Multispecific antibodies, multispecific activatable antibodies, and methods of using the same

Abstract

To provide multispecific antibodies and multispecific activatable antibodies that specifically bind to multiple different antigens or epitopes, and methods of making and using these multispecific antibodies and / or multispecific activatable antibodies in a variety of therapeutic, diagnostic and prophylactic applications.SOLUTION: The present invention provides multispecific activatable antibodies that are multispecific antibodies that include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody such that coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 858,402, filed July 25, 2013, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates generally to multispecific antibodies and multispecific activatable antibodies that specifically bind to multiple different targets or epitopes, and methods for making and using these multispecific and / or multispecific activatable antibodies for a variety of therapeutic, diagnostic, and prophylactic applications. [Background technology]

[0003] Monoclonal antibodies have emerged as molecules for therapeutic intervention in a variety of disorders. However, targeting or neutralization of a single protein is not always sufficient for effective treatment of a particular disorder, limiting the therapeutic use of monospecific monoclonal antibodies. Furthermore, although antibody-based therapies have proven effective treatments for some diseases, in some cases toxicity due to broad target expression has limited their therapeutic effectiveness. Furthermore, antibody-based therapies have shown other limitations, such as rapid clearance from the circulation following administration.

[0004] Thus, there is a need for antibodies and therapeutic agents that allow for the targeting of multiple targets and / or multiple epitopes with a single molecule and also provide increased selectivity for the intended target and reduced administration-based side effects. Summary of the Invention

[0005] The present disclosure provides multispecific antibodies and multispecific activatable antibodies. Multispecific antibodies are antibodies that recognize multiple antigens or epitopes. The multispecific activatable antibodies provided herein are multispecific antibodies that include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody, where coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind 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 circulation, are activated at intended sites of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit target binding at least comparable to that of the corresponding, unmodified multispecific antibody.

[0006] According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies are designed to recruit immune effector cells, also referred to herein as immune effector-recruiting multispecific antibodies and / or immune effector-recruiting multispecific activatable antibodies. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies are designed to recruit leukocytes, also referred to herein as leukemia-recruiting multispecific antibodies and / or leukemia-recruiting multispecific activatable antibodies. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies are designed to recruit T-cells, also referred to herein as T-cell-recruiting multispecific antibodies and / or T-cell-recruiting multispecific activatable antibodies. According to some embodiments, the multispecific antibodies and / or multispecific activatable antibodies recruit surface antigens on leukocytes, such as T cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells. According to some embodiments, the immune effector cells are leukocytes. According to some embodiments, the immune effector cell is a T cell. According to some embodiments, the immune effector cell is a NK cell. According to some embodiments, the immune effector cell is a mononuclear cell, e.g., a bone marrow mononuclear cell. According to some embodiments, the multispecific antibody and / or multispecific activatable antibody is designed to bind or otherwise react with multiple targets and / or multiple epitopes, also referred to herein as a multi-antigen-targeting antibody and / or a multi-antigen-targeting, activatable antibody. As used herein, the terms "target" and "antigen" are used interchangeably.

[0007] According to some embodiments, the immune effector cell-recruiting multispecific antibody comprises a targeting antibody or antigen-binding fragment thereof and an immune effector cell-recruiting antibody or antigen-binding portion thereof. According to some embodiments, the immune effector cell-recruiting multispecific antibody comprises a cancer-targeting antibody or antigen-binding fragment thereof and an immune effector cell-recruiting antibody or antigen-binding portion thereof. According to some embodiments, the immune effector cell-recruiting multispecific antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof and an immune effector cell-recruiting scFv. According to some embodiments, the immune effector cells are leukocytes. According to some embodiments, the immune effector cells are T cells. According to some embodiments, the immune effector cells are NK cells. According to some embodiments, the immune effector cells are bone marrow mononuclear cells.

[0008] According to some embodiments, the T-cell engaging multispecific antibody comprises a targeting antigen or antigen-binding fragment thereof, and a T-cell engaging antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell engaging multispecific antibody comprises a cancer-targeting antigen or antigen-binding fragment thereof, and a T-cell engaging antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell engaging multispecific antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof, and a T-cell engaging scFv. According to some embodiments, the T-cell engaging multispecific antibody comprises an anti-CD3 epsilon (CD3ε, also referred to herein as CD3e and CD3) scFv, and a targeting antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell engaging multispecific antibody comprises an anti-CD3ε scFv, and a cancer-targeting antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell engaging multispecific antibody comprises an anti-CD3ε scFv, and a cancer-targeting antibody or antigen-binding fragment thereof. According to some embodiments, the T-cell binding multispecific antibody comprises an anti-CD3 epsilon (CD3ε) scFv derived from OKT3. According to some embodiments, the T-cell engaging multispecific antibody comprises an anti-CTLA-4 scFv.

[0009] According to some embodiments, the immune effector cell-recruiting multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and an immune effector cell-recruiting antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the immune effector cell-recruiting antibody or antigen-binding portion thereof is masked. According to some embodiments, the immune effector cell-recruiting antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell-recruiting target, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the immune effector cell-recruiting antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first immune effector cell-recruiting target, where AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, where AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the non-immune effector cell-recruiting antibody is a cancer-targeting antibody. According to some embodiments, the immune effector cell-recruiting antibody is an scFv. According to some embodiments, the targeting antibody (e.g., non-immune cell effector antibody) is an IgG, and the immune effector cell-recruiting antibody is an scFv. According to some embodiments, the immune effector cells are leukocytes. According to some embodiments, the immune effector cells are T cells. According to some embodiments, the immune effector cells are NK cells.According to some embodiments, the immune effector cells are bone marrow mononuclear cells.

[0010] According to some embodiments, the T-cell-engaging multispecific activatable antibody of the present disclosure comprises a targeting antibody or antigen-binding fragment thereof and a T-cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the T-cell-engaging antibody or antigen-binding portion thereof is masked. According to some embodiments, the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the T-cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment (AB2) that binds a second target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0011] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises a cancer-targeting antibody or antigen-binding fragment thereof and a T-cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the cancer-targeting antibody or antigen-binding fragment thereof and / or the T-cell-engaging antibody or antigen-binding portion thereof is masked. According to some embodiments, the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind a second cancer-associated target. According to some embodiments, the T-cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0012] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises a cancer-targeting IgG antibody or antigen-binding fragment thereof and a T-cell-engaging scFv, wherein at least one of the cancer-targeting IgG antibody or antigen-binding fragment thereof and / or the T-cell-engaging antibody or antigen-binding portion thereof is masked. According to some embodiments, the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of the MM1 reduces the ability of AB1 to bind the first target. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM2 reduces the ability of AB2 to bind a second cancer-associated target. According to some embodiments, the T-cell engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, wherein said AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind the first target, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein said AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0013] According to some embodiments of the immune effector-recruiting multispecific activatable antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, for example, but not limited to, any of the targets listed in Table 1, such as, but not limited to, EGGR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and another antigen is typically a stimulatory or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. According to some embodiments, the antigen is a stimulatory receptor present on the surface of T cells or NK cells; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. According to some embodiments, the antigen is an inhibitory receptor present on the surface of T cells; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIR. An antibody that confers specificity for a T-cell surface antigen may be substituted with a ligand or ligand domain that binds to an effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9.

