Anti-CD3 antibodies, activatable Anti-CD3 antibodies, multispecific Anti-CD3 antibodies, multispecific activatable Anti-CD3 antibodies, and methods of using the same

Abstract

To provide antibodies and antigen-binding fragments thereof that specifically bind the epsilon chain of CD3, also known as CD3ε.SOLUTION: The anti-CD3ε antibodies and antigen-binding fragments thereof of the disclosure activate T cells via engagement of CD3ε on the T cells. That is, such antibodies agonize, stimulate, activate and / or augment CD3-mediated T cell activation. These antibodies and antigen-binding fragments thereof are referred to herein as "anti-CD3ε antibodies" or "anti-CD3 antibodies". The anti-CD3ε antibodies and antigen-binding fragments thereof of the disclosure include monoclonal antibodies, such as mammalian monoclonal antibodies, primate monoclonal antibodies, fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies, as well as antigen-binding fragments thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 029,325, filed July 25, 2014, the contents of which are incorporated herein by reference in their entirety. Field of Disclosure

[0003] The present invention relates generally to antibodies, activatable antibodies, multispecific antibodies and multispecific activatable antibodies that specifically bind to at least CD3, and methods of making and using these antibodies, activatable antibodies, multispecific antibodies and multispecific activatable antibodies for a variety of therapeutic, diagnostic and prophylactic applications. [Background technology]

[0004] Background to the disclosure

[0005] The CD3 (Cluster of Differentiation 3) T-cell co-receptor is a multimeric protein composed of four distinct polypeptide chains, referred to as the epsilon, gamma, delta, and zeta chains. The CD3 complex serves as the signaling module of the T cell receptor (TCR), noncovalently associating with the antigen-binding a / b chains of the TCR.

[0006] Because direct engagement of CD3 leads to T cell activation, it is a desirable target for a variety of therapeutic and / or diagnostic indications. Thus, there is a need for antibodies and therapeutic agents that target the CD3 / TCR pathway. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind the epsilon chain of CD3, also known as CDBε. The anti-CD3ε antibodies and antigen-binding fragments thereof of the present disclosure activate T cells through engagement of CD3ε on T cells. That is, such antibodies agonize, stimulate, activate, and / or enhance CD3-mediated T cell activation. These antibodies and antigen-binding fragments thereof are referred to herein as "anti-CD3ε antibodies" or "anti-CD3 antibodies." The anti-CD3ε antibodies and antigen-binding fragments thereof of the present disclosure include monoclonal antibodies, such as mammalian monoclonal antibodies, primate monoclonal antibodies, fully human monoclonal antibodies, and humanized monoclonal and chimeric antibodies, and antigen-binding fragments thereof. According to some embodiments, the antibodies and antigen-binding fragments thereof are of the IgG isotype. According to some embodiments, the antibodies and antigen-binding fragments thereof are of the IgG1 isotype. According to some embodiments, the antibodies and antigen-binding fragments thereof have any one of the isotypes disclosed herein. [Means for solving the problem]

[0008] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55).

[0009] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0010] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), and 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 RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR3 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 57). CDR2 sequence; and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0011] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0012] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence that 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 TYAMN (SEQ ID NO: 53); a VH CDR1 sequence that 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 RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); CDR2 sequence; and a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55).

[0013] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 57); CDR2 sequence; and a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0014] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence that 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 TYAMN (SEQ ID NO: 53); a VH CDR1 sequence that 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 RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), and a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56). a VL CDR2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0015] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55). CDR3 sequences, a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0016] In some embodiments, the anti-CD3ε antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-CD3ε antibody, or antigen-binding fragment thereof, comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-CD3ε antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 4 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 2.

[0017] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a variable heavy chain (Hv) comprising 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: 4. In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a variable light chain (Lv) comprising 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: 2. In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a variable heavy chain (Hv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:4 and a variable light chain (Lv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2.

[0018] In some embodiments, the anti-CD3ε antibody is an scFv antibody fragment that binds to CD3ε. In some embodiments, the anti-CD3ε scFv antibody fragment comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD3ε scFv antibody fragment comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-CD3ε scFv antibody fragment 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: 6. In some embodiments, the anti-CD3ε scFv antibody fragment 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: 30.

[0019] In some embodiments, the anti-CD3ε antibody also includes an agent attached to the antibody. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In some embodiments, the agent is attached to the anti-CD3ε antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0020] In some embodiments, anti-CD3ε naturally contains one or more disulfide bonds. In some embodiments, anti-CD3ε antibodies can be engineered to contain one or more disulfide bonds.

[0021] The present disclosure also provides isolated nucleic acid molecules encoding at least a portion of the anti-CD3ε antibodies described herein, and / or one or more nucleic acid molecules encoding the anti-CD3ε described herein, e.g., at least a first nucleic acid encoding at least a portion of the heavy chain of the antibody and a second nucleic acid encoding at least a portion of the light chain of the antibody, and vectors comprising these isolated nucleic acid sequences. The present disclosure also provides methods for producing anti-CD3ε antibodies by culturing cells containing such nucleic acid molecule(s) under conditions that result in expression of the anti-CD3ε antibody. In some embodiments, the cells contain such vectors.

[0022] The present disclosure also provides activatable antibodies and activatable antibody compositions comprising an antibody or antigen-binding fragment thereof (AB) coupled to, or otherwise specifically binding, a masking moiety (MM), such that coupling of the MM reduces the ability of the antibody or antigen-binding fragment thereof (AB) to bind to CD3ε. These activatable antibodies are collectively referred to herein as activatable anti-CD3ε antibodies, and are also referred to herein as anti-CD3ε activatable antibodies or CD3ε activatable antibodies. In some embodiments, the MM is coupled via a sequence comprising a substrate for a protease. For example, the protease is produced by a tumor present in proximity to cells expressing CD3ε. In some embodiments, the protease is produced by a tumor co-localized with cells expressing CD3ε. The activatable anti-CD3ε antibodies provided herein are stable in the blood circulation, are activated at the intended site of therapy and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to CD3ε that is at least comparable to the corresponding unmodified antibody.

[0023] In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55).

[0024] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0025] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof is 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 TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), and 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 a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); CDR2 sequence; and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0026] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof comprises a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:55), a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO:58).

[0027] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence that 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 TYAMN (SEQ ID NO: 53); a VH CDR1 sequence that 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 RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); CDR2 sequence; and a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55).

[0028] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 57); CDR2 sequence; and a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0029] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises a VH CDR1 sequence that comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR1 sequence that comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), and a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56). a VL CDR2 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0030] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof comprises a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55). CDR3 sequences, a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0031] In some embodiments, an activatable antibody comprises a combination of variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequences, variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequences, variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequences, variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequences, variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequences, and variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequences, wherein at least one CDR sequence is a VH CDR1 sequence shown in Table 18; a VH CDR2 shown in Table 18; a VH CDR3 shown in Table 18; a VL CDR1 sequence shown in Table 18; a VL CDR1 sequence shown in Table 18; and the VL CDR2 sequences shown in Table 18.

[0032] In some embodiments, an activatable antibody comprises a combination of VH CDR1 sequences, VH CDR2 sequences, CDR3 sequences, VL CDR1 sequences, VL CDR2 sequences, and VL CDR3 sequences, wherein at least one CDR sequence comprises a VH CDR1 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence shown in Table 18; a VH CDR2 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence shown in Table 18; a VH CDR3 sequence comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence shown in Table 18; VH CDR3 sequences comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence shown in Table 18; VL CDR2 sequences comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence shown in Table 18; and VL CDR3 sequences comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence shown in Table 18. The VL CDR3 sequence is selected from the group consisting of VL CDR3 sequences comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence.

[0033] In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination shown in Table 18.

[0034] In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein each CDR sequence in said combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to its corresponding CDR sequence in the combination shown in Table 18.

[0035] In some embodiments, the activatable anti-CD3ε antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-CD3ε antibody, or antigen-binding fragment thereof, comprises a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-CD3ε antibody, or antigen-binding fragment thereof, comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO: 4 and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO: 2.

[0036] In some embodiments, the activatable anti-CD3ε antibody or antigen-binding fragment thereof comprises a variable heavy chain (Hv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a variable light chain (Lv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2. In some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a variable heavy chain (Hv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:4 and a variable light chain (Lv) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2.

[0037] In some embodiments, the activatable anti-CD3ε antibody is an scFv antibody fragment that binds to CD3ε. In some embodiments, the anti-CD3ε scFv antibody fragment comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD3ε scFv antibody fragment comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-CD3ε scFv antibody fragment 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: 6. In some embodiments, the anti-CD3ε scFv antibody fragment 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: 30.

[0038] In some embodiments, the activatable antibody comprises a heavy chain amino sequence selected from the group consisting of the heavy chain sequences shown in Table 17. In some embodiments, the activatable antibody comprises a light chain amino sequence selected from the group consisting of the light chain sequences shown in Table 17. In some embodiments, the activatable antibody comprises a heavy chain amino sequence selected from the group consisting of the heavy chain sequences shown in Table 17, and a light chain amino sequence selected from the group consisting of the light chain sequences shown in Table 17.

[0039] In some embodiments, the activatable antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 17. In some embodiments, the activatable antibody comprises a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of the light chain sequences shown in Table 17. In some embodiments, the activatable antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 17, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of the light chain sequences shown in Table 17.

[0040] In some embodiments, the MM has a dissociation constant, ie, a dissociation constant at equilibrium, that is a higher Kd for binding to AB than the Kd for binding of AB to CD3ε.

[0041] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB is less than or equal to d It has.

[0042] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d is greater than or equal to K of binding to AB d It has.

[0043] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB is approximately equal to d It has.

[0044] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB is less thand It has.

[0045] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times less than d In some embodiments, the MM has a Kd for binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 times higher than the Kd for binding of AB to CD3ε.

[0046] In some embodiments, the MM has an affinity for binding to AB that is lower than the affinity for binding of AB to CD3ε.

[0047] In some embodiments, the MM has an affinity for binding to AB that is equal to or less than the affinity for binding of AB to CD3ε.

[0048] In some embodiments, the MM has an affinity for binding to AB that is approximately equal to the affinity for binding of AB to CD3ε.

[0049] In some embodiments, the MM has an affinity for binding to AB that is greater than or equal to the affinity for binding of AB to CD3ε.

[0050] In some embodiments, the MM has an affinity for binding to AB that is greater than the affinity for binding of AB to CD3ε.

[0051] In some embodiments, the MM has a K of binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times lower than the affinity of binding of AB to CD3ε. dIn some embodiments, the MM has a K of binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 times lower than the affinity of binding of AB to CD3ε. d In some embodiments, the MM has a binding affinity to AB that is 2-20 times lower than the binding affinity of AB to CD3ε. In some embodiments, the MM is not covalently bound to AB and, at equimolar concentrations to AB, does not inhibit the binding of AB to CD3ε.

[0052] In some embodiments, the MM does not interfere with or compete with the AB for binding to CD3ε when the activatable antibody is present in a cleaved state.

[0053] In some embodiments, the MM is a polypeptide of 40 amino acids or less in length.

[0054] In some embodiments, the MM polypeptide sequence differs from that of CD3ε. In some embodiments, the MM polypeptide sequence is 50% or less identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is 25% or less identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence is 10% or less identical to any natural binding partner of AB.

[0055] In some embodiments, the MM comprises a sequence selected from the group consisting of the sequences shown in Tables 7 or 8.

[0056] In some embodiments, the protease is produced by a tumor present in the vicinity of cells expressing CD3ε in a tissue and / or is produced by a tumor co-localized with cells expressing CD3ε in a tissue, wherein the protease cleaves a CM in the activatable antibody when the activatable antibody is exposed to the protease. In some embodiments, the CM is a polypeptide up to 15 amino acids in length. In some embodiments, the CM is a substrate for a protease selected from the proteases listed in Table 3. In some embodiments, the CM is a substrate for a protease selected from the group consisting of uPA, legumain, matriptase (also referred to herein as MT-SP1 or MTSP1), ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, MMP-14, and any of those shown in Table 3. In some embodiments, the CM is a substrate for a protease selected from the group consisting of uPA, legumain, and matriptase. In some embodiments, the CM is a substrate for a matrix metalloproteinase (MMP).

[0057] In some embodiments, the antigen-binding fragment thereof 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.

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

[0059] In some embodiments, the anti-CD3ε antibody also includes an agent attached to the antibody. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In some embodiments, the agent is attached to the anti-CD3ε antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0060] The present disclosure also provides multispecific antibodies that bind to the epsilon chain of CD3 (CD3ε) and a second target, wherein the antibodies comprise a first antibody or antigen-binding fragment thereof (AB1) that binds to the epsilon chain of CD3 (CD3ε) and a second antibody or antigen-binding fragment thereof (AB2) that binds to the second target, wherein AB1 comprises a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55); and a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56). a VL CDR1 sequence; a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0061] In some embodiments, the activatable antibodies described herein, in their activated state, bind CD3ε and comprise (i) an antibody or antigen-binding fragment thereof (AB) that specifically binds to CD3ε; (ii) a masking moiety (MM) that inhibits binding of the AB to CD3ε when the activatable antibody is present in an uncleaved state; and (iii) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the MM is coupled to the AB via the CM.

[0062] In some embodiments, the activatable antibody, in its uncleaved state, has the following structural configuration from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM.

[0063] According to some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM.

[0064] According to some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB.

[0065] According to some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. According to some embodiments, the two connecting peptides need not be identical to each other.

[0066] According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 59) and (GGGS) n (SEQ ID NO:60), where n is at least one integer. According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:61), GGSGG (SEQ ID NO:62), GSGSG (SEQ ID NO:63), GSGGG (SEQ ID NO:64), GGGSG (SEQ ID NO:65), and GSSSG (SEQ ID NO:66).

[0067] In some embodiments, the activatable antibody comprises an antibody or antigen-binding fragment thereof that specifically binds CD3ε. In some embodiments, the antibody or antigen-binding fragment thereof that binds CD3ε is a monoclonal antibody, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single domain heavy chain antibody, and single domain light chain antibody. In some embodiments, such an antibody or antigen-binding fragment thereof that binds CD3ε is a rodent (e.g., mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

[0068] In some embodiments, AB has a dissociation constant of about 100 nMm or less for binding to CD3ε.

[0069] In some embodiments, the MM has a dissociation constant, i.e., a K for binding to AB that is higher than the K for binding of AB to CD3ε. d The dissociation constant at equilibrium is

[0070] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB is less than or equal to d It has.

[0071] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d is greater than or equal to K of binding to AB d It has.

[0072] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB is approximately equal to d It has.

[0073] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB is less than d It has.

[0074] In some embodiments, the MM is a nucleotide sequence that is a function of the K of AB binding to CD3ε. d K of binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times less than d In some embodiments, the MM has a K d K of binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 times higher than d It has.

[0075] In some embodiments, the MM has an affinity for binding to AB that is lower than the affinity for binding of AB to CD3ε.

[0076] In some embodiments, the MM has an affinity for binding to AB that is equal to or less than the affinity for binding of AB to CD3ε.

[0077] In some embodiments, the MM has an affinity for binding to AB that is approximately equal to the affinity for binding of AB to CD3ε.

[0078] In some embodiments, the MM has an affinity for binding to AB that is greater than or equal to the affinity for binding of AB to CD3ε.

[0079] In some embodiments, the MM has an affinity for binding to AB that is greater than the affinity for binding of AB to CD3ε.

[0080] In some embodiments, the MM has a Kd for binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000-fold lower than the affinity of AB binding to CD3ε. In some embodiments, the MM has a Kd for binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000-fold lower than the affinity of AB binding to CD3ε. In some embodiments, the MM has an affinity for binding to AB that is 2-20-fold lower than the affinity of AB binding to CD3ε. In some embodiments, the MM, which is not covalently bound to AB and at equimolar concentrations to AB, does not inhibit AB binding to CD3ε.

[0081] In some embodiments, the MM does not interfere with or compete with AB binding to CD3ε when the activatable antibody is present in a cleaved state.

[0082] In some embodiments, the MM is a polypeptide about 20 to 40 amino acids in length. In some embodiments, the MM is a polypeptide greater than 40 amino acids in length.

[0083] In some embodiments, the MM polypeptide sequence differs from that of CD3ε. In some embodiments, the MM polypeptide sequence is 50% or less identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence differs from that of CD3ε, and the MM polypeptide sequence is 25% or less identical to any natural binding partner of AB. In some embodiments, the MM polypeptide sequence differs from that of CD3ε, and the MM polypeptide sequence is 10% or less identical to any natural binding partner of AB.

[0084] In some embodiments, the coupling of MM is determined by the dissociation constant (K d ) is the K of AB when it is not coupled to MM towards CD3ε d This reduces the ability of AB to bind to CD3ε so that it is at least 20-fold higher than that of AB.

[0085] In some embodiments, the coupling of MM is determined by the dissociation constant (K d ) is the K of AB when it is not coupled to MM towards CD3ε d This reduces the ability of AB to bind to CD3ε to at least 40-fold higher than that of AB.

[0086] In some embodiments, the coupling of MM is determined by the dissociation constant (K d ) is the K of AB when it is not coupled to MM towards CD3ε d This reduces the ability of AB to bind to CD3ε so that it is at least 100-fold higher than that of AB.

[0087] In some embodiments, the coupling of MM is determined by the dissociation constant (K d ) is the K of AB when it is not coupled to MM towards CD3ε d This reduces the ability of AB to bind to CD3ε so that it is at least 1000-fold higher than that of AB.

[0088] In some embodiments, the coupling of MM is determined by the dissociation constant (K d ) is the K of AB when it is not coupled to MM towards CD3ε d This reduces the ability of AB to bind to CD3ε to at least 10,000-fold higher than that of AB.

[0089] In some embodiments, when assayed in vitro in the presence of CD3ε using a target displacement assay, such as the assay described in WO 2010 / 081173, the contents of which are incorporated herein by reference in their entirety, MM reduces the ability of AB to bind to CD3ε by at least 90% when the CM is uncleaved compared to when the CM is cleaved.

