Anti-CD3 antibodies, activatable anti-CD3 antibodies, multispecific anti-CD3 antibodies, multispecific activatable anti-CD3 antibodies, and methods for 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 on 25 July 2014, the contents of which are incorporated herein by reference in their entirety. Areas of disclosure

[0003] The present invention generally relates to antibodies, activatable antibodies, multispecific antibodies, and multispecific activatable antibodies that specifically bind to at least CD3, as well as methods for producing and using such antibodies, activatable antibodies, multispecific antibodies, and multispecific activatable antibodies for various therapeutic, diagnostic, and prophylactic applications. [Background technology]

[0004] Background of 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 ε, γ, δ, and ζ chains. The CD3 complex acts as a signaling module for the T cell receptor (TCR), non-covalently associating with the antigen-binding a / b chains of the T cell receptor.

[0006] Since CD3's direct involvement leads to T cell activation, it is a desired target for various therapeutic and / or diagnostic indicators. Therefore, antibodies and therapeutic agents targeting the CD3 / TCR pathway are needed. [Overview of the project] [Problems that the invention aims to solve]

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

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

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

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

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

[0012] According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where at least one of the VH CDR1, VH CDR2, and VH CDR3 sequences 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); and VH CDR1 contains 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). The VH CDR3 sequence is selected from a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR2 sequence and the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55).

[0013] According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences is: VL CDR1 sequence containing 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); VL CDR1 sequence containing 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). The VL CDR3 sequence is selected from a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR2 sequence and the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0014] According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment is a combination of VH CDR1 sequence, VH CDR2 sequence and VH CDR3 sequence, where at least one of the VH CDR1 sequence, VH CDR2 sequence and VH CDR3 sequence is a VH CDR1 sequence containing 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); and a VH CDR1 sequence containing 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). A VH CDR3 sequence selected from a CDR2 sequence and 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 VL CDR1, VL CDR2, and VL CDR3 sequences, wherein at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences 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). The CDR1 sequence; a VL CDR2 sequence containing 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 selected from 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] According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment is 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). It includes a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence and 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] According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable heavy chain (Hv) containing the amino acid sequence of SEQ ID NO: 4. According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable light chain (Lv) containing the amino acid sequence of SEQ ID NO: 2. According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable heavy chain (Hv) containing the amino acids of SEQ ID NO: 4 and a variable light chain (Lv) containing the amino acid sequence of SEQ ID NO: 2.

[0017] According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable heavy chain (Hv) containing 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. According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable light chain (Lv) containing 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. According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable heavy chain (Hv) containing 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) containing 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.

[0018] According to some embodiments, the anti-CD3ε antibody is an scFv antibody fragment that binds to CD3ε. According to some embodiments, the anti-CD3ε scFv antibody fragment contains the amino acids of SEQ ID NO: 6. According to some embodiments, the anti-CD3ε scFv antibody fragment contains the amino acid sequence of SEQ ID NO: 30. According to some embodiments, the anti-CD3ε scFv antibody fragment contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acids of SEQ ID NO: 6. According to some embodiments, the anti-CD3ε scFv antibody fragment contains 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] According to some embodiments, the anti-CD3ε antibody also includes an agent that is conjugated to the antibody. According to some embodiments, the agent is a therapeutic agent. According to some embodiments, the agent is a detectable portion. According to some embodiments, the detectable portion is a diagnostic agent. According to some embodiments, the agent is conjugated to the anti-CD3ε 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.

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

[0021] This disclosure also provides isolated nucleic acid molecules encoding at least a portion of the anti-CD3ε antibody described herein, and / or one or more nucleic acid molecules encoding anti-CD3ε described herein, for example, 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 a vector comprising the isolated nucleic acid sequences thereof. This disclosure provides a method for producing anti-CD3ε antibodies by culturing cells containing such nucleic acid molecules(s) under conditions that result in the expression of anti-CD3ε antibodies. According to some embodiments, the cells include such vectors.

[0022] This disclosure also provides activatable antibodies and activatable antibody compositions comprising an antibody or antigen-binding fragment (AB) that is coupled to a masking component (MM) such that the coupling of MM reduces the ability of the antibody or antigen-binding fragment (AB) to bind to CD3ε, or conversely, an antibody or antigen-binding fragment (AB) that specifically binds to the bound CD3ε. These activatable antibodies are collectively referred herein as activatable anti-CD3ε antibodies and are also referred herein as anti-CD3ε activatable antibodies or CD3ε activatable antibodies. According to some embodiments, MM is coupled via a sequence containing a protease substrate. For example, the protease is produced by a tumor located in close proximity to cells expressing CD3ε. According to some embodiments, the protease is produced by a tumor co-localizing with cells expressing CD3ε. The activatable anti-CD3ε antibodies provided herein are stable in the bloodstream and are activated at the site of intended treatment and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to CD3ε at least comparable to that of the corresponding unmodified antibody.

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

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

[0025] According to some embodiments, the activatable anti-CD3ε antibody or its antigen-binding fragment is a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where at least one of the VH CDR1, VH CDR2, and VH CDR3 sequences is selected from: a VH CDR1 sequence containing at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence containing at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); and a VH CDR3 sequence containing at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55). Includes a CDR2 sequence; and a VL CDR3 sequence selected from those containing at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

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

[0027] According to some embodiments, an activatable anti-CD3ε antibody or its antigen-binding fragment comprises a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where at least one of the VH CDR1, VH CDR2, and VH CDR3 sequences 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); and the VH CDR1 sequence 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). The VH CDR3 sequence is selected from a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR2 sequence and the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55).

[0028] According to some embodiments, an activatable anti-CD3ε antibody or its antigen-binding fragment comprises a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences is: VL CDR1 sequence containing 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); VL CDR1 sequence containing 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). The VL CDR3 sequence is selected from a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR2 sequence and the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

[0029] According to some embodiments, the activatable anti-CD3ε antibody or its antigen-binding fragment is a combination of VH CDR1 sequence, VH CDR2 sequence and VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, VH CDR2 sequence and VH CDR3 sequence contains a VH CDR1 sequence which 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); and VH CDR1 sequence which 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 selected from a CDR2 sequence and 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 VL CDR1, VL CDR2, and VL CDR3 sequences, wherein at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences 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). The CDR1 sequence; a VL CDR2 sequence containing 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 selected from 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] According to some embodiments, the activatable anti-CD3ε antibody or its antigen-binding fragment is 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). It includes a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence and 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] According to some embodiments, the activatable antibody comprises a combination of variable heavy chain complementarity determination region 1 (VH CDR1, also referred to herein as CDRH1) sequence, variable heavy chain complementarity determination region 2 (VH CDR2, also referred to herein as CDRH2) sequence, variable heavy chain complementarity determination region 3 (VH CDR3, also referred to herein as CDRH3) sequence, variable light chain complementarity determination region 1 (VL CDR1, also referred to herein as CDRL1) sequence, variable light chain complementarity determination region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and variable light chain complementarity determination region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is one of the VH CDR1 sequence shown in Table 18; VH CDR2 sequence shown in Table 18; VH CDR3 sequence shown in Table 18; VL CDR1 sequence shown in Table 18; VL The sequence is selected from the group consisting of CDR2 sequences and VL CDR3 sequences shown in Table 18.

[0032] According to some embodiments, the activatable antibody comprises a combination of VH CDR1 sequence, VH CDR2 sequence, CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence, where at least one CDR sequence is a VH CDR1 sequence containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR1 sequence shown in Table 18; a VH CDR2 sequence containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VH CDR2 sequence shown in Table 18; and the VH CDR1 sequence shown in Table 18. VH CDR3 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR3 sequence; VL CDR1 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR1 sequences shown in Table 18; VL CDR2 sequences containing sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the VL CDR2 sequences shown in Table 18; and VL CDR1 sequences shown in Table 18 The selection is made from a group consisting of VL CDR3 sequences that include sequences that are identical to the CDR3 sequence by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0033] According to some embodiments, the activatable antibody comprises a combination of VH CDR1 sequence, VH CDR2 sequence, CDR3 sequence, VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence, where the combination is one of those shown in Table 18.

[0034] According to some embodiments, the activatable antibody comprises a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where each CDR sequence in the combination 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] According to some embodiments, the activatable anti-CD3ε antibody or its antigen-binding fragment comprises a variable heavy chain (Hv) containing the amino acid sequence of SEQ ID NO: 4. According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable light chain (Lv) containing the amino acid sequence of SEQ ID NO: 2. According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a variable heavy chain (Hv) containing the amino acids of SEQ ID NO: 4 and a variable light chain (Lv) containing the amino acid sequence of SEQ ID NO: 2.

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

[0037] According to some embodiments, the activatable anti-CD3ε antibody is a CD3ε-binding scFv antibody fragment. According to some embodiments, the anti-CD3ε scFv antibody fragment contains the amino acids of SEQ ID NO: 6. According to some embodiments, the anti-CD3ε scFv antibody fragment contains the amino acid sequence of SEQ ID NO: 30. According to some embodiments, the anti-CD3ε scFv antibody fragment contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acids of SEQ ID NO: 6. According to some embodiments, the anti-CD3ε scFv antibody fragment contains 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] According to some embodiments, the activatable antibody includes a heavy chain amino sequence selected from the group consisting of heavy chain sequences shown in Table 17. According to some embodiments, the activatable antibody includes a light chain amino sequence selected from the group consisting of light chain sequences shown in Table 17. According to some embodiments, the activatable antibody includes a heavy chain amino sequence selected from the group consisting of heavy chain sequences shown in Table 17, and a light chain amino sequence selected from the group consisting of light chain sequences shown in Table 17.

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

[0040] According to some embodiments, MM has a dissociation constant in equilibrium, i.e., a Kd of binding to AB that is higher than the Kd of binding to CD3ε.

[0041] According to some embodiments, MM is the K of the AB bond to CD3ε. d The following is the K bond to AB. d It holds.

[0042] According to some embodiments, MM is the K of the AB bond to CD3ε. d That concludes the explanation of K's bond to AB. d It holds.

[0043] According to some embodiments, MM is the K of the AB bond to CD3ε. d K of the bond to AB is approximately equal to d It holds.

[0044] According to some embodiments, MM is the K of the AB bond to CD3ε. d K of the bond to AB is less thand has.

[0045] According to some embodiments, MM is the K of the binding of AB to CD3ε d which is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500 or 1,000 times or less of the K of the binding to AB. d has. According to some embodiments, MM has a Kd of the 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 of the binding of AB to CD3ε.

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

[0047] According to some embodiments, MM has an affinity for binding to AB that is below the affinity for the binding of AB to CD3ε.

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

[0049] According to some embodiments, MM has an affinity for binding to AB that is above the affinity for the binding of AB to CD3ε.

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

[0051] According to some embodiments, MM has a K of the binding to AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500 or 1,000 times lower than the affinity of the binding of AB to CD3ε. dIt has. According to some embodiments, 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 AB to CD3ε. d It has the following characteristics. According to some embodiments, MM has an affinity for binding to AB that is 2 to 20 times lower than the affinity for binding AB to CD3ε. According to some embodiments, MM is not covalently bonded to AB and, at equimolar concentrations with AB, does not inhibit the binding of AB to CD3ε.

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

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

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

[0055] According to some embodiments, MM includes a sequence selected from the group consisting of sequences shown in Table 7 or 8.

[0056] According to some embodiments, the protease is produced by a tumor located near cells expressing CD3ε in a tissue, and / or by a tumor co-localizing with cells expressing CD3ε in a tissue, wherein the protease cleaves CM in the activatable antibody when the activatable antibody is exposed to the protease. According to some embodiments, CM is a polypeptide of up to 15 amino acids in length. According to some embodiments, CM is a substrate of a protease selected from the proteases listed in Table 3. According to some embodiments, CM is a substrate of a protease selected from the group consisting of uPA, regmine, matryptase (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. According to some embodiments, CM is a substrate of a protease selected from the group consisting of uPA, regmine, and matryptase. According to some embodiments, CM is a substrate of a matrix metalloproteinase (MMP).

[0057] According to some embodiments, the antigen-binding fragment is selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, scab, dAb, single-domain heavy chain antibodies, and single-domain light chain antibodies.

[0058] According to some embodiments, the activatable antibody, in its uncleaved state, has the following N-terminal to C-terminal configuration: MM-CM-AB or AB-CM-MM. According to some embodiments, the activatable antibody contains a linked peptide between MM and CM. According to some embodiments, the activatable antibody contains a linked peptide between CM and AB. According to some embodiments, the activatable antibody comprises a first linked peptide (LP1) and a second linked peptide (LP2), where the activatable antibody, in its uncleaved state, has the following N-terminal to C-terminal configuration: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. According to some embodiments, these two linked peptides do not need to be identical to each other. According to some embodiments, each of LP1 and LP2 is a peptide of approximately 1 to 20 amino acids in length.

[0059] According to some embodiments, the anti-CD3ε antibody also includes an agent that is conjugated to the antibody. According to some embodiments, the agent is a therapeutic agent. According to some embodiments, the agent is a detectable portion. According to some embodiments, the detectable portion is a diagnostic agent. According to some embodiments, the agent is conjugated to the anti-CD3ε 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.

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

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

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

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

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

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

[0066] According to some embodiments, at least one of LP1 or LP2 is (GS) n (GGS) n (GSGGS) n (Sequence No. 59) and (GGGS) n The amino acid sequence is selected from the group consisting of (SEQ ID NO: 60) (where n is at least one integer). According to some embodiments, at least one of LP1 or LP2 includes 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] According to some embodiments, the activatable antibody comprises an antibody or its antigen-binding fragment that specifically binds to CD3ε. According to some embodiments, the antibody or its antigen-binding fragment that binds to CD3ε is a monoclonal antibody, a domain antibody, a single-chain antibody, a Fab fragment, an F(ab')2 fragment, scFv, scAb,dAb, a single-domain heavy-chain antibody, and a single-domain light-chain antibody. According to some embodiments, such an antibody or its antigen-binding fragment that binds to CD3ε is a rodent (e.g., mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

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

[0069] According to some embodiments, MM is a dissociation constant, i.e., Kd of the binding of AB to CD3ε is higher than Kd of the binding of AB to AB. d It has a dissociation constant in equilibrium.

[0070] According to some embodiments, MM is the K of the AB bond to CD3ε. d The following is the K bond to AB. d It holds.

[0071] According to some embodiments, MM is the K of the AB bond to CD3ε. d That concludes the explanation of K's bond to AB. d It holds.

[0072] According to some embodiments, MM is the K of the AB bond to CD3ε. d K of the bond to AB is approximately equal to d It holds.

[0073] According to some embodiments, MM is the K of the AB bond to CD3ε. d K of the bond to AB is less than d It holds.

[0074] According to some embodiments, MM is the K of the AB bond to CD3ε. d K for bonding to AB is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500 or 1,000 times less than or equal to the K of AB. d It has. According to some embodiments, MM is the K of the bond of AB to CD3ε. d The K of the bond to AB 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 that. d It holds.

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

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

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

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

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

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

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

[0082] According to some embodiments, MM is a polypeptide of amino acids with a length of approximately 20 to 40. According to some embodiments, MM is a polypeptide of amino acids with a length of 40 or more.

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

[0084] According to some embodiments, the coupling of MM is such that the dissociation constant of AB when coupled to MM toward CD3ε is (K d ) when AB is not coupled to MM toward CD3ε d This reduces the ability of AB to bind to CD3ε to at least 20 times higher than that.

