term "monoclonal antibody" (mAb) or "monoclonal antibody composition" refers to a population of antibody molecules containing only one molecular species of antibody molecules consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibodies are identical in all molecules of the population. MAbs contain an antigen binding site that is capable of immunoreacting with a specific epitope of an antigen, wherein the epitope is characterized by a unique binding affinity for the antigen binding site.

[0300] The term "antigen binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that participates 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 distinct segments within the V regions of the heavy and light chains, called "hypervariable regions," are interposed between more conserved flanking segments called "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequence that naturally occurs between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementarity-determining regions," or "CDRs." The amino acid assignments for each domain are based on the Kabat sequence definitions for proteins of immunological interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia and Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0301] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants are typically composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and typically have specific three-dimensional structural characteristics and specific charge characteristics. For example, antibodies can be generated against the N-terminal or C-terminal peptide of a polypeptide. When the dissociation constant is ≤1 μM; in some embodiments ≤100 nM and in some embodiments ≤10 nM, the antibody is considered to specifically bind to the antigen.

[0302] 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 the immunological binding interaction can be measured in terms of the dissociation constant (K) of the interaction. d ) to represent the smaller K d =A,B,C,D,E,N,S,N,A,B,D ... on ) and the “dissociation rate constant” (K off (See Nature 361: 186-87 (1993)). off / K on The ratio of 2 / 3 eliminates all parameters not related to affinity and is equal to the dissociation constant K. d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). When the binding constant (K d ) is ≤ 1 μM, in some embodiments ≤ 100 nM, in some embodiments ≤ 10 nM, and in some embodiments ≤ 100 pM to about 1 pM, an antibody of the present disclosure is considered to specifically bind to a target.

[0303] As used herein, the term "isolated polynucleotide" shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, depending on its origin, (1) is not associated with all or a portion of a polynucleotide (wherein the "isolated polynucleotide" is found in nature), (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides according to the present disclosure include nucleic acid molecules encoding heavy chain immunoglobulin molecules as set forth herein and nucleic acid molecules encoding light chain immunoglobulin molecules as set forth herein.

[0304] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; the "isolated protein" being, depending on its source or source of derivation, (1) not associated with proteins found in nature, (2) free of other proteins from the same source, e.g., free of murine proteins, (3) expressed by cells from a different species, or (4) not found in nature.

[0305] The term "polypeptide" is used herein as a general term to refer to a natural protein, fragment or analog having a polypeptide sequence. Therefore, natural protein fragments and analogs are species of the polypeptide genus. Polypeptides according to the present disclosure include heavy chain immunoglobulin molecules as described herein and light chain immunoglobulin molecules as described herein, as well as antibody molecules formed by comprising a combination of heavy chain immunoglobulin molecules and light chain immunoglobulin molecules (such as kappa light chain immunoglobulin molecules, and vice versa), as well as fragments and analogs thereof.

[0306] As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that occurs naturally in an organism (including viruses) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory or that is otherwise found naturally is naturally occurring.

[0307] As used herein, the term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is connected in a manner such that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0308] As used herein, the term "control sequence" refers to polynucleotide sequences necessary to achieve expression and processing of the coding sequences to which they are linked. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is necessary for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "polynucleotide" as referred to herein means a nucleotide sequence of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide. The term includes single-stranded and double-stranded forms of DNA.

[0309] The term oligonucleotide mentioned herein comprises naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages.Oligonucleotide is a polynucleotide subset that generally comprises 200 bases or a length of less base.In some embodiments, the length of oligonucleotide is 10 to 60 bases, and in some embodiments, a length is 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 bases.Oligonucleotide is generally single-stranded, for example, for probes, but oligonucleotide can be double-stranded, for example, for building gene mutants.Oligonucleotide of the present disclosure is sense or antisense oligonucleotide.

[0310] The term "naturally occurring nucleotides" as referred to herein include deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as referred to herein include nucleotides with modified or substituted sugar groups, etc. The term "oligonucleotide linkages" as referred to herein include oligonucleotide linkages such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phoshoraniladates, phosphoramidates, etc. See, e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). If desired, the oligonucleotide can include a label for detection.

[0311] As used herein, twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland 7 Mass. (1991)). Stereoisomers (e.g., D-amino acids), non-natural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid and other unconventional amino acids of twenty conventional 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, according to standard usage and convention, the left-hand direction is the amino terminal direction, and the right-hand direction is the carboxyl terminal direction.

[0312] 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' direction of addition of a nascent RNA transcript is referred to as the transcription direction, and the sequence region on the DNA strand having the same sequence as the RNA and extending 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the sequence region on the DNA strand having the same sequence as the RNA and extending 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0313] When applied to polypeptides, the term "substantial identity" means that two peptide sequences 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 when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights.

[0314] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0315] As discussed herein, it is contemplated that the present disclosure encompasses minor changes in the amino acid sequence of an antibody or immunoglobulin molecule, provided that the change in the amino acid sequence is maintained at least 75%, in some embodiments at least 80%, 90%, 95%, and in some embodiments 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid, glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other amino acid families include (i) serine and threonine, which are aliphatic-hydroxyl families; (ii) asparagine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan and tyrosine, which are aromatic families. For example, it is reasonable to expect that isoleucine or valine is isolated and replaced with leucine, aspartic acid is isolated and replaced with glutamic acid, threonine is isolated and replaced with serine, or similarly replaced with structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule, especially when the replacement does not involve amino acids in the framework site. Whether the amino acid change produces a functional peptide can be easily determined by measuring the specific activity of the polypeptide derivative. The determination is described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be easily prepared by those of ordinary skill in the art. Suitable amino and carboxyl termini of fragments or analogs occur near the boundaries of functional domains. Structure and function domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformational domains that occur in other proteins with 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). Therefore, the foregoing examples demonstrate that, according to the present disclosure, those skilled in the art can identify sequence motifs and structural conformations that can be used to define structure and function domains.

[0316] Suitable amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins having sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence (e.g., in portions of the polypeptide outside of one or more domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., amino acid replacements should not tend to disrupt helices present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structure are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0317] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or one or more internal deletions, but wherein the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence derived, for example, from the full-length cDNA sequence. The fragment length is typically at least 5, 6, 8 or 10 amino acids, in some embodiments at least 14 amino acids in length, in some embodiments at least 20 amino acids in length, typically at least 50 amino acids in length, and in some embodiments at least 70 amino acids in length. As used herein, the term "analog" refers to a polypeptide consisting of a segment of at least 25 amino acids, which has substantial identity to a portion of the derived amino acid sequence and has specific binding to the target under suitable binding conditions. Typically, a polypeptide analog comprises conservative amino acid substitutions (or additions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, at least 50 amino acids in length or longer in some embodiments, and can often be as long as a full-length naturally occurring polypeptide.

[0318] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0319] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or attachment to a polypeptide having a biotinyl moiety detectable by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In some cases, the label or marker can also be therapeutic. 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, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags). In some embodiments, the 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 that is capable of inducing a desired therapeutic effect when properly administered to a patient.

[0320] Other chemical terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S. ed., McGraw-Hill, San Francisco (1985)).

[0321] As used herein, "substantially pure" means that the object species is the predominant species present (i.e., it is more abundant on a molar basis than any other individual species in the composition), and in some embodiments, a substantially purified fraction is a composition in which the object species comprises at least about 50% (on a molar basis) of all macromolecular species present.