[0090] In some embodiments, the MM does not interfere with or compete with the AB of the activatable antibody in its cleaved state for binding to the CD3ε target.

[0091] In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of the sequences shown in Tables 7 or 8.

[0092] In some embodiments, the protease is produced by a tumor present in the vicinity of cells expressing CD3ε in a tissue and / or is produced by a tumor co-localized with cells expressing CD3ε in a tissue, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

[0093] In some embodiments, the CM is positioned in the activatable antibody such that binding of the activatable antibody to CD3ε when the activatable antibody is in its uncleaved state is reduced to occur with a dissociation constant at least 20-fold higher than the dissociation constant of unmodified AB binding to CD3ε, while binding to AB3ε when the activatable antibody is in its cleaved state.

[0094] In some embodiments, the CM is positioned in the activatable antibody such that binding of the activatable antibody to CD3ε when the activatable antibody is in its uncleaved state is reduced to occur with a dissociation constant at least 40-fold higher than the dissociation constant of unmodified AB binding to CD3ε, while binding to AB3ε when the activatable antibody is in its cleaved state.

[0095] In some embodiments, the CM is positioned in the activatable antibody such that binding of the activatable antibody to CD3ε when the activatable antibody is in its uncleaved state is reduced to occur with a dissociation constant at least 50-fold higher than the dissociation constant of unmodified AB binding to CD3ε, while binding to AB3ε when the activatable antibody is in its cleaved state.

[0096] In some embodiments, the CM is positioned in the activatable antibody such that binding of the activatable antibody to CD3ε when the activatable antibody is in its uncleaved state is reduced to occur with a dissociation constant at least 100-fold higher than the dissociation constant of unmodified AB binding to CD3ε, while binding to AB3ε when the activatable antibody is in its cleaved state.

[0097] In some embodiments, the CM is positioned in the activatable antibody such that binding of the activatable antibody to CD3ε when the activatable antibody is in its uncleaved state is reduced to occur with a dissociation constant at least 200-fold higher than the dissociation constant of unmodified AB binding to CD3ε, while binding to AB3ε when the activatable antibody is in its cleaved state.

[0098] In some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0099] In some embodiments, the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 67). In some embodiments, the cleavable moiety is selected for use with a particular protease, e.g., a protease known to be produced by tumors located in proximity to cells expressing the target of the activatable antibody, e.g., CD3ε, and / or co-localized with the target of the activatable antibody. For example, cleavable moieties suitable for use with activatable anti-CD3ε antibodies of the present disclosure are cleaved by at least proteases such as urokinase, legumain, and / or matriptase (also referred to herein as MT-SP1 or MTSP1). In some embodiments, suitable cleavable moieties comprise at least one of the following sequences: TGRGPSWV (SEQ ID NO: 68); SARGPSRW (SEQ ID NO: 69); TARGPSFK (SEQ ID NO: 70); LSGRSDNH (SEQ ID NO: 67); GGWHTGRN (SEQ ID NO: 71); HTGRSGAL (SEQ ID NO: 72); PLTGRSGG (SEQ ID NO: 73); AARGPAIH (SEQ ID NO: 74); RGPAFNPM (SEQ ID NO: 75); SSRGPAYL (SEQ ID NO: 76); RGPATPIM (SEQ ID NO: 77); RGPA (SEQ ID NO: 78); GGQPSGMWGW (SEQ ID NO: 79); FPRPLGITGL (SEQ ID NO: 80); VHMPLGFLGP (SEQ ID NO: 81); SPLTGRSG (SEQ ID NO: 82); SAGFSLPA (SEQ ID NO: 83); LAPLGLQRR (SEQ ID NO: 84); SGGPLGVR (SEQ ID NO: 85); and / or PLGL (SEQ ID NO: 86).

[0100] In some embodiments, the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO:67). In some embodiments, the CM comprises the amino acid sequence TGRGPSWV (SEQ ID NO:68). In some embodiments, the CM comprises the amino acid sequence SARGPSRW (SEQ ID NO:69). In some embodiments, the CM comprises the amino acid sequence TARGPSFK (SEQ ID NO:70). In some embodiments, the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO:67). In some embodiments, the CM comprises the amino acid sequence GGWHTGRN (SEQ ID NO:71). In some embodiments, the CM comprises the amino acid sequence HTGRSGAL (SEQ ID NO:72). In some embodiments, the CM comprises the amino acid sequence PLTGRSGG (SEQ ID NO:73). In some embodiments, the CM comprises the amino acid sequence AARGPAIH (SEQ ID NO:74). In some embodiments, the CM comprises the amino acid sequence RGPAFNPM (SEQ ID NO:75). In some embodiments, the CM comprises the amino acid sequence SSRGPAYL (SEQ ID NO:76). In some embodiments, the CM comprises the amino acid sequence RGPATPIM (SEQ ID NO:77). In some embodiments, the CM comprises the amino acid sequence RGPA (SEQ ID NO:78). In some embodiments, the CM comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO:79). In some embodiments, the CM comprises the amino acid sequence FPRPLGITGL (SEQ ID NO:80). In some embodiments, the CM comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO:81). In some embodiments, the CM comprises the amino acid sequence SPLTGRSG (SEQ ID NO:82). In some embodiments, the CM comprises the amino acid sequence SAGFSLPA (SEQ ID NO:83). In some embodiments, the CM comprises the amino acid sequence LAPLGLQRR (SEQ ID NO:84). In some embodiments, the CM comprises the amino acid sequence SGGPLGVR (SEQ ID NO:85). In some embodiments, the CM comprises the amino acid sequence PLGL (SEQ ID NO:86).

[0101] In some embodiments, the CM is a substrate for MMP and comprises the sequences ISSGLLSS (SEQ ID NO: 321); QNQALRMA (SEQ ID NO: 322); AQNLLGMV (SEQ ID NO: 323); STFPFGMF (SEQ ID NO: 324); PVGYTSSL (SEQ ID NO: 325); DWLYWPGI (SEQ ID NO: 326); MIAPVAYR (SEQ ID NO: 327); RPSPMWAY (SEQ ID NO: 328); WATPRPMR (SEQ ID NO: 329); FRLLDWQW (SEQ ID NO: 330); LKAAPRWA (SEQ ID NO: 331); GPSHLLVLT (SEQ ID NO: 332); LPGGLSPW (SEQ ID NO: 333); MGLFSEAG (SEQ ID NO: 334); SPLPLRVP (SEQ ID NO: 335); RMHLRSLG (SEQ ID NO: 336); LAAPLGLL (SEQ ID NO: 337); AVGLLAPP (SEQ ID NO: 338); LLAPSHRA (SEQ ID NO: 339); PAGLWLDP (SEQ ID NO: 340); and / or ISSGLSS (SEQ ID NO: 341).

[0102] In some embodiments, the CM comprises the amino acid sequence ISSGLLSS (SEQ ID NO: 321). In some embodiments, the CM comprises the amino acid sequence QNQALRMA (SEQ ID NO: 322). In some embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ ID NO: 323). In some embodiments, the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 324). In some embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 325). In some embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ ID NO: 326). In some embodiments, the CM comprises the amino acid sequence MIAPVAYR (SEQ ID NO: 327). In some embodiments, the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 328). In some embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ ID NO: 329). In some embodiments, the CM comprises the amino acid sequence FRLLDWQW (SEQ ID NO: 330). In some embodiments, the CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 331). In some embodiments, the CM comprises the amino acid sequence GPSHLLVLT (SEQ ID NO: 332). In some embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQ ID NO: 333). In some embodiments, the CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 334). In some embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ ID NO: 335). In some embodiments, the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 336). In some embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 337). In some embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ ID NO: 338). In some embodiments, the CM comprises the amino acid sequence LLAPSHRA (SEQ ID NO: 339). In some embodiments, the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 340). In some embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ ID NO: 341).

[0103] In some embodiments, the CM is a substrate for thrombin. In some embodiments, the CM is a substrate for thrombin and comprises the sequence GPRSFGL (SEQ ID NO: 896) or GPRSFG (SEQ ID NO: 897). In some embodiments, the CM comprises the amino acid sequence GPRSFGL (SEQ ID NO: 896). In some embodiments, the CM comprises the amino acid sequence GPRSFG (SEQ ID NO: 897).

[0104] In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of NTLSGRSENHSG (SEQ ID NO: 898); NTLSGRSGNHGS (SEQ ID NO: 899); TSTSGRSAMPRG (SEQ ID NO: 900); TSGRSANP (SEQ ID NO: 901); VAGRSMRP (SEQ ID NO: 902); VVPEGRRS (SEQ ID NO: 903); ILPRSPAF (SEQ ID NO: 904); MVLGRSLL (SEQ ID NO: 905); QGRAITFI (SEQ ID NO: 906); SPRSIMLA (SEQ ID NO: 907); and SMLRSMPL (SEQ ID NO: 908).

[0105] In some embodiments, the CM comprises the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 898). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 899). In some embodiments, the CM comprises the amino acid sequence TSTSGRSAMPRG (SEQ ID NO: 900). In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 901). In some embodiments, the CM comprises the amino acid sequence VAGRSMRP (SEQ ID NO: 902). In some embodiments, the CM comprises the amino acid sequence VVPEGRRS (SEQ ID NO: 903). In some embodiments, the CM comprises the amino acid sequence ILPRSPAF (SEQ ID NO: 904). In some embodiments, the CM comprises the amino acid sequence MVLGRSLL (SEQ ID NO: 905). In some embodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ ID NO: 906). In some embodiments, the CM comprises the amino acid sequence SPRSIMLA (SEQ ID NO: 907). In some embodiments, the CM comprises the amino acid sequence SMLRSMPL (SEQ ID NO: 908).

[0106] In some embodiments, the CM is a substrate for neutrophil elastase. In some embodiments, the CM is a substrate for a serine protease. In some embodiments, the CM is a substrate for uPA. In some embodiments, the CM is a substrate for legumain. In some embodiments, the CM is a substrate for matriptase. In some embodiments, the CM is a substrate for a cysteine ​​protease. In some embodiments, the CM is a substrate for a cysteine ​​protease, such as a cathepsin.

[0107] In some embodiments, the CM is a CM1-CM2 substrate and has the sequences ISSGLLSGRSDNH (SEQ ID NO: 909); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 910); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 911); TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 912); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 913); TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 914); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 915); LSGRSDNHGGAVGLLAPP (SEQ ID NO: 916); VHMPLGFLGPGGLSGRSDN H (SEQ ID NO: 917); LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 918); LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 919); LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 920); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 921); LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 922); QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 923); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 924); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 925) and / or ISSGLLSGRSGNH (SEQ ID NO: 926).

[0108] In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSDNH (SEQ ID NO: 909). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 910). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 911). In some embodiments, the CM1-CM2 substrate comprises the sequence TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 912). In some embodiments, the CM1-CM2 substrate comprises the sequence VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 913). In some embodiments, the CM1-CM2 substrate comprises the sequence TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 914). In some embodiments, the CM1-CM2 substrate comprises the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 915). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 916). In some embodiments, the CM1-CM2 substrate comprises the sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 917). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 918). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGSGGSISSGLLSS (SEQ ID NO: 919). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 920). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 921). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSDNHGSGGSQNQALRMA (SEQ ID NO: 922). In some embodiments, the CM1-CM2 substrate comprises the sequence QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 923). In some embodiments, the CM1-CM2 substrate comprises the sequence LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 924).In some embodiments, the CM1-CM2 substrate comprises the sequence QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 925). In some embodiments, the CM1-CM2 substrate comprises the sequence ISSGLLSGRSGNH (SEQ ID NO: 926).

[0109] In some embodiments, the CM is a substrate for a protease selected from the group consisting of those shown in Table 3. In some embodiments, the protease is selected from the group consisting of uPA, legumain, matriptase, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, and MMP-14. In some embodiments, the protease is a cathepsin. In some embodiments, the CM is a substrate for a protease selected from the group consisting of uPA (urokinase plasminogen activator), legumain, and matriptase. In some embodiments, the protease is uPA. In some embodiments, the protease is legumain. In some embodiments, the protease comprises matriptase. In some embodiments, the protease comprises a matrix metalloproteinase (MMP).

[0110] In some embodiments, the CM is a substrate for at least two proteases. In some embodiments, each protease is selected from the group consisting of those shown in Table 3. In some embodiments, the CM is a substrate for at least two proteases, where one of the proteases is selected from the group consisting of uPA, legumain, and matriptase, and the other protease is selected from the group consisting of those shown in Table 3. In some embodiments, the CM is a substrate for at least two proteases selected from the group consisting of uPA, legumain, and matriptase.

[0111] In some embodiments, the activatable antibody comprises at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each polypeptides 15 or fewer amino acids in length. In some embodiments, the first CM and the second CM in the activatable antibody, in their uncleaved state, have the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, at least one of the first CM and the second CM is a polypeptide that also functions as a substrate for a protease selected from the group consisting of uPA, legumain, and matriptase. In some embodiments, the first CM is cleaved in the target tissue by a first cleavage agent selected from the group consisting of uPA, legumain, and matriptase, and the second CM is cleaved in the target tissue by a second cleavage agent. In some embodiments, the other protease is selected from the group consisting of those shown in Table 3. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group consisting of uPA, legumain, and matriptase, and the first CM and the second CM are different substrates of the protease. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group listed in Table 3. According to some embodiments, the first cleavage agent and the second cleavage agent are different proteases. According to some embodiments, the first cleavage agent and the second cleavage agent are produced by a tumor present in proximity to cells expressing the target and / or produced by a tumor co-localized in the target tissue. According to some embodiments, the first CM and the second CM are cleaved by at least one cleavage agent in the target tissue.

[0112] In some embodiments, when the activatable antibody is in an activated or cleaved state, the activatable antibody is exposed to and cleaved by a protease such that the activated antibody comprises light chain amino acids that include at least a portion of the LP2 and / or CM sequence after the protease cleaves the CM.

[0113] In some embodiments, the MM and CM comprise an amino acid sequence selected from the group consisting of those sequences provided herein.

[0114] According to some embodiments, the activatable antibody also comprises a signal peptide. According to some embodiments, the signal peptide is attached to the activatable antibody via a spacer. According to some embodiments, the spacer is attached to the activatable antibody in the absence of the signal peptide. According to some embodiments, the spacer is attached directly to the MM of the activatable antibody.

[0115] In some embodiments, the activatable antibody in its uncleaved state comprises a spacer directly linked to the MM and having the structural arrangement spacer-MM-CM-AB from N- to C-terminus. In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 407). In some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 87). In some embodiments, the spacer comprises at least the amino acid sequence QGQSG (SEQ ID NO: 408). In some embodiments, the spacer comprises at least the amino acid sequence QGQS (SEQ ID NO: 409). In some embodiments, the spacer comprises at least the amino acid sequence QGQ (SEQ ID NO: 410). In some embodiments, the spacer comprises at least the amino acid sequence QG (SEQ ID NO: 411). In some embodiments, the spacer comprises at least the amino acid residue Q. In some embodiments, the MM and the spacer comprise an amino acid sequence selected from the sequences listed in Tables 7 or 8.

[0116] In some embodiments, the activatable antibody comprises a heavy chain selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544 and 546.

[0117] In some embodiments, the activatable antibody comprises a light chain selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516 and 520.

[0118] In some embodiments, the activatable antibody comprises a heavy chain selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544, and 546, and a light chain selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0119] In some embodiments, the activatable antibody comprises a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544, and 546.

[0120] In some embodiments, the activatable antibody comprises a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0121] In some embodiments, the activatable antibody is an antibody selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544, and 546. and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0122] In some embodiments, the activatable antibody comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, the activatable antibody comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:506.

[0123] In some embodiments, the activatable antibody comprises the amino acid sequence of SEQ ID NO: 587 or 588. In some embodiments, the activatable antibody comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 587 or 588.

[0124] In some embodiments, the activatable antibody also includes an agent attached to AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In some embodiments, the agent is attached to AB via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0125] In some embodiments, the activatable antibody also comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0126] 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.

[0127] According to some embodiments, the serum half-life of the multispecific activatable antibody is longer than the half-life of its corresponding multispecific antibody; for example, the pK of the multispecific activatable antibody is longer than the pK of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is similar to the half-life of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 15 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 11 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 10 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 9 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 7 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 5 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 2 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 24 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 20 hours when administered to an organism.According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 18 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 16 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 14 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 10 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 hours when administered to an organism.

[0128] In some embodiments, the activatable anti-CD3ε antibody is monospecific. In some embodiments, the activatable anti-CD3ε antibody is multispecific, for example, by way of non-limiting example, bispecific or trispecific. In some embodiments, the activatable anti-CD3ε antibody is formulated as part of a pro-bispecific T cell engager (BITE) molecule. In some embodiments, the activatable anti-CD3ε antibody is formulated as part of a pro-chimeric antigen receptor (CAR)-modified T cell or other engineered receptor.

[0129] The compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues in the AB without impairing the activity (e.g., masking, activation, or binding activity) of the activatable anti-CD3ε antibody. According to some embodiments, the compositions and methods provided herein allow for the attachment of one or more agents to one or more cysteine ​​residues without reducing or otherwise interfering with one or more disulfide bonds in the MM. The compositions and methods provided herein produce activatable anti-CD3ε antibodies conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic, and / or prophylactic agents, preferably none of which are conjugated to the MM of the activatable anti-CD3ε antibody. The compositions and methods provided herein produce conjugated activatable anti-CD3ε antibodies that retain the ability to effectively and efficiently mask the AB of the activatable anti-CD3ε antibody when the MM is not cleaved. The compositions and methods provided herein generate conjugated activatable anti-CD3ε antibodies in which the activatable antibodies are still activated, i.e., cleaved, in the presence of a protease capable of cleaving the CM.

[0130] According to some embodiments, the anti-CD3ε antibodies and / or activatable anti-CD3ε antibodies described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended use, e.g., cancer. For example, the anti-CD3ε antibodies and / or activatable anti-CD3ε antibodies can be used in conjunction with additional chemotherapeutic or anti-tumor agents.