[0014] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the cancer microenvironment and includes an antibody, e.g., an IgG or scFv, directed against a tumor target and an agonist antibody, e.g., an IgG or scFv directed against a costimulatory receptor expressed on the surface of activated T cells or NK cells, wherein at least one of the cancer-targeting antibody and / or agonist antibody is masked. Examples of costimulatory receptors include, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, and OX40. According to this embodiment, the multispecific activatable antibody, upon activation by tumor-associated proteases, will effectively crosslink and activate T cell- or NK cell-expressed costimulatory receptors in a tumor-dependent manner to enhance the activity of T cells responding to any tumor antigen via their endogenous T cell antigen or NK-activating receptor. The activation-dependent nature of these T cell or NK cell costimulatory receptors focuses the activity of the activated multispecific activatable antibody on tumor-specific T cells, rather than activating all T cells, regardless of their antigen specificity. According to one embodiment, at least the costimulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of autoreactive T cells that may be present in tissues that also express the antigen recognized by the tumor-targeting antibody in the multispecific activatable antibody, but whose activity is limited by the lack of co-receptor recruitment.

[0015] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in the microenvironment of diseases characterized by T cell hyperstimulation, such as, but not limited to, autoimmune or inflammatory diseases. Such multispecific activatable antibodies include antibodies, e.g., IgG or scFv, directed against targets including surface antigens expressed in tissues targeted by T cells in autoimmune inflammatory diseases, and antibodies, e.g., IgG or scFv, directed against inhibitory receptors expressed on the surface of T cells or NK cells, wherein at least one of the disease tissue-targeting antibody and / or T cell receptor antibody is masked. Examples of inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIR. Examples of tissue antigens targeted by T cells in autoimmune diseases include, but are not limited to, surface antigens expressed on myelin or nerve cells in multiple sclerosis, or surface antigens expressed on pancreatic islet cells in type 1 diabetes. According to this embodiment, the multispecific activatable antibodies, when localized to tissues under autoimmune attack or inflammation, become activated and co-recruit T cell or NK cell inhibitory receptors via their endogenous TCRs or activating receptors to suppress the activity of autoreactive T cells responding to any diseased tissue-targeted antigens. According to one embodiment, at least one or more antibodies are masked to prevent suppression of desired T cell responses in non-diseased tissues where the target antigen may also be expressed.

[0016] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CD3 epsilon (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, comprising a second antibody or antigen-binding fragment thereof (AB2), wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target.

[0017] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0018] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CD3ε scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CD3ε scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the CD3ε scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0019] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an OKT3-derived anti-CD3 epsilon (CD3ε) scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the OKT3 scFv or OKT3-derived scFv is masked. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof that binds a second target, comprising a second antibody or antigen-binding fragment thereof (AB2), wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second target.

[0020] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting antibody or antigen-binding fragment thereof is masked. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0021] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an OKT3 scFv or an OKT3-derived scFv and a cancer-targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the OKT3 scFv or OKT3-derived scFv and / or the cancer-targeting IgG antibody or antigen-binding fragment thereof is masked. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε. According to some embodiments, the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target. According to some embodiments, the OKT3 scFv or OKT3-derived scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, wherein the AB1 is conjugated to a masking moiety (MM1), such that the coupling of MM1 reduces the ability of AB1 to bind CD3ε, and the cancer-targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second cancer-associated target, wherein the AB2 is conjugated to a masking moiety (MM2), such that the coupling of MM2 reduces the ability of AB2 to bind the second cancer-associated target.

[0022] According to some embodiments, the T-cell engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv, wherein at least one of the targeting antibody or antigen-binding fragment thereof and / or the anti-CTLA-4 scFv is masked. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4. According to some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target.

[0023] According to some embodiments, the T-cell-engaging multispecific activatable antibody comprises an anti-CTLA-4 scFv and a targeting IgG antibody or antigen-binding fragment thereof, wherein at least one of the anti-CTLA-4 scFv and / or the targeting IgG antibody or antigen-binding portion thereof is masked. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4. According to some embodiments, the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the anti-CTLA-4 scFv comprises a first antibody or antigen-binding fragment thereof (AB1) that binds CTLA-4, wherein the AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind CTLA-4, and the targeting IgG antibody or antigen-binding fragment thereof comprises a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein the AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM2 reduces the ability of AB2 to bind the second target.

[0024] According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody comprises at least a first antibody or antigen-binding fragment thereof that binds a first target and / or a first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and / or a second epitope. According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different targets. According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds multiple different epitopes on the same target. According to some embodiments, the multiple antigen-targeting antibody and / or multiple antigen-targeting activatable antibody binds a combination of multiple different targets and multiple different epitopes on the same target.

[0025] Various embodiments of the multispecific activatable antibodies of the present disclosure are shown in Figures 3A and 5-9. According to some embodiments, an IgG-containing multispecific activatable antibody has a masked IgG variable domain. According to some embodiments, an scFv-containing multispecific activatable antibody has a masked scFv domain. According to some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. According to some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, a multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein each of the IgG variable domain and the scFv domain is coupled to its own masking moiety. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for another target antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for an epitope of the target antigen and another antibody domain has specificity for another epitope of the target antigen.

[0026] In multispecific activatable antibodies, scFvs can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, the carboxyl terminus of the light chain of an IgG activatable antibody, or the carboxyl terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, the amino terminus of the light chain of an IgG activatable antibody, or the amino terminus of both the heavy and light chains of an IgG activatable antibody. In multispecific activatable antibodies, scFvs can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to the antigen-binding domain of an IgG and masks that domain. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to the antigen-binding domain of at least one scFv and masks that domain. According to some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of an IgG, and a masking moiety (MM) linked to a cleavable moiety (CM) is linked to and masks the antigen-binding domain of at least one scFv.

[0027] The present disclosure provides examples of multispecific activatable antibody structures, including, but 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-L2-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; (VL-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); or (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2: (VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2, where VL and VH represent the light and heavy variable domains of the first specificity contained in IgG; VL * and VH * represents the variable domain of the second specificity contained in the scFv; L1 is a linker peptide connecting the masking moiety (MM) and the cleavable moiety (CM); L2 is a linker peptide connecting the cleavable moiety (CM) and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is a light chain constant domain; and CH1, CH2, and CH3 are heavy chain constant domains. The first and second specificities can be against any antigen or epitope.