[0322] Generally, a substantially pure composition will comprise greater than about 80%, 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 necessary homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0323] The term patient includes human and veterinary subjects.

[0324] The activatable antibodies of the present disclosure specifically bind to a given target, such as a human target protein. Also included in the present disclosure are activatable antibodies that bind to the same epitope as the activatable antibodies described herein.

[0325] Those skilled in the art will recognize that, can, without excessive experimentation, by determining whether a monoclonal antibody (e.g., a mouse monoclonal antibody or a humanized antibody) prevents the monoclonal antibody used in the methods described herein from binding to the target to determine whether the former has the same specificity as the latter. If the monoclonal antibody being tested competes with the monoclonal antibody of the present invention, as shown by the reduction in binding of the monoclonal antibody of the present invention, the two monoclonal antibodies are bound to the same or closely related epitopes. A method for determining whether a monoclonal antibody has the specificity of a monoclonal antibody of the present invention is to pre-incubate the monoclonal antibody of the present invention with the target and then add the monoclonal antibody being tested to determine whether the ability of the monoclonal antibody being tested to bind to the target is suppressed. If the monoclonal antibody being tested is suppressed, it is very likely to have the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention.

[0326] Multispecific activatable antibodies

[0327] The present disclosure also provides multispecific activatable antibodies. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize two or more different antigens or epitopes and comprise at least one masking moiety (MM) that is connected to at least one antigen or epitope binding domain of the multispecific antibody such that coupling of the MM reduces the ability of the antigen or epitope binding domain to bind to its target. In some embodiments, the MM is coupled to the antigen or epitope binding domain of the multispecific antibody via a CM1-CM2 substrate that acts as a substrate for at least one MMP protease and at least one SP. The activatable multispecific antibodies provided herein are stable in circulation, activated at the intended therapeutic and / or diagnostic site, but not activated in normal tissue (i.e., healthy tissue), and upon activation, exhibit at least comparable binding to the target as the corresponding unmodified multispecific antibody.

[0328] In some embodiments, multispecific activatable antibodies are designed to engage immune effector cells, also referred to herein as immune effector cell engagement multispecific activatable antibodies. In some embodiments, multispecific activatable antibodies are designed to engage leukocytes, also referred to herein as leukocyte engagement multispecific activatable antibodies. In some embodiments, multispecific activatable antibodies are designed to engage T cells, also referred to herein as T cell engagement multispecific activatable antibodies. In some embodiments, multispecific activatable antibodies engage surface antigens on leukocytes, such as T cells, natural killer (NK) cells, myeloid monocytes, macrophages and / or surface antigens on another immune effector cell. In some embodiments, immune effector cells are leukocytes. In some embodiments, immune effector cells are T cells. In some embodiments, immune effector cells are NK cells. In some embodiments, immune effector cells are monocytes, such as myeloid monocytes. In some embodiments, multispecific activatable antibodies are designed to bind to or otherwise interact with more than one target and / or more than one epitope, also referred to herein as multi-antigen targeting activatable antibodies. As used herein, the terms "target" and "antigen" are used interchangeably.

[0329] In some embodiments, the multispecific activatable antibodies for immune effector cell engagement of the present disclosure comprise 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 to a first immune effector cell engaging target, wherein AB1 is attached to a masking portion (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking portion (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the antibody or antigen-binding fragment thereof that engages an immune effector cell comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to a first immune effector cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, wherein the second antibody or fragment thereof comprises 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 coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the non-immune effector cell engaging antibody is an antibody targeting cancer. 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., a non-immune cell effector antibody) is an IgG, and the immune effector cell engaging antibody is an scFv. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cells are myeloid mononuclear cells.

[0330] In some embodiments, the T cell engaging multispecific activatable antibodies of the present disclosure comprise 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 to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the T cell engaging antibody or antigen binding fragment thereof comprises a first antibody or antigen binding fragment thereof (AB1) that binds a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen binding fragment thereof comprises a second antibody or 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 coupling of MM2 reduces the ability of AB2 to bind the second target.

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

[0332] In some embodiments, the T cell engaging multispecifically 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 to a first T cell engaging target, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to the first target. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to 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, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to 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 thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0333] In some embodiments of the immune effector cell engaging multispecific activatable antibody, one antigen is typically an antigen present on a tumor cell or other cell type associated with the disease, such as, but not limited to, any of the targets listed in Table 2, such as, but not limited to, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and the other antigen is typically a stimulatory or inhibitory receptor present on the surface of a T cell, natural killer (NK) cell, myeloid monocyte, macrophage, and / or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of a T cell or NK cell; Examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also known as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments, the antigen is an inhibitory receptor present on the surface of a T cell; Examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIR. The antibody domain that imparts specificity to a T cell surface antigen may also be substituted with a ligand or ligand domain that binds to a T cell receptor, NK cell receptor, macrophage receptor, and / or other immune effector cell receptors (such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9).

[0334] One embodiment of the present disclosure is a multi-specific activatable antibody, which can be activated in a cancer microenvironment and includes antibodies (such as IgG or scFv) for tumor targets and agonist antibodies (such as IgG or scFv) for costimulatory receptors expressed on the surface of activated T cells or NK cells, wherein at least one of the cancer target antibodies and / or agonist antibodies is masked. Examples of costimulatory receptors include but are not limited to CD27, CD137, GITR, HVEM, NKG2D and OX40. In this embodiment, once activated by tumor-associated proteases, multi-specific activatable antibodies will effectively cross-link and activate the costimulatory receptors expressed by T cells or NK cells in a tumor-dependent manner to enhance the activity of T cells that respond to any tumor antigens through their endogenous T cell antigens or NK activation receptors. The activation-dependent properties of these T cells or NK cell costimulatory receptors will focus the activity of the activated multi-specific activatable antibodies on tumor-specific T cells without activating all T cells that are not dependent on their antigenic specificity. In one embodiment, at least the co-stimulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of autoreactive T cells that may be present in tissues that also express the antigen recognized by the antibody against the tumor target in the multispecific activatable antibody but whose activity is limited due to the lack of co-receptor engagement.

[0335] One embodiment of the present disclosure is a multi-specific activatable antibody that can be activated in a disease characterized by T cell overstimulation (such as, but not limited to, an autoimmune disease or an inflammatory disease microenvironment). Such a multi-specific activatable antibody includes antibodies for targets comprising surface antigens expressed in tissues targeted by T cells in autoimmune or inflammatory diseases, such as IgG or scFv; and antibodies for inhibitory receptors expressed on the surface of T cells or NK cells, such as IgG or scFv, wherein at least one of the disease tissue target antibodies and / or T cell inhibitory receptor antibodies is masked. Examples of inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed 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, when localized in tissues under autoimmune attack or inflammation, the multispecific activatable antibodies are activated and co-engage with T cell or NK cell inhibitory receptors to inhibit the activity of autoreactive T cells that respond to any disease tissue-targeted antigen through their endogenous TCR or activating receptors. In one embodiment, at least one or more antibodies are masked to prevent inhibition of T cell responses in non-disease tissues that may also express the target antigen.

[0336] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-CD3ε (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the targeting antibody or antigen-binding portion thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the 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 coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0337] 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 portion thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0338] 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 portion thereof is masked. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking portion (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking portion (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target. In some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting antibody IgG or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0339] In some embodiments, the T cell engaging multispecifically activatable antibody comprises an anti-CD3ε (CD3ε) scFv derived from OKT3, 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 coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, 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 coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0340] In some embodiments, the T cell-engaging multispecifically 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 portion thereof is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0341] In some embodiments, the T cell-engaging multispecifically 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 portion thereof is masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CD3ε. In some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, wherein the second antibody or fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target. In some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody IgG or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second cancer-associated target.