[0131] According to some embodiments, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody and the additional agent are formulated into a single therapeutic composition and the anti-CD3ε antibody and / or activatable anti-CD3ε antibody and the additional agent are administered simultaneously. According to some embodiments, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody and the additional agent are separate from one another, e.g., each is formulated into separate therapeutic compositions and the anti-CD3ε antibody and / or activatable anti-CD3ε antibody and the additional agent are administered simultaneously, or the anti-CD3ε antibody and / or activatable anti-CD3ε antibody and the additional agent are administered at different times during the therapeutic regimen. For example, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody may be administered prior to the administration of the additional agent, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody may be administered subsequent to the administration of the additional agent, or the anti-CD3ε antibody and / or activatable anti-CD3ε antibody may be administered in alternation. As described herein, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody and the additional agent may be administered in a single dose or in multiple doses.

[0132] The present disclosure also provides isolated nucleic acid molecules encoding at least a portion of the activatable anti-CD3ε antibodies described herein, and vectors comprising those isolated nucleic acid sequences and / or one or more nucleic acid molecules encoding the anti-CD3ε antibodies described herein, e.g., a first nucleic acid encoding at least a portion of the heavy chain of the activatable antibody and a second nucleic acid encoding at least a portion of the light chain of the activatable antibody. The present disclosure also provides methods for producing an activatable antibody by culturing cells containing such nucleic acid molecules under conditions conducive to expression of the activatable antibody. In some embodiments, the cells contain such vectors.

[0133] In some embodiments, the activatable antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 445, 451, 453, 455, 459, 461, 463, 465, 469, 471, 475, 477, 479, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 511, 513, 517, 522, 525, 529, 531, 533, 535, 537, 539, 541, 543, and 545.

[0134] In some embodiments, the activatable antibody is encoded by a light chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 447, 449, 457, 467, 473, 481, 483, 507, 515 and 519.

[0135] In some embodiments, the activatable antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 445, 451, 453, 455, 459, 461, 463, 465, 469, 471, 475, 477, 479, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 511, 513, 517, 522, 525, 529, 531, 533, 535, 537, 539, 541, 543, and 545, and a light chain nucleic acid sequence selected from the group consisting of SEQ ID NOs: 447, 449, 457, 467, 473, 481, 483, 507, 515, and 519.

[0136] In some embodiments, the activatable antibody is encoded by a heavy chain nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 445, 451, 453, 455, 459, 461, 463, 465, 469, 471, 475, 477, 479, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 511, 513, 517, 522, 525, 529, 531, 533, 535, 537, 539, 541, 543, and 545.

[0137] In some embodiments, the activatable antibody is encoded by a light chain nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 447, 449, 457, 467, 473, 481, 483, 507, 515, and 519.

[0138] In some embodiments, the activatable antibody is directed to a sequence selected from the group consisting of SEQ ID NOs: 445, 451, 453, 455, 459, 461, 463, 465, 469, 471, 475, 477, 479, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 511, 513, 517, 522, 525, 529, 531, 533, 535, 537, 539, 541, 543, and 545. and a light chain nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 447, 449, 457, 467, 473, 481, 483, 507, 515, and 519.

[0139] In some embodiments, the activatable antibody is encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 505. In some embodiments, the activatable antibody comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:505.

[0140] The present disclosure also provides a method for producing an activatable antibody that binds to CD3ε in an activated state by (a) culturing cells containing a nucleic acid construct encoding the activatable antibody under conditions that result in expression of the activatable antibody, the activatable antibody comprising a masking moiety (MM), a cleavable moiety (CM), and an antibody or antigen-binding fragment thereof (AB) that specifically binds CD3ε, and (b) recovering the activatable antibody.

[0141] The present disclosure also provides a method for producing an activatable antibody that binds to CD3ε in an activated state by (a) culturing cells 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 cleavable moiety (CM), and an antibody or antigen-binding fragment thereof (AB) that specifically binds CD3ε, (i) wherein the CM is a polypeptide comprising an amino acid sequence that functions as a substrate for a protease; and (ii) wherein the CM is positioned on the activatable antibody such that the MM does not interfere with or compete with the specific binding of the AB to CD3ε when the activatable antibody is in an uncleaved state; and (b) recovering the activatable antibody.

[0142] The present disclosure also provides multispecific antibodies that bind to the epsilon chain of CD3 (CD3ε) and a second target, wherein the antibodies comprise a first antibody or antigen-binding fragment thereof (AB1) that binds to the epsilon chain of CD3 (CD3ε), and a second antibody or antigen-binding fragment thereof (AB2) that binds to the second target, wherein AB1 comprises a VH CDR1 sequence comprising 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 TYAMN (SEQ ID NO: 53); a VH CDR1 sequence comprising 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 RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); a VH CDR3 sequence comprising 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 HGNFGNSYVSWFAY (SEQ ID NO: 55), and a VL CDR1 sequence comprising a combination of a VL CDR2 sequence and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence and the VL CDR3 sequence comprises 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 RSSTGAVTTSNYAN (SEQ ID NO: 56). a CDR1 sequence; a VL CDR2 sequence comprising 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 GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising 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 ALWYSNLWV (SEQ ID NO: 58).

[0143] According to some embodiments, the multispecific anti-CD3ε antibody or antigen-binding fragment thereof comprises a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:54); and a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:55), a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO:58).

[0144] In some embodiments, AB1 comprises a variable heavy chain (Hv) comprising the amino acid sequence of SEQ ID NO:4, and a variable light chain (Lv) comprising the amino acid sequence of SEQ ID NO:2.

[0145] In some embodiments, AB1 comprises a variable heavy chain (Hv) comprising 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:4, and a variable light chain (Lv) comprising 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:2.

[0146] In some embodiments, AB1 comprises an scFv fragment. In some embodiments, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the scFv fragment 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: 6. In some embodiments, the scFv fragment 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: 30.

[0147] In some embodiments, the multispecific antibody comprises an agent attached to the antibody. In some embodiments, the agent is a therapeutic agent, a detectable moiety, or a diagnostic agent. In some embodiments, the agent is attached to the antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0148] The present disclosure also provides multispecific antibodies and multispecific activatable antibodies that bind to at least CD3ε. The multispecific antibodies provided herein are antibodies that recognize multiple antigens or epitopes, where at least one antigen or epitope is CD3ε. The multispecific activatable antibodies provided herein are multispecific antibodies that include at least one masking moiety (MM) 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. According to some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for a protease. The multispecific activatable antibodies provided herein are stable in the blood circulation, are activated at intended sites for therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit target binding at least comparable to that of the corresponding unmodified multispecific antibody.

[0149] According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies are designed to bind to immune effector cells, also referred to herein as immune effector-binding and / or immune effector-engaging multispecific antibodies. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies are designed to bind to leukocytes, also referred to herein as leukocyte-binding and / or leukocyte-binding multispecific activatable antibodies. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies are designed to bind to T-cells, also referred to herein as T-cell-binding and / or T-cell-binding multispecific activatable antibodies. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies bind to a surface antigen on a leukocyte, e.g., a T cell, a natural killer (NK) cell, a myelomononuclear cell, a macrophage, and / or another immune effector cell. According to some embodiments, the immune effector cell is a leukocyte. According to some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are mononuclear cells, e.g., bone marrow mononuclear cells. In some embodiments, the T-cell-binding multispecific antibody and / or the T-cell-binding multispecific activatable antibody binds to CD3ε.

[0150] According to some embodiments, the immune effector cell-binding multispecific antibody comprises a targeting antibody or antigen-binding fragment thereof and an immune effector cell-binding antibody or antigen-binding portion thereof, including an anti-CD3ε antibody or antigen-binding fragment thereof. According to some embodiments, the immune effector cell-binding multispecific antibody comprises a cancer-targeting antibody or antigen-binding fragment thereof and an immune effector cell-binding antibody or antigen-binding portion thereof. According to some embodiments, the immune effector cell-binding multispecific antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof and an immune effector cell-binding scFv. According to some embodiments, the immune effector cell is a leukocyte. According to some embodiments, the immune effector cell is a T cell. According to some embodiments, the immune effector cell is a NK cell. According to some embodiments, the immune effector cell is a bone marrow mononuclear cell.

[0151] According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε antibody or antigen-binding fragment thereof, and a targeting antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε scFv and a targeting antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε antibody or antigen-binding fragment thereof, and a cancer-targeting antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε scFv and a cancer-targeting antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε antibody or antigen-binding fragment thereof, and a cancer-targeting IgG antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell binding multispecific antibody comprises an anti-CD3 epsilon (CD3ε) scFv derived from OKT3. According to some embodiments, the T-cell binding multispecific antibody comprises an anti-CD3 epsilon (CD3ε) scFv derived from SP34 (available from BD Biosciences, catalog number 556610).

[0152] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises a targeting antibody or antigen-binding fragment thereof and an anti-CD3ε antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the anti-CD3ε antibody or antigen-binding portion thereof is masked. According to some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of AB2 to bind to the second target. According to some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety reduces the ability of AB1 to bind to CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment (AB2) that binds to a second target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety reduces the ability of AB2 to bind to the second target.

[0153] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises a cancer-targeting antibody or antigen-binding fragment thereof and an anti-CD3ε antibody or antigen-binding portion thereof, wherein at least one of the cancer-targeting antibody or antigen-binding fragment thereof and / or the anti-CD3ε antibody or antigen-binding portion thereof is masked. According to some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (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 (AB2) that binds to a second cancer-associated target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0154] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof and an anti-CD3ε or antigen-binding fragment thereof, wherein at least one of the cancer-targeting IgG antibody or antigen-binding fragment thereof and / or the anti-CD3ε antibody or antigen-binding portion thereof is masked. According to some embodiments, the anti-CD3ε antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises two antibodies or fragments thereof, including a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the anti-CD3ε or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0155] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an anti-CD3ε scFv and a targeting antibody, or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and the targeting antibody, or antigen-binding fragment thereof, is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment (AB2) that binds to a second target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target.

[0156] According to some embodiments, the T-cell-binding 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 the cancer-targeting antibody, or antigen-binding fragment thereof, is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (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 (AB2) that binds to a second cancer-associated target, wherein coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0157] According to some embodiments, the T-cell-binding 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 the cancer-targeting IgG antibody, or antigen-binding portion thereof, is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises two antibodies or fragments thereof, comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0158] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an anti-CD3 epsilon (CD3ε) scFv derived from OKT3, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the OKT3 scFv or the OKT3-derived scFv is masked. According to some embodiments, the OKT3 scFv or the OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target. According to 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 conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target.

[0159] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a 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. According to 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 conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to 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 conjugated to a masking moiety (MM1) such that coupling of the masking moiety (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 coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0160] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a 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. According to 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 conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to 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 conjugated to a masking moiety (MM1) such that coupling of the masking moiety (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 coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0161] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an anti-CD3 epsilon (CD3ε) scFv derived from SP34, wherein at least one of the targeting IgG antibody or antigen-binding fragment thereof and the SP34 scFv or SP34-derived scFv is masked. According to some embodiments, the SP34 scFv or SP34-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target. According to some embodiments, the SP34 scFv or SP34-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds to a second target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target.

[0162] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises SP34 scFv or SP34-derived scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the SP34 scFv or SP34-derived scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the SP34 scFv or SP34-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the SP34 scFv or SP34-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (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 coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0163] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an SP34 scFv or an SP34-derived scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the SP34 scFv or SP34-derived scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. According to some embodiments, the SP34 scFv or SP34-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the SP34 scFv or SP34-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to CD3ε, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0164] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an anti-CD3 epsilon (CD3ε) scFv comprising the following complementarity determining region (CDR) sequences: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO:53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:54); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:55); a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO:57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO:58), wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the anti-CD3ε scFv is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) having the CDR sequences shown above, i.e., SEQ ID NOs: 53, 54, 55, 56, 57, and 58, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target.According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) comprising the CDR sequences of SEQ ID NOs: 53, 54, 55, 56, 57, and 58, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, wherein coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target.

[0165] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an anti-CD3ε scFv comprising the CDR sequences of SEQ ID NOs: 53, 54, 55, 56, 57, and 58, and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) comprising the CDR sequences of SEQ ID NOs: 53, 54, 55, 56, 57, and 58, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety reduces the ability of AB1 to bind to CD3ε. According to 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 conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) comprising the CDR sequences of SEQ ID NOs: 53, 54, 55, 56, 57, and 58, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety 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 coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0166] According to some embodiments, the T-cell-binding multispecific activatable antibody comprises an anti-CD3ε scFv comprising the CDR sequences of SEQ ID NOs: 53, 54, 55, 56, 57, and 58, and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) comprising the CDR sequences of SEQ ID NOs: 53, 54, 55, 56, 57, and 58, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety reduces the ability of AB1 to bind to CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein AB2 is conjugated to a masking moiety (MM2) such that coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) comprising the CDR sequences of SEQ ID NOs: 53, 54, 55, 56, 57, and 58, wherein AB1 is conjugated to a masking moiety (MM1) such that coupling of the masking moiety reduces the ability of AB1 to bind to CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds to a second cancer-associated target, wherein coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second cancer-associated target.

[0167] According to some embodiments, the multi-antigen targeting antibody and / or 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, wherein at least one of the targets and / or epitopes is CD3ε. According to some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different targets, wherein at least one target is CD3ε. According to some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different epitopes on CD3ε. According to some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds a combination of two or more different targets and two or more different epitopes on the same target, wherein at least one of the targets and / or epitopes is CD3ε.

[0168] Various embodiments of the multispecific activatable antibodies of the present disclosure are shown in Figures 5A-5D, 6A-6F, 7A-7J, 8A-8j, 9A-9J, 10A-10J, 11A-11j, and 12A-12D. According to some embodiments, the multispecific activatable antibody comprising an IgG has a masked IgG variable domain. According to some embodiments, the multispecific activatable antibody comprising an scFv has a masked scFv domain. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein each of the IgG variable domain and the scFv domain is coupled to its own masking moiety. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen, and another antibody domain has specificity for a T-cell surface antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen, and another antibody domain has specificity for another target antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for an epitope of a target antigen, and another antibody domain has specificity for another epitope of the target antigen.

[0169] In multispecific activatable antibodies, scFvs can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, the carboxyl terminus of the light chain of an IgG activatable antibody, or the carboxyl terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, the amino terminus of the light chain of an IgG activatable antibody, or the amino terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks the antigen-binding domain of an IgG. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks the antigen-binding domain of at least one scFv. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of an IgG, and a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of at least one scFv.

[0170] According to some embodiments, a single-chain variable domain specific for binding to CD3ε is fused to the carboxyl terminus of a fully human IgG antibody (targeting antibody) that binds to a cell surface antigen. The scFv fusion can be to the carboxyl terminus of the heavy chain, the carboxyl terminus of the light chain, or both chains. According to some embodiments, a single-chain variable domain specific for binding to CD3ε is fused to the amino terminus of a fully human IgG1 antibody (targeting antibody) that binds to a cell surface antigen. The scFv fusion can be to the amino terminus of the heavy chain, the amino terminus of the light chain, or both chains. The fusion is constructed as a single gene construct and expressed in cultured cells. The targeting antibody can be specific for binding to one or more tumor surface antigens, or any cells that are targeted for elimination. The scFvs are specific for the same or different antigens.

[0171] Other examples of multispecific activatable antibody structures include, but are not limited to, the following: (VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM-L1-MM)2;(VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1-MM)2;(MM-L1-CM-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*)2;(MM-L1-CM-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*)2;(VL-CL)2:(MM-L1-CM-L2-VL*- L3-VH*-L4-VH-CH1-CH2-CH3)2;(VL-CL)2:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(MM-L1-CM-L2-VL-CL)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(MM-L1-CM-L2-VL-CL)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2:(VH-CH1-CH2-CH3)2 ;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2;(VH-CH1-CH2-CH3)2;(MM-L1-CM-L2-VL*-L3-VH*-L4-VL-CL)2;(VH-CH1-CH2-CH3)2;(MM-L1-CM-L2-VH*-L3-VL*-L4-VL-CL)2;(VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2;(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2 ;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*)2:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*)2:(MM-L1-CM-L 2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VL*-L3-VH*)2:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(V L-CL-L4-VL*-L3-VH*)2:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2:( VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL- CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; or (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2, where VL and VH represent the light and heavy variable domains of a first specificity contained in an IgG; VL* and VH* represent the light and heavy variable domains of a second specificity contained in an scFv. L1 represents the variable domain; L1 is a linker peptide connecting the masking moiety (MM) and the cleavable moiety (CM); L2 is a linker peptide connecting the cleavable moiety (CM) and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is a light chain constant domain; and CH1, CH2, and CH3 are heavy chain constant domains. The first and second specificities may be directed against any antigen or epitope.

[0172] According to some embodiments of the T-cell-binding multispecific activatable antibody, one antigen is typically CD3ε and the other is an antigen present on the surface of a tumor cell or other cell type associated with the disease, such as, but not limited to, any of the targets listed in Table 1, including EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor).

[0173] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v4, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v6, which is specific for binding to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which is specific for binding to human and mouse Jagged1 and Jagged2. In some embodiments, the targeting antibody is 4D11v2, which is specific for binding to human and mouse Jagged1 and Jagged2.

[0174] In some embodiments, the targeting antibody can be in the form of an activatable antibody. In some embodiments, the scFv can be in the form of a pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0175] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds CD3ε and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55); and combinations thereof.

[0176] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds CD3ε and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from: a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 57); a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58); and combinations thereof.

[0177] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein specifically bind CD3ε and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising: a VH CDR1 sequence comprising at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence comprising at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); a VH CDR3 sequence comprising at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), a VL CDR1 sequence comprising at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence comprising at least the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0178] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein specifically bind CD3ε and comprise at least a VH CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence that 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 RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); or a VH CDR2 sequence that 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 HGNFGNSYVSWFAY (SEQ ID NO: 55). and a VL CDR3 sequence comprising at least a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence comprising at least a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence comprising at least a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0179] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence SYAMS (SEQ ID NO: 88); a VH CDR2 sequence comprising at least the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 89); a VH CDR3 sequence comprising at least the amino acid sequence DIGGRSAFDY (SEQ ID NO: 90); and combinations thereof.