[0085] According to some embodiments, the coupling of MM is such that the dissociation constant of AB when coupled to MM toward CD3ε is (K d ) when AB is not coupled to MM toward CD3ε d This reduces the ability of AB to bind to CD3ε to at least 40 times higher than that.

[0086] According to some embodiments, the coupling of MM is such that the dissociation constant of AB when coupled to MM toward CD3ε is (K d ) when AB is not coupled to MM toward CD3ε d This reduces the ability of AB to bind to CD3ε to at least 100 times higher than that.

[0087] According to some embodiments, the coupling of MM is such that the dissociation constant of AB when coupled to MM toward CD3ε is (K d ) when AB is not coupled to MM toward CD3ε d This reduces the ability of AB to bind to CD3ε to at least 1000 times higher than before.

[0088] According to some embodiments, the coupling of MM is such that the dissociation constant of AB when coupled to MM toward CD3ε is (K d ) when AB is not coupled to MM toward CD3ε d This reduces the ability of AB to bind to CD3ε to at least 10,000 times higher than before.

[0089] According to some embodiments, when assayed in vitro in the presence of CD3ε using a target substitution assay, for example, the assay described in International Publication No. 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 CM is not cleaved compared to when CM is cleaved.

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

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

[0092] According to some embodiments, the protease is produced by a tumor located near cells expressing CD3ε in the tissue, and / or by a tumor co-localizing with cells expressing CD3ε in the tissue, and the protease cleaves CM in the activatable antibody when the activatable antibody is exposed to the protease.

[0093] According to some embodiments, the binding of the activatable antibody to CD3ε is reduced to a dissociation constant at least 20 times higher than that of unmodified AB binding to CD3ε when the activatable antibody is not cleaved, while the CM is positioned in the activatable antibody to bind to AB3ε when the activatable antibody is cleaved.

[0094] According to some embodiments, the binding of the activatable antibody to CD3ε is reduced to a dissociation constant at least 40 times higher than that of unmodified AB that binds to CD3ε when the activatable antibody is not cleaved, while the CM is positioned in the activatable antibody to bind to AB3ε when the activatable antibody is cleaved.

[0095] According to some embodiments, the binding of the activatable antibody to CD3ε is reduced to a dissociation constant at least 50 times higher than that of unmodified AB that binds to CD3ε when the activatable antibody is not cleaved, while the CM is positioned in the activatable antibody to bind to AB3ε when the activatable antibody is cleaved.

[0096] According to some embodiments, the CM is positioned within the activatable antibody such that when the activatable antibody is not cleaved, the binding of the activatable antibody to CD3ε occurs with a dissociation constant at least 100 times higher than the dissociation constant of unmodified AB that binds to CD3ε, while when the activatable antibody is cleaved, it binds to AB3ε.

[0097] According to some embodiments, the binding of the activatable antibody to CD3ε is reduced to a dissociation constant at least 200 times higher than that of unmodified AB binding to CD3ε when the activatable antibody is not cleaved, while the CM is positioned in the activatable antibody to bind to AB3ε when the activatable antibody is cleaved.

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

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

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

[0101] According to some embodiments, CM is a substrate of MMP and includes the sequence ISSGLLSS (sequence number 321); QNQALRMA (sequence number 322); AQNLLGMV (sequence number 323); STFPFGMF (sequence number 324); PVGYTSSL (sequence number 325); DWLYWPGI (sequence number 326); MIAPVAYR (sequence number 327); RPSPMWAY (sequence number 328); WATPRPMR (sequence number 329); FRLLDWQW (sequence number 330); LKAAPRWA (sequence number 331); GPSHLVLT (sequence number 332); LPGGLSPW (sequence number 333); MGLFSEAG (sequence number 334); SPLPLRVP (sequence number 335); RMHLRSLG (sequence number 336); LAAPLGLL (sequence number 337); AVGLLAPP (sequence number 338); LLAPSHRA (sequence number 339); PAGLWLDP (sequence number 340); and / or ISSGLSS (sequence number 341).

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

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

[0104] According to some embodiments, CM comprises an amino acid sequence selected from the group consisting of NTLSGRSENHSG (sequence number 898); NTLSGRSGNHGS (sequence number 899); TSTSGRSANPRG (sequence number 900); TSGRSANP (sequence number 901); VAGRSMRP (sequence number 902); VVPEGRRS (sequence number 903); ILPRSPAF (sequence number 904); MVLGRSLL (sequence number 905); QGRAITFI (sequence number 906); SPRSIMLA (sequence number 907); and SMLRSMPL (sequence number 908).

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

[0106] According to some embodiments, CM is a substrate of neutrophil elastase. According to some embodiments, CM is a substrate of serine protease. According to some embodiments, CM is a substrate of uPA. According to some embodiments, CM is a substrate of legmaine. According to some embodiments, CM is a substrate of matryptase. According to some embodiments, CM is a substrate of cysteine ​​protease. According to some embodiments, CM is a substrate of cysteine ​​protease, such as cathepsin.

[0107] According to some embodiments, CM is a CM1-CM2 substrate, and the sequence is 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 (sequence code 917); LSGRSDNHGGVHMPLGFLGP (sequence code 918); LSGRSDNHGGSGGSISSGLLSS (sequence code 919); LSGRSGNHGGSGGSISSGLLSS (sequence code 920); ISSGLLSSGGSGGSLSGRSGNH (sequence code 921); LSGRSDNHGGSGGSQNQALRMA (sequence code 922); QNQALRMAGGSGGSLSGRSDNH (sequence code 923); LSGRSGNHGGSGGSQNQALRMA (sequence code 924); QNQALRMAGGSGGSLSGRSGNH (sequence code 925) and / or ISSGLLSGRSGNH (sequence code 926).

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

[0109] According to some embodiments, CM is a substrate of 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, legmine, matryptase, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, and MMP-14. According to some embodiments, the protease is cathepsin. According to some embodiments, CM is a substrate of a protease selected from the group consisting of uPA (urokinase plasminogen activator), legmine, and matryptase. According to some embodiments, the protease is uPA. According to some embodiments, the protease is legmine. According to some embodiments, the protease includes matryptase. According to some embodiments, the protease includes matrix metalloproteinase (MMP).

[0110] According to some embodiments, CM is a substrate of 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, CM is a substrate of at least two proteases, where one protease is selected from the group consisting of uPA, legmine, and matryptase, and the other proteases are selected from the group consisting of those shown in Table 3. According to some embodiments, CM is a substrate of at least two proteases selected from the group consisting of uPA, legmine, and matryptase.

[0111] According to some embodiments, the activatable antibody comprises at least a first CM and a second CM. According to some embodiments, the first CM and the second CM are polypeptides of 15 amino acids or less in length. According to some embodiments, the first CM and the second CM in the activatable antibody, in their uncleaved state, have the following N-terminal to C-terminal configuration: MM-CM1-CM2-AB or AB-CM2-CM1-MM. 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, legmine, and matryptase. According to some embodiments, the first CM is cleaved in target tissue by a first cleavage agent selected from the group consisting of uPA, legmine, and matryptase, and the second CM is cleaved in target tissue by a second cleavage agent. According to some embodiments, the other proteases are selected from the group consisting of those shown in Table 3. According to some embodiments, the first and second cleaving agents are identical proteases selected from the group consisting of uPA, regmine, and matriptase, and the first CM and second CM are substrates different from the proteases. According to some embodiments, the first and second cleaving agents are identical proteases selected from the group listed in Table 3. According to some embodiments, the first and second cleaving agents are different proteases. According to some embodiments, the first and second cleaving agents are produced by a tumor located in close proximity to cells expressing the target and / or by a tumor co-localizing in the target tissue. According to some embodiments, the first and second CMs are cleaved in the target tissue by at least one cleaving agent.

[0112] According to some embodiments, when an activatable antibody is in an activated or cleaved state, the activatable antibody is exposed to a protease and cleaved thereby, such that the activated antibody contains light chain amino acids including at least a portion of the LP2 and / or CM sequence after the protease has cleaved the CM.

[0113] According to some embodiments, MM and CM include amino acid sequences selected from the group consisting of those sequences provided herein.

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

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

[0116] According to some embodiments, the activatable antibodies are 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, 546, 879 and Beauty 883 Includes a heavy chain selected from the group consisting of the following:

[0117] According to some embodiments, the activatable antibodies are SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516 、 520 、881 Includes a light chain selected from the group consisting of the following.

[0118] According to some embodiments, the activatable antibodies are 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 、546、879 and 883 A heavy chain selected from the group consisting of, and Sequence IDs: 448, 450, 458, 468, 474, 482, 484, 508, 516 、 520 and 881 Includes a light chain selected from the group consisting of the following.

[0119] According to some embodiments, the activatable antibody contains 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] According to 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] According to some embodiments, the 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. It comprises a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence, 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] According to some embodiments, the activatable antibody comprises the amino acid sequence of SEQ ID NO: 506. According to 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] According to some embodiments, the activatable antibody comprises the amino acid sequence of SEQ ID NO: 587 or 588. According to 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] According to some embodiments, the activatable antibody also includes an agent bound to AB. According to some embodiments, the agent is a therapeutic agent. According to some embodiments, the agent is a detectable portion. According to some embodiments, the detectable portion is a diagnostic agent. According to some embodiments, the agent is bound to AB via a linker. According to some embodiments, the linker is a cleavable linker. According to some embodiments, the linker is a non-cleavable linker.

[0125] According to some embodiments, the activatable antibody also includes a detectable portion. According to some embodiments, the detectable portion is a diagnostic agent.

[0126] According to some embodiments, the activatable antibody AB contains one or more disulfide bonds in nature. According to some embodiments, AB can be constructed to contain one or more disulfide bonds.

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

[0128] According to some embodiments, the activatable anti-CD3ε antibody is monospecific. According to some embodiments, the activatable anti-CD3ε antibody is multispecific, for example, bispecific or tripspecific in non-limiting examples. According to some embodiments, the activatable anti-CD3ε antibody is formulated as part of a pro-bispecific T cell engager (BITE) molecule. According to 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 enable the binding of one or more agents to one or more cysteine ​​residues in AB without impairing the activity (e.g., masking, activating, or binding activity) of the activatable anti-CD3ε antibody. According to some embodiments, the compositions and methods provided herein enable the binding of one or more agents to one or more cysteine ​​residues without reducing or interfering with one or more disulfide bonds in MM. The compositions and methods provided herein generate an activatable anti-CD3ε antibody that is bound to one or more agents, for example, various therapeutic, diagnostic, and / or prophylactic agents, preferably none of which are bound to the MM of the activatable anti-CD3ε antibody. The compositions and methods provided herein generate a bound activatable anti-CD3ε antibody that retains the ability to effectively and efficiently mask AB of the activatable anti-CD3ε antibody while MM is not cleaved. The compositions and methods provided herein involve an activatable antibody generating a conjugated activatable anti-CD3ε antibody that is still activated, i.e., cleaved, in the presence of a protease capable of cleaving 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 those for the intended use, e.g., in-house pharmaceuticals and / or surgical agents for cancer. For example, the anti-CD3ε antibodies and / or activatable anti-CD3ε antibodies may be used in conjunction with additional chemotherapeutic or antitumor agents.

[0131] According to some embodiments, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody, and additional agents are formulated into a single therapeutic composition, and the anti-CD3ε antibody and / or activatable anti-CD3ε antibody, and additional agents are administered simultaneously. According to some embodiments, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody, and additional agents are separate from each other, for example, each being formulated into separate therapeutic compositions, and the anti-CD3ε antibody and / or activatable anti-CD3ε antibody, and additional agents are administered simultaneously, or the anti-CD3ε antibody and / or activatable anti-CD3ε antibody, and additional agents are administered at different time points during the therapeutic regimen. For example, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody may be administered before the administration of the additional agent, or after the administration of the additional agent, or the anti-CD3ε antibody and / or activatable anti-CD3ε antibody may be administered alternately. Where described herein, the anti-CD3ε antibody and / or activatable anti-CD3ε antibody and the additional agent may be administered in single or multiple doses.

[0132] This disclosure also provides isolated nucleic acid molecules encoding at least a portion of the activatable anti-CD3ε antibodies described herein, and vectors comprising the isolated nucleic acid sequences and / or one or more nucleic acid molecules encoding the anti-CD3ε antibodies described herein, for example, 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. This disclosure provides a method for producing activatable antibodies by culturing cells containing such nucleic acid molecules under conditions that lead to the expression of the activatable antibody. According to some embodiments, the cells include such vectors.

[0133] According to some embodiments, the activatable antibodies are 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 、545、878 and 882 Encoded by a heavy chain nucleic acid sequence selected from the group consisting of the following:

[0134] According to some embodiments, the activatable antibodies are SEQ ID NOs: 447, 449, 457, 467, 473, 481, 483, 507, 515 、 519 and 880 It is encoded by a light chain nucleic acid sequence selected from the group consisting of the following:

[0135] According to some embodiments, the activatable antibodies are 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 , 545, 878 and 882 A heavy chain nucleic acid sequence selected from the group consisting of the following, and SEQ ID NOs: 447, 449, 457, 467, 473, 481, 483, 507, 515 、 519 and 880 It is encoded by a light chain nucleic acid sequence selected from the group consisting of the following:

[0136] According to 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, 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] According to 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] According to some embodiments, the activatable antibody is used in response 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. The sequences are 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 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 sequence numbers: 447, 449, 457, 467, 473, 481, 483, 507, 515, and 519.

[0139] According to some embodiments, the activatable antibody is encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 505. According to 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 disclosure also provides a method for producing an activatable antibody that binds to CD3ε in an activated state, by (a) culturing cells comprising a nucleic acid construct encoding an activatable antibody under conditions that result in the expression of the activatable antibody (the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or its antigen-binding fragment (AB) that specifically binds to CD3ε), and (b) recovering the activatable antibody.

[0141] The Disclosure also provides a method for producing an activatable antibody that binds to CD3ε in an activated state, comprising (a) culturing cells comprising a nucleic acid construct encoding an activatable antibody under conditions that result in the expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or antigen-binding fragment (AB) that specifically binds to 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, when the activatable antibody is in an uncleaved state, the MM does not interfere with or compete with the specific binding of AB to CD3ε; and (b) recovering the activatable antibody.

[0142] This disclosure also provides a multispecific antibody that binds to the epsilon chain of CD3 (CD3ε) and a second target, wherein the antibody comprises a first antibody or its antigen-binding fragment (AB1) that binds to the epsilon chain of CD3 (CD3ε), and a second antibody or its antigen-binding fragment (AB2) that binds to a second target, wherein AB1 comprises a VH CDR1 sequence containing 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); and a VH CDR1 sequence containing 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). VH CDR3 sequence containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the CDR2 sequence and the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55), and VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence combination, wherein at least one of the VL CDR1 sequence, VL CDR2 sequence and VL CDR3 sequence contains 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). The CDR1 sequence is selected from a VL CDR2 sequence containing 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 containing 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, a multispecific anti-CD3ε antibody or its antigen-binding fragment comprises a VH CDR1 sequence containing at least the amino acid sequence TYAMN (SEQ ID NO: 53); a VH CDR2 sequence containing at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 54); a VH CDR3 sequence containing at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 55); a VL CDR1 sequence containing at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 56); a VL CDR2 sequence containing at least the amino acid sequence GTNKRAP (SEQ ID NO: 57); and a VL CDR3 sequence containing at least the amino acid sequence ALWYSNLWV (SEQ ID NO: 58).