[0028] According to some embodiments of the T-cell engaging multispecific activatable antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, such as, but not limited to, any of the targets listed in Table 1, such as EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or The antigen may be, but is not limited to, CD71 (transferrin receptor), and another antigen may typically be a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of T-cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain that provides specificity for a T-cell surface antigen may also be replaced with a ligand or ligand domain that binds to a T-cell receptor, an NK-cell receptor, a macrophage receptor and / or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2 or TNFSF9. According to some embodiments of the multi-antigen targeted activatable antigen, one antigen is selected from the target group listed in Table 1, and another antigen is selected from the target group listed in Table 1.

[0029] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v4, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v6, which is specific for binding to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which is specific for binding to human and mouse Jagged1 and Jagged2. In some embodiments, the targeting antibody is 4D11v2, which is specific for binding to human and mouse Jagged1 and Jagged2.

[0030] In some embodiments, the targeting antibody can be in the form of an activatable antibody. In some embodiments, the scFv can be in the form of a pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0031] According to some embodiments, the scFv is specific for binding CD3ε and is or is derived from an antibody or fragment thereof that binds CD3ε, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. According to some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).

[0032] According to some embodiments, the anti-CD3ε scFv comprises a sequence selected from the group consisting of those sequences shown in Table 7 and / or Example 5. According to some embodiments, the anti-CD3ε scFv comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in Table 7 and / or Example 5.

[0033] According to some embodiments, the scFv is specific for binding one or more T-cells, one or more NK cells, and / or one or more macrophages. According to some embodiments, the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.

[0034] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence SYAMS (SEQ ID NO: 6); a VH CD2 sequence comprising at least the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 7); a VH CDR3 sequence comprising at least the amino acid sequence DIGGRSAFDY (SEQ ID NO: 8); and combinations thereof.

[0035] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RASQSISSY (SEQ ID NO: 9); a VL CD2 sequence comprising at least the amino acid sequence AASSLQS (SEQ ID NO: 10); a VL CDR3 sequence comprising at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 11); and combinations thereof.

[0036] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence and the VH CDR3 sequence comprises a VH CDR1 sequence that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SYAMS (SEQ ID NO: 6); a VH CD2 sequence comprising a sequence that is 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 8); a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 8); and combinations thereof.

[0037] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a Jagged target, e.g., Jagged1 and / or Jagged2, and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence and the VL CDR3 sequence comprises a VL CDR1 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSISSY (SEQ ID NO: 9); a VL CDR2 sequence comprising a sequence that is 97%, 98%, 99% or more identical to the amino acid sequence QQTVVAPPL (SEQ ID NO: 11); a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQTVVAPPL (SEQ ID NO: 11); and combinations thereof.

[0038] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein specifically bind a Jagged target, e.g., Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises at least the amino acid sequence SYAMS (SEQ ID NO: 6); the VH CDR2 sequence comprises at least the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 7); the VH CDR3 sequence comprises at least the amino acid sequence DIGGRSAFDY (SEQ ID NO: 8); the VL CDR1 sequence comprises at least the amino acid sequence RASQSISSY (SEQ ID NO: 9); the VL CD2 sequence comprises at least the amino acid sequence AASSLQS (SEQ ID NO: 10); and the VL CDR3 sequence comprises at least the amino acid sequence AASSLQS (SEQ ID NO: 11). The CDR3 sequence comprises at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 11).

[0039] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein specifically bind a Jagged target, e.g., Jagged1 and / or Jagged2, and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence SYAMS (SEQ ID NO: 6); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:8); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSISSY (SEQ ID NO:9); the VL CD2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQ ID NO:10). and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQTVVAPPL (SEQ ID NO: 11).

[0040] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds epidermal growth factor receptor (EGFR) and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence and the VH CDR3 sequence is selected from a VH CDR1 sequence comprising at least the amino acid sequence NYGVH (SEQ ID NO: 12); a VH CD2 sequence comprising at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 13); a VH CDR3 sequence comprising at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 14); and combinations thereof.

[0041] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence and the VL CDR3 sequence is selected from a VL CDR1 sequence comprising at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 15); a VL CD2 sequence comprising at least the amino acid sequence KYASESIS (SEQ ID NO: 16); a VL CDR3 sequence comprising at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 17); and combinations thereof.

[0042] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence and a VH CDR3 sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence and the VH CDR3 sequence comprises a VH CDR1 sequence that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 12); a VH CD2 sequence comprising a sequence that is 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 14); a VH CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 14); and combinations thereof.

[0043] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds EGFR and comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence and a VL CDR3 sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence and the VL CDR3 sequence comprises a VL CDR1 sequence that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 15); a VL CD2 sequence comprising a sequence that is 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 17); a VL CDR3 sequence comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 17); and combinations thereof.

[0044] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises at least the amino acid sequence NYGVH (SEQ ID NO: 12); the VH CDR2 sequence comprises at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 13); the VH CDR3 sequence comprises at least the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 14); the VL CDR1 sequence comprises at least the amino acid sequence RASQSIGTNIH (SEQ ID NO: 15); the VL CD2 sequence comprises at least the amino acid sequence KYASESIS (SEQ ID NO: 16); and the VL CDR3 sequence comprises at least the amino acid sequence QQNNNWPTT (SEQ ID NO: 17).

[0045] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein specifically bind EGFR and comprise at least a first antibody or antigen-binding fragment thereof (AB1) comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 12); and the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 13). the VH CDR3 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 14); the VL CDR1 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 15); and the VL CD2 sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ ID NO: 16). and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 17).

[0046] According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence selected from the group consisting of those sequences shown in Table 7 and / or Example 5, including Table 11 herein. According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a light chain amino acid sequence selected from the group consisting of those sequences shown in Table 7 and / or Example 5, including Table 11 herein. According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence selected from the group consisting of those sequences shown in Table 7 and / or Example 5, including Table 11 herein, and a light chain amino acid sequence selected from the group consisting of those sequences shown in Table 7 and / or Example 5, including Table 11 herein.

[0047] According to some embodiments, multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in Table 7, including Table 11, and / or Example 5 herein. According to some embodiments, multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in Table 7, including Table 11, and / or Example 5 herein. According to some embodiments, the multispecific antibodies, multispecific activatable antibodies, conjugated multispecific antibodies and / or conjugated multispecific activatable antibodies provided herein comprise at least a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence selected from the group consisting of those sequences shown in Table 7, including Table 11, and / or Example 5 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 Table 7, including Table 11, and / or Example 5 herein.

[0048] According to some embodiments, the multispecific antibody and / or multispecific activatable antibody also includes an agent conjugated to the AB. According to some embodiments, the agent is an anti-tumor agent. According to some embodiments, the agent is a toxin or a fragment thereof. According to some embodiments, the agent is conjugated to the multispecific antibody via a linker. According to some embodiments, the linker is a non-cleavable linker. According to some embodiments, the agent is a microtubule inhibitor. According to some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent, or a DNA intercalator, or other DNA damaging agent. According to some embodiments, the linker is a cleavable linker. According to some embodiments, the agent is an agent selected from the group listed in Table 4. According to some embodiments, the agent is trastatin. According to some embodiments, the agent is an auristatin or a derivative thereof. According to some embodiments, the agent is auristatin E or a derivative thereof. According to some embodiments, the agent is monomethyl auristatin E (MMAE). According to some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0049] According to some embodiments, the multispecific and / or multispecific activatable antibody can also comprise a detectable moiety, which, according to some embodiments, is a diagnostic agent.