[0342] In some embodiments, the T cell engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the anti-CTLA-4 scFv is masked. In some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CTLA-4, wherein AB1 is attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to CTLA-4. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the 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 coupling 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, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0343] 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 coupling of MM1 reduces the ability of AB1 to bind to CTLA-4. In some embodiments, the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, which comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is attached to a masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target. In some embodiments, the 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 coupling of MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting antibody IgG 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 coupling of MM2 reduces the ability of AB2 to bind the second target.

[0344] In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody comprises at least a first antibody or antigen-binding fragment thereof that binds to a first target and / or a first epitope and a second antibody or antigen-binding fragment thereof that binds to a second target and / or a second epitope. In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody binds to two or more different targets. In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody binds to two or more different epitopes on the same target. In some embodiments, the multi-antigen targeting antibody and / or the multi-antigen targeting activatable antibody binds to a combination of two or more different targets and two or more different epitopes on the same target.

[0345] In some embodiments, a multispecific activatable antibody comprising an IgG has a masked IgG variable domain. In some embodiments, a multispecific activatable antibody comprising an scFv has a masked scFv domain. In some embodiments, a multispecific activatable antibody has an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking portion. In some embodiments, a multispecific activatable antibody has an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking portion. In some embodiments, a multispecific activatable antibody has an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking portion, and at least one of the scFv domains is coupled to a masking portion. In some embodiments, a multispecific activatable antibody has an IgG variable domain and an scFv domain, wherein each of the IgG variable domain and the scFv domain is coupled to its own masking portion. In some embodiments, one antibody domain of the multispecific activatable antibody is specific for a target antigen, and another antibody domain is specific for a T cell surface antigen. In some embodiments, one antibody domain of a multispecifically activatable antibody is specific for a target antigen, and another antibody domain is specific for another target antigen. In some embodiments, one antibody domain of a multispecifically activatable antibody is specific for an epitope of a target antigen, and another antibody domain is specific for another epitope of the target antigen.

[0346] In a multi-specific activatable antibody, the scFv may be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, the carboxyl terminus of the light chain of an IgG activatable antibody, or the carboxyl terminus of the heavy and light chains of an IgG activatable antibody. In a multi-specific activatable antibody, the scFv may be fused to the amino terminus of the heavy chain of an IgG activatable antibody, the amino terminus of the light chain of an IgG activatable antibody, or the amino terminus of the heavy and light chains of an IgG activatable antibody. In a multi-specific activatable antibody, the scFv 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, the masking portion (MM) connected to the CM1-CM2 substrate is attached to the antigen-binding domain of IgG and masks it. In some embodiments, the masking portion (MM) connected to the CM1-CM2 substrate is attached to the antigen-binding domain of at least one scFv and masks it. In some embodiments, the masking moiety (MM) linked to the CM1-CM2 substrate is attached to and masks the antigen binding domain of the IgG, and the masking moiety (MM) linked to the CM1-CM2 substrate is attached to and masks the antigen binding domain of at least one scFv.

[0347] The present disclosure provides examples of multispecific activatable antibody structures, which include, but are not limited to, the following: (VL-CL)2: (VH-CH1-CH2-CH3-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2; (VL-CL)2: (VH-CH1-CH2-CH3-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2; (MM-LP1-CM1-CM2 substrate-LP2-VL-CL)2: (VH-CH1-CH2-CH3-L4-VH*-L3-VL*)2; (MM-LP1-CM1-CM2 substrate-LP2-VL- CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*)2;(VL-CL)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(VL-CL)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(MM-LP1-CM1-CM2 substrate-LP2-VL-CL)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(MM-LP1-CM1-CM2 substrate-L P2-VL-CL)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(VH-CH1-CH2-CH3)2;(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2;(MM-LP1-CM1-C M2 substrate-LP2-VH*-L3-VL*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VL*)2:(MM-LP1-CM1-CM2 substrate-LP2-VL*-L3- VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*)2: (MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*)2: (MM-LP1-CM1-CM2 substrate-LP2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*)2: (MM-LP1-CM1-CM2 substrate-LP2-VH*-L3-VL*-L4-VH-CH1-CH2- CH3)2; (VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM)2: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-LP2-CM1-CM2 substrate-LP1-MM )2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; or(VL-CL-L4- VL*-L3-VH*-LP2-CM1-CM2 substrate-LP1-MM)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2, wherein: VL and VH represent the light and heavy variable domains with a first specificity contained in IgG; VL* and VH* represent the variable domains with a second specificity contained in scFv; LP1 is a linker peptide connecting the masking portion (MM) and the CM1-CM2 substrate; LP2 is a linker peptide connecting the CM1-CM2 substrate and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody with the first specificity to the antibody with the second specificity; CL is a light chain constant domain;CH1, CH2, and CH3 are heavy chain constant domains. The first specificity and the second specificity can be directed against any antigen or epitope.

[0348] 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 2, such as, but not limited to, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and the other antigen is typically a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of a T cell, natural killer (NK) cell, myeloid monocyte, macrophage, and / or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, (也称为TNFRSF9) , CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3 or VISTA. The antibody domain that gives T cell surface antigen specificity can also be bound to T cell receptor, NK cell receptor, macrophage receptor and / or other immune effector cell receptor (such as but not limited to B7-1, B7-2, B7H3, PD-L1, PD-L2 or TNFSF9) ligand or ligand domain replacement. In some embodiments of multi-antigen targeted activatable antibodies, one antigen is selected from the group of targets listed in Table 2, and another antigen is selected from the group of targets listed in Table 2.

[0349] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which has specificity for binding to EGFR. In some embodiments, the targeting antibody is C225, which has specificity for binding to EGFR. In some embodiments, the targeting antibody is C225v4, which has specificity for binding to EGFR. In some embodiments, the targeting antibody is C225v6, which has specificity for binding to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which has specificity for binding to human and mouse Jagged1 and Jagged2. In some embodiments, the targeting antibody is 4D11v2, which has specificity for binding to human and mouse Jagged1 and Jagged2.

[0350] In some embodiments, the targeting antibody may be in the form of an activatable antibody. In some embodiments, one or more scFvs may be in the form of a Pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0351] In some embodiments, the scFv is specific for binding to CD3ε and is or is derived from an antibody or fragment thereof that binds to CD3ε, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, the scFv is specific for binding to CTLA-4 (also referred to herein as CTLA and CTLA4).

[0352] In some embodiments, the anti-CTLA-4 scFv comprises the following amino acid sequence:

[0353]

[0354] 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.

[0355] In some embodiments, the anti-CD3ε scFv comprises the following amino acid sequence:

[0356]

[0357] 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.

[0358] In some embodiments, the scFv is specific for binding to one or more T cells, one or more NK cells, and / or one or more macrophages. In some embodiments, the scFv is specific for binding to a target selected from the group consisting of: B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.