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

[0145] According to some embodiments, AB1 includes a variable heavy chain (Hv) containing 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) containing 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] According to some embodiments, AB1 includes an scFv fragment. According to some embodiments, the scFv fragment includes the amino acid sequence of SEQ ID NO: 6. According to some embodiments, the scFv fragment includes the amino acid sequence of SEQ ID NO: 30. According to some embodiments, the scFv fragment includes 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. According to some embodiments, the scFv fragment includes 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] According to some embodiments, a multispecific antibody comprises an agent conjugated to the antibody. According to some embodiments, the agent is a therapeutic agent, a detectable portion, or a diagnostic agent. According to some embodiments, the agent is conjugated to the 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.

[0148] This 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 comprising at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody, such that the coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind 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 and are activated at the site of intended therapeutic and / or diagnostic use, but not in normal, i.e., healthy tissue, and, if activated, exhibit binding to a target at least comparable to that of its corresponding unmodified multispecific antibody.

[0149] According to some embodiments, a multispecific antibody and / or multispecific activatable antibody, also referred herein as an immune effector-engaging multispecific antibody and / or immune effector-engaging multispecific activatable antibody, is designed to bind to immune effector cells. According to some embodiments, a multispecific antibody and / or multispecific activatable antibody, also referred herein as a leukocyte-binding multispecific antibody and / or leukocyte-binding multispecific activatable antibody, is designed to bind to leukocytes. According to some embodiments, a multispecific antibody and / or multispecific activatable antibody, also referred herein as a T-cell-binding multispecific antibody and / or T-cell-binding multispecific activatable antibody, is designed to bind to T cells. According to some embodiments, a multispecific antibody and / or multispecific activatable antibody binds to surface antigens on leukocytes, such as T cells, natural killer (NK) cells, bone marrow mononuclear cells, macrophages, and / or other immune effector cells. 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 cells are NK cells. According to some embodiments, the immune effector cells are mononuclear cells, such as bone marrow mononuclear cells. According to some embodiments, the T-cell-binding multispecific antibody and / or T-cell-binding multispecific activatable antibody binds to CD3ε.

[0150] According to some embodiments, the immune effector cell-binding multispecific antibody comprises a targeted antibody or its antigen-binding fragment, and an immune effector cell-binding antibody or its antigen-binding moiety containing an anti-CD3ε antibody or its antigen-binding fragment. According to some embodiments, the immune effector cell-binding multispecific antibody comprises a cancer-targeting antibody or its antigen-binding fragment, and an immune effector cell-binding antibody or its antigen-binding moiety. According to some embodiments, the immune effector cell-binding multispecific antibody comprises a cancer-targeting IgG antibody or its antigen-binding fragment, 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 an 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 its antigen-binding fragment, and a targeted antibody or its antigen-binding fragment. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε scFv, and a targeted antibody or its antigen-binding fragment. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε antibody or its antigen-binding fragment, and a cancer-targeting antibody or its antigen-binding fragment. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε scFv and a cancer-targeting antibody or its antigen-binding fragment. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε antibody or its antigen-binding fragment, and a cancer-targeting IgG antibody or its antigen-binding fragment. According to some embodiments, the T-cell-binding multispecific antibody comprises an anti-CD3ε scFv, and a cancer-targeting IgG antibody or its antigen-binding fragment. According to some embodiments, the T-cell binding multispecific antibody comprises anti-CD3 epsilon (CD3ε)scFv derived from OKT3. According to some embodiments, the T-cell binding multispecific antibody comprises anti-CD3 epsilon (CD3ε)scFv derived from SP34 (available from BD Biosciences, catalog number 556610).

[0152] According to some embodiments, the T-cell-binding multiple-specific activatable antibody comprises a targeted antibody or its antigen-binding fragment, and an anti-CD3ε antibody or its antigen-binding moiety, wherein at least one of the targeted antibody or its antigen-binding fragment and / or the anti-CD3ε antibody or its antigen-binding moiety is masked. According to some embodiments, the anti-CD3ε antibody or its antigen-binding fragment comprises a first antibody or its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound to the 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 targeted antibody or its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second target, wherein AB2 is bound to the masking moiety (MM2) such that coupling of MM2 reduces the ability of AB2 to bind to the second target. According to some embodiments, an anti-CD3ε antibody or its antigen-binding fragment comprises a first antibody or its antigen-binding fragment that binds to CD3ε, wherein AB1 is bound to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and a targeted antibody or its antigen-binding fragment comprises a second antibody or its fragment that includes a second antibody or antigen-binding fragment (AB2) that binds to a second target, wherein AB2 is bound to a masking component (MM2) such that coupling of the masking component (MM2) reduces the ability of AB2 to bind to the second target.

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

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

[0155] According to some embodiments, the T-cell-binding multiple-specific activatable antibody comprises an anti-CD3ε scFv and a targeted antibody, or its antigen-binding fragment, wherein at least one of the anti-CD3ε scFv and the targeted antibody, or its antigen-binding fragment, is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound 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 targeted antibody or its antigen-binding fragment comprises a second antibody or its fragment (AB2) that binds to a second target, wherein AB2 is bound 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 its antigenic fragment (AB1) that binds to CD3ε, wherein AB1 is bound to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the targeted antibody or its antigenic binding fragment comprises a second antibody or its fragment containing a second antibody or antigenic binding fragment (AB2) that binds to a second target, wherein AB2 is bound 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 multiple-specific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting antibody, or its antigen-binding fragment, wherein at least one of the anti-CD3ε scFv and the cancer-targeting antibody, or its antigen-binding fragment, is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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 its antigenic fragment (AB1) that binds to CD3ε, wherein AB1 is bound 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 its antigenic-binding fragment comprises a second antibody or its fragment containing a second antibody or antigenic-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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.

[0157] According to some embodiments, the T-cell-binding multiple-specific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeted IgG antibody, or an antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and the cancer-targeted IgG antibody, or the antigen-binding moiety thereof, is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound to the 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-targeted IgG antibody or its antigen-binding fragment comprises a second antibody or its fragment, comprising a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound to the 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 its antigen fragment (AB1) that binds to CD3ε, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its fragment containing a second antibody or antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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.

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

[0160] According to some embodiments, the T-cell-binding multiple-specific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv, and a targeted IgG antibody or its antigen-binding fragment, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeted IgG antibody or its antigen-binding moiety is masked. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound to the 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-targeted IgG antibody or its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound to the 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 its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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.

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

[0163] According to some embodiments, the T-cell-binding multiple-specific activatable antibody comprises an SP34 scFv or an SP34-derived scFv and a cancer-targeting IgG antibody or its antigen-binding fragment, wherein at least one of the SP34 scFv or SP34-derived scFv and / or the cancer-targeting IgG antibody or its antigen-binding fragment is masked. According to some embodiments, the SP34 scFv or SP34-derived scFv comprises a first antibody or its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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 its antigen-binding fragment (AB1) that binds to CD3ε, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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.

[0164] According to some embodiments, the T-cell-binding multispecifically 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 targeted antibody or its antigen-binding fragment and / or anti-CD3ε scFv is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or its antigen-binding fragment (AB1) having the CDR sequences shown above, i.e., SEQ ID NOs: 53, 54, 55, 56, 57, and 58, wherein AB1 is bound 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 targeted antibody or its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second target, wherein AB2 is bound 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 its antigen-binding fragment (AB1) containing the CDR sequences of SEQ ID NOs. 53, 54, 55, 56, 57, and 58, wherein AB1 is bound to a masking moiety (MM1) such that coupling of the masking moiety (MM1) reduces the ability of AB1 to bind to CD3ε, and the targeted antibody or its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second target, wherein AB2 is bound to a masking moiety (MM2) such that 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 multiple-specific activatable antibody comprises an anti-CD3ε scFv containing the CDR sequences of SEQ ID NOs. 53, 54, 55, 56, 57, and 58, and a cancer-targeting antibody or its antigen-binding fragment, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting antibody or its antigen-binding fragment is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or its antigen-binding fragment (AB1) containing the CDR sequences of SEQ ID NOs. 53, 54, 55, 56, 57, and 58, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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 its antigen-binding fragment (AB1) containing the CDR sequences of SEQ ID NOs. 53, 54, 55, 56, 57, and 58, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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.

[0166] According to some embodiments, the T-cell-binding multiple-specific activatable antibody comprises an anti-CD3ε scFv containing the CDR sequences of SEQ ID NOs. 53, 54, 55, 56, 57, and 58, and a cancer-targeting IgG antibody or its antigen-binding fragment, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting IgG antibody or its antigen-binding fragment is masked. According to some embodiments, the anti-CD3ε scFv comprises a first antibody or its antigen-binding fragment (AB1) containing the CDR sequences of SEQ ID NOs. 53, 54, 55, 56, 57, and 58, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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 its antigen-binding fragment (AB1) containing the CDR sequences of SEQ ID NOs. 53, 54, 55, 56, 57, and 58, wherein AB1 is bound 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 its antigen-binding fragment comprises a second antibody or its antigen-binding fragment (AB2) that binds to a second cancer-associated target, wherein AB2 is bound 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.

[0167] According to some embodiments, a multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody comprises at least a first antibody or its antigen-binding fragment that binds to a first target and / or a first epitope, and a second antibody or its antigen-binding fragment that binds to a second target and / or a second epitope, where at least one of the targets and / or epitopes is CD3ε. According to some embodiments, a multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different targets, where at least one of the targets is CD3ε. According to some embodiments, a multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different epitopes on CD3ε. According to some embodiments, a multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to a combination of two or more different targets and two or more different epitopes on the same target, where at least one of the targets and / or epitopes is CD3ε.

[0168] Various embodiments of the multispecifically activatable antibodies of this 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, a multispecifically activatable antibody containing IgG has a masked IgG-potential domain. According to some embodiments, a multispecifically activatable antibody containing scFv has a masked scFv domain. According to some embodiments, the multispecifically activatable antibody has both an IgG-variable domain and an scFv domain, where at least one of the IgG-variable domains is coupled to the masking portion. According to some embodiments, the multispecifically activatable antibody has both an IgG-variable domain and an scFv domain, where at least one of the scFv domains is coupled to the masking portion. According to some embodiments, the multispecifically activatable antibody has both an IgG-variable domain and an scFv domain, where at least one of the IgG-variable domains is coupled to the masking portion, and at least one of the scFv domains is coupled to the masking portion. According to some embodiments, the multispecifically activatable antibody has both an IgG variable domain and an scFv domain, where each of the IgG variable domain and the scFv domain is coupled to its own masking portion. According to some embodiments, one antibody domain of the multispecifically 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 multispecifically 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 multispecifically activatable antibody has specificity for an epitope of the target antigen, and another antibody domain has specificity for another epitope of the target antigen.

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

[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 (targeted antibody) that binds to a cell surface antigen. The scFv fusion may 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 (targeted antibody) that binds to a cell surface antigen. The scFv fusion may 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 targeted antibody may be specific to binding to one or more tumor surface antigens, or to any cells that are targeted for removal. The scFv is specific to the same or different antigens.

[0171] Other examples of multi-specifically 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 first specificity light and heavy variable domains contained in IgG; VL* and VH* represent the second specificity variable domains contained in scFv. The variable domains are represented; L1 is a linker peptide that links the masking portion (MM) and the cleavable portion (CM); L2 is a linker peptide that links the cleavable portion (CM) and the antibody; L3 is a linker peptide that links the variable domain of scFv; L4 is a linker peptide that links the first-specific antibody to the second-specific antibody; CL is the light chain constant domain; and CH1, CH2, and CH3 are the heavy chain constant domains. The first and second specificities may be for any antigen or epitope.

[0172] According to some embodiments of T-cell-binding multispecific activatable antibodies, one antigen is typically CD3ε, and the others are antigens present on the surface of tumor cells or other disease-associated cell types, such as any target listed in Table 1, e.g., EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), but are not limited to these.

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

[0174] According to some embodiments, the targeted antibody may exist in the form of an activatable antibody. According to some embodiments, the scFv may exist in the form of a pro-scFv (see, for example, International Publication No. 2009 / 025846 and International Publication No. 2010 / 081173).

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

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

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

[0179] According to some embodiments, the multispecific antibodies and / or multispecifically activatable antibodies provided herein include at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to Jagged targets, e.g., Jagged1 and / or Jagged2, and comprises a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, where at least one of the VH CDR1, VH CDR2, and VH CDR3 sequences 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 antibodies and / or multispecifically activatable antibodies provided herein include at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to Jagged targets, e.g., Jagged1 and / or Jagged2, and includes a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, where at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences is selected from: a VL CDR1 sequence containing at least the amino acid sequence RASQSISSY (SEQ ID NO: 91); a VL CDR2 sequence containing at least the amino acid sequence AASSLQS (SEQ ID NO: 92); a VL CDR3 sequence containing at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 93); and combinations thereof.

[0181] According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies provided herein include at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to Jagged targets, e.g., Jagged1 and / or Jagged2, and includes a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, wherein at least one of the VH CDR1, VH CDR2, and VH CDR3 sequences is: VH CDR1 sequence containing 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 SYAMS (SEQ ID NO: 88); and VH CDR1 sequence containing 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 SIDPEGRQTYYADSVKG (SEQ ID NO: 89). A CDR2 sequence; a VH CDR3 sequence containing 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 are selected.

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

[0183] According to some embodiments, the multispecific antibodies and / or multispecifically activatable antibodies provided herein include at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to Jagged targets, such as Jagged1 and / or Jagged2, and comprises a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where 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 antibodies and / or multispecific activatable antibodies provided herein include at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to Jagged targets, e.g., Jagged1 and / or Jagged2, and includes a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences, where the VH CDR1 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 SYAMS (SEQ ID NO: 88); the VH CDR2 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 SIDPEGRQTYYADSVKG (SEQ ID NO: 89); VH The CDR3 sequence contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 90); the VL CDR1 sequence contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences to the amino acid sequence RASQSISSY (SEQ ID NO: 91); the VL CDR2 sequence contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences to the amino acid sequence AASSLQS (SEQ ID NO: 92); and VL The CDR3 sequence contains at least a sequence that is identical to the amino acid sequence QQTVVAPPL (SEQ ID NO: 93) by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0185] According to some embodiments, the multispecific antibodies and / or multispecifically activatable antibodies provided herein include at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to the epidermal growth factor receptor (EGFR) and comprises a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, wherein at least one of the VH CDR1, VH CDR2, and VH CDR3 sequences is selected from: 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 antibodies and / or multispecifically activatable antibodies provided herein include at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to EGFR and includes a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, wherein at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences is selected from: a VL CDR1 sequence containing at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 97); a VL CDR2 sequence containing at least the amino acid sequence AASSLQS (SEQ ID NO: 98); a VL CDR3 sequence containing at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 99); and combinations thereof.

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

[0188] According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies provided herein specifically bind to EGFR and comprise at least a first antibody or its antigen-binding fragment (AB1) comprising a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, wherein at least one of the VL CDR1, VL CDR2, and VL CDR3 sequences is: VL CDR1 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 RASQSIGTNIH (SEQ ID NO: 97); VL CDR1 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 KYASESIS (SEQ ID NO: 98). A CDR2 sequence; a VL CDR3 sequence containing 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 are selected.

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

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

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

[0192] According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies provided herein include 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 table provided herein. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies provided herein include 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 table provided herein. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies provided herein include 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 table 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 table provided herein.

[0193] According to some embodiments, the multispecific antibody and / or multispecifically activatable antibody also includes an agent bound to AB. According to some embodiments, the agent is a therapeutic agent. According to some embodiments, the agent is a detectable portion. According to some embodiments, the detectable portion is a diagnostic agent. The agent is bound 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, a multispecific antibody and / or a multispecific activatable antibody may also include a detectable portion. According to some embodiments, the detectable portion is a diagnostic agent.