[0050] According to some embodiments, multispecific and / or multispecific activatable antibodies naturally comprise one or more disulfide bonds. According to some embodiments, multispecific and / or multispecific activatable antibodies can be engineered to comprise one or more disulfide bonds.

[0051] The present disclosure also provides isolated nucleic acid molecules encoding the multispecific antibodies and / or multispecific activatable antibodies described herein, and vectors comprising those isolated nucleic acid sequences. The present disclosure provides methods for producing multispecific antibodies by culturing cells comprising such nucleic acid molecules under conditions conducive to expression of the antibodies. According to some embodiments, the cells comprise such vectors.

[0052] The present disclosure also provides multispecific antibody and / or multispecific activatable antibody compositions comprising at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that binds a second target or a second epitope, wherein at least AB1 is coupled to or otherwise bound to a masking moiety (MM1), such that coupling of MM1 reduces the ability of AB1 to bind its target. According to some embodiments, MM1 is coupled to AB1 via a first cleavable moiety (CM1) sequence that comprises a substrate for a protease, e.g., a protease that co-localizes with the target of AB1 at a therapeutic or diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, are activated at the intended site of therapy and / or diagnosis, but not in normal, i.e., healthy tissue, and, when activated, exhibit binding of AB1 to the target at least comparable to that of the corresponding unmodified multispecific antibody.

[0053] According to some embodiments, the multispecific activatable antibody comprises a linking peptide between MM1 and CM1.

[0054] According to some embodiments, the multispecific activatable antibody comprises a linking peptide between CM1 and AB1.

[0055] According to some embodiments, the activatable antibody comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and at least a portion of the multispecific activatable antibody, in its uncleaved state, has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. According to some embodiments, the two connecting peptides need not be identical to each other.

[0056] According to some embodiments, at least one of LP1 or LP2 is (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 18) and (GGGS) n (SEQ ID NO:19), where n is at least one integer. According to some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:20), GGSGG (SEQ ID NO:21), GSGSG (SEQ ID NO:22), GSGGG (SEQ ID NO:23), GGGSG (SEQ ID NO:24), and GSSSG (SEQ ID NO:25).

[0057] According to some embodiments, the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or a first epitope, and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds a second target or a second epitope. According to some embodiments, each AB in the multispecific activatable antibody is independently selected from the group consisting of monoclonal antibodies, domains, antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single-domain heavy chain antibodies, and single-domain light chain antibodies. According to some embodiments, each AB in the multispecific activatable antibody is a rodent (e.g., mouse or rat), chimeric, humanized, or fully human monoclonal antibody.

[0058] According to some embodiments, each AB in the multispecific activatable antibody has an equilibrium dissociation constant of about 100 nM or less for binding to its corresponding target or epitope.

[0059] According to some embodiments, MM1 has an equilibrium dissociation constant for binding to its corresponding AB that is higher than the equilibrium dissociation constant for binding of AB to its corresponding target or epitope.

[0060] According to some embodiments, MM1 has an equilibrium dissociation constant for binding to its corresponding AB that is no more than the equilibrium dissociation constant for binding of AB to its corresponding target or epitope.

[0061] According to some embodiments, MM1 does not block or compete with the corresponding AB for binding to its corresponding target or epitope when the multispecific activatable antibody is present in a cleaved state.

[0062] According to some embodiments, MM1 is a polypeptide about 2-40 amino acids in length. According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 or less amino acids in length.

[0063] According to some embodiments, MM1 has a polypeptide sequence that differs from the sequence of the target of its corresponding AB.

[0064] According to some embodiments, MM1 has a polypeptide sequence that is 50% or less identical to any of the natural binding partners of its corresponding AB. According to some embodiments, MM1 has a polypeptide sequence that is 25% or less identical to any of the natural binding partners of its corresponding AB. According to some embodiments, MM1 has a polypeptide sequence that is 10% or less identical to any of the natural binding partners of its corresponding AB.

[0065] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 20 times higher than

[0066] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 40 times higher than

[0067] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitoped At least 100 times higher than

[0068] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 1000 times higher than

[0069] According to some embodiments, the coupling of MM1 reduces the ability of its corresponding AB to bind to its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM1 towards its corresponding target or epitope d At least 10,000 times higher than

[0070] According to some embodiments, MM1 is an amino acid sequence selected from the MMs shown in the Examples provided herein.

[0071] According to some embodiments, the multispecific activatable antibody comprises at least a second masking moiety (MM2), which inhibits binding of AB2 to its target when the multispecific activatable antibody is in an uncleaved state, and a second cleavable moiety (CM2), coupled to AB2, which functions as a substrate for a second protease. According to some embodiments, CM2 is a polypeptide 15 amino acids or less in length. According to some embodiments, the second protease is co-localized with a second target or epitope in a tissue, and the second protease cleaves CM2 on the multispecific activatable antibody when the multispecific activatable antibody is exposed to the second protease. According to some embodiments, the first protease and the second protease are co-localized with the first target or epitope and the second target or epitope in a tissue. According to some embodiments, the first protease and the second protease are the same protease. According to some embodiments, CM1 and CM2 are different substrates for 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.

[0072] According to some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has an equilibrium dissociation constant for binding to its corresponding AB that is higher than the equilibrium dissociation constant for binding to its corresponding target or epitope of AB.

[0073] According to some embodiments, each MM in the multispecific activatable antibody, e.g., MM1 and at least MM2, has an equilibrium dissociation constant for binding to its corresponding AB that is less than or equal to the equilibrium dissociation constant for binding to its corresponding target or epitope of AB.

[0074] According to some embodiments, each MM in the multispecific activatable antibody does not interfere with or compete with its corresponding AB for binding to the corresponding target or epitope when the multispecific activatable antibody is in a cleaved state.

[0075] According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide about 2-40 amino acids in length. According to some embodiments, each MM in the multispecific activatable antibody is a polypeptide 40 amino acids or less in length.

[0076] According to some embodiments, each MM in the multispecific activatable antibody has a polypeptide sequence that differs from the sequence of the target of the corresponding AB.

[0077] According to some embodiments, each MM in the multispecific activatable antibody has a polypeptide sequence that is no more than 50% identical to any natural binding partner of its corresponding AB. According to some embodiments, each MM in the multispecific activatable antibody has a polypeptide sequence that is no more than 25% identical to any natural binding partner of its corresponding AB. According to some embodiments, each MM in the multispecific activatable antibody has a polypeptide sequence that is no more than 10% identical to any natural binding partner of its corresponding AB.