[0359] In some embodiments, the multispecific, activatable antibody further comprises an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the multispecific, activatable antibody via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker comprising at least one CM1-CM2 substrate sequence. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid-damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA-damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is riocetin or a derivative thereof. In some embodiments, the agent is riocetin E or a derivative thereof. In some embodiments, the agent is monomethylriocetin E (MMAE). In some embodiments, the agent is monomethylriocetin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0360] In some embodiments, the multispecifically activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0361] In some embodiments, a multispecifically activatable antibody naturally contains one or more disulfide bonds. In some embodiments, a multispecifically activatable antibody can be engineered to contain one or more disulfide bonds.

[0362] The present disclosure also provides an isolated nucleic acid molecule encoding a multispecific activatable antibody described herein, and a vector comprising such an isolated nucleic acid sequence. The present disclosure provides a method for producing a multispecific activatable antibody by culturing a cell under conditions that result in expression of the activatable antibody, wherein the cell comprises such a nucleic acid molecule. In some embodiments, the cell comprises such a vector.

[0363] The present disclosure also provides a method for producing the multispecific, activatable antibody of the present disclosure, which is performed by the following steps: (a) culturing cells 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.

[0364] The present disclosure also provides multispecific activatable antibodies and / or multispecific activatable antibody compositions comprising at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a first target or a first epitope and a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target or a second epitope, wherein at least AB1 is coupled to or otherwise attached to a masking moiety (MM1) such that coupling of MM1 reduces the ability of AB1 to bind to its target. In some embodiments, MM1 is coupled to AB1 via a CM1-CM2 substrate of a MMP and SP, wherein at least one of the MMP and SP is co-localized with the target of AB1 at a therapeutic site or a diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, activated at the intended therapeutic and / or diagnostic site, but not activated in normal tissue (i.e., healthy tissue), and upon activation, exhibit at least comparable target binding to AB1 as the corresponding unmodified multispecific antibody.

[0365] In some embodiments, the multispecifically activatable antibody comprises a linker peptide between MM1 and the CM1-CM2 substrate.

[0366] In some embodiments, the multispecifically activatable antibody comprises a linker peptide between the CM1-CM2 substrate and AB1.

[0367] 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.

[0368] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of: (GS) n 、(GGS) n 、(GSGGS) n (SEQ ID NO: 381) and (GGGS) n (SEQ ID NO: 382), wherein n is an integer of at least 1. 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).

[0369] In some embodiments, the activatable antibody comprises a connecting peptide (LP') between CM1 and CM2.

[0370] In some embodiments, the activatable antibody comprises a first connecting peptide (LP1), a second connecting peptide (LP2), and a connecting peptide (LP') between CM1 and CM2, and at least a portion of the multispecific activatable antibody 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.

[0371] In some embodiments, LP' is GG. In some embodiments, LP' is GGSGGS (SEQ ID NO: 218).

[0372] In some embodiments, the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a first target or a first epitope and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds to a second target or a second epitope. In some embodiments, each AB in the multispecific activatable antibody is independently selected from the group consisting of: a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., mouse or rat) antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0373] In some embodiments, each AB in the multispecifically activatable antibody binds to its corresponding target or epitope with a dissociation constant of about 100 nM or less.

[0374] In some embodiments, the dissociation constant of the MM1 binding to its corresponding AB is greater than the dissociation constant of the AB to its corresponding target or epitope.

[0375] In some embodiments, the dissociation constant of the MM1 binding to its corresponding AB is no greater than the dissociation constant of the AB to its corresponding target or epitope.

[0376] In some embodiments, when the multispecifically activatable antibody is in a cleaved state, MM1 does not interfere with or compete for binding of its corresponding AB to the corresponding target or epitope.

[0377] In some embodiments, MM1 is a polypeptide of about 2 to 40 amino acids in length. In some embodiments, each of the MMs in the multispecifically activatable antibody is a polypeptide of no more than 40 amino acids in length.

[0378] In some embodiments, the polypeptide sequence of MM1 differs from the sequence of the target of the corresponding AB.

[0379] In some embodiments, the polypeptide sequence of MM1 is no more than 50% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of MM1 is no more than 25% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of MM1 is no more than 10% identical to any natural binding partner of the corresponding AB.

[0380] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K) of the AB coupled to MM1 for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to MM1 against its corresponding target or epitope d At least 20 times larger.

[0381] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K) of the AB coupled to MM1 for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to MM1 against its corresponding target or epitope d At least 40 times larger.

[0382] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K) of the AB coupled to MM1 for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to MM1 against its corresponding target or epitope d At least 100 times larger.

[0383] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K) of the AB coupled to MM1 for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to MM1 against its corresponding target or epitope d At least 1000 times larger.

[0384] In some embodiments, the coupling of MM1 reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K) of the AB coupled to MM1 for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to MM1 against its corresponding target or epitope d At least 10,000 times larger.

[0385] In some embodiments, MM1 is an amino acid sequence selected from the MMs disclosed herein.

[0386] 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') coupled to AB2, wherein CM' is a CM1-CM2 substrate or a polypeptide that serves as a substrate for a second protease. In some embodiments, CM' is a polypeptide of no more than 15 amino acids in length. In some embodiments, CM' is a CM1-CM2 substrate, wherein CM1 and CM2 in the CM1-CM2 substrate are each independently no more than 15 amino acids in length.

[0387] In some embodiments, the MMP protease, SP protease, and / or second protease are co-localized with the second target or epitope in a tissue, and when the multispecifically activatable antibody is exposed to the MMP protease, SP protease, and / or second protease, the MMP protease, SP protease, and / or second protease cleaves the CM in the multispecifically activatable antibody. In some embodiments, the MMP protease, SP protease, and / or second protease are co-localized with the first target or epitope and the 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, SP protease, and / or second protease are not the same MMP protease and are not the same SP protease. In some embodiments, the CM1-CM2 substrate and CM′ are different substrates for the same MMP protease and the same SP protease. In some embodiments, the protease that cleaves CM′ is selected from the group consisting of those shown in Table 1.

[0388] In some embodiments, the MMs (eg, MM1 and at least MM2) in the multispecifically activatable antibody each bind to its corresponding AB with a dissociation constant that is greater than the dissociation constant between the AB and its corresponding target or epitope.

[0389] In some embodiments, the MMs in the multispecifically activatable antibody each bind to its corresponding AB with a dissociation constant that is no greater than the dissociation constant of the AB and its corresponding target or epitope.

[0390] In some embodiments, when the multispecifically activatable antibody is in the cleaved state, each MM in the multispecifically activatable antibody does not interfere with or compete for binding of its corresponding AB to the corresponding target or epitope.

[0391] In some embodiments, each MM in a multispecifically activatable antibody is a polypeptide of about 2 to 40 amino acids in length. In some embodiments, each MM in a multispecifically activatable antibody is a polypeptide of no more than 40 amino acids in length.

[0392] In some embodiments, the polypeptide sequence of each of the MMs in the multispecifically activatable antibody differs from the sequence of the target of the corresponding AB.

[0393] In some embodiments, the polypeptide sequence of each MM in a multispecifically activatable antibody is no more than 50% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of each MM in a multispecifically activatable antibody is no more than 25% identical to any natural binding partner of the corresponding AB. In some embodiments, the polypeptide sequence of each MM in a multispecifically activatable antibody is no more than 10% identical to any natural binding partner of the corresponding AB.

[0394] In some embodiments, the respective coupling of the MM reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K) of the AB coupled to the MM for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to the MM against its corresponding target or epitope d At least 20 times larger.