[0195] According to some embodiments, multispecific antibodies and / or multispecifically activatable antibodies naturally contain one or more disulfide bonds. According to some embodiments, multispecific antibodies and / or multispecifically activatable antibodies can be manipulated to contain one or more disulfide bonds.

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

[0197] The disclosure also provides a multispecific antibody and / or multispecifically activatable antibody composition comprising at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to a first target or first epitope, and a second antibody or its antigen-binding fragment (AB2) that binds to a second target or second epitope, wherein at least AB1 is coupled to or otherwise attached to a masking moiety (MM1) such that the coupling of the masking moiety (MM1) 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 containing a substrate for a protease, for example, a protease produced by a tumor located in close proximity to cells expressing the target, and / or a protease produced by a tumor co-localizing with the target of AB1 at the therapeutic or diagnostic site in the subject. The multispecifically activatable antibodies provided herein are stable in the bloodstream and are activated at the site of intended treatment and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the AB1 target at least comparable to that of the corresponding unmodified multispecific antibody.

[0198] According to some embodiments, the multispecifically activatable antibody includes a linked peptide between MM1 and CM1.

[0199] According to some embodiments, the multispecifically activatable antibody contains a linked peptide between CM1 and AB1.

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

[0201] According to some embodiments, at least one of LP1 or LP2 is (GS) n (GGS) n (GSGGS) n (Sequence No. 59) and (GGGS) n The amino acid sequence is selected from the group consisting of (SEQ ID NO: 60) (where n is at least one integer). According to some embodiments, at least one of LP1 or LP2 includes 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 multispecifically activatable antibody comprises at least a first antibody or its antigen-binding fragment (AB1) that specifically binds to a first target or first epitope, and a second antibody or its antibody-binding fragment (AB2) that specifically binds to a second target or second epitope. According to some embodiments, each AB in the multispecifically activatable antibody is independently selected from the group consisting of monoclonal antibodies, domain antibodies, single-chain antibodies, Fab fragments, F(ab')2 fragments, scFv, scAb, dAb, single-domain heavy-chain antibodies, and single-domain light-chain antibodies. According to some embodiments, each AB in the multispecifically 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 a multispecifically activatable antibody has a dissociation constant of about 100 nM or less in order to bind to its corresponding target or epitope.

[0204] According to some embodiments, MM1 is the dissociation constant, i.e., the K of the binding of AB to its corresponding target or epitope. d K of the bond to AB is higher than d It has a dissociation constant in equilibrium.

[0205] According to some embodiments, MM1 is the binding of AB to its corresponding target or epitope. d The following is the K bond to AB. d It holds.

[0206] According to some embodiments, MM1 is the binding of AB to its corresponding target or epitope. d That concludes the explanation of K's bond to AB. d It holds.

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

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

[0209] According to some embodiments, MM1 is the binding of AB to its corresponding target or epitope. d The Kd of binding to AB is not 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times higher than . According to some embodiments, MM1 has a K of binding of AB to its corresponding target or epitope. d It has a Kd 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 [the specified value].

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

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

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

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

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

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

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

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

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

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

[0220] According to some embodiments, the dissociation constant (K) of AB when coupled to MM1 toward its corresponding target or epitope is determined by the following mechanism. d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d The coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope by at least 20 times.

[0221] According to some embodiments, the dissociation constant (K) of AB when coupled to MM1 toward its corresponding target or epitope is determined by the following mechanism. d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d The coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope by at least 40 times.

[0222] According to some embodiments, the dissociation constant (K) of AB when coupled to MM1 toward its corresponding target or epitope is determined by the following mechanism.d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d The coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope by at least 100 times.

[0223] According to some embodiments, the dissociation constant (K) of AB when coupled to MM1 toward its corresponding target or epitope is determined by the following mechanism. d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d The coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope by at least 1000 times.

[0224] According to some embodiments, the dissociation constant (K) of AB when coupled to MM1 toward its corresponding target or epitope is determined by the following mechanism. d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d The coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope by at least 10,000 times.

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

[0226] According to some embodiments, the multispecific activatable antibody comprises at least a second masking moiety (MM2) that inhibits the binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state, and a second cleavable moiety (CM2) coupled to AB2 that functions as a substrate for a second protease. According to some embodiments, CM2 is a polypeptide of 15 or fewer amino acids in length. According to some embodiments, the second protease is produced by a tumor present in proximity to a cell expressing the target and / or by a tumor co-existing with a second target or epitope in a tissue, and the second protease cleaves CM2 in 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 present in proximity to a cell expressing the target and / or by a tumor co-existing with a 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] According to some embodiments, each MM in the multispecific activatable antibody has a dissociation constant, i.e., the K d d of the binding of AB to its corresponding target or epitope, which is higher than the Kd of the binding of AB to the AB.

[0228] According to some embodiments, each MM in the multispecific activatable antibody has a K d of the binding of AB to its corresponding target or epitope, which is dhas.

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

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

[0231] [[ID=1​​​​​​​​​​​​​​​​​​​​​​​​According to some embodiments, each MM in a multispecific activatable antibody has an affinity for binding to AB, which is less than or equal to the affinity for binding AB to its corresponding target or epitope.

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

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

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

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

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

[0240] According to some embodiments, each MM in a multispecifically activatable antibody is a polypeptide of amino acids with a length of approximately 2 to 40. According to some embodiments, each MM in a multispecifically activatable antibody is a polypeptide of 40 amino acids or less.

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

[0242] According to some embodiments, each MM in a multispecifically activatable antibody has a polypeptide sequence that is 50% or less identical to any native binding partner of its corresponding AB. According to some embodiments, each MM in a multispecifically activatable antibody has a polypeptide sequence that is 25% or less identical to any native binding partner of its corresponding AB. According to some embodiments, each MM in a multispecifically activatable antibody has a polypeptide sequence that is 10% or less identical to any native binding partner of its corresponding AB.

[0243] According to some embodiments, when MM1 is coupled toward its corresponding target or epitope, the dissociation constant of AB (K d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d Each coupling of MMs reduces the ability of its corresponding AB to bind to its target or epitope by at least 20 times.

[0244] According to some embodiments, when MM1 is coupled toward its corresponding target or epitope, the dissociation constant of AB (K d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d Each coupling of MMs reduces the ability of its corresponding AB to bind to its target or epitope by at least 40 times.

[0245] According to some embodiments, when MM1 is coupled toward its corresponding target or epitope, the dissociation constant of AB (K d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d Each coupling of MMs reduces the ability of its corresponding AB to bind to its target or epitope by at least 100 times.

[0246] According to some embodiments, when MM1 is coupled toward its corresponding target or epitope, the dissociation constant of AB (K d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d Each coupling of MMs reduces the ability of its corresponding AB to bind to its target or epitope by at least 1000 times.

[0247] According to some embodiments, when MM1 is coupled toward its corresponding target or epitope, the dissociation constant of AB (K d ) but when MM1 is not coupled toward its corresponding target or epitope, K of AB d Each coupling of MMs reduces the ability of its corresponding AB to bind to its target or epitope by at least 10,000 times.

[0248] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 20 times higher than the K d of AB2 when coupled to MM2. According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 20 times lower than the K d of AB2 when coupled to MM2.

[0249] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 40 times higher than the K d of AB2 when coupled to MM2. According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 40 times lower than the K d of AB2 when coupled to MM2.

[0250] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 100 times higher than the K d of AB2 when coupled to MM2. According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 100 times lower than the K d of AB2 when coupled to MM2.

[0251] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 1,000 times higher than the K d of AB2 when coupled to MM2. According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 1,000 times lower than the K d of AB2 when coupled to MM2.

[0252] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 10,000 times higher than the K d of AB2 when coupled to MM2. According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind to its target or epitope, and as a result, when coupled to MM1, the dissociation constant (K d ) of AB1 is at least 10,000 times lower than the K d of AB2 when coupled to MM2.

[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, the protease that cleaves the first cleavable portion (CM1) sequence is produced by a tumor that is present in proximity to a cell expressing the target and / or by a tumor that co-localizes with the target of AB1 in a multi-specific activatable antibody in a tissue, and the protease cleaves CM1 in the multi-specific activatable antibody when the multi-specific activatable antibody is exposed to the protease.

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

[0256] According to some embodiments, in the uncleaved state, the binding of a multispecifically activatable antibody to a target in one region of the AB region is reduced to occur with a dissociation constant at least 20 times higher than that of the unmodified AB that binds to that target, while in the cleaved state, each CM, e.g., CM1 and at least CM2, is arranged in the multispecifically activatable antibody so that AB binds to its target.

[0257] According to some embodiments, in the uncleaved state, the binding of a multispecifically activatable antibody to a target in one region of the AB region is reduced to occur with a dissociation constant at least 40 times higher than that of the unmodified AB that binds to that target, while in the cleaved state, each CM, e.g., CM1 and at least CM2, is arranged in the multispecifically activatable antibody so that AB binds to its target.

[0258] According to some embodiments, in the uncleaved state, the binding of a multispecifically activatable antibody to a target in one region of the AB region is reduced to occur with a dissociation constant at least 50 times higher than that of the unmodified AB that binds to that target, while in the cleaved state, each CM, e.g., CM1 and at least CM2, is arranged in the multispecifically activatable antibody so that AB binds to its target.

[0259] According to some embodiments, in the uncleaved state, the binding of a multispecifically activatable antibody to a target in one region of the AB region is reduced to occur with a dissociation constant at least 100 times higher than that of the unmodified AB that binds to that target, while in the cleaved state, each CM, for example CM1 and at least CM2, is arranged in the multispecifically activatable antibody so that AB binds to its target.

[0260] According to some embodiments, in the uncleaved state, the binding of a multispecifically activatable antibody to a target in one region of the AB region is reduced to occur with a dissociation constant at least 200 times higher than that of the unmodified AB that binds to that target, while in the cleaved state, each CM, for example CM1 and at least CM2, is arranged in the multispecifically activatable antibody so that AB binds to its target.

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

[0262] According to some embodiments, at least one CM in a 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 specific protease, for example, a protease known to be produced by a tumor located in close proximity to cells expressing the target and / or by a tumor co-localizing with at least one target of the multispecific activatable antibody. For example, a suitable cleavable moiety for use with the multispecific activatable antibody of this disclosure is cleaved by at least a protease, for example, urokinase, regmine and / or matryptase (also referred herein as MT-SP1 or MTSSP1). According to some embodiments, a suitable cleavable portion includes at least one of the following sequences: TGRGPSWV (sequence number 68); SARGPSRW (sequence number 69); TARGPSFK (sequence number 70); LSGRSDNH (sequence number 67); GGWHTGRN (sequence number 71); HTGRSGAL (sequence number 72); PLTGRSGG (sequence number 73); AARGPAIH (sequence number 74); RGPAFNPM (sequence number 75); SSRGPAYL (sequence number 76); RGPATPIM (sequence number 77); RGPA (sequence number 78); GGQPSGMWGW (sequence number 79); FPRPLGITGL (sequence number 80); VHMPLGFLGP (sequence number 81); SPLTGRSG (sequence number 82); SAGFSLPA (sequence number 83); LAPLGLQRR (sequence number 84); SGGPLGVR (sequence number 85); and / or PLGL (sequence number 86).

[0263] According to some embodiments, each CM in a multispecific activatable antibody is a substrate of 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, regmine, matryptase, 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, cathepsin S, but is not limited to that. According to some embodiments, each CM in a multispecific activatable antibody is a substrate of a protease selected from the group consisting of uPA (urokinase plasminogen activator), regmine, and matryptase. According to some embodiments, the protease includes uPA. According to some embodiments, the protease includes regmine. According to some embodiments, the protease includes matryptase. According to some embodiments, the protease includes matrix metalloproteinases (MMPs).

[0264] According to some embodiments, at least one CM in a multispecific activatable antibody is a substrate of 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 a multispecific activatable antibody is a substrate of at least two proteases, where one of the proteases is selected from the group consisting of uPA, regmine, and matryptase, 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 a multispecific activatable antibody is a substrate of at least two proteases selected from the group consisting of uPA, regmine, and matryptase.

[0265] According to some embodiments, at least one of the proteases capable of cleaving each CM in a multispecifically activatable antibody is the same protease. According to some embodiments, the same protease capable of cleaving both CMs in a multispecifically 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 a multispecifically activatable antibody is selected from the group consisting of uPA, regmine, and matryptase.

[0266] According to some embodiments, at least two proteases capable of cleaving each CM in a multispecifically activatable antibody are identical proteases. According to some embodiments, the two identical proteases capable of cleaving both CMs in a multispecifically activatable antibody are selected from the group shown in Table 3. According to some embodiments, at least one of the at least two proteases capable of cleaving both CMs in a multispecifically activatable antibody is selected from the group consisting of uPA, regmine, and matryptase, and the other of the at least two proteases is selected from the group shown in Table 3. According to some embodiments, at least two proteases capable of cleaving both CMs in a multispecifically activatable antibody are selected from the group consisting of uPA, regmine, and matryptase.

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

[0268] According to some embodiments, a multispecifically 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 that links MM to AB. According to some embodiments, CM1 is part of a cleavable linker that links MM1 to AB1, and CM2 is part of another cleavable linker that links MM2 to AB2. According to some embodiments, a multispecifically activatable antibody comprises two or more CMs. According to some embodiments, such a multispecifically activatable antibody comprises two or more CMs and two or more MMs. According to some embodiments, CM1 and CM2 are polypeptides with a length of 15 amino acids or less. 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, regmine, and matryptase. 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, legmine, and matryptase, and the second CM is cleaved in the target tissue by a second cleavage agent. According to some embodiments, other proteases are selected from the group consisting of those shown in Table 3. According to some embodiments, the first and second cleavage agents are the same proteases selected from the group consisting of those listed in Table 3, and the first CM and second CM are different substrates for the proteases. According to some embodiments, the first and second cleavage agents are the same proteases selected from the group consisting of uPA, legmine, and matryptase, and the first CM and second CM are different substrates for the proteases. According to some embodiments, the first and second cleavage agents are the same proteases selected from the group listed in Table 3, and the first CM and second CM are the same substrate. According to some embodiments, the first and second cleavage agents are different proteases.According to some embodiments, the first and second cleavage agents are different proteases selected from the group consisting of those shown in Table 3. According to some embodiments, the first and second cleavage agents are produced by a tumor located in close proximity to cells expressing the target and / or by a tumor co-localizing in the target tissue. According to some embodiments, the first and second CMs are cleaved in the target tissue by at least one cleavage agent.

[0269] According to some embodiments, the multispecific activatable antibody is exposed to and cleaved by a protease such that, while the activatable antibody is in an activated or cleaved state, the multispecific activatable antibody contains a light chain amino acid sequence that includes at least a portion of the LP2 and / or CM sequence after the protease has cleaved the CM.

[0270] According to some embodiments, the multispecifically activatable antibody also includes a signal peptide. According to some embodiments, the signal peptide is bound to the multispecifically activatable antibody via a spacer. According to some embodiments, the spacer is bound to the multispecifically activatable antibody in the absence of the signal peptide. According to some embodiments, the spacer is directly bound to at least one of the multispecifically activatable antibody's multiplier molecules (MMs).

[0271] According to some embodiments, the multispecifically activatable antibody in its uncleaved state is directly bound to the first MM and includes a spacer having the structural configuration of spacer-MM1-CM-AB1 from the N-terminus to the C-terminus. According to some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 87).