[0078] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 20 times higher than

[0079] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 40 times higher than

[0080] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 100 times higher than

[0081] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 1000 times higher than

[0082] According to some embodiments, the coupling of each MM reduces the ability of its corresponding AB to bind its target or epitope, resulting in a decrease in the dissociation constant (K d ) is the K of AB when not coupled to MM towards its corresponding target or epitope d At least 10,000 times higher than

[0083] According to some embodiments, each MM is an amino acid sequence selected from the MMs shown in the examples provided herein.

[0084] According to some embodiments, a protease that cleaves the first cleavable moiety (CM1) sequence co-localizes in a tissue with the target of AB1 in the multispecific activatable antibody, and the protease cleaves CM1 in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0085] According to some embodiments, the multispecific activatable antibody comprises multiple cleavable subsequences, and a protease that cleaves at least one cleavable subsequence is co-localized in a tissue with a target of at least one region of the AB region in the multispecific activatable antibody, and the protease cleaves the CM in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

[0086] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 20-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0087] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 40-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0088] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 50-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0089] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 100-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0090] According to some embodiments, each CM, e.g., CM1 and at least CM2, is located on the multispecific activatable antibody such that, under uncleaved conditions, binding of the multispecific activatable antibody to a target in one of the AB regions is reduced with an equilibrium dissociation constant that is at least 200-fold higher than the equilibrium dissociation constant of the unmodified AB binding to that target, whereas under cleaved conditions, the AB binds its target.

[0091] According to some embodiments, each CM in the multispecific activatable antibody is a polypeptide up to 15 amino acids in length.

[0092] According to some embodiments, at least one CM in the multispecific activatable antibody comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 26). According to some embodiments, at least one cleavable moiety is selected for use with a particular protease, e.g., a protease known to co-localize with at least one target of the multispecific activatable antibody. For example, suitable cleavable moieties for use in the multispecific activatable antibodies of the present disclosure are cleaved by at least proteases such as urokinase, legumain, and / or matriptase (also referred to herein as MT-SP1 or MTSSP1). According to some embodiments, suitable cleavable moieties comprise at least one of the following sequences: TGRGPSWV (SEQ ID NO:27); SARGPSRW (SEQ ID NO:28); TARGPSFK (SEQ ID NO:29); LSGRSDNH (SEQ ID NO:26); GGWHTGRN (SEQ ID NO:30); HTGRSGAL (SEQ ID NO:31); PLTGRSGG (SEQ ID NO:32); AARGPAIH (SEQ ID NO:33); RGPAFNPM (SEQ ID NO:34); SSRGPAYL (SEQ ID NO:35); RGPATPIM (SEQ ID NO:36); RGPA (SEQ ID NO:37); GGQPSGMWGW (SEQ ID NO:38); FPRPLGITGL (SEQ ID NO:39); VHMPLGFLGP (SEQ ID NO:40); SPLTGRSG (SEQ ID NO:41); SAGFSLPA (SEQ ID NO:42); LAPLGLQRR (SEQ ID NO:43); SGGPLGVR (SEQ ID NO:44); and / or PLGL (SEQ ID NO:45).

[0093] According to some embodiments, each CM in the multispecific activatable antibody is a substrate for a protease selected from the group consisting of those shown in Table 3. According to some embodiments, the protease is selected from the group consisting of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, neutrophil elastase, MMP-7, MMP-9, MMP-12, MMP-13, and MMP-14. The protease is a cathepsin, such as, but not limited to, cathepsin S. According to some embodiments, each CM in the multispecific activatable antibody is a substrate for a protease selected from the group consisting of uPA (urokinase plasminogen activator), legumain, and MT-SP1 (matriptase). According to some embodiments, the protease includes uPA. According to some embodiments, the protease includes legumain. According to some embodiments, the protease includes MT-SP1. According to some embodiments, the protease includes a matrix metalloproteinase (MMP).

[0094] According to some embodiments, at least one CM in the multispecific activatable antibody is a substrate for at least two proteases. According to some embodiments, each protease is selected from the group consisting of those shown in Table 3. According to some embodiments, at least one CM in the multispecific activatable antibody is a substrate for at least two proteases, where one of the proteases is selected from the group consisting of uPA, legumain, and MT-SP1, and the other protease is selected from the group consisting of those shown in Table 3. According to some embodiments, at least one CM in the multispecific activatable antibody is a substrate for at least two proteases selected from the group consisting of uPA, legumain, and MT-SP1.

[0095] According to some embodiments, a multispecific activatable antibody comprises at least a first CM (CM1) and a second CM (CM2). According to some embodiments, CM1 and CM2 are part of a single cleavable linker linking the MM to the AB. According to some embodiments, CM1 is part of a cleavable linker linking MM1 to AB1, and CM2 is part of another cleavable linker linking MM2 to AB2. According to some embodiments, a multispecific activatable antibody comprises two or more CMs. According to some embodiments, such a multispecific activatable antibody comprises two or more CMs and two or more MMs. According to some embodiments, CM1 and CM2 are each polypeptides 15 or fewer amino acids in length. According to some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of those listed in Table 3. According to some embodiments, at least one of the first CM and the second CM is a polypeptide that also functions as a substrate for a protease selected from the group consisting of uPA, legumain, and MT-SP1. According to some embodiments, the first CM is cleaved in the target tissue by a first cleavage agent selected from the group consisting of uPA, legumain, and MT-SP1, and the second CM is cleaved in the target tissue by a second cleavage agent. According to some embodiments, the other protease is selected from the group consisting of those shown in Table 3. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group consisting of those listed in Table 3, and the first CM and the second CM are different substrates for the enzyme. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group consisting of uPA, legumain, and MT-SP1, and the first CM and the second CM are different substrates for the enzyme. According to some embodiments, the first cleavage agent and the second cleavage agent are the same protease selected from the group listed in Table 3, and the first CM and the second CM are the same substrate. According to some embodiments, the first cleavage agent and the second cleavage agent are different proteases.According to some embodiments, the first cleaving agent and the second cleaving agent are different proteases selected from the group consisting of those shown in Table 3. According to some embodiments, the first cleaving agent and the second cleaving agent are co-localized in the target tissue. According to some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

[0096] According to some embodiments, the multispecific activatable antibody is exposed to a protease and cleaved, such that under activated or cleaved conditions, the activated multispecific activatable antibody comprises a light chain amino acid sequence that includes at least a portion of the LP2 and / or CM sequence after the protease cleaves the CM.

[0097] According to some embodiments, the multispecific activatable antibody also comprises a signal peptide. According to some embodiments, the signal peptide is conjugated to the multispecific activatable antibody via a spacer. According to some embodiments, the spacer is conjugated to the multispecific activatable antibody in the absence of a signal peptide. According to some embodiments, the spacer is directly attached to at least one of the MMs of the multispecific activatable antibody.