[0395] In some embodiments, the respective coupling of the MM reduces the ability of the corresponding AB to bind to its target or epitope, such that the dissociation constant (K) of the AB coupled to the MM for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to the MM against its corresponding target or epitope d At least 40 times larger.

[0396] In some embodiments, the respective coupling of the MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB coupled to the MM for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to the MM against its corresponding target or epitope d At least 100 times larger.

[0397] In some embodiments, the respective coupling of the MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB coupled to the MM for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to the MM against its corresponding target or epitope d At least 1000 times larger.

[0398] In some embodiments, the respective coupling of the MM reduces the ability of the corresponding AB to bind its target or epitope, such that the dissociation constant (K) of the AB coupled to the MM for its corresponding target or epitope is d ) is greater than the K of the AB not coupled to the MM against its corresponding target or epitope d At least 10,000 times larger.

[0399] In some embodiments, each MM is an amino acid sequence selected from the MMs disclosed herein.

[0400] In some embodiments, a protease that cleaves the CM1-CM2 substrate sequence is co-localized with the target of AB1 in the multispecific, activatable antibody in a tissue, and when the multispecific, activatable antibody is exposed to the protease, the MMP protease and / or the SP protease (i.e., at least one of the MMP protease and the SP protease) cleaves the CM1-CM2 substrate in the multispecific, activatable antibody.

[0401] In some embodiments, the multispecifically activatable antibody comprises more than one CM1-CM2 substrate sequence, and an MMP protease and / or SP protease that cleaves at least one CM1-CM2 substrate sequence is co-localized with the target of at least one AB region in the multispecifically activatable antibody in a tissue, and when the multispecifically activatable antibody is exposed to the protease, the MMP protease and / or SP protease cleaves the CM1-CM2 substrate in the multispecifically activatable antibody.

[0402] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least two-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0403] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least three-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0404] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least four times greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0405] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least five times greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0406] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least ten-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0407] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least 20-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0408] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least 40-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0409] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least 50-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0410] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least 100-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0411] In some embodiments, each CM1-CM2 substrate is positioned in the multispecifically activatable antibody such that, in the uncleaved state, the multispecifically activatable antibody has reduced binding to a target of one AB region with a dissociation constant at least 200-fold greater than the dissociation constant for binding of the unmodified AB to its target, while in the cleaved state, the AB binds to its target.

[0412] The present disclosure also provides compositions and methods comprising multispecific activatable antibodies, the multispecific activatable antibodies comprising at least a first antibody or antibody fragment (AB1) that specifically binds to a target and a second antibody or antibody fragment (AB2), wherein at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that reduces the ability of AB1 to bind to its target. In some embodiments, each AB is coupled to an MM that reduces the ability of its corresponding AB to bind to each target. For example, in embodiments of bispecific activatable antibodies, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind to its target, and AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind to its target. In some embodiments, a multispecifically activatable antibody comprises more than two AB regions; in such embodiments, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target, AB3 is coupled to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target, and so on for each AB in the multispecifically activatable antibody.

[0413] In some embodiments, the multispecific, activatable antibody further comprises a CM1-CM2 substrate that is a substrate for an MMP protease and an SP protease, wherein 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) that specifically binds to a target and a second antibody or antibody fragment (AB2), wherein at least the first AB in the multispecific, activatable antibody is coupled to a masking moiety (MM1) that reduces the ability of AB1 to bind to its target via a first CM1-CM2 substrate. In some bispecific, activatable antibody embodiments, AB1 is coupled to MM1 via a first CM1-CM2 substrate, and AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind to its target via a second CM1-CM2 substrate. In some embodiments, the multispecifically activatable antibody comprises more than two AB regions; in some of these embodiments, AB1 is coupled to MM1 via a first CM1-CM2 substrate, AB2 is coupled to MM2 via a second CM1-CM2 substrate, and AB3 is coupled to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target via a third CM1-CM2 substrate, and so on for each AB in the multispecifically activatable antibody.

[0414] Activatable antibodies with a non-binding steric portion or a binding partner for a non-binding steric portion

[0415] The present disclosure also provides activatable antibodies comprising a non-binding space portion (NB) or a binding partner (BP) of the non-binding space portion, wherein the BP recruits or otherwise attracts the NB to the activatable antibody. The activatable antibodies provided herein include, for example, activatable antibodies comprising a non-binding space portion (NB), a CM1-CM2 substrate, and an antibody or antibody fragment (AB) that binds to a target; activatable antibodies comprising a binding partner (NB) of the non-binding space portion, a CM1-CM2 substrate, and an AB; and activatable antibodies comprising a BP that has recruited a NB, a CM1-CM2 substrate, and an AB that binds to a target. Activatable antibodies in which the NB is covalently linked to the CM1-CM2 substrate and AB of the activatable antibody or associated by interacting with a BP covalently linked to the CM1-CM2 substrate and AB of the activatable antibody are referred to herein as "activatable antibodies containing NBs." Activatable or switchable means that when the activatable antibody is in an inhibited, masked or uncleaved state (i.e., a first conformation), the activatable antibody exhibits a first level of binding to the target, and when the activatable antibody is in an uninhibited, unmasked and / or cleaved state (i.e., a second conformation, i.e., an activated antibody), the activatable antibody exhibits a second level of binding to the target, wherein the second target binding level is greater than the first target binding level. Activatable antibody compositions can exhibit increased bioavailability and more favorable biodistribution compared to conventional antibody therapies.

[0416] In some embodiments, if the AB is not masked or otherwise inhibited from binding to non-therapeutic and / or non-diagnostic sites, the activatable antibody provides reduced toxicity and / or adverse side effects that would otherwise result from binding to such sites.

[0417] In one embodiment, the activatable antibody comprises a non-binding spacer portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein the NB is a polypeptide that does not specifically bind to the AB; the CM1-CM2 substrate is a polypeptide that comprises a substrate (S) for an enzyme; the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target; and the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme. As used herein and throughout, the term polypeptide refers to any polypeptide comprising at least two amino acid residues, including larger polypeptides, full-length proteins, and fragments thereof, and the term polypeptide is not limited to single-chain polypeptides and may include multi-unit (e.g., multi-chain) polypeptides. In the case where the polypeptide is short in length (e.g., less than 50 amino acids in total), the terms peptide and polypeptide are used interchangeably herein, and in the case where the polypeptide is long in length (e.g., 50 amino acids or greater), the terms polypeptide and protein are used interchangeably herein.

[0418] In one embodiment, the activatable antibody comprises a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide of up to 50 amino acids in length that comprises a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with binding of the AB to the target, and in the cleaved state, the NB does not interfere with binding of the AB to the target; and (iv) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. For example, the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate each independently have a length of up to 15 amino acids.

[0419] In one embodiment, the activatable antibody comprises a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that, in the uncleaved state, the NB interferes with binding of the AB to the target, and in the cleaved state, the NB does not interfere with binding of the AB to the target; (iv) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (v) the activatable antibody has the following structural arrangement from N-terminus to C-terminus in the uncleaved state: NB-CM1-CM2 substrate-AB or AB-CM1-CM2 substrate-NB.

[0420] In one embodiment, the activatable antibody comprises a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein (i) the NB comprises a polypeptide that does not specifically bind to the AB; (ii) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) for an enzyme; (iii) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with binding of the AB to the target, and in the cleaved state, the NB does not interfere with binding of the AB to the target, and wherein the NB in ​​the uncleaved activatable antibody reduces the ability of the AB to bind to the target by at least 50%, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, compared to the ability of the cleaved AB to bind to the target; and (iv) the NB does not inhibit cleavage of the CM1-CM2 substrate by the enzyme. For example, a reduction in the ability of the AB to bind to a target is determined using an assay as described herein or an in vitro target displacement assay (e.g., such as the assays described in PCT Publication Nos. WO 2009 / 025846 and WO 2010 / 081173).