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

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

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

[0275] According to some embodiments, the multispecific antibodies 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 those for the intended use, e.g., active pharmaceutical and / or surgical agents for cancer. For example, the multispecific antibodies and / or multispecific activatable antibodies may be used in conjunction with additional chemotherapy or antitumor agents.

[0276] According to some embodiments, the multispecific antibody and / or multispecific activatable antibody, and additional agents are formulated into a single therapeutic composition, and the multispecific antibody and / or multispecific activatable antibody, and additional agents are administered simultaneously. According to some embodiments, the multispecific antibody and / or multispecific activatable antibody, and additional agents are separate from each other, for example, each being formulated into separate therapeutic compositions, and the multispecific antibody and / or multispecific activatable antibody, and additional agents are administered simultaneously, or at different time points 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 after the administration of the additional agent, or the multispecific antibody and / or multispecific activatable antibody is administered alternately. As described herein, the anti-multispecific antibody and / or multispecific activatable antibody, and additional agents are administered in single doses or multiple doses.

[0277] This disclosure also provides isolated nucleic acid molecules encoding at least a portion of the multispecific antibodies and / or multispecifically activatable antibodies described herein, for example, a first nucleic acid encoding at least a portion of the heavy chain of the multispecific antibodies and / or multispecifically activatable antibodies, and / or a second nucleic acid encoding at least a portion of the light chain of the multispecific antibodies and / or multispecifically activatable antibodies, and vectors comprising such isolated nucleic acid sequences. This disclosure provides a method for producing multispecific antibodies and / or multispecifically activatable antibodies by culturing cells containing such nucleic acid molecules under conditions that lead to the expression of multispecific antibodies and / or multispecifically activatable antibodies. According to some embodiments, the cells include such vectors.

[0278] The Disclosure also provides a method for producing the multispecific antibody and / or the multispecific activatable antibody of the Disclosure, comprising (a) culturing cells comprising a nucleic acid construct encoding a multispecific antibody and / or a multispecific activatable antibody under conditions that lead to the expression of the multispecific antibody and / or the multispecific activatable antibody, and (b) recovering the multispecific antibody and / or the multispecific activatable antibody.

[0279] The Disclosure also provides methods for treating, preventing, slowing the progression of, or otherwise alleviating the symptoms of one or more medical conditions, or improving symptoms associated with a medical condition, by administering the anti-CD3ε antibody, activatable anti-CD3ε antibody, multispecific antibody, and / or multispecific activatable antibody of the Disclosure that specifically binds CD3ε to a subject for which treatment or prevention is desired. The subject to be treated is, for example, a human or other mammal. According to some embodiments, the subject is a non-human mammal, such as a non-human primate, a pet (e.g., a cat, a dog, a horse), an agricultural animal, a working animal, or a zoo animal. According to some embodiments, the subject is a rodent.

[0280] This disclosure also provides a method for inducing target-dependent T-cell activation and target cell death in subjects where such induction is desired, by administering the multispecifically activatable antibody of this disclosure, wherein when the multispecifically activatable antibody is cleaved, for example, each masking moiety in the multispecifically activatable antibody is no longer bound or, on the other hand, not associated with its corresponding AB domain; and when the multispecifically activatable antibody is not cleaved, for example, at least one masking moiety in the multispecifically activatable antibody is bound or, on the other hand, associated with its corresponding AB domain, then target-dependent T-cell activation and death of target cells are reduced or, on the other hand, inhibited. Any multispecifically activatable antibody described herein is suitable for use in such a method. Subjects to be treated are, for example, humans or other mammals. According to some embodiments, subjects are non-human mammals, for example, non-human primates, companion animals (e.g., cats, dogs, horses), agricultural animals, working animals or zoo animals. According to some embodiments, subjects are rodents.

[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 those methods and embodiments of use, may be administered to a patient at any stage of development. For example, such antibodies, activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies may be administered to a patient with cancer at any stage from early to metastatic. Terms, subjects, and patients are used interchangeably herein.

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

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

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

[0285] According to 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. According to some embodiments, the additional agent(s) are radiotherapy, either alone or in combination with another additional agent(s), e.g., chemotherapeutic agents or antineoplastic agents. According to some embodiments, the additional agent(s) are vaccines, oncoviruses, and / or CD-activators, e.g., Toll-like receptor (TLR) agonists and / or α-CD40 (but not limited to these). According to some embodiments, the additional agent(s) are tumor-targeting antibodies designed to kill tumors via ADCCs or via direct binding to toxins (e.g., antibody-drug conjugates (ADCs)).

[0286] According to 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. According to some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. According to some embodiments, the kinase inhibitor is crizotinib. According to some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of IDO inhibitors, α-CSF1R inhibitors, α-CCR4 inhibitors, TGF-beta, bone marrow-derived suppressor cells, or T-regulatory cells. According to some embodiments, the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM. According to some embodiments, the checkpoint inhibitor is an antibody that binds to a target selected from CTLA-4, PD-1, and / or PD-L1. According to 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. According to some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody, e.g., Yervoy™. According to some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, e.g., Opdivo™ and / or Keytruda™.

[0287] According to some embodiments, the inhibitor is a CTLA-4 inhibitor. According to some embodiments, the inhibitor is a LAG-3 inhibitor. According to some embodiments, the inhibitor is a PD-1 inhibitor. According to some embodiments, the inhibitor is a PDL1 inhibitor. According to some embodiments, the inhibitor is a TIGIT inhibitor. According to some embodiments, the inhibitor is a TIM-3 inhibitor. According to some embodiments, the inhibitor is a B7H4 inhibitor. According to some embodiments, the inhibitor is a Vista inhibitor. According to some embodiments, the inhibitor is a B-RAFi inhibitor. According to some embodiments, the inhibitor is a MEKi inhibitor. According to some embodiments, the inhibitor is a Btk inhibitor. According to some embodiments, the inhibitor is ibrutinib. According to some embodiments, the inhibitor is crizotinib. According to some embodiments, the inhibitor is an IDO inhibitor. According to some embodiments, the inhibitor is an α-CSF1R inhibitor. According to some embodiments, the inhibitor is an α-CCR4 inhibitor. According to some embodiments, the inhibitor is TGF-β. According to some embodiments, the inhibitor is bone marrow-derived suppressor cells. According to some embodiments, the inhibitor is T-regulatory cells.

[0288] According to some embodiments, the agonist is Ox40. According to some embodiments, the agonist is GITR. According to some embodiments, the agonist is CD137. According to some embodiments, the agonist is ICOS. According to some embodiments, the agonist is CD27. According to some embodiments, the agonist is HVEM.

[0289] According to some embodiments, the additional agent is another antibody or its antigen-binding fragment, another conjugated antibody or its antigen-binding fragment, another activatable antibody or its antigen-binding fragment, and / or another conjugated activatable antibody or its antigen-binding fragment. According to some embodiments, the additional agent is another antibody or its antigen-binding fragment, another conjugated antibody or its antigen-binding fragment, another activatable antibody or its antigen-binding fragment, and / or another conjugated activatable antibody or its antigen-binding fragment, for the same target as the first antibody or its antigen-binding fragment, for example, PDL1. According to some embodiments, the additional agent is another antibody or its antigen-binding fragment, another conjugated antibody or its antigen-binding fragment, another activatable antibody or its antigen-binding fragment, and / or another conjugated activatable antibody or its antigen-binding fragment, for a target different from the target of the first antibody or its antigen-binding fragment, the first conjugated antibody or its fragment, and / or another conjugated activatable antibody or its antigen-binding fragment.

[0290] According to some embodiments, the anti-CD3ε antibody and / or activatable antibody is administered during and / or after treatment in combination with one or more additional agents, such as chemotherapeutic agents, anti-inflammatory agents, and / or immunosuppressants. According to some embodiments, the anti-CD3ε antibody and / or activatable antibody and the additional agents are formulated into a single therapeutic composition and administered simultaneously. On the other hand, the anti-CD3ε antibody and / or activatable antibody and the additional agents are separate from each other, for example, each being formulated into a separate therapeutic composition and administered simultaneously, or administered at different time points during the treatment regimen. For example, the anti-CD3ε antibody and / or activatable antibody may be administered before the administration of the additional agent, the anti-CD3ε antibody and / or activatable antibody may be administered after the administration of the additional agent, or the anti-CD3ε antibody and / or activatable antibody and the additional agent may be administered alternately. As described herein, the anti-CD3ε antibody and / or activatable antibody and the additional agent may be administered as a single dose or multiple doses.

[0291] According to some embodiments, the anti-CD3ε antibody and / or activatable antibody, and additional agents are administered simultaneously. For example, the anti-CD3ε antibody and / or activatable antibody, and additional agents may be formulated into a single composition or administered as multiple separate compositions. According to some embodiments, the anti-CD3ε antibody and / or activatable antibody, and additional agents are administered sequentially or at different time points during a treatment regimen.

[0292] The Disclosure also provides methods and kits for the use of anti-CD3ε antibodies, activatable anti-CD3ε antibodies, multispecific antibodies that specifically bind to CD3ε, and / or multispecific activatable antibodies of the Disclosure that specifically bind to CD3ε, for various diagnostic and / or prophylactic signs. For example, the Disclosure provides for contacting an activatable antibody or multispecific activatable antibody with a subject or sample, wherein (i) the MM1 is a peptide that inhibits the binding of AB1 to the target, wherein the MM1 does not have the amino acid sequence of the naturally occurring binding partner of AB1 and is not a modified form of the naturally occurring binding partner of AB1; and (b) where, in an uncleaved, inactive state, the MM1 interferes with the specific binding of AB1 to the target, and in a cleaved, activated state, the MM1 interferes with the specific binding of AB1 to the target. (i) not to, or not to compete with; and (ii) measuring the level of activated antibody or activated multispecifically activatable antibody in a subject or sample, wherein a detectable level of activated antibody or activated multispecifically activatable antibody in the subject or sample suggests the presence of the cleavage agent and target in the subject or sample, and the absence of a detectable level of activated antibody or activated multispecifically activatable antibody in the subject or sample suggests the absence and / or insufficient presence of the cleavage agent, target, or both of the cleavage agent and target in the subject or sample, thereby providing a method and kit for detecting the presence or absence of a cleavage agent and a target of interest in a 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 bound. According to some embodiments, the activatable antibody, or activatable multispecific activatable antibody, is not bound to the agent. According to some embodiments, the activatable antibody, or activatable multispecific activatable antibody, includes a detectable label. According to some embodiments, the detectable label is placed on AB1. According to some embodiments, the level of the activatable antibody, or activatable multispecific activatable antibody in a subject or sample is achieved using a secondary reagent containing a detectable label that specifically binds to the activatable antibody, or activatable multispecific activatable antibody. According to some embodiments, the secondary reagent is an antibody containing a detectable label.

[0294] According to some embodiments of the methods and kits, the activatable antibody, or multispecific activatable antibody, includes a detectable label. According to some embodiments of the methods and kits, the detectable label includes an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. According to some embodiments of the methods and kits, the imaging agent includes 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 includes iodine, gadolinium, or iron oxide. According to some embodiments of the methods and kits, the enzyme includes horseradish peroxidase, alkaline phosphatase, or β-galactosidase. According to some embodiments of those 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 t-dimer 2 (RFP t-dimer 2), HCRED, or europium derivatives. According to some embodiments of those methods and kits, the luminescence label comprises N-methylacridium derivatives. According to some embodiments of those methods, the label comprises Alexa Fluor® label, e.g., Alex Fluor® 680 or Alexa Fluor® 750. According to some embodiments of those methods and kits, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0295] According to some embodiments of those methods and kits, the subject is mammals. According to some embodiments of those methods and kits, the subject is humans. According to some embodiments, the subject is non-human mammals, such as non-human primates, pets (e.g., cats, dogs, horses), agricultural animals, working animals, or zoo animals. According to some embodiments, the subject is rodents.

[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, on the other hand, to refine a suitable patient population for treatment with the multispecific activatable antibody of the Disclosure. For example, patients who are positive for both the protease and target that cleave the substrate at the first cleavable portion (CM1) in the multispecific activatable antibody tested in these methods are identified as suitable candidates for treatment with such CM1-containing activatable antibody or multispecific activatable antibody. Similarly, patients who are negative for both the protease and target that cleave the substrate at CM1 in the activatable antibody or multispecific activatable antibody, as tested using these methods, may be identified as suitable candidates for other forms of treatment.

[0298] According to some embodiments, methods or kits are used to identify or narrow down a patient population suitable for treatment with the activatable antibody and / or multispecific activatable antibody of the present disclosure, and subsequently by administering the activatable antibody and / or multispecific activatable antibody to the target of interest. For example, a patient who is positive for both the protease and target that cleave the substrate at the first cleavable portion (CM1) in the activatable antibody and / or multispecific activatable antibody tested in those methods is identified as a suitable candidate for treatment with such CM1-containing activatable antibody and / or multispecific activatable antibody, and the patient is then administered a therapeutically effective amount of the tested activatable antibody and / or multispecific activatable antibody. Similarly, a patient who is negative for either or both of the protease and target that cleave the substrate at CM1 in the activatable antibody and / or multispecific activatable antibody, as tested using those methods, may be identified as a suitable candidate for another form of treatment.

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

[0300] The pharmaceutical compositions of this disclosure may include the multispecific antibodies and / or multispecific activatable antibodies and carriers of this disclosure. These pharmaceutical compositions may be included in a kit, for example, a diagnostic kit.