[0098] According to some embodiments, the multispecific activatable antibody in its uncleaved state is directly linked to the first MM and comprises a spacer N-terminally to C-terminally having the structural arrangement spacer-MM1-CM-AB1. According to some embodiments, the spacer comprises at least the amino acid sequence QGQSGQ (SEQ ID NO:46).

[0099] According to some embodiments, the serum half-life of the multispecific activatable antibody is longer than the half-life of its corresponding multispecific antibody; for example, the pK of the multispecific activatable antibody is longer than the pK of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is similar to the half-life of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 15 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 11 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 10 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 9 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 7 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 5 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 2 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 24 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 20 hours when administered to an organism.According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 18 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 16 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 14 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 10 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 hours when administered to an organism.

[0100] The present disclosure also provides compositions and methods including multispecific activatable antibodies comprising at least a first antibody or fragment (AB1) that specifically binds a target and a second antibody or fragment (AB2), wherein at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that reduces the ability of AB1 to bind its target. According to some embodiments, each AB is coupled to a MM that reduces the ability of its corresponding AB to bind its respective target. For example, according to embodiments of a multispecific activatable antibody, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, and AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target. According to some embodiments, the multispecific activatable antibody comprises two or more AB regions; according to such embodiments, AB1 is coupled to a first masking moiety (MM1) that reduces the ability of AB1 to bind its target, AB2 is coupled to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target, AB3 is coupled to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody.

[0101] According to some embodiments, the multispecific activatable antibody further comprises at least one cleavable moiety (CM) that is a substrate for a protease, where the CM links the MM to the AB. For example, according to some embodiments, the multispecific activatable antibody comprises at least a first antibody or antibody fragment (AB1) and a second antibody or antibody fragment (AB2) that specifically bind a target, wherein at least the first AB in the multispecific activatable antibody is coupled via a cleavable moiety (CM1) to a masking moiety (MM1) that reduces the ability of AB1 to bind its target. According to some multispecific activatable antibody embodiments, AB1 is coupled to MM1 via CM1, and AB2 is coupled via a second cleavable moiety (CM2) to a second masking moiety (MM2) that reduces the ability of AB2 to bind its target. According to some embodiments, the multispecific activatable antibody comprises two or more AB regions; according to some of those embodiments, AB1 is coupled to MM1 via CM1, AB2 is coupled to MM2 via CM2, and AB3 is coupled via a third cleavable moiety (CM3) to a third masking moiety (MM3) that reduces the ability of AB3 to bind its target, and similarly for each AB in the multispecific activatable antibody.

[0102] The compositions and methods provided herein allow for the conjugation of one or more agents to one or more cysteine ​​residues in any of the AB regions without impairing the activity (e.g., masking, activation, or binding activity) of the multispecific activatable antibody. According to some embodiments, the compositions and methods provided herein allow for the conjugation of one or more agents to one or more cysteine ​​residues in any of the AB regions without reducing or otherwise interfering with one or more disulfide bonds within any of the MMs. The compositions and methods provided herein produce multispecific activatable antibodies conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic, and / or prophylactic agents, preferably none of which are conjugated to any of the MMs of the multispecific activatable antibody. The compositions and methods provided herein produce conjugated multispecific activatable antibodies in which each MM retains the ability to effectively and efficiently mask its corresponding AB of the multispecific activatable antibody in the uncleaved state. The compositions and methods provided herein produce conjugated multispecific activatable antibodies in which the activatable antibody is still activated, i.e., cleaved, in the presence of a protease capable of cleaving the CM.

[0103] Although a multispecific activatable antibody has at least one conjugation point for an agent, in the methods and compositions provided herein, fewer than all possible conjugation points are available for conjugation to an agent. According to some embodiments, one or more conjugation points are sulfur atoms involved in disulfide bonds. According to some embodiments, one or more conjugation points are sulfur atoms involved in interchain disulfide bonds. According to some embodiments, one or more conjugation points are sulfur atoms involved in interchain sulfide bonds, but not intrachain disulfide bonds. According to some embodiments, one or more conjugation points are sulfur atoms of cysteine ​​or other amino acid residues that contain sulfur atoms. Such residues can occur naturally in antibody structures or can be incorporated into antibodies by site-directed mutagenesis, chemical conversion, or misincorporation of unnatural amino acids.

[0104] Also provided are methods for preparing conjugates of multispecific activatable antibodies having one or more interchain disulfide bonds in one or more ABs and one or more intrachain disulfide bonds in the corresponding MMs, and for which a drug reactive with free thiols is provided. The methods generally involve partially reducing the interchain disulfide bonds in the activatable antibody with a reducing agent, e.g., TCEP; and conjugating a drug reactive with free thiols to the partially reduced activatable antibody. As used herein, the term "partial reduction" refers to a situation in which a multispecific activatable antibody is contacted with a reducing agent and fewer than all disulfide bonds, e.g., fewer than all possible conjugation sites, are reduced. According to some embodiments, less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of all possible conjugation sites are reduced.

[0105] According to yet another embodiment, a method is provided for reducing an agent, e.g., a drug, and conjugating it to a multispecific activatable antibody, resulting in selection under the displacement of the agent. The method involves reducing the multispecific activatable antibody with a reducing agent, thereby unreducing any conjugation sites on either the masking portion or other non-AB portions of the activatable antibody, and conjugating the agent to interchain thiols in one or more AB regions of the multispecific activatable antibody. The conjugation sites are selected to allow for the desired displacement of the agent, allowing conjugation to occur at the desired site. For example, the reducing agent is TCEP. The reduction reaction conditions, such as the ratio of reducing agent to multispecific activatable antibody, the length of incubation, the temperature during incubation, the pH of the reduction reaction solution, etc., are determined by identifying conditions that produce a conjugated activatable antibody that retains the ability to effectively and efficiently mask the ABs of the activatable antibody under conditions where the MM is not cleaved. The ratio of reducing agent to multispecific activatable antibody will vary depending on the activatable antibody. According to some embodiments, the ratio of reducing agent:multispecific activatable antibody is about 20:1-1:1, about 10:1-1:1, about 9:1-1:1, about 8:1-1:1, about 7:1-1:1, about 6:1-1:1, about 5:1-1:1, about 4:1-1:1, about 3:1-1:1, about 2:1-1:1, about 20:1-1:1.5, about 10:1-1:1.5, about 9:1-1:1.5, about 8:1-1:1.5, about 7:1-1:1.5, about 6:1-1:1.5, about 5:1-1:1.5, about 4:1-1:1.5, about 3:1-1:1.5, about 2:1-1:1.5, about The ratio may be in the range of 1.5:1-1:1.5 or about 1:1-1:1.5. According to some embodiments, the ratio is in the range of about 5:1-1.5:1. According to some embodiments, the ratio is in the range of about 4:1-1:1. According to some embodiments, the ratio is in the range of about 4:1-1.4:1. According to some embodiments, the ratio is in the range of about 8:1 to about 1:1. According to some embodiments, the ratio is in the range of about 2.5:1-1:1.