[0180] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RASQSISSY (SEQ ID NO: 91); a VL CDR2 sequence comprising at least the amino acid sequence AASSLQS (SEQ ID NO: 92); a VL CDR3 sequence comprising at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 93); and combinations thereof.

[0181] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence; a VH CDR1 sequence that 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 SYAMS (SEQ ID NO: 88); a VH CDR1 sequence that 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 SIDPEGRQTYYADSVKG (SEQ ID NO: 89); a CDR2 sequence; a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 90); and combinations thereof.

[0182] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises at least a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSISSY (SEQ ID NO: 91); a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQ ID NO: 92); a CDR2 sequence; a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 93); and combinations thereof.

[0183] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises at least the amino acid sequence SYAMS (SEQ ID NO: 88), the VH CDR2 sequence comprises at least the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 89), the VH CDR3 sequence comprises at least the amino acid sequence DIGGRSAFDY (SEQ ID NO: 90), the VL CDR1 sequence comprises at least the amino acid sequence RASQSISSY (SEQ ID NO: 91), the VL CDR2 sequence comprises at least the amino acid sequence AASSLQS (SEQ ID NO: 92), and the VL CDR3 sequence comprises at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 93).

[0184] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein specifically bind a Jagged target, e.g., Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SYAMS (SEQ ID NO: 88); 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 SIDPEGRQTYYADSVKG (SEQ ID NO: 89); the CDR3 sequence comprises at least a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:90); the VL CDR1 sequence comprises at least a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSISSY (SEQ ID NO:91); the VL CDR2 sequence comprises at least a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQ ID NO:92); and the VL 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 QQTVVAPPL (SEQ ID NO: 93).

[0185] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds epidermal growth factor receptor (EGFR) and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence NYGVH (SEQ ID NO: 94); a VH CDR2 sequence comprising at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 95); a VH CDR3 sequence comprising at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 96); and combinations thereof.

[0186] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 97); a VL CDR2 sequence comprising at least the amino acid sequence AASSLQS (SEQ ID NO: 98); a VL CDR3 sequence comprising at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 99); and combinations thereof.

[0187] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence comprises at least: a VH CDR1 sequence that 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: 94); a VH CDR1 sequence that 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: 95); CDR2 sequence; a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 96); and combinations thereof.

[0188] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) that comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence comprises at least: a VL CDR1 sequence that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 97); a VL CDR1 sequence that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO: 98); a CDR2 sequence; a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 99); and combinations thereof.

[0189] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, 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:94), the VH CDR2 sequence comprises at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO:95), the VH CDR3 sequence comprises at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO:96), the VL CDR1 sequence comprises at least the amino acid sequence RASQSIGTNIH (SEQ ID NO:97), the VL CDR2 sequence comprises at least the amino acid sequence KYASESIS (SEQ ID NO:98), and the VL CDR3 sequence comprises at least the amino acid sequence QQNNNWPTT (SEQ ID NO:99).

[0190] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, 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: 94); 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: 95); the CDR3 sequence comprises at least 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:96); the VL CDR1 sequence comprises at least a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO:97); the VL CDR2 sequence comprises at least a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO:98); and the VL The CDR3 sequence includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 99).

[0191] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein. According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a light chain amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein. According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein, and a light chain amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein.

[0192] According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein. According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein. According to some embodiments, the multispecific and / or multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in the Tables provided herein.

[0193] According to some embodiments, the multispecific antibody and / or multispecific activatable antibody also includes an agent attached to AB. According to some embodiments, the agent is a therapeutic agent. According to some embodiments, the agent is a detectable moiety. According to some embodiments, the detectable moiety is a diagnostic agent. The agent is attached to the multispecific antibody via a linker. According to some embodiments, the linker is a cleavable linker. According to some embodiments, the linker is a non-cleavable linker.

[0194] According to some embodiments, the multispecific and / or multispecific activatable antibody can also comprise a detectable moiety, which, according to some embodiments, is a diagnostic agent.

[0195] According to some embodiments, the multispecific and / or multispecific activatable antibody naturally contains one or more disulfide bonds. According to some embodiments, the multispecific and / or multispecific activatable antibody can be engineered to contain one or more disulfide bonds.

[0196] The present disclosure also provides isolated nucleic acid molecules encoding at least a portion of the multispecific and / or multispecific activatable antibodies described herein, e.g., a first nucleic acid encoding at least a portion of a heavy chain of a multispecific and / or multispecific activatable antibody and a second nucleic acid encoding at least a portion of a light chain of a multispecific and / or multispecific activatable antibody, as well as vectors comprising these isolated nucleic acid molecules. The present disclosure also provides methods for producing multispecific antibodies by culturing cells containing such nucleic acid molecules under conditions conducive to expression of the antibody. According to some embodiments, the cells contain such vectors.

[0197] The present disclosure also provides multispecific antibody and / or multispecific activatable antibody compositions comprising at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that binds a second target or a second epitope, wherein at least AB1 is coupled or otherwise attached to a masking moiety (MM1) such that coupling of the masking moiety reduces the ability of AB1 to bind to its target. According to some embodiments, MM1 is coupled to AB1 via a first cleavable moiety (CM1) sequence that comprises a substrate for a protease, for example, a protease produced by a tumor present in proximity to cells expressing the target and / or produced by a tumor co-localized with the target of AB1 at a therapeutic or diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in the blood circulation, are activated at intended sites of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the AB1 target that is at least comparable to that of the corresponding unmodified multispecific antibody.

[0198] According to some embodiments, the multispecific activatable antibody comprises a linking peptide between MM1 and CM1.

[0199] According to some embodiments, the multispecific activatable antibody comprises a linking peptide between CM1 and AB1.

[0200] According to some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and at least a portion of the multispecific activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. According to some embodiments, the two connecting peptides need not be identical to each other.

[0201] According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 59) and (GGGS) n (SEQ ID NO:60), where n is at least one integer. According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:61), GGSGG (SEQ ID NO:62), GSGSG (SEQ ID NO:63), GSGGG (SEQ ID NO:64), GGGSG (SEQ ID NO:65), and GSSSG (SEQ ID NO:66).

[0202] According to some embodiments, the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds a second target or a second epitope. According to some embodiments, each AB in the multispecific activatable antibody is independently selected from the group consisting of monoclonal antibodies, domain antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single-domain heavy chain antibodies, and single-domain light chain antibodies. According to some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

[0203] According to some embodiments, each AB in the multispecific activatable antibody has a dissociation constant of about 100 nM or less for binding to its corresponding target or epitope.

[0204] In some embodiments, MM1 determines the dissociation constant, i.e., the K of binding of AB to its corresponding target or epitope. d K of binding to AB is higher than d The dissociation constant at equilibrium is

[0205] In some embodiments, MM1 determines the K of binding of AB to its corresponding target or epitope. d K of binding to AB is less than or equal to d It has.

[0206] In some embodiments, MM1 determines the K of binding of AB to its corresponding target or epitope. d is greater than or equal to K of binding to AB d It has.

[0207] In some embodiments, MM1 determines the K of binding of AB to its corresponding target or epitope. d K of binding to AB is approximately equal to d It has.

[0208] In some embodiments, MM1 determines the K of binding of AB to its corresponding target or epitope. d K of binding to AB is less than d It has.

[0209] In some embodiments, MM1 determines the K of binding of AB to its corresponding target or epitope. d In some embodiments, MM1 has a Kd for binding to AB that is no greater than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times higher than the Kd for binding of AB to its corresponding target or epitope. d has a Kd for binding to AB that is 1 to 5, 2 to 5, 2 to 10, 5 to 10, 5 to 20, 5 to 50, 5 to 100, 10 to 100, 10 to 1,000, 20 to 100, 20 to 1,000, or 100 to 1,000 times higher than

[0210] In some embodiments, MM1 has an affinity for binding to AB that is lower than the affinity for binding of AB to its corresponding target or epitope.

[0211] In some embodiments, MM1 has an affinity for binding to AB that is equal to or less than the affinity for binding of AB to its corresponding target or epitope.

[0212] In some embodiments, MM1 has an affinity for binding to AB that is approximately equal to the affinity for binding of AB to its corresponding target or epitope.

[0213] In some embodiments, MM1 has an affinity for binding to AB that is equal to or greater than the affinity for binding of AB to its corresponding target or epitope.

[0214] In some embodiments, MM1 has an affinity for binding to AB that is greater than the affinity for binding of AB to its corresponding target or epitope.

[0215] In some embodiments, the MM1 has a Kd for binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000-fold lower than the affinity of AB for binding to its corresponding target or epitope. In some embodiments, the MM has a Kd for binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000-fold lower than the affinity of AB for binding to its corresponding target or epitope. In some embodiments, the MM has an affinity for binding to AB that is 2-20-fold lower than the affinity of AB for binding to its corresponding target or epitope. In some embodiments, MM that is not covalently linked to AB and at equimolar concentrations to AB does not inhibit binding of AB to its corresponding target or epitope.

[0216] In some embodiments, MM1 does not interfere with or compete with its corresponding AB for binding to its corresponding target or epitope when the multiactivatable antibody is present in a cleaved state.

[0217] According to some embodiments, MM1 is a polypeptide about 2 to 40 amino acids in length. According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 or less amino acids in length.

[0218] According to some embodiments, MM1 has a polypeptide sequence that differs from the sequence of the target of its corresponding AB.

[0219] According to some embodiments, MM1 has a polypeptide sequence that is 50% or less identical to any of the natural binding partners of its corresponding AB. According to some embodiments, MM1 has a polypeptide sequence that is 25% or less identical to any of the natural binding partners of its corresponding AB. According to some embodiments, MM1 has a polypeptide sequence that is 10% or less identical to any of the natural binding partners of its corresponding AB.

[0220] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d Coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope so that the binding is at least 20-fold higher than the binding of the corresponding AB.

[0221] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d Coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope so that it is at least 40-fold higher than the MM1.

[0222] According to some embodiments, the dissociation constant (Kd ) is not coupled to MM1 towards its corresponding target or epitope, d Coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope so that the binding is at least 100-fold higher than the binding of MM1.

[0223] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d Coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope so that it is at least 1000-fold higher than the binding of MM1.

[0224] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d Coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope so that it is at least 10,000 times higher than the binding of MM1.

[0225] According to some embodiments, MM1 is an amino acid sequence selected from the MMs shown in the Examples provided herein.

[0226] According to some embodiments, the multispecific activatable antibody comprises at least a second masking moiety (MM2), which inhibits binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state, and a second cleavable moiety (CM2), coupled to AB2, which functions as a substrate for a second protease. According to some embodiments, CM2 is a polypeptide 15 amino acids or less in length. According to some embodiments, the second protease is produced by a tumor located in proximity to cells expressing the target and / or co-localized with the second target or epitope in a tissue, and the second protease cleaves CM2 on the multispecific activatable antibody when the multispecific activatable antibody is exposed to the second protease. According to some embodiments, the first protease and the second protease are produced by a tumor located in proximity to cells expressing the target and / or co-localized with the second target or epitope in a tissue. According to some embodiments, the first protease and the second protease are the same protease. According to some embodiments, CM1 and CM2 are different substrates of the same protease. According to some embodiments, the protease is selected from the group consisting of those shown in Table 3. According to some embodiments, the first protease and the second protease are different proteases. According to some embodiments, the first protease and the second protease are different proteases selected from the group consisting of those shown in Table 3.

[0227] In some embodiments, each MM in the multispecific activatable antibody has a dissociation constant, i.e., the K of binding of the AB to its corresponding target or epitope. d has a dissociation constant at equilibrium, the Kd for binding to AB, that is higher than

[0228] In some embodiments, each MM in the multispecific activatable antibody has a K of AB binding to its corresponding target or epitope. d K of binding to AB is less than or equal to dIt has.

[0229] In some embodiments, each MM in the multispecific activatable antibody has a K for binding to AB that is equal to or greater than the K for binding of AB to its corresponding target or epitope. d It has.

[0230] In some embodiments, each MM in the multispecific activatable antibody has a K of AB binding to its corresponding target or epitope. d K of binding to AB is approximately equal to d It has.

[0231] In some embodiments, each MM in the multispecific activatable antibody has a K of AB binding to its corresponding target or epitope. d K of binding to AB is less than d It has.

[0232] In some embodiments, each MM in the multispecific activatable antibody has a K of AB binding to its corresponding target or epitope. d K of binding to AB is not 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times higher than d In some embodiments, each MM in the multispecific activatable antibody has a K of AB binding to its corresponding target or epitope. d K of binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 times higher than d It has.

[0233] In some embodiments, each MM in the multispecific activatable antibody has an affinity for binding to AB that is lower than the affinity for binding of AB to its corresponding target or epitope.

[0234] In some embodiments, each MM in the multispecific activatable antibody has an affinity for binding to AB that is equal to or less than the affinity for binding of AB to its corresponding target or epitope.

[0235] In some embodiments, each MM in the multispecific activatable antibody has an affinity for binding to AB that is approximately equal to the affinity for binding of AB to its corresponding target or epitope.

[0236] In some embodiments, each MM in the multispecific activatable antibody has an affinity for binding to AB that is equal to or greater than the affinity for binding of AB to its corresponding target or epitope.

[0237] In some embodiments, each MM in the multispecific activatable antibody has an affinity for binding to AB that is greater than the affinity for binding of AB to its corresponding target or epitope.

[0238] In some embodiments, each MM in the multispecific activatable antibody has a K of binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times lower than the affinity of binding of AB to its corresponding target or epitope. d According to some embodiments, each MM in the multispecific activatable antibody has a K of binding to AB that is 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 times lower than the affinity of binding of AB to its corresponding target or epitope. d In some embodiments, each MM in the multispecific activatable antibody has an affinity for binding to AB that is 2-20 times lower than the affinity for binding of AB to its corresponding target or epitope. In some embodiments, a MM that is not covalently bound to AB and at an equimolar concentration to AB does not inhibit binding of AB to its corresponding target or epitope.

[0239] In some embodiments, each MM in the multispecific activatable antibody does not interfere with or compete with its corresponding AB for binding to its corresponding target or epitope when the multispecific activatable antibody is present in a cleaved state.

[0240] According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide about 2 to 40 amino acids in length. According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 amino acids or less in length.

[0241] According to some embodiments, each MM in the multispecific activatable antibody has a polypeptide sequence that differs from the sequence of the target of its corresponding AB.

[0242] According to some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 50% identical to any natural binding partner of its corresponding AB. According to some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 25% identical to any natural binding partner of its corresponding AB. According to some embodiments, each MM in a multispecific activatable antibody has a polypeptide sequence that is no more than 10% identical to any natural binding partner of its corresponding AB.

[0243] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d The coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope so that the coupling is at least 20-fold higher than the MM.

[0244] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d The coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope so that the coupling is at least 40-fold higher than the MM.

[0245] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d The coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope so that the coupling is at least 100-fold higher than the MM.

[0246] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d The coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope so that the coupling is at least 1000-fold higher than the binding of the corresponding AB.

[0247] According to some embodiments, the dissociation constant (K d ) is not coupled to MM1 towards its corresponding target or epitope, d The coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope so that the coupling is at least 10,000 times higher than the binding of the corresponding AB.

[0248] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d at least 20 times lower than

[0249] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d at least 40 times lower than

[0250] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d is at least 100 times lower than

[0251] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d at least 1,000 times lower than

[0252] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a lower dissociation constant (K d ) is the K of AB2 when coupled to MM2 d at least 10,000 times lower than

[0253] According to some embodiments, each MM is an amino acid sequence selected from the MMs shown in the examples provided herein.

[0254] According to some embodiments, a protease that cleaves the first cleavable moiety (CM1) sequence is produced by a tumor that is in proximity to cells that express the target and / or is produced by a tumor that co-localizes in a tissue with the target of AB1 in the multispecific activatable antibody, and the protease cleaves CM1 in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0255] According to some embodiments, the multispecific activatable antibody comprises multiple cleavable subsequences, and a protease that cleaves at least one cleavable subsequence is produced by a tumor that is in proximity to cells that express the target and / or is produced by a tumor that co-localizes in a tissue with the target of the AB in the multispecific activatable antibody, and the protease cleaves the CM in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0256] According to some embodiments, each CM, e.g., CM1 and at least CM2, is positioned in the multispecific activatable antibody such that under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions occurs with a dissociation constant that is at least 20-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, AB binds to its target.

[0257] According to some embodiments, each CM, e.g., CM1 and at least CM2, is positioned in the multispecific activatable antibody such that under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions occurs with a dissociation constant that is at least 40-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, AB binds to its target.

[0258] According to some embodiments, each CM, e.g., CM1 and at least CM2, is positioned in the multispecific activatable antibody such that under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions occurs with a dissociation constant at least 50-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, AB binds to its target.

[0259] According to some embodiments, each CM, e.g., CM1 and at least CM2, is positioned in the multispecific activatable antibody such that under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions occurs with a dissociation constant that is at least 100-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, AB binds to its target.

[0260] In some embodiments, each CM, e.g., CM1 and at least CM2, is positioned in the multispecific activatable antibody such that under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions occurs with a dissociation constant that is at least 200-fold higher than the dissociation constant of the unmodified AB binding to that target, while under cleaved conditions, AB binds to its target.

[0261] According to some embodiments, each CM in the multispecific activatable antibody is a polypeptide up to 15 amino acids in length.