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

[0302] [Figure 1] Figure 1 shows the general mask selection population nomenclature used in this specification. [Figure 2] Figures 2A and 2B are a series of graphs showing the ability of SP34 in scFv form 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 mouse sP34-2 IgG1 binding as a function of concentration. [Figure 3] Figures 3A–3D are a series of graphs showing the ability of such masking moieties to reduce the ability of activatable anti-CD3ε antibodies containing the masking moieties of this disclosure to bind to CD3ε on Jurkat T cells. Each activatable antibody is labeled by the name of its masking moiety. The antibody SP34scFv(LvHv)-Fc is identified by LvHv. [Figure 4] Figures 4A, 4B, and 4C are a series of diagrams illustrating the various antibodies and activatable antibodies of this disclosure. Figure 4A shows a component fragment for the scFv-Fc antibody. Figure 4B shows the scFv(LvHv)-Fc and scFv(HvLv)-Fc antibodies of this disclosure. Figure 4C shows an activatable antibody containing the scFv(LvHv)-Fc or scFv(HvLv)-Fc antibody of this disclosure. [Figure 5]Figures 5A, 5B, 5C, and 5D are a series of diagrams illustrating the multispecific antibodies and multispecific activatable antibodies of the present disclosure. Figure 5A shows the multispecific antibody of the present disclosure, including an Ig antibody and scFv. Figure 5B shows the multispecific antibody of the present disclosure with the primary antibody portion masked. Figure 5C shows the multispecific activatable antibody of the present disclosure with the secondary scFv portion masked. Figure 5D shows the multispecific activatable antibody of the present disclosure with the primary antibody and secondary scFv portion masked. [Figure 6] Figures 6A, 6B, 6C, 6D, 6E, and 6F are a series of diagrams illustrating various embodiments of the multispecifically activatable antibody of this disclosure. [Figure 7] Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, and 7J are schematic diagrams of a selection of possible sequences of the multispecific antibodies of this disclosure. [Figure 8] Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 8I, and 8J are schematic diagrams of a selected set of possible sequences of the multispecific antibodies of this disclosure. In particular, these figures show multispecific activatable antibodies in which the primary antigen-binding site is masked (i.e., activatable) and additional antigen-binding domains are masked. [Figure 9] Figures 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, 9I, and 9J are schematic diagrams of a series 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 schematic diagrams of a series of arrays of multispecific activatable antibodies in which the secondary antigen-binding domain is masked and additional antigen-binding domains (one or more) are not masked. [Figure 11] Figures 11A, 11B, 11C, 11D, 11E, 11F, 11G, 11H, 11I, and 11J are schematic diagrams of a series of arrays of multispecific activatable antibodies in which most, but not all, antigen-binding domains are masked, and at least one additional antigen-binding domain is not masked. [Figure 12] Figures 12A, 12B, 12C, and 12D are schematic diagrams of a series of arrays of multispecifically activatable antibodies in which the primary antigen-binding domain and another antigen-binding domain are masked, while the remaining antigen-binding domain(s) are not masked. [Figure 13] Figures 13A and 13B are a series of graphs showing the binding of SP34 scFv-Fc to CD3ε-positive Jurkat cells. Figure 13A is a graph showing the orientation ability of both SP34 scFv to CD3ε-Jurkat cells. Figure 13B is a graph showing the binding of SP34-2IgG. All forms of SP34 bind with similar EC50 values. [Figure 14] Figures 14A and 14B are a series of graphs showing the ability of such masking moieties to reduce the ability of activatable anti-CD3ε antibodies containing the masking moieties of this disclosure to bind to CD3ε on Jurkat T cells. [Figure 15] Figures 15A, 15B, 15C, and 15D are a series of graphs showing that the CD3ε masking peptide alters the EC50 of CD3ε binding to an EGFR-masked, multispecific, activatable antibody that utilizes two versions of SP34 scFv, while maintaining effective EGFR masking. [Figure 16] Figures 16A and 16B are a series of figures showing that the cytotoxicity EC50 of CD3ε and EGFR-masked multispecific activatable antibodies is altered compared to the unmasked control. This was evident from two versions of SP34 scFv, namely scFv in CI023 and CI024 versus scFv in CI011 and CI010. [Figure 17] Figures 17A and 17B are a series of graphs showing that in multispecifically activatable antibodies, EGFR-bound EC50 is unaffected by the presence or absence of both CD3ε masks; similarly, CD3ε EC50 is unaffected by the presence or absence of the EGFR mask. [Figure 18]Figures 18A and 18B are a series of graphs showing that the maximum change in cytotoxic EC50 is obtained by masking both EGFR and CD3 binding. Furthermore, the cytotoxicity of multispecific antibodies and multispecific activatable antibodies is EGFR-dependent. [Figure 19] Figure 19 is a graph showing that the maximum change in activated EC50 is obtained by masking both EGFR and CD3 binding. [Figure 20] Figures 20A and 20B show that the CD3-masked multispecific antibody exhibits negligible target-independent activation, as evidenced by the CD69+ frequency analysis shown in Figure 20A and the average CD69 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 multispecifically activatable CI011 (M-EGFR / M-hCD3) antibody. Cell death by all multispecifically activatable antibodies is restored by uPA activation. [Figure 22] Figure 22 is a graph showing the ability of double-masked, multispecifically activatable antibodies containing different substrates to alter EC50 cytotoxicity. [Figure 23] Figure 23 is a graph showing that bispecific CI005 administered at 0.1 mg / kg twice over 7 days, and CI059 (BiTE) administered at 0.5 mg / kg / day for 8 days, inhibited the growth of HT-29Luc2 xenograft tumors. [Figure 24] Figure 24 is a graph showing that the CI048 antibody limited 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] Figure 25 is a graph showing the dose-response of the CI048 bispecific antibody for the elimination of HT-29Luc2 xenograft tumors. [Figure 26]Figure 26 is a graph showing that a 0.3 mg / kg dose of CI048 bispecific antibody eliminated xenograft tumors, and a 0.1 mg / kg dose of CI040 bispecific activatable antibody limited the growth of HT-29Luc2 xenograft tumors. [Figure 27] Figure 27 is a graph showing that a 1.0 mg / kg dose of CI040 bispecific activatable antibody eliminated HT-29Luc2 xenograft tumors, and a 0.3 mg / kg dose of CI048 bispecific antibody limited the growth of HT-29Luc2 xenograft tumors. [Figure 28] Figure 28 is a graph showing that a 1.0 mg / kg dose of CI011 bispecific activatable antibody significantly inhibited the growth of HT-29Luc2 xenograft tumors (p<0.05). [Figure 29] Figure 29 is a graph showing dose-response plots of CI048, CI011, and CI048 for serum alanine aminotransferase (ALT) concentrations 48 hours after administration. [Figure 30] Figure 30 shows that a mouse CD3ε mask incorporated into an EGFR-masked multispecific activatable antibody alters the EC50 of binding to mouse CD3 while maintaining effective EGFR masking. [Figure 31] Figure 31 shows that the death of IL6R+Molp-8 cells is maximally attenuated by masking both IL6R and CD3 binding. [Modes for carrying out the invention]

[0303] The disclosure of the present invention provides antibodies, activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies that conjugate at least the epsilon chain (CD3ε) of CD3. As used herein, an activatable antibody is an antibody comprising a masking moiety (MM) that is conjugated to the antigen- or epitope-binding domain of the antibody such that MM coupling 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 comprising at least one masking moiety (MM) that is conjugated to at least one antigen- or epitope-binding domain of the multispecific antibody such that MM coupling 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 bloodstream and are activated at the site of intended treatment and / or diagnosis, but not in normal, i.e., healthy tissue, and, if activated, exhibit binding to targets at least equivalent to those of their corresponding unmodified multispecific antibodies.

[0304] The antibodies, activated activatable antibodies, multispecific antibodies and / or activated multispecific activatable antibodies of this disclosure are < μ1M, according to some embodiments, < 100 nM, < 10 nM or < The coupling constant (K) of 1nM d ) and binds to CD3ε.

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

[0306] Non-restrictive examples of multispecific antibodies include bispecific antibodies, triplicate antibodies, quadruplicate antibodies, and other multispecific antibodies. Multispecific antibodies provided herein are also polyvalent; as used herein, polyvalent means the total number of binding sites on the antibody, regardless of whether the binding sites recognize the same or different antigens or epitopes. Non-restrictive examples of multispecific activatable antibodies include bispecific activatable antibodies, triplicate activatable antibodies, quadruplicate activatable antibodies, and other multispecific activatable antibodies. Multispecific activatable antibodies provided herein are also polyvalent.

[0307] According to some embodiments, a multispecific antibody or fragment thereof and / or a multispecific activatable antibody or fragment thereof is designed to bind to T cells and / or other immune effector cells. A multispecific activatable antibody or fragment thereof that binds to T cells is also referred to herein as a T-cell-binding multispecific antibody or fragment thereof and / or a T-cell-binding multispecific activatable antibody or fragment thereof. A multispecific activatable antibody or fragment thereof that binds to immune effector cells is also referred to herein as an immune effector cell-binding multispecific antibody or fragment thereof and / or an immune effector cell-binding multispecific activatable antibody or fragment thereof. According to some embodiments, a multispecific antibody or fragment thereof and / or a multispecific activatable antibody or fragment thereof is designed to bind to multiple targets and / or multiple epitopes, or to interact with them, and is also referred to herein as a multi-antigen-targeting antibody or fragment thereof and / or a multi-antigen-targeting activatable antibody or fragment 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 another target antigen. According to some embodiments, one antibody domain of the multispecific antibody or fragment thereof has specificity for another epitope of the target antigen.

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

[0310] According to some embodiments, the multispecifically activatable antibody is a T-cell-binding multispecifically activatable antibody comprising a targeted antibody or its antigen-binding fragment and a T-cell-binding antibody or its antigen-binding moiety, wherein the T-cell-binding antibody or its antigen-binding fragment comprises a first antibody or its antigen-binding fragment (AB1) that binds to a first T-cell-binding target, wherein AB1 is bound to a masking moiety (MM1) via a non-cleavable linker such that the coupling of MM reduces the ability of AB1 to bind to the first target, and the targeted antibody or its antigen-binding fragment is not masked. definition

[0311] Unless otherwise defined, scientific and technical terms used in connection with this disclosure will have meanings that are commonly understood by those skilled in the art. The terms “a” or “an” 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” may be used interchangeably. Furthermore, unless required by context, singular terms will also include plural forms, and plural terms will also include singular forms. In general, the nomenclature used in connection with and in connection with cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein is 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 carried out according to the manufacturer's specifications, or as commonly achieved in the nuclear art, or as described herein. The aforementioned techniques and procedures are generally carried out in accordance with conventional techniques well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature, experimental procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques for chemical synthesis, chemical analysis, pharmaceutical formulation, formulation and delivery, and patient treatment are employed.

[0312] When used in accordance with this disclosure, the following terms will be understood to have the following meanings unless otherwise indicated:

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

[0314] The basic antibody structural unit is known to include a tetramer. Each tetramer consists of two identical polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminus of each chain defines an invariant 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 in the properties of the heavy chain present in the molecule. A particular class has subclasses, such as IgG1, IgG2, IgG3, IgG4, and others. Furthermore, in humans, the light chain can be a κ chain or a λ chain.

[0315] The terms "monoclonal antibody" (mAb) or "monoclonal antibody composition," as used herein, refer to a group of antibody molecules comprising only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining region (CDR) of a monoclonal antibody is identical in all molecules of the group. An MAb contains an antigen-binding site that can react immunologically with a specific epitope of an antigen characterized by its unique binding affinity to that site.

[0316] The term "antigen-binding site" or "binding portion" refers to a part 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 branched stretches within the V regions of the H and light chains, referred to as "hyperrariable regions," are interposed between more conserved flanking stretches known as "framework regions" or FRs. Thus, the term "FR" refers to amino acid sequences found naturally between and adjacent to the hypervariable regions in immunoglobulins. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to which it is bound, and the three individual hypervariable regions of the heavy and light chains are called "complementarity-determining regions" or "CDRs." The assignment of amino acids to each domain follows the definitions of 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” encompasses any protein determinant capable of specifically binding to immunoglobulins, scFv, or T-cell receptors. Epitope determinants typically consist of a chemically active surface group of a molecule, such as an amino acid or sugar side chain, and usually possess specific conformational and charge properties. For example, antibodies are elevated to the N-terminal or C-terminal peptide of a polypeptide. Antibodies have a dissociation constant of < If the concentration is 1 μM, for example, according to some embodiments, < 100 nM, and according to some embodiments, < When the concentration is 10 nM, it is said to specifically bind to the antigen.

[0318] As used herein, the terms “specific binding,” “immunological binding,” and “immunological binding properties” refer to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction may be expressed by the dissociation constant (Kd) of the interaction, where a smaller K d This indicates a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally influence the rates in both directions. Thus, the "on rate constant" (K on ) and "off rate constant" (K off Both of these can be determined by calculating the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The ratio allows for the cancellation of all parameters unrelated to affinity, and the dissociation constant K d This is equal to (see Davies et al. (1990) Annual Rev Biochem 59:439-473 in general). When the antibodies of this disclosure are measured by assays known to those skilled in the art, such as radioligand binding assays or similar assays, the equilibrium binding constant (K) is equal to (K). d )but, < 1 μM, for example, according to some embodiments, < 100 nM, according to some embodiments, < 10 nM, and according to some embodiments, < It is said that when the concentration is between 100 pM and approximately 1 pM, it specifically binds to EGFR.

[0319] The term “isolated polynucleotide,” as used herein, means a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, and depending on its origin, “isolated polynucleotide” means (1) not relating to any or all polynucleotides found in nature, (2) being operablely ligated to a polynucleotide that is not ligated in nature, or (3) not naturally occurring as part of a large sequence. The polynucleotides of this disclosure include nucleic acid molecules encoding heavy-chain immunoglobulin molecules as shown herein, and nucleic acid molecules encoding light-chain immunoglobulin molecules as shown herein.

[0320] As used herein, the term “isolated protein” means a protein of cDNA, recombinant RNA, or synthetic origin, or any combination thereof, and depending on its origin or source of origin, an “isolated protein” is (1) unrelated to any protein found in nature, (2) not related to any other protein from the same source, such as a mouse protein, (3) expressed by cells from a different species, or (4) not found in nature.

[0321] The term "polypeptide" is used herein as a genetic term to refer to natural protein fragments or sequence analogs of polypeptides. Thus, natural protein fragments and analogs are species of the genus Polypeptide. The polypeptides of this disclosure include heavy-chain immunoglobulin molecules and light-chain immunoglobulin molecules as shown herein, as well as antibody molecules formed by combinations including heavy-chain immunoglobulin molecules with light-chain immunoglobulin molecules, such as kappa-light-chain immunoglobulin, and vice versa, and also their fragments and analogs.

[0322] As used herein, the term "naturally-occurring" means, where applicable to the purpose, that the purpose may be found in nature. For example, polypeptides or polynucleotide sequences present in organisms (including viruses) that can be isolated from natural sources and that have not been intentionally modified by humans in the laboratory or otherwise are naturally-occurring.

[0323] The term "operably linked," as used herein, means that the positions of components described in this way are related in a way that allows them to function in their intended manner. A control sequence "operably linked" to a code sequence is linked in such a way that the expression of the code sequence is achieved under conditions that it can be matched with the control sequence.

[0324] The term "control sequence," as used herein, means a polynucleotide sequence necessary to influence the expression and processing of a linked coding sequence. The nature of such a control sequence varies depending on the host organism in prokaryotes, and such a control sequence generally includes a promoter, ribosome binding site, and transcription termination sequence in eukaryotes, and generally includes a promoter and transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence. The term "polynucleotide," as used herein, means a nucleotide, ribonucleotide, or deoxynucleotide, or any modified form of any type of nucleotide, consisting of at least 10 bases in length. This term encompasses single-stranded and double-stranded DNA.

[0325] As used herein, the term polynucleotide encompasses naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide bonds. An oligonucleotide is a polynucleotide subset generally containing 200 or fewer bases. According to some embodiments, an oligonucleotide is 10 to 60 bases long, for example, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases long, according to some embodiments. Oligonucleotides are usually single-stranded, for example, for probes, but may be double-stranded, for example, for use in constructing gene variants. The oligonucleotides of this disclosure are either sense or antisense oligonucleotides.

[0326] As used herein, the term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. As used herein, the term "modified nucleotides" includes nucleotides having modified or substituted sugar groups, etc. As used herein, the term "oligonucleotide linkages" includes oligonucleotide links such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniradiates, phosphoronmidates, and similar. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. 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 may optionally include labels for detection.

[0327] Where used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland 7 Mass. (1991)). The 20 conventional amino acids, unnatural amino acids, e.g., α-substituted amino acids, N-alkyl amino acids, lactic acid, and stereoisomers of other unconventional amino acids (e.g., D-amino acids) may also be suitable components for the polypeptides of this 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, according to standard usage and convention, the left direction is the amino-terminal direction and the right direction is the carboxy-terminal direction.

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

[0329] When applied to polypeptides, the term “substantial identity” means that two peptide sequences share at least 80% sequence identity, for example, at least 90% sequence identity according to some embodiments, at least 95% sequence identity according to some embodiments, and at least 99% sequence identity according to some embodiments, when optimally aligned by programmed GAP or BESTFIT using default gap weights.

[0330] According to some embodiments, the non-identical residue positions differ due to conserved amino acid substitutions.