[0106] According to some embodiments, methods are provided for reducing interchain disulfide bonds in one or more AB regions of a multispecific activatable antibody and conjugating an agent, e.g., a thiol-containing agent, e.g., a drug, to the resulting interchain thiols to selectively locate the agent on the AB. The methods generally involve partially reducing one or more AB regions with a reducing agent to form at least two interchain thiols, but not all possible interchain thiols, in the activatable antibody; and conjugating the agent to the interchain thiols of the partially reduced AB. For example, one or more AB regions of a multispecific activatable antibody are partially reduced at a desired ratio of reducing agent:activatable antibody at about 37°C for about 1 hour. According to some embodiments, the ratio of reducing agent:activatable antibody is about 20:1-1:1, about 10:1-1:1, about 9:1-1:1, about 8:1-1:1, about 7:1-1:1, about 6:1-1:1, about 5:1-1:1, about 4:1-1:1, about 3:1-1:1, about 2:1-1:1, about 20:1-1:1.5, about 10:1-1:1.5, about 9:1-1:1.5, about 8:1-1:1.5, about 7:1-1:1.5, about 6:1-1:1.5, about 5:1-1:1.5, about 4:1-1:1.5, about 3:1-1:1.5, about 2:1-1:1.5, or The ratio will be in the range of about 1:1 to 1:1.5. According to some embodiments, the ratio is in the range of about 5:1 to 1.5:1. According to some embodiments, the ratio is in the range of about 4:1 to 1:1. According to some embodiments, the ratio is in the range of about 4:1 to 1:1. According to some embodiments, the ratio is in the range of about 4:1 to 1.4:1. According to some embodiments, the ratio is in the range of about 8:1 to about 1:1. According to some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0107] The thiol-containing reagent can be, for example, cysteine ​​or N-acetylcysteine. The reducing agent can be, for example, TCEP. According to some embodiments, the reduced activatable antibody can be purified prior to conjugation, for example, using column chromatography, dialysis, or diafiltration. Alternatively, the reduced antibody is not purified after partial reduction and prior to conjugation.

[0108] The disclosure also provides a partially reduced multispecific activatable antibody in which at least one interchain disulfide bond in the multispecific activatable antibody has been reduced with a reducing agent without disrupting any intrachain disulfide bonds in the multispecific activatable antibody, wherein the multispecific activatable antibody comprises at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds to a standard, a first masking moiety (MM1) that, under uncleaved conditions, inhibits binding of the AB of the multispecific activatable antibody to the target, a first cleavable moiety (CM1) coupled to AB1, the CM1 being a polypeptide that functions as a substrate for a protease, and a second antibody or antigen-binding fragment thereof (AB2) that specifically binds to a second target. According to some embodiments, the MM1 is coupled to AB1 via CM1. According to some embodiments, one or more interchain disulfide bonds in the multispecific activatable antibody are not disrupted by the reducing agent. According to some embodiments, one or more interchain disulfide bonds of MM1 in the multispecific activatable antibody are not disrupted by the reducing agent. According to some embodiments, the reducing agent is TCEP.

[0109] According to some embodiments, the multispecific and / or multispecific activatable antibodies described herein are used in conjunction with one or more additional agents or combinations of additional agents. Suitable additional agents include current pharmaceutical and / or surgical treatments for the intended use, e.g., cancer. For example, the multispecific and / or multispecific activatable antibodies may be used in conjunction with additional chemotherapeutic or anti-tumor agents.

[0110] According to some embodiments, the multispecific antibody and / or multispecific activatable antibody and the additional agent are formulated into a single therapeutic composition, and the multispecific antibody and / or multispecific activatable antibody and the additional agent are administered simultaneously. Alternatively, the multispecific antibody and / or multispecific activatable antibody and the additional agent are separate from one another, e.g., each is formulated into a separate therapeutic composition, and the multispecific antibody and / or multispecific activatable antibody and the additional agent are administered simultaneously, or the multispecific antibody and / or multispecific activatable antibody and the additional agent are administered at different times during the treatment regimen. For example, the multispecific antibody and / or multispecific activatable antibody is administered before the administration of the additional agent, the multispecific antibody and / or multispecific activatable antibody is administered subsequent to the administration of the additional agent, or the multispecific antibody and / or multispecific activatable antibody are administered alternately. As described herein, the anti-multispecific antibody and / or multispecific activatable antibody and the additional agent may be administered in a single dose or multiple doses.

[0111] The present disclosure also provides isolated nucleic acid molecules encoding the multispecific and / or multispecific activatable antibodies described herein, and vectors comprising those isolated nucleic acid sequences. The present disclosure provides methods for producing multispecific and / or multispecific activatable antibodies by culturing cells comprising such nucleic acid molecules under conditions conducive to expression of the multispecific and / or multispecific activatable antibodies. According to some embodiments, the cells comprise such vectors.

[0112] The present disclosure also provides methods for producing a multispecific antibody and / or a multispecific activatable antibody of the present disclosure by (a) culturing a cell comprising a nucleic acid construct encoding the multispecific antibody and / or the multispecific activatable antibody under conditions conducive to expression of the multispecific antibody and / or the multispecific activatable antibody, and (b) recovering the multispecific antibody and / or the multispecific activatable antibody.

[0113] The present disclosure also provides methods for treating, preventing, delaying the progression of, or otherwise alleviating the symptoms of, or ameliorating the symptoms associated with, one or more conditions by administering a multispecific antibody and / or multispecific activatable antibody of the present disclosure to a subject in whom such treatment or prevention is desired. The subject to be treated can be, for example, a human or other mammal. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), an agricultural animal, a work animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0114] The present disclosure also provides a method of inducing target-dependent T-cell activation and killing of target cells by administering a multispecific activatable antibody of the present disclosure to a subject in whom such induction is desired, wherein target-dependent T-cell activation and killing of target cells occurs when the multispecific activatable antibody is in a cleaved state, e.g., when each masking moiety in the multispecific activatable antibody is no longer bound or otherwise associated with its corresponding AB domain, and target-dependent T-cell activation and killing of target cells is reduced or otherwise inhibited when the multispecific activatable antibody is in an uncleaved state, e.g., when at least one masking moiety in the multispecific activatable antibody is bound or otherwise associated with its corresponding AB domain. Any of the multispecific activatable antibodies described herein are suitable for use in such methods. The subject to be treated may be, for example, a human or other mammal. According to some embodiments, the subject is a non-human mammal, e.g., a non-human primate, a companion animal (e.g., a cat, dog, horse), an agricultural animal, a work animal, or a zoo animal. According to some embodiments, the subject is a rodent.