[0262] According to some embodiments, at least one CM in the multispecific activatable antibody comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 67). According to some embodiments, at least one cleavable moiety is selected for use with a particular protease, such as a protease known to be produced by tumors located in proximity to cells expressing the target and / or co-localized with at least one target of the multispecific activatable antibody. For example, suitable cleavable moieties for use in the multispecific activatable antibodies of the present disclosure are cleaved by at least proteases such as urokinase, legumain, and / or matriptase (also referred to herein as MT-SP1 or MTSSP1). According to some embodiments, suitable cleavable moieties comprise at least one of the following sequences: TGRGPSWV (SEQ ID NO: 68); SARGPSRW (SEQ ID NO: 69); TARGPSFK (SEQ ID NO: 70); LSGRSDNH (SEQ ID NO: 67); GGWHTGRN (SEQ ID NO: 71); HTGRSGAL (SEQ ID NO: 72); PLTGRSGG (SEQ ID NO: 73); AARGPAIH (SEQ ID NO: 74); RGPAFNPM (SEQ ID NO: 75); SSRGPAYL (SEQ ID NO: 76); RGPATPIM (SEQ ID NO: 77); RGPA (SEQ ID NO: 78); GGQPSGMWGW (SEQ ID NO: 79); FPRPLGITGL (SEQ ID NO: 80); VHMPLGFLGP (SEQ ID NO: 81); SPLTGRSG (SEQ ID NO: 82); SAGFSLPA (SEQ ID NO: 83); LAPLGLQRR (SEQ ID NO: 84); SGGPLGVR (SEQ ID NO: 85); and / or PLGL (SEQ ID NO: 86).

[0263] According to some embodiments, each CM in the multispecific activatable antibody is a substrate for a protease selected from the group consisting of those shown in Table 3. According to some embodiments, the protease is selected from the group consisting of uPA, legumain, matriptase, ADAM17, BMP-1, TMPRSS3, TMPRSS4, neutrophil elastase, MMP-7, MMP-9, MMP-12, MMP-13, and MMP-14. The protease is a cathepsin, for example, but not limited to, cathepsin S. According to some embodiments, each CM in the multispecific activatable antibody is a substrate for a protease selected from the group consisting of uPA (urokinase plasminogen activator), legumain, and matriptase. According to some embodiments, the protease includes uPA. According to some embodiments, the protease includes legumain. According to some embodiments, the protease includes matriptase. According to some embodiments, the protease includes a matrix metalloproteinase (MMP).

[0264] According to some embodiments, at least one CM in the multispecific activatable antibody is a substrate for at least two proteases. According to some embodiments, each protease is selected from the group consisting of those shown in Table 3. According to some embodiments, at least one CM in the multispecific activatable antibody is a substrate for at least two proteases, where one of the proteases is selected from the group consisting of uPA, legumain, and matriptase, and the other protease is selected from the group consisting of those shown in Table 3. According to some embodiments, at least one CM in the multispecific activatable antibody is a substrate for at least two proteases selected from the group consisting of uPA, legumain, and matriptase.

[0265] According to some embodiments, at least one of the proteases capable of cleaving each CM in the multispecific activatable antibody is the same protease. According to some embodiments, the same protease capable of cleaving both CMs in the multispecific activatable antibody is selected from the group consisting of those shown in Table 3. According to some embodiments, the same protease capable of cleaving both CMs in the multispecific activatable antibody is selected from the group consisting of uPA, legumain, and matriptase.

[0266] According to some embodiments, at least two of the proteases capable of cleaving each CM in the multispecific activatable antibody are the same two proteases. According to some embodiments, the same two proteases capable of cleaving both CMs in the multispecific activatable antibody are selected from the group consisting of those shown in Table 3. According to some embodiments, at least one of the at least two proteases capable of cleaving both CMs in the multispecific activatable antibody is selected from the group consisting of uPA, legumain, and matriptase, and the other of the at least two proteases is selected from the group consisting of those shown in Table 3. According to some embodiments, the at least two proteases capable of cleaving both CMs in the multispecific activatable antibody are selected from the group consisting of uPA, legumain, and matriptase.

[0267] According to some embodiments, at least one of the proteases capable of cleaving CM1 is different from at least one of the proteases capable of cleaving CM2 in the multispecific activatable antibody. According to some embodiments, there is at least one protease capable of cleaving CM1 in the multispecific activatable antibody but not CM2. According to some embodiments, there is at least one protease capable of cleaving CM2 in the multispecific activatable antibody but not CM1. According to some embodiments, at least one of the proteases capable of cleaving CM1 in the multispecific activatable antibody but not CM2 is selected from the group consisting of those shown in Table 3. According to some embodiments, at least one of the proteases capable of cleaving CM1 in the multispecific activatable antibody but not CM2 is selected from the group consisting of uPA, legumain, and matriptase.

[0268] According to some embodiments, the multispecific activatable antibody comprises at least a first CM (CM1) and a second CM (CM2). According to some embodiments, CM1 and CM2 are part of a single cleavable linker linking the MM to the AB. According to some embodiments, CM1 is part of a cleavable linker linking MM1 to AB1, and CM2 is part of another cleavable linker linking MM2 to AB2. According to some embodiments, the multispecific activatable antibody comprises two or more CMs. According to some embodiments, such a multispecific activatable antibody comprises two or more CMs and two or more MMs. According to some embodiments, CM1 and CM2 are each polypeptides 15 or fewer amino acids in length. According to some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of those listed in Table 3. According to some embodiments, at least one of the first CM and the second CM is a polypeptide that also functions as a substrate for a protease selected from the group consisting of uPA, legumain, and matriptase. According to some embodiments, the first CM is cleaved in the target tissue by a first cleavage agent selected from the group consisting of uPA, legumain, and matriptase, and the second CM is cleaved in the target tissue by a second cleavage agent. According to some embodiments, the other protease is selected from the group consisting of those shown in Table 3. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group consisting of those listed in Table 3, and the first CM and the second CM are different substrates for the protease. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group consisting of uPA, legumain, and matriptase, and the first CM and the second CM are different substrates for the protease. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group listed in Table 3, and the first CM and the second CM are the same substrate. According to some embodiments, the first cleavage agent and the second cleavage agent are different proteases.According to some embodiments, the first cleaving agent and the second cleaving agent are different proteases selected from the group consisting of those shown in Table 3. According to some embodiments, the first cleaving agent and the second cleaving agent are produced by a tumor that is in proximity to cells that express the target and / or are produced by tumors that are co-localized in the target tissue. According to some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

[0269] According to some embodiments, the multispecific activatable antibody is exposed to a protease and cleaved such that under activated or cleaved conditions, the multispecific activatable antibody comprises a light chain amino acid sequence that comprises at least a portion of the LP2 and / or CM sequence after the protease has cleaved the CM.

[0270] According to some embodiments, the multispecific activatable antibody also comprises a signal peptide. According to some embodiments, the signal peptide is attached to the multispecific activatable antibody via a spacer. According to some embodiments, the spacer is attached to the multispecific activatable antibody in the absence of the signal peptide. According to some embodiments, the spacer is attached directly to at least one of the MMs of the multispecific activatable antibody.

[0271] According to some embodiments, the multispecific activatable antibody in its uncleaved state is directly linked to the first MM and comprises a spacer N-terminally to C-terminally having the structural arrangement spacer-MM1-CM-AB1. According to some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO: 87).

[0272] According to some embodiments, the serum half-life of the multispecific activatable antibody is longer than the half-life of its corresponding multispecific antibody; for example, the pK of the multispecific activatable antibody is longer than the pK of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is similar to the half-life of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 15 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 11 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 10 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 9 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 7 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 5 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 2 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 24 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 20 hours when administered to an organism.According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 18 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 16 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 14 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 10 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 hours when administered to an organism.

[0273] The present disclosure also provides compositions and methods including multispecific activatable antibodies comprising at least a first antibody or fragment (AB1) that specifically binds a target and a second antibody or 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. According to some embodiments, each AB is coupled to a MM that reduces the ability of its corresponding AB to bind to its respective target. For example, according to embodiments of a bispecific activatable antibody, 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. According to some embodiments, the multispecific activatable antibody comprises two or more AB regions; according to such embodiments, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind to its target, AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind to its target, AB3 is coupled to a third masking moiety (MM3) that reduces the ability of AB3 to bind to its target, and so on for each AB in the multispecific activatable antibody.

[0274] According to some embodiments, the multispecific activatable antibody further comprises at least one cleavable moiety (CM) that is a substrate for a protease, where the CM links the MM to the AB. For example, according to some embodiments, the multispecific activatable antibody comprises at least a first antibody or antibody fragment (AB1) and a second antibody or antibody fragment (AB2) that specifically bind a target, wherein at least the first AB in the multispecific activatable antibody is coupled via a cleavable moiety (CM1) to a masking moiety (MM1) that reduces the ability of AB1 to bind to its target. According to some bispecific activatable antibody embodiments, AB1 is coupled to MM1 via CM1, and AB2 is coupled via a second cleavable moiety (CM2) to a second masking moiety (MM2) that reduces the ability of AB2 to bind to its target. According to some embodiments, the multispecific activatable antibody comprises two or more AB regions; according to some of those embodiments, AB1 is coupled to MM1 via CM1, AB2 is coupled to MM2 via CM2, and AB3 is coupled via a third cleavable moiety (CM3) to a third masking moiety (MM3) that reduces the ability of AB3 to bind to its target, and similarly for each AB in the multispecific activatable antibody.

[0275] According to some embodiments, the multispecific and / or multispecific activatable antibodies described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended use, e.g., cancer. For example, the multispecific and / or multispecific activatable antibodies may be used in conjunction with additional chemotherapeutic or anti-tumor agents.

[0276] According to some embodiments, the multispecific antibody and / or multispecific activatable antibody and the additional agent are formulated into a single therapeutic composition, and the multispecific antibody and / or multispecific activatable antibody and the additional agent are administered simultaneously. According to some embodiments, the multispecific antibody and / or multispecific activatable antibody and the additional agent are separate from one another, e.g., each is formulated into a separate therapeutic composition, and the multispecific antibody and / or multispecific activatable antibody and the additional agent are administered simultaneously, or the multispecific antibody and / or multispecific activatable antibody and the additional agent are administered at different times during the therapeutic regimen. For example, the multispecific antibody and / or multispecific activatable antibody is administered before the administration of the additional agent, the multispecific antibody and / or multispecific activatable antibody is administered subsequent to the administration of the additional agent, or the multispecific antibody and / or multispecific activatable antibody are administered alternately. As described herein, the anti-multispecific antibody and / or multispecific activatable antibody and the additional agent are administered in a single dose or in multiple doses.

[0277] The present disclosure also provides isolated nucleic acid molecules encoding at least a portion of the multispecific and / or multispecific activatable antibodies described herein, e.g., a first nucleic acid encoding at least a portion of a heavy chain of the multispecific antibody and / or multispecific activatable antibody, and / or a second nucleic acid encoding at least a portion of a light chain of the multispecific antibody and / or multispecific activatable antibody, and vectors comprising those isolated nucleic acid sequences. The present disclosure also provides methods for producing multispecific and / or multispecific activatable antibodies by culturing cells comprising such nucleic acid molecules under conditions conducive to expression of the multispecific and / or multispecific activatable antibody. According to some embodiments, the cells comprise such vectors.

[0278] The present disclosure also provides methods for producing the multispecific antibodies and / or multispecific activatable antibodies of the present disclosure by (a) culturing cells containing a nucleic acid construct encoding the multispecific antibody and / or multispecific activatable antibody under conditions conducive to expression of the multispecific antibody and / or multispecific activatable antibody, and (b) recovering the multispecific antibody and / or multispecific activatable antibody.

[0279] The present disclosure also provides methods for treating, preventing, delaying the progression of, or otherwise alleviating the symptoms of, or ameliorating the symptoms associated with, one or more disease states by administering to a subject in need of treatment or prevention an anti-CD3ε antibody, activatable anti-CD3ε antibody, multispecific antibody that specifically binds CD3ε, and / or multispecific activatable antibody of the present disclosure that specifically binds CD3ε. The subject to be treated can be, for example, a human or other mammal. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), an agricultural animal, a work animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0280] The present disclosure also provides a method of inducing target-dependent T-cell activation and killing of target cells by administering a multispecific activatable antibody of the present disclosure to a subject in whom such induction is desired, wherein target-dependent T-cell activation and killing of target cells is reduced or otherwise inhibited when the multispecific activatable antibody is in a cleaved state, e.g., when each masking moiety in the multispecific activatable antibody is no longer bound to or otherwise associated with its corresponding AB domain, and when the multispecific activatable antibody is in an uncleaved state, e.g., when at least one masking moiety in the multispecific activatable antibody is bound to or otherwise associated with its corresponding AB domain. Any of the multispecific activatable antibodies described herein is suitable for use in such methods. The subject to be treated may be, for example, a human or other mammal. In some embodiments, the subject is a non-human mammal, e.g., a non-human primate, a companion animal (e.g., a cat, dog, horse), an agricultural animal, a work animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0281] The anti-CD3ε antibodies, activatable anti-CD3ε antibodies, multispecific antibodies that specifically bind CD3ε, and / or multispecific activatable antibodies of the disclosure that specifically bind CD3ε used in any of these method and use embodiments can be administered to patients at any stage. For example, such antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies can be administered to patients suffering from cancer at any stage, from early stage to metastatic. The terms subject and patient are used interchangeably herein.

[0282] Anti-CD3ε antibodies, activatable anti-CD3ε antibodies, multispecific antibodies that specifically bind CD3ε, and / or multispecific activatable antibodies that specifically bind CD3ε of the present disclosure may be used in therapeutic regimens, including neoadjuvant therapy.

[0283] Anti-CD3ε antibodies, activatable anti-CD3ε antibodies, multispecific antibodies that specifically bind CD3ε, and / or multispecific activatable antibodies of the present disclosure that specifically bind CD3ε can be administered alone or in combination with one or more additional agents, such as small molecule inhibitors, other antibody-based therapeutics, polypeptide- or peptide-based therapeutics, nucleic acid-based therapeutics, and / or other biologics. In some embodiments, the antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies are administered in combination with one or more additional agents, such as, but not limited to, chemotherapeutic agents, including alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics, and any other nucleic acid inhibitors. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, the additional agent is an antimetabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, e.g., a platinum-based chemotherapeutic agent, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, e.g., a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a targeted agent, e.g., another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteasome inhibitor, e.g., bortezomib or carfilzomib. In some embodiments, the additional agent is an immunomodulatory agent, e.g., lenalidomide or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered standard of care by those of skill in the art. In some embodiments, the additional agent is a chemotherapeutic agent known to those of skill in the art. In some embodiments, the antibody, activatable antibody, multispecific antibody, and / or multispecific activatable antibody and the additional agent(s) are formulated in a single composition. In some embodiments, the antibody, activatable antibody, multispecific antibody, and / or multispecific activatable antibody and the additional agent(s) are administered as two or more separate compositions.In some embodiments, the antibody, activatable antibody, multispecific antibody, and / or multispecific activatable antibody and the additional agent(s) are administered simultaneously. In some embodiments, the antibody, activatable antibody, multispecific antibody, and / or multispecific activatable antibody and the additional agent(s) are administered separately.

[0284] In some embodiments, the additional agent(s) is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, Abraxane (i.e., albumin-bound paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

[0285] In some embodiments, the additional agent(s) are checkpoint inhibitors, kinase inhibitors, agents that target inhibitors in the tumor microenvironment, and / or T cell or NK agonists. In some embodiments, the additional agent(s) are radiation therapy alone or in combination with another additional agent(s), such as a chemotherapeutic agent or an anti-neoplastic agent. In some embodiments, the additional agent(s) are vaccines, oncoviruses, and / or CD40-activating agents, such as, but not limited to, Toll-like receptor (TLR) agonists and / or α-CD40. In some embodiments, the additional agent(s) are tumor-targeting antibodies designed to kill tumors via ADCC or via direct conjugation to a toxin (e.g., antibody-drug conjugates (ADCs)).

[0286] In some embodiments, the checkpoint inhibitor is selected from the group consisting of CTLA-4, LAG-3, PD-1, PDL1, TIGIT, TIM-3, B7H4, 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 inhibitor, α-CSF1R inhibitor, α-CCR4 inhibitor, TGF-beta, 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. In some embodiments, the checkpoint inhibitor is an antibody that binds to a target selected from CTLA-4, PD-1, and / or PD-L1. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody, and / or a combination thereof. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, such as Yervoy™. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, such as Opdivo™ and / or Keytruda™.

[0287] 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 PDL1 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 a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T-regulatory cell.

[0288] 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.

[0289] In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activatable antibody or antigen-binding fragment thereof, and / or another conjugated activatable antibody or antigen-binding fragment thereof. In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activatable antibody or antigen-binding fragment thereof, and / or another conjugated activatable antibody or antigen-binding fragment thereof, directed against the same target as the first antibody or antigen-binding fragment thereof, the first conjugated antibody or fragment thereof, the activatable antibody or antigen-binding fragment thereof, and / or the conjugated activatable antibody or antigen-binding fragment thereof, e.g., directed against PDL1. In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activatable antibody or antigen-binding fragment thereof, and / or another conjugated activatable antibody or antigen-binding fragment thereof, directed to a target different from that of the first antibody or antigen-binding fragment thereof, the first conjugated antibody or fragment thereof, the activatable antibody or antigen-binding fragment thereof, and / or the conjugated activatable antibody or antigen-binding fragment thereof.

[0290] In some embodiments, the anti-CD3ε antibody and / or activatable antibody is administered in combination with one or more additional agents, e.g., chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressive agents, during and / or after treatment. In some embodiments, the anti-CD3ε antibody and / or activatable antibody and the additional agent are formulated into a single therapeutic composition, and the anti-CD3ε antibody and / or activatable antibody and the additional agent are administered simultaneously. Alternatively, the anti-CD3ε antibody and / or activatable antibody and the additional agent are separate from one another, e.g., each is formulated into a separate therapeutic composition, and the anti-CD3ε antibody and / or activatable antibody and the additional agent are administered simultaneously, or the anti-CD3ε antibody and / or activatable antibody and the additional agent are administered at different times during the therapeutic regimen. For example, the anti-CD3ε antibody and / or activatable antibody may be administered prior to administration of the additional agent, the anti-CD3ε antibody and / or activatable antibody may be administered subsequent to administration of the additional agent, or the anti-CD3ε antibody and / or activatable antibody and the additional agent may be administered in alternation. As described herein, the anti-CD3ε antibody and / or activatable antibody and the additional agent may be administered in a single dose, or multiple doses.

[0291] In some embodiments, the anti-CD3ε antibody and / or activatable antibody and the additional agent are administered simultaneously. For example, the anti-CD3ε antibody and / or activatable antibody and the additional agent can be formulated into a single composition or administered as multiple separate compositions. In some embodiments, the anti-CD3ε antibody and / or activatable antibody and the additional agent are administered sequentially or at different times during the treatment regimen.