[0331] Where discussed herein, small variations in the amino acid sequence of antibody or immunoglobulin molecules are intended to be covered by this disclosure, provided that the variation in the amino acid sequence is maintained by at least 75%, for example, by at least 80%, 90%, 95%, and by 99% according to some embodiments. In particular, conserved amino acid substitutions are intended. Conserved substitutions occur within the amino acid family associated with 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) non-charged 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 have a major effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid in the skeletal region. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. The assay is described in detail herein.Fragments or analogues of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. According to some embodiments, the amino- and carboxy-terminuses of the fragment or analogue are located near the boundaries of the functional domain. 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 above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with this disclosure.

[0332] According to some embodiments, amino acid substitutions are those substitutions that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogues. Analogues may include various mutations in sequences other than naturally occurring peptide sequences. For example, one or more amino acid substitutions (e.g., conserved amino acid substitutions) may be made in naturally occurring sequences (in the polypeptide portion outside the domain that forms intermolecular contacts). Conserved amino acid substitutions should not substantially alter the structural properties of the parent sequence (e.g., amino acid substitutions should not tend to disrupt helices present in the parent sequence or to break other types of secondary structures that characterize the parent sequence). Examples of secondary and tertiary structures of polypeptides recognized in the art are described in *Principles of Proteins, Structure and Molecules* (Creighton, Ed., WH Freeman and Company, New York (1984)); *Introduction to Protein Structure* (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and *Thornton et al. Nature 354:105 (1991)*.

[0333] The term "polypeptide fragment," as used herein, means a polypeptide having an amino-terminus and / or carboxyl-terminus deletion and / or one or more internal deletions, but whose remaining amino acid sequence is identical to its corresponding position in a naturally occurring sequence, for example, one inferred from a complete-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids long; for example, according to some embodiments, at least 14 amino acids long; according to some embodiments, at least 20 amino acids long; typically at least 50 amino acids long; and according to some embodiments, 70 amino acids long. The term "analog," as used herein, means a polypeptide consisting of at least 25 amino acid segments that have substantial identity with respect to a portion of the inferred amino acid sequence and have specific binding to EGFR under appropriate binding conditions. Typically, polypeptide analogs contain conserved amino acid substitutions (or additions or deletions) to a naturally occurring sequence. The analogs are typically at least 20 amino acids long, for example, at least 50 or more amino acids long according to some embodiments, and are often the same length as a full-length naturally occurring polypeptide.

[0334] The term "agent" is used herein to refer to a compound, a mixture of compounds, a biomolecule, or an extract produced from a biomaterial.

[0335] As used herein, the terms “label” or “labeled” mean the incorporation of a detectable marker, for example, by the incorporation of a radiolabeled amino acid or by the binding of a biotinyl moiety to a polypeptide that can be detected by a marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity detectable by optical or colorimetric methods). Various methods for labeling polypeptides and glycoproteins are known and can be used in the art. Examples of labeling 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 labeling (e.g., fluorophores, rhodamine, lanthanide phosphors), enzyme labeling (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope labeling). According to some embodiments, the labeling is conjugated by spacer arms of varying lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein means a compound or composition that, when correctly administered to a patient, can induce a desired therapeutic effect.

[0336] As used herein, “substantially pure” means a composition in which the species of interest is the dominant species (i.e., in abundance on a molar basis, more abundant than any other individual species in the composition) and is substantially purified, where the species of interest constitutes at least about 50% (on a molar basis) of all polymer species in which the species of interest is present.

[0337] Generally, a substantially pure composition will contain about 80% or more of all polymer species present in the composition, and according to some embodiments, about 85%, 90%, and 99% or more. According to some embodiments, the target species is purified to an essentially homogeneous degree (contaminating species in the composition cannot be detected by conventional detection methods), where the composition essentially consists of a single polymer species.

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

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

[0340] Typical multispecific antibodies and / or multispecifically activatable antibodies of this disclosure include, for example, those shown in the examples provided herein, and their variants.

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

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

[0343] According to some non-restrictive embodiments, at least one of the A and B antibodies of the multispecific antibody is derived from the antibodies listed in Table 2. [Table 2-1] [Table 2-2]

[0344] Typical antibodies, activatable antibodies, and their antigen-binding fragments that bind to CD3ε in this disclosure include CD3ε-binding sequences described in Examples, for example, Examples 2 and 4. Typical multispecific antibodies and multispecific activatable antibodies include multispecific activatable antibodies described in Examples, for example, Example 4.

[0345] According to some embodiments, the multispecifically activatable antibody contains 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] According to some embodiments, the multispecifically activatable antibody contains 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 multispecifically 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 multispecifically activatable antibody contains a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid 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] According to some embodiments, the multispecifically activatable antibody contains a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0350] According to some embodiments, the multispecifically 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. The heavy chain sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the specified amino acid; and the light chain sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid selected from the group consisting of SEQ ID NOs: 448, 450, 458, 468, 474, 482, 484, 508, 516, and 520.

[0351] According to some embodiments, the multispecifically activatable antibody contains the amino acid sequence of SEQ ID NO: 506. According to some embodiments, the multispecifically activatable antibody contains 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] A typical AB that binds to CD3ε in this disclosure includes the CD3ε binding sequence shown below: CD3Hv Lv EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL(Sequence ID 587) CD3 LvHv QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (Sequence ID 588)

[0353] According to some embodiments, the activatable anti-CD3ε antibody includes or comprises a heavy chain derived from the heavy chain amino acid sequences shown in Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes or comprises a light chain derived from the light chain amino acid sequences shown in Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a heavy chain including or derived from the heavy chain amino acid sequences shown in Table 17, and a light chain including or derived from the light chain amino acid sequences shown in Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group A in Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group B in Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group C in Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes 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. According to some embodiments, the activatable anti-CD3ε antibody includes 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. According to some embodiments, the activatable anti-CD3ε antibody includes 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. According to some embodiments, the activatable anti-CD3ε antibody includes 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. According to some embodiments, the activatable anti-CD3ε antibody includes 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. According to 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.According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in group J of Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in group K of Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in group L of Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in group M of Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in group N of Table 17. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in group O of Table 17. According to 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. According to 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 chain (VH) and variable light chain (VL) 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] According to some embodiments, the activatable anti-CD3ε antibody comprises or is derived from a commercially available antibody, for example, an anti-CD3 antibody from Boehringer Mannheim Corp. (Indianapolis, Ind.; catalog number 1273 485).

[0356] According to some embodiments, the activatable anti-CD3ε antibody comprises or is derived from an antibody produced, secreted, or otherwise generated by a hybridoma, for example, the hybridoma disclosed in U.S. Patent No. 4,361,549 and / or No. 4,658,019, and deposited with the American Type Culture Collection under accession number ATCC CRL8001OKT-3.

[0357] According to some embodiments, the activatable anti-CD3ε antibody is produced, secreted, or otherwise derived from the antibody produced by a hybridoma, for example, the hybridoma disclosed in PCT International Publication No. 1995 / 16037 and deposited with Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM ACC2152.

[0358] According to some embodiments, the activatable anti-CD3ε antibody comprises or is derived from an antibody produced, secreted, or otherwise generated by a hybridoma, for example, the hybridoma disclosed in PCT International Publication No. 1991 / 01752 and deposited in the American Type Culture Collection under accession number HB10,166.

[0359] According to some embodiments, the activatable anti-CD3ε antibody includes a combination of VL CDR sequences selected from the group consisting of the CDR sequences shown in Table 18, the combinations thereof shown in Table 18, and / or a combination of VH CDR sequences selected from the group consisting of the combinations thereof shown in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group R in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group R in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group S in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in group S in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in group T in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of light chain CDR sequences selected from the group consisting of combinations shown in Group T in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in Group U in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of light chain CDR sequences selected from the group consisting of combinations shown in Group U in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in Group V in Table 18. According to some embodiments, the activatable anti-CD3ε antibody includes a combination of light chain CDR sequences selected from the group consisting of combinations shown in Group V in Table 18.According to some embodiments, the activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in Group W in Table 18. According to some embodiments, the activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of combinations shown in Group W in Table 18. According to some embodiments, the activatable anti-CD3ε antibody comprises a combination of heavy chain CDR sequences selected from the group consisting of combinations shown in Group X in Table 18. According to some embodiments, the activatable anti-CD3ε antibody comprises a combination of light chain CDR sequences selected from the group consisting of 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 this disclosure specifically bind to at least mammalian targets. According to some embodiments, such ABs bind to mammalian CD3ε. According to some embodiments, such ABs bind to human targets. According to some embodiments, such ABs bind to non-human primate targets. ABs that bind to the same epitopes as the antibodies and / or activated, activatable antibodies described herein are also included in this disclosure. ABs that compete with the antibodies and / or activated, activatable antibodies described herein for binding to targets, e.g., human targets, are also included in this disclosure. ABs that cross-compete with the antibodies and / or activated, activatable antibodies described herein for binding to targets, e.g., human targets, are also included in this disclosure.

[0362] According to some embodiments, the AB in the multispecific activatable antibody of this disclosure specifically binds to at least a CD3ε target, e.g., mammalian CD3ε. According to some embodiments, such AB binds to mammalian CD3ε. According to some embodiments, such AB binds to human CD3ε. According to some embodiments, such AB binds to non-human primate CD3ε. AB that binds to the same CD3ε epitope as the antibody and / or activated activatable antibody described herein is also included in this disclosure. AB that competes with the anti-CD3ε antibody and / or activated anti-CD3ε activatable antibody described herein for binding to a CD3ε target, e.g., human CD3ε, is also included in this disclosure. AB that cross-competes with the anti-CD3ε antibody and / or activated anti-CD3ε activatable antibody described herein for binding to a CD3ε target, e.g., human CD3ε, is also included in this disclosure.

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

[0364] C225v5 antibody heavy chain nucleotide sequence: C225v5 antibody heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(Sequence ID 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* (Sequence ID 105)

[0367] C225v6 antibody heavy chain nucleotide sequence: C225v6 antibody heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (Sequence ID 107)

[0368] C225 antibody heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (Sequence ID 523)

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

[0370] Av1 antibody heavy chain amino acid sequence: QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGQGSL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 108)

[0371] Av1 antibody light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 109)

[0372] According to some embodiments, AB binds to a Jagged target, for example, Jagged1, Jagged2, or both. According to some embodiments, AB that binds to a Jagged target comprises one or more heavy and / or light chain sequences as shown below.

[0373] 4D11 light chain arrangement: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 110)

[0374] 4D11 heavy chain sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 111)

[0375] 4D11v2 double-stranded array: EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 112)

[0376] 4D11v2 light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 113)

[0377] According to some embodiments, at least one of the A and B of a multispecific antibody and / or multispecific activatable antibody conjugates a jagged target and includes one or more variable heavy chain and / or variable light chain sequences as described below.

[0378] Variable light chain amino chain sequence Lc4 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 114)

[0379] Variable heavy chain amino chain sequence Hc4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS(Sequence ID 115)

[0380] Variable light chain amino chain sequence Lc5 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 116)

[0381] Variable heavy chain amino chain sequence Hc5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYHGQFDYWGQGTLVTVSS(Sequence ID 117)

[0382] Variable light chain amino chain sequence Lc7 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 118)

[0383] Variable heavy chain amino chain sequence Hc7 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS(Sequence ID 119)

[0384] Variable light chain amino chain sequence Lc8 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 120)

[0385] Variable heavy chain amino chain sequence Hc8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHIGRTNPFDYWGQGTLVTVSS(Sequence ID 121)

[0386] Variable light chain amino chain sequence Lc13 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 122)

[0387] Variable heavy chain amino chain sequence Hc13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 123)

[0388] Variable light chain amino chain sequence Lc16 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 124)

[0389] Variable heavy chain amino chain sequence Hc16 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYYGQFDYWGQGTLVTVSS(Sequence ID 125)

[0390] Variable light chain amino chain sequence Lc19 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 126)

[0391] Variable heavy chain amino chain sequence Hc19 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS(Sequence ID 127)

[0392] Variable light chain amino chain sequence Lc21 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 128)

[0393] Variable heavy chain amino chain sequence Hc21 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS(Sequence ID 129)

[0394] Variable light chain amino chain sequence Lc24 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 130)

[0395] Variable heavy chain amino chain sequence Hc24 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 131)

[0396] Variable light chain amino chain sequence Lc26 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 132)

[0397] Variable heavy chain amino chain sequence Hc26 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS(Sequence ID 133)

[0398] Variable light chain amino chain sequence Lc27 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 134)

[0399] Variable heavy chain amino chain sequence Hc27 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFYGQFDYWGQGTLVTVSS(Sequence ID 135)

[0400] Variable light chain amino chain sequence Lc28 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 136)

[0401] Variable heavy chain amino chain sequence Hc28 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS(Sequence ID 137)

[0402] Variable light chain amino chain sequence Lc30 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 138)

[0403] Variable heavy chain amino chain sequence Hc30 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAAAFDYWGQGTLVTVSS(Sequence ID 139)

[0404] Variable light chain amino chain sequence Lc31 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 140)

[0405] Variable heavy chain amino chain sequence Hc31 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS(Sequence ID 141)

[0406] Variable light chain amino chain sequence Lc32 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 142)

[0407] Variable heavy chain amino chain sequence Hc32 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 143)

[0408] Variable light chain amino chain sequence Lc37 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 144)

[0409] Variable heavy chain amino chain sequence Hc37 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPHNGQFDYWGQGTLVTVSS(Sequence ID 145)

[0410] Variable light chain amino chain sequence Lc39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 146)

[0411] Variable heavy chain amino chain sequence Hc39 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 147)

[0412] Variable light chain amino chain sequence Lc40 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR (Sequence ID 148)

[0413] Heavy chain amino acid sequence Hc40 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS(Sequence ID 149)

[0414] Variable light chain amino chain sequence Lc47 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR(Sequence ID 150)

[0415] Variable heavy chain amino chain sequence Hc47 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 151)

[0416] Variable 4B2 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQGTKVEIKR(Sequence ID 152)

[0417] Variable 4B2 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS(Sequence ID 153)

[0418] Variable 4D11 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKR(Sequence ID 154)

[0419] Variable 4D11 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS(Sequence ID 155)

[0420] Variable 4E7 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQGTKVEIKR (Sequence ID 156)

[0421] Variable 4E7 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 157)

[0422] Variable 4E11 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQGTKVEIKR(Sequence ID 158)

[0423] Variable 4E11 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS(Sequence ID 159)

[0424] Variable 6B7 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR(Sequence ID 160)

[0425] Variable 6B7 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 161)

[0426] Variable 6F8 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR(Sequence ID 162)

[0427] Variable 6F8 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS(Sequence ID 163)

[0428] The antibodies and / or activatable antibodies of this disclosure specifically bind to a predetermined target, such as a human target protein, such as human CD3ε. Anti-CD3ε antibodies, activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies that bind to the same epitopes as the anti-CD3ε antibodies, activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies described herein are also included in this disclosure. 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 to bind to CD3ε, such as human CD3ε, are also included in this disclosure. 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 to bind to CD3ε, such as human CD3ε, are also included in this disclosure. 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 CD3ε to, for example, human CD3ε, are also included in this disclosure.

[0429] Those skilled in the art will recognize that if an anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody, and / or multispecific activatable antibody has the same or similar specificity as the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody, and / or multispecific activatable antibody of this disclosure, it can be determined without excessive experimentation by confirming whether the former prevents the latter from binding to a target. 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 this disclosure, as indicated by the reduced binding by the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody, and / or multispecific activatable antibody of this disclosure, then the anti-CD3ε antibody, anti-CD3ε activatable antibody, and both of these multispecific antibodies and / or multispecific activatable 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 the 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 together with a typically reactive soluble target, and then add the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody to be tested in order to determine whether the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody to be tested inhibits the ability of the anti-CD3ε antibody, anti-CD3ε activatable antibody, multispecific antibody and / or multispecific activatable antibody 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, it is likely that it 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 this disclosure.