[0421] In one embodiment, the activatable antibody comprises a binding partner (BP) of a non-binding steric portion (NB); a CM1-CM2 substrate; and an antibody or antibody fragment (AB) that specifically binds to a target, wherein the BP is a polypeptide that binds to the NB when exposed to the NB; the NB does not specifically bind to the AB; the CM1-CM2 substrate is a polypeptide comprising a substrate (S) for an enzyme; the CM1-CM2 substrate is positioned such that, in the uncleaved state in the presence of the NB, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target and the BP does not interfere with the binding of the AB to the target; and the NB and BP do not inhibit 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.

[0422] In some examples of any of these activatable antibody embodiments, the activatable antibody is formulated as a composition. In some of these embodiments, the composition further comprises a NB, wherein the NB is co-formulated with an activatable antibody comprising a BP, a CM1-CM2 substrate, and an AB. In some 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.

[0423] 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.

[0424] In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a substrate (S) of a protease. In some examples of any of these activatable antibody embodiments, the protease is co-localized with the tissue, and when the activatable antibody is exposed to the protease, the protease cleaves the CM1-CM2 substrate in the activatable antibody. In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide having a length of up to 50 amino acids. In some examples of any of these activatable antibody embodiments, the CM1-CM2 substrate is a polypeptide comprising a substrate (S) having a length of up to 15 amino acids (e.g., 3 amino acids long, 4 amino acids long, 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, or 15 amino acids long).

[0425] In some examples of any of these activatable antibody embodiments, the activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: NB-CM1-CM2 substrate-AB, AB-CM1-CM2 substrate-NB, BP-CM1-CM2 substrate-AB, or AB-CM1-CM2 substrate-BP. In embodiments where the activatable antibody comprises a BP and a corresponding NB is present for the activatable antibody, the activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: NB:BP-CM1-CM2-AB, NB:BP-CM2-CM1-AB, AB-CM1-CM2-BP:NB, or AB-CM2-CM1-BP:NB, where ":" indicates an interaction, e.g., binding, between the NB and the BP.

[0426] In some examples of any of these activatable antibody embodiments, the activatable antibody comprises an antibody or antigen-binding fragment thereof that specifically binds to a given target and is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such a target-binding antibody or immunologically active fragment thereof is a mouse antibody, other rodent antibodies, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0427] In some examples of any of these activatable antibody embodiments, the activatable antibody comprises a variable heavy chain region comprising an amino acid sequence set forth herein and a variable light chain region comprising an amino acid sequence set forth herein. In some embodiments, the activatable antibody comprises a variable heavy chain region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence set forth herein and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence set forth herein.

[0428] In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an anti-tumor agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the agent is dolastatin. In some embodiments, the agent is riocetin or a derivative thereof. In some embodiments, the agent is riocetin E or a derivative thereof. In some embodiments, the agent is monomethyl riocetin E (MMAE). In some embodiments, the agent is monomethyl riocetin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0429] In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0430] In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises a spacer. In some examples of any of these activatable antibody embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some examples of any of these activatable antibody embodiments, the spacer is directly attached to the MM of the activatable antibody.

[0431] In some embodiments, the serum half-life of the activatable antibody is longer than the serum half-life of the corresponding antibody; for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 15 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 12 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 11 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 10 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 9 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 8 days. In some embodiments, when administered to an organism, the serum half-life of the activatable antibody is at least 7 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 6 days. In some examples of any of these activatable antibody embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 5 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 4 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 3 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 2 days. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 24 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 20 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 18 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 16 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 14 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 12 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 10 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 8 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 6 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 4 hours. In some embodiments, the serum half-life of the activatable antibody when administered to an organism is at least 3 hours.

[0432] The present disclosure also provides an isolated nucleic acid molecule encoding any of these activatable antibodies, and vectors comprising these isolated nucleic acid sequences. The present disclosure provides a method for producing an activatable antibody by culturing cells under conditions that result in expression of the activatable antibody, wherein the cells comprise such a nucleic acid sequence. In some embodiments, the cells comprise such a vector.

[0433] The dissociation constant (K) of the activatable antibody containing NB against the target d ) is greater than the K of the AB against the target not associated with NB or NB:BP d The dissociation constant (K) of the activatable antibody containing NB against the target d ) is greater than the K of the parental AB against the target d For example, activatable antibodies containing NBs are directed against the target's K d Compared with the K of AB against target not associated with NB or NB:BP d or the K of the parental 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, or between 5-10 times, 10-100 times, 10-1,000 times, 10-10,000 times, 10-100,000 times, 10-1,000,000 times, 10-10,000,000 times, 100-1,000 times, 1 00-10,000 times, 100-100,000 times, 100-1,000,000 times, 100-10,000,000 times, 1,000-10,000 times, 1,000-10,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 10,000-10,000,000 times, 100,000-1,000,000 times or between 100,000-10,000,000 times. In contrast, the binding affinity of the activatable antibody containing a NB for the target is lower than the binding affinity of an AB not associated with a NB or NB:BP for the target or lower than the binding affinity of the parental AB for the target. For example, the binding affinity of the activatable antibody containing a NB for a target is lower than the binding affinity of an AB not associated with a NB or NB:BP for the target, or is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 fold or more, or is between 5-10 fold, 10-100 fold, 10-1,000 fold, 10-10,000 fold, 10-100,000 fold, 10-1 ... ,000 times, 10-10,000,000 times, 100-1,000 times, 100-10,000 times, 100-1,000,000 times, 100-10,000,000 times, 1,000-10,000,000 times, 1,000-10,000 times, 1,000-10,000,000 times, 1,000-1,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 10,000-10,000,000 times, 100,000-1,000,000 times or between 100,000-10,000,000 times.

[0434] When the activatable antibody containing the NB is in the presence of the target, specific binding of the AB to the target is reduced or inhibited compared to specific binding of the AB not associated with the NB or NB:BP. When the activatable antibody containing the NB is in the presence of the target, specific binding of the AB to the target is reduced or inhibited compared to specific binding of the parental AB to the target. The ability of the activatable antibody containing a NB to bind to a target is reduced, e.g., by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% compared to binding of an AB not associated with a NB or NB:BP, or binding of a parental AB to the target, when measured in vitro and / or in vivo for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or longer.

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

[0436] In some examples of any of these activatable antibody embodiments, the activatable antibody comprises an agent conjugated to the AB to produce an activatable antibody conjugate. In some embodiments of the activatable antibody conjugate, the agent is a therapeutic agent. In some embodiments, the agent is a diagnostic agent. In some embodiments, the agent is a detectable marker. In some embodiments of the activatable antibody conjugate, the agent is an anti-tumor agent. In some embodiments of the activatable antibody conjugate, the agent is a toxin or a fragment thereof. In some embodiments of the activatable antibody conjugate, the agent is conjugated to the AB via a linker. In some embodiments of the activatable antibody conjugate, the linker is a cleavable linker. In some embodiments, the agent is conjugated to the AB via a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalating agent, or other DNA damaging agent. In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is dolastatin. In some embodiments, the agent is riocetin or a derivative thereof. In some embodiments, the agent is riocetin E or a derivative thereof. In some embodiments, the agent is monomethyloxetine E (MMAE). In some embodiments, the agent is monomethyloxetine D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is duocarmycin or a derivative thereof. In some embodiments, the agent is calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.