[0292] The present disclosure also provides methods and kits for using anti-CD3ε antibodies, activatable anti-CD3ε antibodies, multispecific antibodies that specifically bind CD3ε, and / or multispecific activatable antibodies of the present disclosure that specifically bind CD3ε for various diagnostic and / or prophylactic indications. For example, the present disclosure provides a method for contacting an activatable antibody or multispecific activatable antibody comprising at least a first masking moiety (MM1), a first cleavable moiety (CM1) that is cleaved by a cleaving agent, and a first antigen-binding domain or fragment thereof (AB1) that specifically binds a desired target with a subject or sample, (a) wherein the MM1 is a peptide that inhibits binding of AB1 to the target, and wherein the MM1 does not have the amino acid sequence of a naturally occurring binding partner of AB1 and is not a modified form of the natural binding partner of AB1; and (b) wherein, in an uncleaved, non-activated state, the MM1 interferes with specific binding of AB1 to the target, and in a cleaved, activated state, the MM1 interferes with specific binding of AB1 to the target. and (ii) measuring the level of the activated antibody or activated multispecific activatable antibody in the subject or sample, wherein a detectable level of the activated antibody or activated multispecific activatable antibody in the subject or sample indicates that the cleavage agent and target are present in the subject or sample, and the absence of a detectable level of the activated antibody or activated multispecific activatable antibody in the subject or sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and / or insufficiently present in the subject or sample.

[0293] According to some embodiments, the activatable antibody or activatable multispecific activatable antibody is an activatable antibody or activatable multispecific activatable antibody to which a therapeutic agent is conjugated. According to some embodiments, the activatable antibody or activatable multispecific activatable antibody is not conjugated to an agent. According to some embodiments, the activatable antibody or activatable multispecific activatable antibody comprises a detectable label. According to some embodiments, the detectable label is disposed on AB1. According to some embodiments, measuring the level of the activatable antibody or activatable multispecific activatable antibody in a subject or sample is achieved using a secondary reagent that specifically binds to the activatable antibody or activatable multispecific activatable antibody and comprises a detectable label. According to some embodiments, the secondary reagent is an antibody that comprises a detectable label.

[0294] According to some embodiments of the methods and kits, the activatable antibody or multispecific activatable antibody comprises a detectable label. According to some embodiments of the methods and kits, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. According to some embodiments of the methods and kits, the imaging agent comprises a radioisotope. According to some embodiments of the methods and kits, the radioisotope is indium or technetium. According to some embodiments of the methods and kits, the contrast agent comprises iodine, gadolinium, or iron oxide. According to some embodiments of the methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. According to some embodiments of the methods and kits, 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. According to some embodiments of the methods and kits, the luminescent label comprises an N-methylacridium derivative. According to some embodiments of the methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. According to some embodiments of the methods and kits, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0295] In some embodiments of the methods and kits, the subject is a mammal. In some embodiments of the methods and kits, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), an agricultural animal, a work animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0296] According to some embodiments of these methods, the method is an in vivo method. According to some embodiments of these methods, the method is an in situ method. According to some embodiments of these methods, the method is an ex vivo method. According to some embodiments of these methods, the method is an in vitro method.

[0297] According to some embodiments of these methods, the methods or kits are used to identify or otherwise refine a patient population suitable for treatment with a multispecific activatable antibody of the present disclosure. For example, patients who test positive for both the protease that cleaves the substrate and the target in the first cleavable moiety (CM1) in the multispecific activatable antibody tested in these methods are identified as suitable candidates for treatment with an activatable antibody or multispecific activatable antibody that contains such a CM1. Similarly, patients who test negative using these methods for both the protease that cleaves the substrate and the target in the CM1 in the activatable antibody or multispecific activatable antibody can be identified as suitable candidates for another form of treatment.

[0298] According to some embodiments, methods or kits are used to identify or otherwise narrow down patient populations suitable for treatment with the activatable antibodies and / or multispecific activatable antibodies of the present disclosure, followed by treatment by administering the activatable antibodies and / or multispecific activatable antibodies to subjects in need thereof. For example, patients who test positive for both the protease that cleaves the substrate and the target at the first cleavable moiety (CM1) in the activatable antibodies and / or multispecific activatable antibodies tested in these methods are identified as suitable candidates for treatment with the activatable antibodies and / or multispecific activatable antibodies that contain such a CM1, and the patients are then administered a therapeutically effective amount of the tested activatable antibodies and / or multispecific activatable antibodies. Similarly, patients who test negative using these methods for either or both the protease that cleaves the substrate and the target at the CM1 in the activatable antibodies and / or multispecific activatable antibodies can be identified as suitable candidates for another form of treatment.

[0299] According to some embodiments, such patients may be tested with other activatable antibodies, or conjugated activatable antibodies, or multispecific activatable antibodies and / or conjugated multispecific activatable antibodies, for example, an activatable antibody, or conjugated activatable antibodies, or multispecific activatable antibodies and / or conjugated multispecific activatable antibodies, that comprise a CM that is cleaved by the patient at the site of disease, until a suitable activatable antibody, or conjugated activatable antibodies, or multispecific activatable antibodies and / or conjugated multispecific activatable antibodies for treatment is identified. According to some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody, or conjugated activatable antibody, or multispecific activatable antibody and / or conjugated multispecific activatable antibody for the patient that tests positive.

[0300] Pharmaceutical compositions of the disclosure can include the multispecific and / or multispecific activatable antibodies of the disclosure and a carrier. These pharmaceutical compositions can be included in kits, such as diagnostic kits.

[0301] Those skilled in the art will recognize that the antibodies of the present disclosure have a variety of uses. For example, the proteins of the present disclosure are used as therapeutic agents for various disorders. The antibodies of the present disclosure are also used as reagents in diagnostic kits or as diagnostic tools, or they can be used in competitive assays to generate therapeutic reagents. [Brief explanation of the drawings]

[0302] [Figure 1] FIG. 1 illustrates the general mask selection population nomenclature used herein. [Figure 2] Figures 2A and 2B are a series of graphs showing the ability of the scFv form of SP34 to bind to cynomolgus monkey CD3ε. Figure 2A shows SP34scFv(LvHv)-Fc and SP34scFv(HvLv)-Fc binding as a function of Fc fusion protein concentration. Figure 2B shows murine sP34-2 IgG1 binding as a function of concentration. [Figure 3] 3A-3D are a series of graphs showing the ability of masking moieties of the present disclosure to reduce the ability of activatable anti-CD3ε antibodies comprising such masking moieties to bind to CD3ε on Jurkat T cells. Each activatable antibody is labeled by its masking moiety name. Antibody SP34scFv(LvHv)-Fc is identified by LvHv. [Figure 4] Figures 4A, 4B, and 4C are a series of diagrams showing various antibodies and activatable antibodies of the present disclosure. Figure 4A shows constituent fragments for an scFv-Fc antibody. Figure 4B shows scFv(LvHv)-Fc and scFv(HvLv)-Fc antibodies of the present disclosure. Figure 4C shows an activating antibody comprising an scFv(LvHv)-Fc or scFv(HvLv)-Fc antibody of the present disclosure. [Figure 5]Figures 5A, 5B, 5C, and 5D are a series of diagrams illustrating multispecific antibodies and multispecific activatable antibodies of the present disclosure. Figure 5A illustrates a multispecific antibody of the present disclosure comprising an Ig antibody and an scFv. Figure 5B illustrates a multispecific antibody of the present disclosure in which the primary antibody portion is masked. Figure 5C illustrates a multispecific activatable antibody of the present disclosure in which the secondary scFv portion is masked. Figure 5D illustrates a multispecific activatable antibody of the present disclosure in which the primary antibody and secondary scFv portions are masked. [Figure 6] 6A, 6B, 6C, 6D, 6E and 6F are a series of diagrams illustrating various embodiments of multispecific activatable antibodies of the present disclosure. [Figure 7] Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 71 and 7J are a series of schematic diagrams of a selected set of possible permutations of multispecific antibodies of the present disclosure. [Figure 8] 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 81, and 8J are a series of schematic diagrams of a selected set of possible permutations of multispecific antibodies of the present disclosure. In particular, the diagrams show multispecific activatable antibodies in which the primary antigen-binding site is masked (i.e., activatable) and the additional antigen-binding domain is masked. [Figure 9] Figures 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, 91 and 9J are a series of schematic diagrams of arrays of multispecific activatable antibodies in which all antigen binding domains are masked. [Figure 10] Figures 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I and 10J are a series of schematic diagrams of arrays of multispecific activatable antibodies in which the secondary antigen-binding domain is masked and the additional antigen-binding domain(s) is / are unmasked. [Figure 11] Figures 11A, 11B, 11C, 11D, 11E, 11F, 11G, 11H, 11I and 11J are a series of schematic diagrams of arrays of multispecific activatable antibodies in which most, if not all, of the antigen-binding domains are masked and at least one additional antigen-binding domain is unmasked. [Figure 12] Figures 12A, 12B, 12C and 12D are a series of schematic diagrams of arrays of multispecific activatable antibodies in which a primary antigen-binding domain and another antigen-binding domain are masked and the remaining antigen-binding domain(s) are unmasked. [Figure 13] Figures 13A and 13B are a series of graphs showing SP34 scFv-Fc binding to CD3ε positive Jurkat cells. Figure 13A is a graph showing the ability of both orientations of SP34 scFv to bind to CD3ε Jurkat cells. Figure 13B is a graph showing binding of SP34-2 IgG. All forms of SP34 bind with similar EC50 values. [Figure 14] 14A and 14B are a series of graphs showing the ability of a masking moiety of the present disclosure to reduce the ability of an activatable anti-CD3ε antibody comprising such a masking moiety to bind to CD3ε on Jurkat T cells. [Figure 15] Figures 15A, 15B, 15C, and 15D are a series of graphs showing that a CD3ε masking peptide alters the CD3ε binding EC50 of an EGFR-masked multispecific activatable antibody utilizing two versions of SP34 scFv while retaining effective EGFR masking. [Figure 16] Figures 16A and 16B are a series of graphs showing that the cytotoxicity EC50 of CD3ε and EGFR-masked multispecific activatable antibodies was altered compared to the unmasked control, as was evident for two versions of SP34 scFv, namely, scFvs CI023 and CI024 versus scFvs CI011 and CI010. [Figure 17] Figures 17A and 17B are a series of graphs showing that for a multispecific activatable antibody, the EGFR binding EC50 is not affected by the presence or absence of both CD3ε masks; similarly, the CD3ε EC50 is not affected by the presence or absence of the EGFR mask. [Figure 18]Figures 18A and 18B are a series of graphs showing that the greatest change in cytotoxicity EC50 is obtained by masking both EGFR and CD3 binding. Furthermore, the cytotoxicity of multispecific and multispecific activatable antibodies is EGFR-dependent. [Figure 19] FIG. 19 is a graph showing that the greatest change in activation EC50 is obtained by masking both EGFR and CD3 binding. [Figure 20] Figures 20A and 20B show that CD3-masked multispecific antibodies exhibit negligible target-independent activation, as evidenced by the CD69+ frequency analysis shown in Figure 20A and the CD69 mean fluorescence intensity shown in Figure 20B. [Figure 21] Figures 21A, 21B, 21C, and 21D are a series of graphs showing that uPA activation restores binding of the multispecific activatable CI011 (M-EGFR / M-hCD3) antibody. Cell killing of all multispecific activatable antibodies is restored by uPA activation. [Figure 22] FIG. 22 is a graph showing the ability of dual-masked multispecific activatable antibodies with different substrates to vary EC50 cytotoxicity. [Figure 23] FIG. 23 is a graph showing that bispecific CI005 administered at 0.1 mg / kg twice over seven days and CI059 administered at 0.5 mg / kg / day for eight days (BiTE) prevented the growth of HT-29Luc2 xenograft tumors. [Figure 24] Figure 24 is a graph showing that CI048 antibody restricted the establishment of HT-29Luc2 xenograft tumors, and CI011 inhibited the growth of HT-29Luc2 xenograft tumors. The difference in effect between PBS and CI011 is significant (p<0.05). [Figure 25] FIG. 25 is a graph showing the dose response of CI048 bispecific antibody for the elimination of HT-29Luc2 xenograft tumors. [Figure 26]Figure 26 is a graph showing that CI048 bispecific antibody at 0.3 mg / kg eliminated xenograft tumors and CI040 bispecific activatable antibody at 0.1 mg / kg restricted the growth of HT-29Luc2 xenograft tumors. [Figure 27] Figure 27 is a graph showing that CI040 bispecific activatable antibody at 1.0 mg / kg eliminated HT-29Luc2 xenograft tumors and CI048 bispecific antibody at 0.3 mg / kg restricted the growth of HT-29Luc2 xenograft tumors. [Figure 28] FIG. 28 is a graph showing that CI011 bispecific activatable antibody at 1.0 mg / kg significantly inhibited the growth of HT-29Luc2 xenograft tumors (p<0.05). [Figure 29] FIG. 29 is a graph showing dose-response plots of CI048, CI011, and CI048 on serum alanine aminotransferase (ALT) levels 48 hours after administration. [Figure 30] FIG. 30 shows that incorporating a mouse CD3ε mask into an EGFR-masked multispecific activatable antibody alters the EC50 of binding to mouse CD3 while retaining effective EGFR masking. [Figure 31] FIG. 31 shows that killing of IL6R+ Molp-8 cells is maximally attenuated by masking both IL6R and CD3 binding. DETAILED DESCRIPTION OF THE INVENTION

[0303] The present disclosure provides antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies that bind at least the epsilon chain of CD3 (CD3ε). As used herein, an activatable antibody is an antibody that comprises a masking moiety (MM) attached to an antigen- or epitope-binding domain of the antibody such that coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind to its target. As used herein, a multispecific antibody is an antibody that recognizes multiple different antigens or epitopes, and a multispecific activatable antibody is a multispecific antibody that comprises at least one masking moiety (MM) attached 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. The activatable antibodies and / or activatable multispecific antibodies provided herein are stable in the blood circulation, are activated at the intended site of therapy and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the target at least equivalent to the corresponding unmodified multispecific antibody.

[0304] The antibodies, activated activatable antibodies, multispecific antibodies and / or activated multispecific activatable antibodies of the disclosure are < μΜ, in some embodiments, < 100nM, < 10 nM or < Binding constant (K d ) and binds to CD3ε.

[0305] The anti-CD3ε antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies of the present disclosure act to activate T cells through binding of CD3ε on T cells. That is, such antibodies agonize, stimulate, activate, and / or enhance CD3-mediated T cell activation. The biological activity of CD3 includes T cell activation and other signaling, for example, through the interaction between CD3 and the antigen-binding subunit of the T cell receptor (TCR). For example, anti-CD3ε antibodies, activating activatable antibodies, multispecific antibodies, and / or activating multispecific activatable antibodies activate T cells, completely or partially, through binding of CD3ε on T cells by agonizing, stimulating, activating, or otherwise enhancing CD3-mediated T cell activation.

[0306] Non-limiting examples of multispecific antibodies include bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and other multispecific antibodies. The multispecific antibodies provided herein are also multivalent; as used herein, multivalent refers to the total number of binding sites on the antibody, regardless of whether the binding sites recognize the same or different antigens or epitopes. Non-limiting examples of multispecific activatable antibodies include bispecific activatable antibodies, trispecific activatable antibodies, tetraspecific activatable antibodies, and other multispecific activatable antibodies. The multispecific activatable antibodies provided herein are also multivalent.

[0307] According to some embodiments, the multispecific antibodies or fragments thereof and / or multispecific activatable antibodies or fragments thereof are designed to bind to T cells and / or other immune effector cells. Multispecific activatable antibodies or fragments thereof that bind to T cells are also referred to herein as T cell-binding multispecific antibodies or fragments thereof and / or T-cell-binding multispecific activatable antibodies or fragments thereof. Multispecific activatable antibodies or fragments thereof that bind to immune effector cells are referred to herein as immune effector cell-binding multispecific antibodies or fragments thereof and / or immune effector cell-binding multispecific activatable antibodies or fragments thereof. According to some embodiments, the multispecific antibodies or fragments thereof and / or multispecific activatable antibodies or fragments thereof are designed to bind to or otherwise interact with multiple targets and / or multiple epitopes, and are also referred to herein as multi-antigen-targeting antibodies or fragments thereof and / or multi-antigen-targeting activatable antibodies or fragments thereof.

[0308] According to some embodiments, the multispecific antibody or fragment thereof comprises an IgG domain and an scFv domain. According to some embodiments, the multispecific antibody or fragment thereof comprises an IgG variable domain and a csFv domain. According to some embodiments, one antibody domain of the multispecific antibody or fragment thereof has specificity for a target antigen, and another antibody domain has specificity for a T-cell surface antigen. According to some embodiments, one antibody domain of the multispecific antibody or fragment thereof has specificity for a target antigen, and another antibody domain has specificity for a different target antigen. According to some embodiments, one antibody domain of the multispecific antibody or fragment thereof has specificity for a different epitope of the target antigen.

[0309] According to some embodiments, a multispecific activatable antibody is constructed to include a masking moiety (MM) coupled to an antibody or antigen-binding fragment thereof (AB) via a non-cleavable linker. For example, according to some embodiments, the multispecific activatable antibody is a T-cell-binding multispecific activatable antibody comprising a targeting antibody or antigen-binding fragment thereof and a T-cell-binding antibody or antigen-binding fragment thereof, wherein the T-cell-binding antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to a first T-cell-binding target, where AB1 is coupled to a masking moiety (MM1) via a non-cleavable linker such that coupling of the MM 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 antigen-binding fragment thereof that binds to a second target, where AB2 is coupled to a masking moiety (MM2) via a cleavable linker such that coupling of the MM reduces the ability of AB2 to bind to the second target.