[0431] Anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies are produced, for example, by 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 by any of the many techniques recognized in the art for antibody production and / or purification.

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

[0433] Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and similar materials. Convenient vectors are those encoding functionally complete human CH or CL immunoglobulin sequences, along with appropriate restriction sites constructed to readily insert and express any VH or VL sequence. In such vectors, splicing occurs between the splice donor site in the inserted J region and the splice receptor site following the human C region, as well as in splice regions present within human CH exons. Polyadenylation and transcriptional arrest occur at innate chromosomal sites downstream of the coding region. The resulting antibodies can be ligated to any strong promoter, such as retroviral LTRs, e.g., the SV-40 initial promoter (Okayama et al. Mol.Cell.Bio.3:280 (1983)), the Rous sarcoma virus LTR (Gorman et al. PNAS79:6777 (1982)), and the Moloney-Masu leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Alternatively, the innate Ig promoter and similar promoters may be used for recognition.

[0434] Furthermore, anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies and / or multispecific activatable antibodies may be generated using techniques well known in the art, via display-type techniques such as phage display, retrovirus display, ribosome display and other techniques (but not limited to these), and the resulting molecules may be subjected to further maturation, such as affinity maturation, using 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), Russel 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 is desirable to modify the anti-CD3ε antibodies, anti-CD3ε activatable antibodies, multispecific antibodies, and / or multispecific activatable antibodies of this disclosure with respect to effector function in order to enhance or reduce such function in improving the efficacy of the antibody in recruiting CD3+ cells against one or more targets related to the disease being treated, such as targets expressed on tumor cells. For example, a cysteine ​​residue is introduced into the Fc region, thereby enabling the formation of an interchain disulfide bond in this region. The heterodimer antibodies thus produced have improved internalization ability and / or enhanced complement-mediated cell death and antibody-dependent 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, antibodies having a dual Fc region and thereby possessing enhanced complement lysis and ADCC capabilities can be constructed (see Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)). According to some embodiments, Fc maturation is performed, the glycosylation site is removed, and thereby the Fc function is reduced. Activatable antibodies and multispecific activatable antibodies

[0436] The activatable antibodies provided herein comprise an antibody or antibody fragment thereof (collectively referred to as AB throughout this disclosure) that specifically binds to a target and / or epitope, wherein the AB is modified by a masking moiety (MM). The multiple activatable antibodies provided herein comprise at least a first antibody or antibody fragment thereof (collectively referred to as AB1 throughout this disclosure) that specifically binds to a first target and / or first epitope, and a second antibody or antibody fragment thereof (collectively referred to as AB2 throughout this disclosure) that specifically binds to a second target and / or second epitope, wherein at least one of the ABs is modified by a masking moiety (MM). According to some embodiments, each AB in the multiple specific activatable antibody is modified by its own masking moiety.

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

[0438] K of AB modified by MM against the target d This refers to AB not modified by MM, or the K of the parent AB relative to the target. dRather than that, 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, 10-10,000, 10-1,000,000, 10-10,000,000, 10- 1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-10,000,000, 10,000-100,000, 10,000-100,000, 10,000-10,000,000, 10,000-10,000,000, or 100,000-10,000,000 times higher. Conversely, the binding affinity of MM-modified AB to its 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, 1 0-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-10,000,000, 100-10,000,000, 1,000-10,000, 1,000-10,000,000, 1,000-10,000,000, 10,000-100,000, 10,000-10,000,000, 10,000-10,000,000, 100,000-10,000,000 or 100,000-10,000,000 times lower.

[0439] The dissociation constant (K) of MM with respect to AB in activatable antibodies d ) is generally the K of AB against the target.d It is higher than the dissociation constant (K) of MM for at least one of AB in a multispecifically activatable antibody. d ) is generally the K of AB against the target d Higher than . K of MM relative to AB d , is the K of AB against the target d It 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 higher than AB. Conversely, the binding affinity of MM to AB is generally lower than the binding affinity of AB to the target. The binding affinity of MM to 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 AB to the target.

[0440] In an activatable antibody, if AB is modified by MM and in the presence of its target, the specific binding of AB to its target is reduced or inhibited compared to the specific binding of unmodified AB or the specific binding of parental AB to its target. In a multiple-specific activatable antibody, if at least one AB is modified by MM and in the presence of its target, the specific binding of AB to its target is reduced or inhibited compared to the specific binding of unmodified AB or the specific binding of parental AB to its target. When compared to the binding of AB unmodified by MM to a target or the binding of parental AB to a target, the ability of AB modified by MM 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% over 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer, as measured in vivo or in vitro assays.

[0441] MM inhibits the binding of AB to its target in an activatable antibody. MM inhibits the binding of AB to at least one of its targets in a multispecific activatable antibody. MM binds to the antigen-binding domain in AB and inhibits the binding of AB to its target. MM can sterically inhibit the binding of AB to its target. MM allosterically inhibits the binding of AB to its target. According to these embodiments, when AB is modified or coupled to MM and in the presence of a target, as measured in an in vivo or in vitro assay, 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 For longer periods, binding of AB to targets is either nonexistent, substantially nonexistent, 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% compared to binding of AB to targets unmodified by MM, parent AB, or AB not coupled to MM.

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

[0443] When at least one of the AB groups in a multispecifically activatable antibody is coupled to or modified by MM, MM “masks,” reduces, or inhibits the specific binding of AB to its target. When at least one of the AB groups in a multispecifically activatable antibody is coupled to or modified by MM, such coupling or modification can result in a structural change that reduces or inhibits the ability of AB to specifically bind to its target.

[0444] In activatable antibodies, if at least one AB is coupled to MM or modified by MM, at least a portion of the activatable antibody can be represented by the following formula, in order from the amino (N) terminal region to the carboxyl (C) terminal region: (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) Here, MM is the masking portion, AB is the antibody or its antibody fragment, and L is the linker. According to many embodiments, it is desirable to insert one or more linkers, such as flexible linkers, into the composition to provide flexibility.

[0445] In a multispecifically activatable antibody, if at least one AB is coupled to MM or modified by MM, at least a portion of the multispecifically activatable antibody can be represented by the following formula, in order from the amino (N) terminal region to the carboxyl (C) terminal region: (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) Here, MM is the masking portion, AB is the antibody or its antibody fragment, and L is the linker. According to many embodiments, it is desirable to insert one or more linkers, such as flexible linkers, into the composition to provide flexibility.

[0446] According to certain embodiments, MM is not a natural binding partner of AB. According to some embodiments, MM does not contain any natural binding partner of AB or has no homology to such a partner. According to other embodiments, MM is identical to any natural binding partner of AB by at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. According to some embodiments, MM is identical to any natural binding partner of AB by only 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. According to some embodiments, MM is identical to any natural binding partner of AB by only 25%. According to some embodiments, MM is only 50% identical to any natural binding partner of AB. According to some embodiments, MM is only 20% identical to any natural binding partner of AB. According to some embodiments, MM is only 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 by a masking moiety (MM) and also including one or more cleavable moieties (CMs). Such an activatable antibody and / or multispecific activatable antibody exhibits activatable / switchable binding of the AB to a target. The activatable antibody and / or multispecific activatable antibody generally comprises 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 the protease of interest.

[0448] The elements in activatable antibodies and / or multispecific activatable antibodies are positioned such that each MM and CM, when cleaved (or relatively active) and in the presence of the target, has its corresponding AB binding to the target, but when cleaved (or relatively inactive) in the presence of the target, the specific binding of the AB to its target is reduced or inhibited. The specific binding of the AB to its target can be reduced by inhibition or masking of the AB's ability to specifically bind to its target by the MM.

[0449] K of each AB modified by MM and CM against the target d This refers to AB that is not modified by MM and CM, or the K of the parent AB relative to the target. dRather than that, 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, 10-10,000, 10-1,000,000, 10-10,000,000, 10- 1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-10,000,000, 10,000-100,000, 10,000-100,000, 10,000-10,000,000, 10,000-10,000,000, or 100,000-10,000,000 times higher. Conversely, the binding affinity of each AB modified by MM and CM to its 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, 10-10 000, 10~1,000,000, 10~10,000,000, 100~1,000, 100~10,000, 100~100,000, 100~1,000,000, 100~10,000,000, 1,000~10,000, 1,000~100,000, 1,000~10,000,000, 10,000~100,000, 10,000~10,000,000, 10,000~10,000,000, 100,000~1,000,000 or 100,000~10,000,000 times lower.

[0450] If at least one AB is modified by MM and CM, and is present in the presence of its target but not in the presence of a modifier (e.g., a protease), then 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 by MM and CM, or the specific binding of the parent AB to its target. Compared to the binding of parental AB or the binding of AB unmodified by MM and CM to a target, the ability of AB modified by MM and CM to bind to a target may decrease by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% over 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer, as measured in vivo or in vitro assays.

[0451] As used herein, the term "cleaved state" means the state of an activatable antibody and / or a multispecific activatable antibody after modification of the CM by a protease. The term "uncleaved state" means the state of an activatable antibody and / or a multispecific activatable antibody in the absence of cleavage of the CM by a protease. As discussed above, the term "activatable antibody" is used herein to refer to an activatable antibody in both its uncleaved (native) state and its cleaved state. According to some embodiments, it will be apparent to those skilled in the art that 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., if the MM is not covalently bound to the activ...

Claims

1. An activatable anti-CD3ε antibody (AB) is formed by linking a cleavable portion (CM) and a masking portion (MM) that are substrates of a protease, The activatable anti-CD3ε antibody has the following structural configuration from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM; The aforementioned MM is, It contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, and 391; Having one or more features selected from the group consisting of, Activatable anti-CD3ε antibody.

2. The activatable anti-CD3εAB according to claim 1, wherein the CM is a polypeptide up to 15 amino acids in length.

3. The activatable anti-CD3εAB according to claim 1, wherein the arrangement of the CM in the activatable anti-CD3ε antibody reduces the binding of the activatable anti-CD3ε antibody to CD3ε, resulting in a dissociation constant at least 20 times higher than the dissociation constant of the anti-CD3ε antibody's binding to CD3ε when the anti-CD3ε antibody is not bound to the CM and MM.

4. The activatable anti-CD3εAB according to claim 1, wherein in an in vitro targeted substitution assay, the MM reduces the binding ability of the activatable anti-CD3ε antibody to CD3ε by at least 90%.

5. The dissociation constant (K) of the activatable anti-CD3ε antibody with respect to CD3ε when bound to the aforementioned MM. ｄ ) when the activatable anti-CD3ε antibody is not bound to the MM, K against CD3ε ｄ The activatable anti-CD3εAB according to claim 1, wherein the MM reduces the binding ability of the activatable anti-CD3ε antibody to CD3ε to at least 20 times higher than the MM.

6. An activatable anti-CD3ε antibody (AB) is formed by linking a cleavable portion (CM) and a masking portion (MM) that are substrates of a protease, The activatable anti-CD3ε antibody has the following structural configuration from the N-terminus to the C-terminus: MM-CM-AB or AB-CM-MM; The activatable anti-CD3ε antibody, (a) Dissociation constant (K) of the binding of the MM to the activatable anti-CD3ε antibody ｄ ) is the K of the binding of the activatable anti-CD3ε antibody to CD3ε. ｄ It must be less than twice the amount; (b) The binding affinity of the MM to the activatable anti-CD3ε antibody is half the binding affinity of the activatable anti-CD3ε antibody to CD3ε; (c) In an in vitro target substitution assay, the MM reduces the binding ability of the activatable anti-CD3ε antibody to CD3ε by at least 90%; (d) Dissociation constant (K) of the activatable anti-CD3ε antibody with respect to CD3ε when bound to MM ｄ ) when the activatable anti-CD3ε antibody is not bound to the MM, K against CD3ε ｄ The MM reduces the binding ability of the activatable anti-CD3ε antibody to CD3ε to at least 20 times higher than; (e) In an in vitro target substitution assay, the MM reduces the binding ability of the activatable anti-CD3ε antibody to CD3ε by at least 90%; and, (f) The arrangement of the CM in the activatable anti-CD3ε antibody reduces the binding of the activatable anti-CD3ε antibody to CD3ε, resulting in a dissociation constant at least 20 times higher than the dissociation constant of the anti-CD3ε antibody binding to CD3ε when the anti-CD3ε antibody is not bound to the CM and MM; Having one or more features selected from the group consisting of, Here, MM includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, and 391. Activatable anti-CD3ε antibody.

7. The binding of MM to the activatable anti-CD3ε antibody ｄ However, the binding of the activatable anti-CD3ε antibody to CD3ε is K ｄ The activatable anti-CD3εAB according to claim 6, which is less than twice the amount of the above.

8. The activatable anti-CD3εAB according to claim 6, wherein the affinity of the MM for binding to the activatable anti-CD3ε antibody is half the affinity of the activatable anti-CD3ε antibody for binding to CD3ε.

9. The activatable anti-CD3εAB according to claim 6, wherein in an in vitro targeted substitution assay, the MM reduces the binding ability of the activatable anti-CD3ε antibody to CD3ε by at least 90%.

10. The dissociation constant (K ｄ ) of the activatable anti-CD3ε antibody when bound to the MM for CD3ε is at least 20-fold higher than the K ｄ of the activatable anti-CD3ε antibody for CD3ε when not bound to the MM, such that the MM reduces the binding ability of the activatable anti-CD3ε antibody for CD3ε. The activatable anti-CD3ε AB according to claim 6.

11. The activatable anti-CD3εAB according to claim 6, wherein in an in vitro targeted substitution assay, the MM reduces the binding ability of the activatable anti-CD3ε antibody to CD3ε by at least 90%.

12. The activatable anti-CD3εAB according to claim 6, wherein the arrangement of the CM in the activatable anti-CD3ε antibody reduces the binding of the activatable anti-CD3ε antibody to CD3ε, resulting in a dissociation constant at least 20 times higher than the dissociation constant of the binding of the anti-CD3ε antibody to CD3ε when the anti-CD3ε antibody is not bound to the CM and MM.

13. The activatable anti-CD3εAB according to claim 6, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-86, 321-341, and 896-926.

14. The activatable anti-CD3εAB according to claim 6, further comprising a linked peptide between the MM and the CM.

15. The activatable anti-CD3εAB according to claim 14, wherein the linked peptide has a length of 1 to 20 amino acids.

16. The activatable anti-CD3εAB according to claim 6, wherein the activatable anti-CD3ε antibody has the following structural configuration from the N-terminus to the C-terminus: MM-CM-AB;

17. The activatable anti-CD3εAB according to claim 6, wherein the activatable anti-CD3ε antibody has the following structural configuration from the N-terminus to the C-terminus: AB-CM-MM;

18. A composition comprising an activatable anti-CD3εAB according to any one of claims 1 to 17 and a carrier.

19. A nucleic acid encoding an activatable anti-CD3εAB according to any one of claims 1 to 17.

20. An expression vector comprising the nucleic acid described in claim 19.

21. A method for producing an activatable anti-CD3εAB, comprising culturing cells containing the expression vector described in claim 20 and expressing the activatable anti-CD3εAB.

22. An activatable anti-CD3εAB according to any one of claims 1 to 17, or the composition according to claim 18, for use in the treatment of cancer in a subject.

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