[0437] 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 contain two Abs that can bind to the same or different targets. In specific embodiments, the dual-target activatable antibody contains a bispecific antibody or antibody fragment.

[0438] Design a dual-target binding activatable antibody so that it has a CM1-CM2 substrate that can be cleaved by a cleavage agent, and the cleavage agent is co-localized in the target tissue with one or two targets that can bind to the AB of the activatable antibody. Dual-target binding activatable antibodies with more than one AB for the same or different targets can be designed so that they have more than one CM1-CM2 substrate, wherein the first CM1-CM2 substrate can be cleaved by a cleavage agent in the first target tissue, and wherein the second CM1-CM2 substrate can be cleaved by a cleavage agent in the second target tissue, wherein one or more targets are bound to the AB of the activatable antibody. In one embodiment, the first target tissue and the second target tissue are spatially separated, for example, at different locations in the organism. In one embodiment, the first target tissue and the second target tissue are the same tissue separated in time, 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 late-stage tumor.

[0439] The present disclosure also provides nucleic acid molecules encoding the activatable antibodies described herein. The present disclosure also provides vectors comprising these nucleic acids. The activatable antibodies described herein are produced by culturing cells under conditions that result in expression of the activatable antibodies, wherein the cells comprise these nucleic acid molecules or vectors.

[0440] The present disclosure also provides a method for producing an activatable antibody. In one embodiment, the method comprises the following steps: (a) culturing a cell comprising a nucleic acid construct encoding the activatable antibody under conditions that result in expression of the activatable antibody, wherein the activatable antibody comprises (i) a non-binding steric portion (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) of an enzyme; (3) the CM1-CM2 substrate is positioned such that in the uncleaved state, the NB interferes with the binding of the AB to the target, and in the cleaved state, the NB does not interfere with the binding of the AB to the target; and (4) the NB does not inhibit the cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody.

[0441] In some embodiments, the method comprises the following steps: (a) culturing a cell comprising a nucleic acid construct encoding the activatable antibody under conditions that result in expression of the activatable antibody, wherein the activatable antibody comprises (i) a binding partner (BP) of a non-binding steric portion (NB); (ii) a CM1-CM2 substrate; and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds to a target, wherein (1) the NB does not specifically bind to the AB; (2) the CM1-CM2 substrate is a polypeptide comprising a substrate (S) for an enzyme; (3) the CM1-CM2 substrate is positioned such that in the uncleaved state in which the NB is present, the NB interferes with binding of the AB to the target, and in the 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; and (4) the NB and the BP do not inhibit cleavage of the CM1-CM2 substrate by the enzyme; and (b) recovering the activatable antibody. In some examples of this embodiment, the BP of the activatable antibody is bound to the NB.

[0442] Uses of activatable antibodies and conjugated activatable antibodies

[0443] It will be understood that administration of the therapeutic entities according to the present disclosure will be administered with suitable carriers, excipients, and other agents incorporated into formulations to provide improved transfer, delivery, tolerability, and the like. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 of Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as Lipofectin) and lipofectin. TM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. Any of the above mixtures may be suitable for treatments and therapies according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerable 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 form...

Claims

1. An isolated polypeptide comprising a tandem substrate, wherein the tandem substrate comprises at least a first cleavable portion CM1 which is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable portion CM2 which is a substrate for at least one serine protease (SP), wherein CM1 comprises the amino acid sequence ALAHGLF as shown in SEQ ID NO: 1 and CM2 comprises the amino acid sequence APRS as shown in SEQ ID NO: 131, and wherein the N-terminus to C-terminus of the tandem substrates are arranged as CM1-CM2 or CM2-CM1; Wherein CM1 and CM2 are directly linked to each other so that the tandem substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 and 32.

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

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

4. The isolated polypeptide of any one of claims 1 to 3, wherein the tandem substrate comprises the amino acid sequence shown in SEQ ID NO:

31.

5. The isolated polypeptide of any one of claims 1 to 3, wherein the tandem substrate comprises the amino acid sequence shown in SEQ ID NO:

32.

6. An isolated polypeptide comprising: Antibody or antigen-binding fragment thereof AB; A tandem substrate comprising at least a first cleavable portion CM1 that is a substrate for at least one matrix metalloproteinase MMP, and at least a second cleavable moiety CM2 which is a substrate for at least one serine protease SP, wherein CM1 comprises the amino acid sequence ALAHGLF as shown in SEQ ID NO: 1 and CM2 comprises the amino acid sequence APRS as shown in SEQ ID NO: 131, and wherein the N-terminus to C-terminus of the tandem substrates are arranged as CM1-CM2 or CM2-CM1; Wherein CM1 and CM2 are directly linked to each other so that the tandem substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 and 32.

7. The isolated polypeptide of claim 6, wherein the CM2 comprises a substrate for a SP enzyme selected from the group consisting of urokinase, matriptase, and neutrophil elastase.

8. The isolated polypeptide of claim 6, wherein the MMP is MMP2, MMP9 or MMP14.

9. The isolated polypeptide of any one of claims 6 to 8, wherein the tandem substrate comprises the amino acid sequence shown in SEQ ID NO:

31.

10. The isolated polypeptide of any one of claims 6 to 8, wherein the tandem substrate comprises the amino acid sequence shown in SEQ ID NO:

32.

11. The isolated polypeptide of any one of claims 6 to 8, wherein at least one of the MMP and SP is co-localized with a target in a tissue.

12. The isolated polypeptide of any one of claims 6 to 8, wherein the antigen binding fragment is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

13. The isolated polypeptide of any one of claims 6 to 8, wherein the AB is linked to the CM1.

14. The isolated polypeptide of claim 13, wherein the AB is directly linked to the CM1. The isolated polypeptide of claim 13 , wherein the AB is linked to the CM1 via a linker peptide.

16. The isolated polypeptide of any one of claims 6 to 8, wherein the AB is linked to CM2.

17. The isolated polypeptide of claim 16, wherein the AB is directly linked to CM2.

18. The isolated polypeptide of claim 16, wherein the AB is linked to the CM2 via a linker peptide.

19. The isolated polypeptide according to any one of claims 6 to 8, wherein the AB comprises a light chain variable region of an antibody or antigen-binding fragment thereof, and wherein the CM1 or the CM2 is connected to the N-terminus of the light chain variable region of the AB.

20. The isolated polypeptide according to any one of claims 6 to 8, wherein the AB comprises a heavy chain variable region of an antibody or antigen-binding fragment thereof, and wherein the CM1 or the CM2 is connected to the N-terminus of the heavy chain variable region of the AB.

21. The isolated polypeptide according to any one of claims 6 to 8, wherein the isolated polypeptide comprises a masking moiety MM.

22. The isolated polypeptide of claim 21, wherein the MM binds to the AB with a dissociation constant greater than the dissociation constant of the AB binding to the target.

23. The isolated polypeptide of claim 21, wherein the MM is a polypeptide of no more than 40 amino acids in length.

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

25. The isolated polypeptide of claim 24, wherein the isolated polypeptide comprises a connecting peptide between the MM and the CM1.

26. The isolated polypeptide of claim 24, wherein the isolated polypeptide comprises a connecting peptide between CM2 and the AB.