[0310] According to some embodiments, the multispecific activatable antibody is a T-cell-binding multispecific activatable antibody comprising a targeting antibody or antigen-binding fragment thereof and a T-cell-binding antibody or antigen-binding portion thereof, wherein the T-cell-binding antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to a first T-cell-binding target, wherein AB1 is linked to a masking moiety (MM1) via a non-cleavable linker such that coupling of the MM reduces the ability of AB1 to bind to the first target, and wherein the targeting antibody or antigen-binding fragment thereof is unmasked. definition

[0311] Unless otherwise defined, scientific and technical terms used in connection with this disclosure will have the meanings commonly understood by those of ordinary skill in the art. The terms "a" or "an" entity refer to one or more entities. For example, "a compound" refers to one or more compounds. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Furthermore, unless otherwise required by context, singular terms will include plurals, and plural terms will include the singular. Generally, the nomenclature used in connection with and in connection with the cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization techniques described herein is that 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 manufacturer's specifications, or as commonly accomplished in the nuclear art, or as described herein. The foregoing techniques and procedures are generally performed according to conventional techniques well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are 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 patients.

[0312] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0313] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with, or with a lower affinity (K) with, other polypeptides. d >10 -6 ) means to bind. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, fully human, domain antibodies, single chain, Fab and F(ab')2 fragments, scFv, and a Fab expression library.

[0314] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Generally, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chain present in the molecule. Specific classes have subclasses, e.g., IgG1, IgG2, IgG3, IgG4, and others. Furthermore, in humans, the light chains can be kappa or lambda chains.

[0315] The term "monoclonal antibody" (mAb) or "monoclonal antibody composition," as used herein, refers to a population of antibody molecules containing only one species of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0316] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the H and light chain V regions, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions" or FRs. Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the 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 antigen to be bound, and each of the three hypervariable regions of the heavy and light chains is called a "complementarity-determining region" or "CDR." The amino acid assignment to each domain follows the definitions in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0317] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific conformational characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of polypeptides. Antibodies can be derived from proteins with a dissociation constant of < 1 μM, for example, according to some embodiments: < 100 nM, and according to some embodiments, < If it is 10 nM, it is said to specifically bind the antigen.

[0318] As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, where the smaller the K d represents a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measurement of the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, the "on rate constant" (K on ) and the "off rate constant" (K off Both K and K can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The ratio of α to β allows for the cancellation of all parameters not related to affinity, and the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present disclosure have an equilibrium binding constant (K d )but, < 1 μM, for example, according to some embodiments, < 100 nM, according to some embodiments < 10 nM, and according to some embodiments, < When the concentration is 100 pM to about 1 pM, it is said to specifically bind to EGFR.

[0319] The term "isolated polynucleotide," as used herein, means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an "isolated polynucleotide" is one that (1) is not associated with all or a portion of a polynucleotide with which it is found in nature, (2) is operably linked to a polynucleotide with which it is not linked in nature, or (3) is not found in nature as part of a larger sequence. Polynucleotides of the present disclosure include nucleic acid molecules encoding heavy chain immunoglobulin molecules as set forth herein, and nucleic acid molecules encoding light chain immunoglobulin molecules as set forth herein.

[0320] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; by its origin or source, an "isolated protein" is one that: (1) is not associated with a protein found in nature; (2) is free from other proteins from the same source, e.g., mouse protein; (3) is expressed by cells from a different species; or (4) does not occur in nature.

[0321] The term "polypeptide" is used herein as a genetic term to refer to a naturally occurring protein fragment or analog of a polypeptide sequence. Naturally occurring protein fragments and analogs are therefore species of the polypeptide genus. Polypeptides of the present disclosure include heavy chain immunoglobulin molecules as set forth herein, and light chain immunoglobulin molecules as set forth herein, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules together with light chain immunoglobulin molecules, e.g., kappa light chain immunoglobulins, and vice versa, and fragments and analogs thereof.

[0322] As used herein, the term "naturally-occurring," as applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified by humans, in the laboratory or otherwise, is naturally-occurring.

[0323] The term "operably linked," as used herein, means that the positions of the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0324] The term "control sequence," as used herein, refers to polynucleotide sequences necessary to affect the expression and processing of ligated coding sequences. The nature of such control sequences varies depending on the host organism in prokaryotes; generally, such control sequences include a promoter, ribosomal binding site, and transcription termination sequence in eukaryotes. The term "control sequence" is intended to include, at a minimum, all components whose presence is essential 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 used herein, refers to a sequence of nucleotides, ribonucleotides or deoxynucleotides, of at least 10 bases in length, or modified forms of either type of nucleotide. The term encompasses single- and double-stranded forms of DNA.

[0325] The term "polynucleotide," as used herein, encompasses naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides generally containing 200 or fewer bases in length. In some embodiments, oligonucleotides are 10-60 bases in length, e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length in some embodiments. Oligonucleotides are typically single-stranded, e.g., for probes, but oligonucleotides can be double-stranded, e.g., for use in constructing gene mutants. Oligonucleotides of the present disclosure can be either sense or antisense oligonucleotides.

[0326] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkages" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoronmidate, and the like. See, e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). Oligonucleotides can, if desired, contain a label for detection.

[0327] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers of the 20 conventional amino acids, unnatural amino acids, such as α-substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids (e.g., D-amino acids), may also be suitable components for the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, δ-N-methylarginine, and other similar amino acids, and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0328] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of the nascent RNA transcript is referred to as the transcription direction sequence with the DNA strand having the same sequence as the RNA, and the 5' to 5' end direction of the RNA transcript is referred to as the "upstream sequences," i.e., the region of sequences on the DNA strand having the same sequence as the RNA, and the 3' to 3' end direction of the RNA transcript is referred to as the "downstream sequences."

[0329] The term "substantial identity," as applied to polypeptides, means that two peptide sequences, when optimally aligned with the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity; for example, according to some embodiments, at least 90% sequence identity, according to some embodiments, at least 95% sequence identity, and according to some embodiments, at least 99% sequence identity.

[0330] According to some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0331] As discussed herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are intended to be encompassed by the present disclosure, provided that the variation in amino acid sequence is maintained at least 75%, e.g., according to some embodiments, at least 80%, 90%, 95%, and according to some embodiments, 99%. Conservative amino acid substitutions are particularly contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic-hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. It is reasonable to predict that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the backbone. Whether an amino acid change results in a functional peptide can be easily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by one of skill in the art. According to some embodiments, the amino- and carboxy-termini of the fragment or analog reside near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequences to public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Methods for identifying proteins that fold into known three-dimensional structures are known (Bowie et al., Science 253:164 (1991)). Thus, the foregoing examples demonstrate that one of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present disclosure.

[0332] According to some embodiments, 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 mutations of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence (in portions of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., amino acid substitutions should not tend to disrupt helices present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Principles of Proteins, Structure and Molecules (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0333] The term "polypeptide fragment," as used herein, refers to a polypeptide having an amino- and / or carboxy-terminal deletion and / or one or more internal deletions, but in which the remaining amino acid sequence is identical to the corresponding positions in a naturally occurring sequence, e.g., as deduced from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, e.g., at least 14 amino acids in length, in some embodiments, at least 20 amino acids in length, usually at least 50 amino acids in length, and in some embodiments, at least 70 amino acids in length. The term "analog," as used herein, refers to a polypeptide comprised of a segment of at least 25 amino acids that has substantial identity to a portion of the deduced amino acid sequence and has specific binding to EGFR under appropriate binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, e.g., in some embodiments at least 50 or more amino acids in length, and can often be as long as a full-length naturally occurring polypeptide.

[0334] The term "agent" is used herein to denote a compound, a mixture of compounds, a biopolymer, or an extract made from biomaterials.

[0335] 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 by attachment to the polypeptide of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected optically or colorimetrically). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., fluorophores, rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug," as used herein, refers to a compound or composition capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0336] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and the substantially purified fraction is a composition in which the target species accounts for at least about 50% (on a molar basis) of all macromolecular species present.

[0337] Generally, a substantially pure composition will comprise greater than about 80%, and in some embodiments, greater than about 85%, 90%, and 99% of all macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity (contaminating species in the composition cannot be detected by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0338] The term "patient" includes human and veterinary subjects.

[0339] Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)). Multispecific and multispecific activatable antibodies

[0340] Exemplary multispecific and / or multispecific activatable antibodies of the present disclosure include, for example, those shown in the Examples provided herein, and variants thereof.

[0341] According to some non-limiting embodiments, at least one of the ABs in the multispecific antibody is specific for CD3ε, and at least one other AB is a binding partner for any of the targets listed in Table 1. [Table 1-1] [Table 1-2]

[0342] According to some non-limiting embodiments, at least one of the ABs of the multispecific antibody is derived from a sequence shown in the examples provided herein.

[0343] According to some non-limiting embodiments, at least one of the ABs of the multispecific antibody is derived from an antibody listed in Table 2. [Table 2-1] [Table 2-2]

[0344] Exemplary antibodies, activatable antibodies, and antigen-binding fragments thereof that bind to CD3ε of the present disclosure include the CD3ε-binding sequences described in the Examples, e.g., Examples 2 and 4. Exemplary multispecific antibodies and multispecific activatable antibodies include the multispecific activatable antibodies described in the Examples, e.g., Example 4.

[0345] According to some embodiments, the multispecific activatable antibody comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544 and 546.

[0346] In some embodiments, the multispecific activatable antibody comprises a light chain sequence selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516 and 520.

[0347] According to some embodiments, the multispecific activatable antibody comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544, and 546; and a light chain sequence selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0348] According to some embodiments, the multispecific activatable antibody comprises a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544, and 546.

[0349] In some embodiments, the multispecific activatable antibody comprises a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0350] In some embodiments, the multispecific activatable antibody is selected from the group consisting of SEQ ID NOs: 446, 452, 454, 456, 460, 462, 464, 466, 470, 472, 476, 478, 480, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 510, 512, 514, 518, 524, 526, 530, 532, 534, 536, 538, 540, 542, 544 and 546. a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520; and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0351] In some embodiments, the multispecific activatable antibody comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, the multispecific activatable antibody comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:506.

[0352] Exemplary ABs that bind to CD3ε of the present disclosure include the CD3ε binding sequence shown below: CD3HvLv EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL (SEQ ID NO: 587) CD3LvHv QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 588)

[0353] In some embodiments, an activatable anti-CD3ε antibody comprises a heavy chain comprising or derived from a heavy chain amino acid sequence shown in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a light chain comprising or derived from a light chain amino acid sequence shown in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a heavy chain comprising or derived from a heavy chain amino acid sequence shown in Table 17, and a light chain comprising or derived from a light chain amino acid sequence shown in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of heavy chain variable region sequences and light chain variable region sequences from the combinations shown in Group A in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of heavy chain variable region sequences and light chain variable region sequences from the combinations shown in Group B in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of heavy chain variable region sequences and light chain variable region sequences from the combinations shown in Group C in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group D of Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group E of Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group F of Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group G of Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group H of Table 17. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of heavy chain variable region sequences and light chain variable region sequences from the combinations shown in Group I in Table 17.In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group J in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group K in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group L in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group M in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group N in Table 17. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group O in Table 17. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group P in Table 17. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group Q in Table 17.

[0354] Table 17. Variable heavy (VH) and variable light (VL) chain sequences for CD3ε-binding activatable antibodies [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12]

[0355] In some embodiments, the activatable anti-CD3ε antibody comprises or is derived from a commercially available antibody, such as an anti-CD3 antibody from Boehringer Mannheim Corp. (Indianapolis, Ind.; catalog number 1273 485).

[0356] In some embodiments, the activatable anti-CD3ε antibody comprises or is derived from an antibody produced, secreted, or otherwise generated by a hybridoma, such as the hybridoma disclosed in U.S. Pat. Nos. 4,361,549 and / or 4,658,019 and deposited with the American Type Culture Collection under accession number ATCC CRL8001OKT-3.

[0357] In some embodiments, the activatable anti-CD3ε antibody comprises or is derived from an antibody produced, secreted, or otherwise generated by a hybridoma, such as the hybridoma disclosed in PCT Publication WO 1995 / 16037 and deposited at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM ACC2152.

[0358] In some embodiments, the activatable anti-CD3ε antibody comprises or is derived from an antibody produced, secreted, or otherwise generated by a hybridoma, such as the hybridoma disclosed in PCT Publication WO 1991 / 01752 and deposited with the American Type Culture Collection under Accession No. HB10,166.

[0359] In some embodiments, an activatable anti-CD3ε antibody comprises the CDR sequences shown in Table 18, a combination of VL CDR sequences selected from the group consisting of combinations thereof shown in Table 18, and / or a combination of VH CDR sequences selected from the group consisting of combinations thereof shown in Table 18. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in Group R in Table 18. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of combinations shown in Group R in Table 18. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in Group S in Table 18. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of combinations shown in Group S in Table 18. In some embodiments, an activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in Group T in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group T in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group U in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group U in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group V in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group V in Table 18.In some embodiments, the activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group W in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group W in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group X in Table 18. In some embodiments, the activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group X in Table 18.

[0360] Table 18. Additional CDR sequences for antibodies that bind to CD3ε and activatable antibodies [Table 4-1] [Table 4-2] [Table 4-3]

[0361] The ABs in the multispecific activatable antibodies of the present disclosure specifically bind at least a mammalian target. In some embodiments, such ABs bind mammalian CD3ε. In some embodiments, such ABs bind a human target. In some embodiments, such ABs bind a non-human primate target. ABs that bind to the same epitope as the antibodies and / or activated, activatable antibodies described herein of the present disclosure are also included in the present disclosure. ABs that compete with the antibodies and / or activated, activatable antibodies described herein for binding to a target, e.g., a human target, are also included in the present disclosure. ABs that cross-compete with the antibodies and / or activated, activatable antibodies described herein for binding to a target, e.g., a human target, are also included in the present disclosure.

[0362] In some embodiments, the ABs in the multispecific activatable antibodies of the present disclosure specifically bind at least a CD3ε target, e.g., a mammalian CD3ε. In some embodiments, such ABs bind mammalian CD3ε. In some embodiments, such ABs bind human CD3ε. In some embodiments, such ABs bind non-human primate CD3ε. ABs that bind to the same CD3ε epitope as the antibodies of the present disclosure and / or the activating anti-CD3ε activatable antibodies described herein are also included in the present disclosure. ABs that compete with the anti-CD3ε antibodies and / or activating anti-CD3ε activatable antibodies described herein for binding to a CD3ε target, e.g., human CD3ε, are also included in the present disclosure. ABs that cross-compete with the anti-CD3ε antibodies and / or activating anti-CD3ε activatable antibodies described herein for binding to a CD3ε target, e.g., human CD3ε, are also included in the present disclosure.

[0363] In some embodiments, at least one of the ABs in the multispecific antibody and / or multispecific activatable antibody binds to epidermal growth factor receptor (EGFR). In some embodiments, the AB that binds to EGFR comprises one or more of the heavy and / or light chain sequences shown below.

[0364] C225v5 antibody heavy chain nucleotide sequence: C225v5 antibody heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 101)

[0365] C225v5 antibody light chain nucleotide sequence : CAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 102) C225v5 antibody light chain amino acid sequence : QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 103)

[0366] C225v4 antibody heavy chain nucleotide sequence: C225v4 antibody heavy chain amino acid sequence : QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 105)

[0367] C225v6 antibody heavy chain nucleotide sequence: C225v6 antibody heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 107)

[0368] C225 antibody heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 523)

[0369] In some embodiments, at least one of the ABs in the multispecific antibody and / or multispecific activatable antibody binds to the interleukin-6 receptor (IL-6R). In some embodiments, the AB that binds to IL-6R comprises one or more of the heavy and / or light chain sequences shown below.

[0370] Av1 antibody heavy chain amino acid sequence: QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGQGSL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108)

[0371] Av1 antibody light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 109)

[0372] In some embodiments, the AB binds to a Jagged target, e.g., Jagged 1, Jagged 2, or both. In some embodiments, the AB that binds to a Jagged target comprises one or more of the heavy and / or light chain sequences shown below.

[0373] 4D11 light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 110)

[0374] 4D11 heavy chain sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 111)

[0375] 4D11v2 heavy chain sequence: EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 112)

[0376] 4D11v2 light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113)

[0377] In some embodiments, at least one of the ABs of the multispecific antibody and / or multispecific activatable antibody binds a Jagged target and comprises one or more of the variable heavy and / or variable light chain sequences described below.