27. The isolated polypeptide of claim 24, wherein the isolated polypeptide comprises A connecting peptide between the MM and the CM1; and A connecting peptide between the CM2 and the AB.

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

29. The isolated polypeptide of claim 28, wherein the isolated polypeptide comprises a connecting peptide between the MM and the CM2.

30. The isolated polypeptide of claim 28, wherein the isolated polypeptide comprises a connecting peptide between CM1 and the AB.

31. The isolated polypeptide of claim 28, wherein the isolated polypeptide comprises A connecting peptide between the MM and the CM2; and A connecting peptide between the CM1 and the AB.

32. The isolated polypeptide of claim 21, wherein the AB comprises a light chain variable region of an antibody or antigen-binding fragment thereof, and wherein the CM1 or the CM2 is connected to the N-terminus of the light chain variable region of the AB.

33. The isolated polypeptide of claim 21, wherein the AB comprises a heavy chain variable region of an antibody or antigen-binding fragment thereof, and wherein the CM1 or the CM2 is connected to the N-terminus of the heavy chain variable region of the AB.

34. The isolated polypeptide of claim 21, wherein the isolated polypeptide comprises a first connecting peptide LP1 and a second connecting peptide LP2, and wherein the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus in an 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.

35. The isolated polypeptide of claim 34, wherein the two connecting peptides are different from each other.

36. The isolated polypeptide of claim 34 or 35, wherein each of LP1 and LP2 is a peptide of 1 to 20 amino acids in length.

37. The isolated polypeptide of claim 34 or 35, wherein the isolated polypeptide comprises a third connecting peptide LP' between CM1 and CM2.

38. An isolated polypeptide as described in claim 34 or 35, wherein LP1 or LP2 comprises the following amino acid sequence: GGSG as shown in SEQ ID NO:202, GGSGG as shown in SEQ ID NO:203, GGSSG as shown in SEQ ID NO:204, GSGGG as shown in SEQ ID NO:205, GGGSG as shown in SEQ ID NO:206, or GSSSG as shown in SEQ ID NO:

207.

39. The isolated polypeptide of claim 34 or 35, wherein LP1 comprises the following amino acid sequence: GSSGGSGGSGGSG as shown in SEQ ID NO:208, GSSGGSGGSGG as shown in SEQ ID NO:209, GSSGGSGGSGG as shown in SEQ ID NO:210, GSSGGSGGSGGS as shown in SEQ ID NO:211, GSSGGSGGSGGSGGGS as shown in SEQ ID NO:212, or GSSGGSGGSGS as shown in SEQ ID NO:213, GGGSSGGS as shown in SEQ ID NO:214, or GGGS as shown in SEQ ID NO:

215.

40. The isolated polypeptide of claim 34 or 35, wherein LP2 comprises the following amino acid sequence: GSS, GGS, GGGS as shown in SEQ ID NO:215, GSSGT as shown in SEQ ID NO:216, or GSSG as shown in SEQ ID NO:

217.

41. An isolated polypeptide as described in claim 34 or 35, wherein LP1 comprises the following amino acid sequence: GSSGGSGGSGGSG as shown in SEQ ID NO:208 or GGGSSGGS as shown in SEQ ID NO:214, and wherein LP2 comprises the following amino acid sequence: GGS or GGGS as shown in SEQ ID NO:

215.

42. The isolated polypeptide of claim 34 or 35, wherein the AB comprises a light chain variable region of an antibody or antigen-binding fragment thereof, and Wherein the LP2 is connected to the N-terminus of the light chain variable region of the AB.

43. The isolated polypeptide of claim 34, wherein the AB comprises a heavy chain variable region of an antibody or antigen-binding fragment thereof, and Wherein the LP2 is connected to the N-terminus of the heavy chain variable region of the AB.

44. The isolated polypeptide of claim 22, wherein the amino acid sequence of the MM is different from the amino acid sequence of the target and is no more than 10% identical to the amino acid sequence of the natural binding partner of the AB.

45. The isolated polypeptide of claim 22, wherein the MM does not interfere with or compete for the binding of the AB to the target in the cleaved state.

46. ​​The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the cleavability of MMP9 and MMP14 to the first cleavable moiety CM1 is each at least 80%.

47. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the cleavability of MMP9 and MMP14 to the first cleavable moiety CM1 is each at least 85%.

48. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the cleavage of MMP9 and MMP14 to the first cleavable moiety CM1 is each at least 90%.

49. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the in vivo stability of the first cleavable moiety CM1 is less than 25% activation.

50. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the cleavage of the tandem substrate by MMP9 and MMP14 is each at least 30%.

51. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the cleavability of the tandem substrate by MMP9 and MMP14 is each at least 50%.

52. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the cleavability of the tandem substrate by MMP9 and MMP14 is each at least 70%.

53. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the tandem substrate is each cleavable by at least 50% by MMP9 and MMP14, and the tandem substrate is cleavable by at least 50% by neutrophil elastase.

54. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the tandem substrate is each cleavable by at least 70% by MMP9 and MMP14, and the tandem substrate is cleavable by at least 70% by neutrophil elastase.

55. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the in vivo stability of the first cleavable moiety CM1 is less than 40% activation.

56. The isolated polypeptide of any one of claims 1 to 3 and 6 to 8, wherein the in vivo stability of the first cleavable moiety CM1 is less than 30% activation.

57. A conjugated polypeptide comprising the isolated polypeptide of any one of claims 6 to 56 conjugated to an agent, wherein the agent is selected from the group consisting of: a) toxins or fragments thereof; b) microtubule inhibitors; c) nucleic acid damaging agents; d) Dolastatin or its derivatives; e) riocetine or its derivatives; f) maytansinoids or their derivatives; g) Duocarmycin or its derivatives; h) calicheamicin or its derivatives; i) pyrrolobenzodiazepine or its derivatives; j) riocetine E or its derivatives; k) monomethyloxetine E (MMAE); l) monomethyloxetine D (MMAD); m) Maytansinoid DM1; n) maytansinoid DM4; and o) Detectable moiety.

58. The conjugated polypeptide of claim 57, wherein the detectable moiety is a diagnostic agent.

59. A pharmaceutical composition comprising: The isolated polypeptide of any one of claims 6 to 56; and Carrier.

60. The pharmaceutical composition of claim 59, comprising an additional agent.

61. The pharmaceutical composition of claim 60, wherein the additional agent is a therapeutic agent.

62. An isolated nucleic acid molecule encoding the isolated polypeptide of any one of claims 6 to 56.

63. A vector comprising the isolated nucleic acid molecule of claim 62.

64. A method of producing an antibody or an activatable antibody by culturing a cell under conditions resulting in the expression of an isolated polypeptide as described in any one of claims 6 to 56, wherein the cell comprises an isolated nucleic acid molecule as described in claim 62.

65. Use of the isolated polypeptide of any one of claims 6 to 56 or the pharmaceutical composition of any one of claims 59 to 61 in the preparation of a medicament for treating a condition or disease, alleviating the symptoms of the condition or disease, or delaying the progression of the condition or disease, wherein the medicament comprises a therapeutically effective amount of the isolated polypeptide of any one of claims 6 to 56 or the pharmaceutical composition of any one of claims 59 to 61; wherein the condition or disease is cancer.

66. The use of claim 65, wherein the medicament comprises an additional agent.

67. The use of claim 66, wherein the additional agent is a therapeutic agent.

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