[0378] Variable light chain amino acid sequence Lc4 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 114)

[0379] Variable heavy chain amino acid sequence Hc4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 115)

[0380] Variable light chain amino acid sequence Lc5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 116)

[0381] Variable heavy chain amino acid sequence Hc5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYHGQFDYWGQGTLVTVSS (SEQ ID NO: 117)

[0382] Variable light chain amino acid sequence Lc7 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 118)

[0383] Variable heavy chain amino acid sequence Hc7 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 119)

[0384] Variable light chain amino acid sequence Lc8 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 120)

[0385] Variable heavy chain amino acid sequence Hc8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHIGRTNPFDYWGQGTLVTVSS (SEQ ID NO: 121)

[0386] Variable light chain amino acid sequence Lc13 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 122)

[0387] Variable heavy chain amino acid sequence Hc13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 123)

[0388] Variable light chain amino acid sequence Lc16 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 124)

[0389] Variable heavy chain amino acid sequence Hc16 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYYGQFDYWGQGTLVTVSS (SEQ ID NO: 125)

[0390] Variable light chain amino acid sequence Lc19 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 126)

[0391] Variable heavy chain amino acid sequence Hc19 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 127)

[0392] Variable light chain amino acid sequence Lc21 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 128)

[0393] Variable heavy chain amino acid sequence Hc21 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 129)

[0394] Variable light chain amino acid sequence Lc24 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 130)

[0395] Variable heavy chain amino acid sequence Hc24 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 131)

[0396] Variable light chain amino acid sequence Lc26 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 132)

[0397] Variable heavy chain amino acid sequence Hc26 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 133)

[0398] Variable light chain amino acid sequence Lc27 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 134)

[0399] Variable heavy chain amino acid sequence Hc27 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFYGQFDYWGQGTLVTVSS (SEQ ID NO: 135)

[0400] Variable light chain amino acid sequence Lc28 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 136)

[0401] Variable heavy chain amino acid sequence Hc28 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 137)

[0402] Variable light chain amino acid sequence Lc30 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 138)

[0403] Variable heavy chain amino acid sequence Hc30 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 139)

[0404] Variable light chain amino acid sequence Lc31 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 140)

[0405] Variable heavy chain amino acid sequence Hc31 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 141)

[0406] Variable light chain amino acid sequence Lc32 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 142)

[0407] Variable heavy chain amino acid sequence Hc32 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 143)

[0408] Variable light chain amino acid sequence Lc37 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 144)

[0409] Variable heavy chain amino acid sequence Hc37 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPHNGQFDYWGQGTLVTVSS (SEQ ID NO: 145)

[0410] Variable light chain amino acid sequence Lc39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 146)

[0411] Variable heavy chain amino acid sequence Hc39 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 147)

[0412] Variable light chain amino acid sequence Lc40 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 148)

[0413] Heavy chain amino acid sequence Hc40 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS (SEQ ID NO: 149)

[0414] Variable light chain amino acid sequence Lc47 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (SEQ ID NO: 150)

[0415] Variable heavy chain amino acid sequence Hc47 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 151)

[0416] Variable 4B2 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQGTKVEIKR (SEQ ID NO: 152)

[0417] Variable 4B2 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 153)

[0418] Variable 4D11 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKR (SEQ ID NO: 154)

[0419] Variable 4D11 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 155)

[0420] Variable 4E7 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQGTKVEIKR (SEQ ID NO: 156)

[0421] Variable 4E7 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 157)

[0422] Variable 4E11 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQGTKVEIKR (SEQ ID NO: 158)

[0423] Variable 4E11 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS (SEQ ID NO: 159)

[0424] Variable 6B7 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR (SEQ ID NO: 160)

[0425] Variable 6B7 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 161)

[0426] Variable 6F8 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR (SEQ ID NO: 162)

[0427] Variable 6F8 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS (SEQ ID NO: 163)

[0428] The antibodies and / or activatable antibodies of the present disclosure specifically bind a predetermined target, e.g., a human target protein, e.g., human CD3ε. Also included in the present disclosure are anti-CD3ε antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies that bind to the same epitope as the anti-CD3ε antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies described herein. Also included in the present disclosure are anti-CD3ε antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies that compete with the anti-CD3ε antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies described herein for binding to CD3ε, e.g., human CD3ε. Also included in the present disclosure are anti-CD3ε antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies that compete with the anti-CD3ε antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies described herein for binding to CD3ε, e.g., human CD3ε. Also included in the disclosure are anti-CD3ε antibodies, activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies that cross-compete with the anti-CD3ε antibodies, activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies described herein for binding to CD3ε, e.g., human CD3ε.

[0429] Those skilled in the art will recognize that it is possible to determine without undue experimentation if an anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody has the same or similar specificity as an anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody of the present disclosure by determining whether the former prevents binding to the target by the latter. If the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody being tested competes with the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody of the present disclosure, as indicated by decreased binding by the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody of the present disclosure, then the two anti-CD3ε antibodies bind to the same or closely related epitopes.

[0430] One embodiment for determining whether an anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody has the same or similar specificity as an anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody of the present disclosure is to pre-incubate the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody of the present disclosure with the soluble target it is normally reactive with, and then add the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody being tested to determine whether the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody being tested inhibits its ability to bind to the target. If the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody being tested is inhibited, then it likely has the same or functionally equivalent epitope specificity as the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody of the present disclosure.

[0431] Anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies are produced, for example, using the procedures described in the Examples provided below. Anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies can also be produced using any of a number of art-recognized techniques for antibody production and / or purification.

[0432] Antibody fragments, such as Fv, F(ab')2, and Fab, for use in anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies can be prepared by cleavage of the intact protein, for example, by protease or chemical cleavage. Alternatively, a truncated gene can be engineered. For example, a chimeric gene encoding a portion of the F(ab')2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the heavy chain followed by a translation stop codon to generate a truncated molecule.

[0433] Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. Convenient vectors are those encoding functionally complete human CH or CL immunoglobulin sequences with appropriate restriction sites engineered to allow for easy insertion and expression of any VH or VL sequence. In such vectors, splicing occurs between the splice donor site in the inserted J region and the splice acceptor site following the human C region, as well as at splice regions present within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. The resulting antibody can be linked to any strong promoter, such as a retroviral LTR, such as the SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. PNAS 79:6777 (1982)), and Moloney Mouse Leukemia Virus LTR (Grosschedl et al. Cell 41:885 (1985)). It will be appreciated that native Ig promoters and the like can also be used.

[0434] Furthermore, anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies can be generated via display-type technologies, including but not limited to, phage display, retroviral display, ribosome display and other techniques, using techniques well known in the art, and the resulting molecules can be subjected to further maturation, e.g., affinity maturation, e.g., techniques well known in the art. Wright et al. Crit. Reviews in Immunol. 12125-168 (1992), Hanes and Pluckthun PNAS USA 94:4937-4942 (1997) (ribosome display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol. 17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russell et al. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH;10:80-8A(1992), and U.S. Patent No. 5,733,743.

[0435] It may be desirable to modify the anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies of the present disclosure with respect to effector function to enhance or reduce such function, thereby improving the effectiveness of the antibody in recruiting CD3+ cells to one or more targets associated with the disease being treated, e.g., targets expressed on tumor cells. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. Heterodimeric antibodies thus generated have improved internalization capabilities and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). According to some embodiments, an antibody can be constructed which has dual Fc regions and thereby can have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)). According to some embodiments, Fc maturation is performed to remove glycosylation sites, thereby reducing Fc functionality. Activatable antibodies and multispecific activatable antibodies

[0436] The activatable antibodies provided herein include antibodies or antibody fragments thereof (collectively referred to throughout this disclosure as AB) that specifically bind a target and / or epitope, wherein the ABs are modified with masking moieties (MM). The multi-activatable antibodies provided herein include at least a first antibody or antibody fragment thereof (collectively referred to throughout this disclosure as AB1) that specifically binds a first target and / or a first epitope, and a second antibody or antibody fragment thereof (collectively referred to throughout this disclosure as AB2) that specifically binds a second target and / or a second epitope, wherein at least one of the ABs is modified with a masking moiety (MM). According to some embodiments, each AB in a multispecific activatable antibody is modified with its own masking moiety.

[0437] When at least one of the ABs of an activatable antibody or the ABs in a multispecific activatable antibody is modified with a MM and in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to its target.

[0438] K of MM-modified AB against target d is the K of the AB unmodified by MM or the parent AB against the target dor more than 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 or more, or between 5 and 10, 10 and 100, 10 and 1,000, 10 and 10,000, 10 and 100,000, 10 and 1,000,000, 10 and 10,000,000, 10 and 10,000,000 or more 1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of an AB modified with a MM to a target is at 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 or more, or 5-10, 10-100, 10-1,000, 10-10 ... 0 to 1,000,000, 10 to 10,000,000, 100 to 1,000, 100 to 10,000, 100 to 100,000, 100 to 1,000,000, 100 to 10,000,000, 1,000 to 10,000,000, 1,000 to 10,000,000, 10,000 to 100,000, 10,000 to 1,000,000, 10,000 to 10,000,000, 100,000 to 1,000,000 or 100,000 to 10,000,000 times lower.

[0439] The dissociation constant (K) of MM to AB in the activatable antibody d ) is generally the K of AB against the targetd The dissociation constant (K d ) is generally the K of AB against the target d Higher than AB. d is the K of AB against the target d The binding affinity of the MM to the AB is generally 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 higher than the binding affinity of the MM to the AB. Conversely, the binding affinity of the MM to the AB is generally lower than the binding affinity of the AB to the target. The binding affinity of the MM to the AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times lower than the binding affinity of the AB to the target.

[0440] When the ABs in an activatable antibody are modified with a MM and in the presence of their target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB not modified with a MM or the specific binding of the parent AB to its target. When at least one of the ABs in a multispecific activatable antibody is modified with a MM and in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB not modified with a MM or the specific binding of the parent AB to its target. The ability of the AB to bind to a target when modified with a MM, when compared to the binding of an AB not modified with a MM or the binding of the parent AB to a target, may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, as measured in an in vivo or in vitro assay.

[0441] The MM inhibits the binding of the AB in the activatable antibody to its target. The MM inhibits the binding of the AB in the multispecific activatable antibody to at least one of its targets. The MM binds the antigen-binding domain in the AB and inhibits the binding of the AB to its target. The MM can sterically inhibit the binding of the AB to its target. The MM allosterically inhibits the binding of the AB to its target. According to these embodiments, when the AB is modified or coupled to the MM and in the presence of the target, the AB binds to the MM 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, as measured in an in vivo or in vitro assay. For periods greater than this, there is no or substantially no binding of the AB to the target, or only 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% of the binding of the AB to the target compared to the binding of the AB unmodified with the MM, the parent AB, or the AB not coupled to the MM.

[0442] When the AB in an activatable antibody is coupled to or modified with a MM, the MM "masks," or reduces, or otherwise inhibits, the specific binding of the AB to its target. When the AB in an activatable antibody is coupled to or modified with a MM, such coupling or modification can result in a conformational change that reduces or inhibits the ability of the AB to specifically bind to its target.

[0443] When at least one of the ABs in a multispecific activatable antibody is coupled to or modified with a MM, the MM "masks," or reduces, or otherwise inhibits, the specific binding of the AB to its target. When at least one of the ABs in a multispecific activatable antibody is coupled to or modified with a MM, such coupling or modification can result in a conformational change that reduces or inhibits the ability of the AB to specifically bind its target.

[0444] In an activatable antibody, when at least one AB is coupled to or modified with a MM, at least a portion of the activatable antibody can be represented, in order from the amino (N)-terminal region to the carboxyl (C)-terminal region, by the following formula: (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) where MM is a masking moiety, AB is an antibody or antibody fragment thereof, and L is a linker. In many embodiments, it is desirable to include one or more linkers, e.g., flexible linkers, in the composition to provide flexibility.

[0445] In a multispecific activatable antibody, when at least one AB is coupled to or modified with a MM, at least a portion of the multispecific activatable antibody can be represented, in order from the amino (N)-terminal region to the carboxyl (C)-terminal region, by the following formula: (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) where MM is a masking moiety, AB is an antibody or antibody fragment thereof, and L is a linker. In many embodiments, it is desirable to include one or more linkers, e.g., flexible linkers, in the composition to provide flexibility.

[0446] According to certain embodiments, the MM is not a natural binding partner of AB. According to some embodiments, the MM does not include or contain homology to any natural binding partner of AB. According to other embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0447] According to some embodiments, the activatable antibody and / or multispecific activatable antibody comprises an AB modified with a MM and also comprising one or more cleavable moieties (CM). Such activatable antibodies and / or multispecific activatable antibodies exhibit activatable / switchable binding of the AB to a target. Activatable antibodies and / or multispecific activatable antibodies generally comprise at least one antibody or antibody fragment (AB) modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). According to some embodiments, the CM comprises an amino acid sequence that acts as a substrate for a protease of interest.

[0448] The elements in the activatable antibody and / or multispecific activatable antibody are positioned so that each MM and CM is positioned such that, in a cleaved (or relatively active) state and in the presence of a target, its corresponding AB binds to the target, but in an uncleaved (or relatively inactive) state in the presence of a target, specific binding of the AB to its target is reduced or inhibited. Specific binding of an AB to its target can be reduced by inhibiting or masking the ability of the AB to specifically bind its target by the MM.

[0449] K of each AB modified by MM and CM against the target d is the K of the AB unmodified by MM and CM, or the parent AB, against the target. dor more than 5, 10, 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 or more, or between 5 and 10, 10 and 100, 10 and 1,000, 10 and 10,000, 10 and 100,000, 10 and 1,000,000, 10 and 10,000,000, 10 and 10,000,000 or more 1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of each AB modified with MM and CM to the target is at 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 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100, 000, 10 to 1,000,000, 10 to 10,000,000, 100 to 1,000, 100 to 10,000, 100 to 100,000, 100 to 1,000,000, 100 to 10,000,000, 1,000 to 10,000,000, 1,000 to 100,000, 10,000 to 100,000, 10,000 to 1,000,000, 10,000 to 10,000,000, 100,000 to 1,000,000 or 100,000,000 times lower.

[0450] When at least one AB is modified with a MM and a CM and in the presence of its target but not in the presence of a modifying agent (e.g., a protease), the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM and a CM or the specific binding of the parent AB to its target. When compared to the binding of the parent AB or the binding of an AB that is not modified with a MM and CM to a target, the ability of the AB when modified with a MM and CM 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% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84 or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150 or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, as measured in an in vivo or in vitro assay.

[0451] As used herein, the term cleaved state refers to the state of an activatable antibody and / or multispecific activatable antibody after modification of the CM by a protease. As used herein, the term uncleaved state refers to the state of an activatable antibody and / or multispecific activatable antibody in the absence of cleavage of the CM by a protease. As discussed above, the term "activatable antibodies" is used herein to refer to an activatable antibody in both its uncleaved (native) state and its cleaved state. It will be apparent to those skilled in the art that, according to some embodiments, a cleaved activatable antibody lacks MM due to cleavage of the CM by a protease, resulting in the release of at least MM (e.g., when the MM is not attached to the activatable antibody by a covalent bond (e.g., a disulfite bond between cysteine ​​residues)). As used herein, the term cleaved state refers to the state of a multispecific activatable antibody after modification of the CM by a protease. The term "uncleaved state," as used herein, refers to the state of a multispecific activatable antibody in the absence of cleavage of the CM by a protease. As discussed above, the term "...

Claims

1. 1. An activatable antibody that, in its activated state, binds to the epsilon chain of CD3 (CD3ε), said activatable antibody comprising: An antibody or antigen-binding fragment thereof (AB) that specifically binds to CD3ε, (a) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 53), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54), a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56), a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 57), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 58); or (b) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO: 850), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54), a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 851), a VL CDR1 comprising the amino acid sequence GSSTGAVTSGYYPN (SEQ ID NO: 847), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 848), and a VL CDR3 comprising the amino acid sequence ALWYSNRWV (SEQ ID NO: 849); The AB; a masking moiety (MM) that inhibits binding of the AB to CD3ε when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to said AB, wherein said CM is a polypeptide that functions as a substrate for a protease; wherein (i) the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 371-391, or (ii) the AB comprises the amino acid sequence of SEQ ID NO: 587; Said activatable antibody.

2. The activatable antibody of claim 1, wherein the AB comprises the amino acid sequence of SEQ ID NO:587, and the MM has a dissociation constant for binding to the AB selected from the group consisting of: higher than the dissociation constant of the AB for CD3ε; equivalent to the dissociation constant of the AB for CD3ε; and lower than the dissociation constant of the AB for CD3ε.

3. The activatable antibody of claim 1, wherein the AB comprises the amino acid sequence of SEQ ID NO:587 and the MM does not interfere with or compete with the AB binding to CD3ε when the activatable antibody is in a cleaved state.

4. 2. The activatable antibody of claim 1, wherein the AB comprises the amino acid sequence of SEQ ID NO:587 and the MM is a polypeptide of 40 amino acids or less in length.

5. The activatable antibody of claim 1, wherein the AB comprises the amino acid sequence of SEQ ID NO:587 and the MM comprises an amino acid sequence that is different from the amino acid sequence of CD3ε, and the MM sequence is less than 50% identical to any natural binding partner of the AB.

6. The activatable antibody of claim 1, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 371-391.

7. The activatable antibody of claim 1, wherein the protease is produced by a tumor in proximity to cells expressing CD3ε in a tissue and / or is produced by a tumor that co-localizes with CD3ε in a tissue, and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

8. The activatable antibody of claim 1 , wherein the CM is a polypeptide up to 15 amino acids in length.

9. The activatable antibody of claim 1, wherein the CM is a substrate for a protease selected from the group consisting of the proteases shown in Table 3, or the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-86, 321-341 and 896-926. 【Table 3】

10. 2. The activatable antibody of claim 1, wherein the antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, and a scab.

11. 2. The activatable antibody of claim 1, wherein the activatable antibody in its uncleaved state has the following structural sequence from N-terminus to C-terminus: MM-CM-AB or AB-CM-MM.

12. The activatable antibody of claim 1 , wherein the activatable antibody comprises a linking peptide between the MM and the CM.

13. The activatable antibody of claim 1 , wherein the activatable antibody comprises a connecting peptide between the CM and the AB.

14. The activatable antibody of any one of claims 1 to 13, wherein the AB comprises the amino acid sequence of SEQ ID NO:

587.

15. The activatable antibody of claim 1 or any one of claims 6 to 13, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 371 to 391, and the AB comprises the amino acid sequence of SEQ ID NO:

588.

16. 16. The activatable antibody of any one of claims 1 to 15, comprising an agent attached to the AB, wherein the agent is a therapeutic agent, an anti-tumor agent, a toxin or fragment thereof, a detectable moiety or a diagnostic agent, and / or the agent is attached to the AB via a linker, wherein the linker is a cleavable linker or a non-cleavable linker.

17. The activatable antibody of any one of claims 1 to 16, wherein the activatable antibody is bispecific.

18. A pharmaceutical composition comprising an activatable antibody according to any one of claims 1 to 17 and a carrier.

19. 20. The pharmaceutical composition of claim 18 comprising an additional agent.

20. 20. The pharmaceutical composition of claim 19, wherein the additional agent is a therapeutic agent.

21. An isolated nucleic acid molecule encoding an activatable antibody according to any one of claims 1 to 17.

22. A vector comprising the isolated nucleic acid molecule of claim 21.

23. 23. A method for producing an activatable antibody that specifically binds at least the epsilon chain of CD3 (CD3ε), the method comprising culturing a cell under conditions that result in expression of the activatable antibody, the cell comprising the isolated nucleic acid molecule of claim 21 or the vector of claim 22.

24. An activatable antibody according to any one of claims 1 to 17, an activatable antibody produced by the method of claim 23, or a pharmaceutical composition according to any one of claims 18 to 20, for treating cancer in a subject.

25. The activatable antibody or pharmaceutical composition for treating cancer of claim 24 , wherein the subject is a human.

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