[0115] The multispecific antibodies and / or multispecific activatable antibodies used in any of these method and use embodiments can be administered at any stage of disease and / or at any stage where T-cell activation and target cell killing are desired. For example, such multispecific antibodies and / or multispecific activatable antibodies can be administered to patients with cancer at any stage, from early to metastatic. The terms subject and patient can be used interchangeably herein. The multispecific antibodies and / or multispecific activatable antibodies used in any of these method and use embodiments can be used in treatment regimens, including neoadjuvant therapy. The multispecific antibodies and / or multispecific activatable antibodies used in any of these method and use embodiments can be administered alone or in combination with one or more additional agents, such as small molecule inhibitors, other antibody-based therapies, polypeptide-based therapies, nucleic acid-based therapies, and / or other biologics.

[0116] The present disclosure also provides methods and kits for the use of multispecific antibodies and / or multispecific activatable antibodies for various diagnostic and / or prophylactic indications. For example, the present disclosure ..., including (i) contacting a subject or sample with a multispecific activatable antibody comprising a first masking portion (MM1), a first cleavage portion (CM1) that is cleaved by a cleavage agent, and a first antigen-binding domain or fragment thereof (AB1) that specifically binds a target of interest, and a second antigen-binding domain or fragment thereof (AB2) that specifically binds a second target and / or second epitope, (a) wherein said MM1 is a peptide that inhibits binding of AB1 to the target, and MM2 does not have the amino acid sequence of a naturally occurring binding partner of AB1 and is not a modified form of the natural binding partner of AB1; and (b) wherein under uncleaved, non-activating conditions, MM1 interferes with specific binding of AB1 to the target, and under cleaved, active conditions, MM1 interferes with specific binding of AB1 to the target. Under activated conditions, MM1 does not interfere with or compete with the specific binding of AB1 to the target; and (ii) provides methods and kits for detecting the presence or absence of a cleavage agent and a target of interest in a subject or sample by measuring the level of activated multispecific activatable antibody in the subject or sample, wherein in (ii), a detectable level of activated multispecific activatable antibody in the subject or sample indicates that the cleavage agent and the target are present in the subject or sample, and the absence of a detectable level of activated multispecific activatable antibody in the subject or sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and / or insufficiently present in the subject or sample.

[0117] According to some embodiments, the activatable multispecific activatable antibody is an activatable multispecific activatable antibody conjugated to a therapeutic agent. According to some embodiments, the activatable multispecific activatable antibody is not conjugated to an agent. According to some embodiments, the activatable multispecific activatable antibody comprises a detectable label. According to some embodiments, the detectable label is located on AB1. According to some embodiments, measuring the level of the activatable multispecific activatable antibody in a subject or sample is achieved using a secondary reagent that specifically binds to the activated multispecific activatable antibody and comprises a detectable label. According to some embodiments, the secondary reagent is an antibody that comprises a detectable label.

[0118] According to some embodiments of the methods and kits, the activatable multispecific activatable antibody comprises a detectable label. According to some embodiments of the methods and kits, the detectable label comprises an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. According to some embodiments of the methods and kits, the imaging agent comprises a radioisotope. According to some embodiments of the methods and kits, the radioisotope is indium or technetium. According to some embodiments of the methods and kits, the contrast agent comprises iodine, gadolinium, or iron oxide. According to some embodiments of the methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. According to some embodiments of the methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (MRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. According to some embodiments of the methods and kits, the luminescent label comprises an N-methylacridinium derivative. According to some embodiments of the methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. According to some embodiments of the methods and kits, the ligand-based label comprises biotin, avidin, streptavidin, or one or more haptens.

[0119] In some embodiments of the methods and kits, the subject is a mammal. In some embodiments of the methods and kits, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), an agricultural animal, a work animal, or a zoo animal. In some embodiments, the subject is a rodent.

[0120] 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.

[0121] According to some embodiments of these methods, the methods or kits are used to identify or otherwise further refine patient populations suitable for treatment with the multispecific activatable antibodies of the present disclosure. For example, patients who test positive for both the target and the protease that cleaves the substrate at the first cleavable moiety (CM1) in the multispecific activatable antibody tested in these methods are identified as suitable candidates for treatment with the multispecific activatable antibody that contains such a CM1. Similarly, patients who test negative using these methods for both the target and the protease that cleaves the substrate at the CM1 in the multispecific activatable antibody may be identified as suitable candidates for another form of treatment.

[0122] According to some embodiments, methods or kits are used to identify or otherwise narrow down a patient population suitable for treatment with a multispecific activatable antibody and / or conjugated multispecific activatable antibody of the present disclosure (e.g., a multispecific activatable antibody conjugated with a therapeutic agent), followed by treatment by administering the multispecific antibody and / or multispecific activatable antibody and / or conjugated multispecific activatable antibody to a subject in need thereof. For example, a patient testing positive for both the target and the protease that cleaves the substrate at the first cleavable moiety (CM1) in the multispecific activatable antibody and / or conjugated multispecific activatable antibody tested in the methods is identified as a suitable candidate for treatment with such a CM1-containing multispecific activatable antibody and / or a conjugated multispecific activatable antibody containing such a CM1, and the patient is then administered a therapeutically effective amount of the tested multispecific activatable antibody and / or conjugated multispecific activatable antibody. Similarly, patients who test negative using these methods for either or both of the target and the protease that cleaves the substrate in the multispecific activatable antibody and / or CM1 in the conjugated multispecific activatable antibody can be identified as suitable candidates for another form of treatment.

[0123] According to some embodiments, such patients can be tested with other multispecific antibodies and / or multispecific activatable antibodies, and / or conjugated multispecific activatable antibodies until a suitable multispecific activatable antibody and / or conjugated multispecific activatable antibody for treatment is identified, e.g., a multispecific activatable antibody and / or conjugated multispecific activatable antibody that contains a CM that is cleaved by the patient at the site of disease. According to some embodiments, the patient is then administered a therapeutically effective amount of the multispecific activatable antibody and / or conjugated multispecific activatable antibody for the patient that tests positive.

[0124] Pharmaceutical compositions of the disclosure can include the multispecific and / or multispecific activatable antibodies of the disclosure and a carrier. These pharmaceutical compositions can be included in kits, such as diagnostic kits.

[0125] Those skilled in the art will recognize that the antibodies of the present disclosure have a variety of uses. For example, the proteins of the present disclosure are used as therapeutic agents for various disorders. The antibodies of the present disclosure can also be used as reagents in diagnostic kits or as diagnostic tools, or they can be used in competitive assays to generate therapeutic reagents. [Brief explanation of the drawings]

[0126] [Figure 1A] FIG. 1A is an illustration showing various bispecific antibody formats (adapted from Chan and Carter, Nat. Rev. Immunol. 2010). [Figure 1B] FIG. 1B is an illustration showing various bispecific antibody formats (adapted from Chan and Carter, Nat. Rev. Immunol. 2010). [Figure 2] Figures 2A-2C are a series of diagrams showing various multispecific antibody formats suitable for use in the compositions and methods of the present disclosure. In bispecific antibodies, scFvs can be fused to the carboxyl terminus of the heavy chain (Hc) of an IgG antibody (Figure 2A); the carboxyl terminus of the light chain (Lc) (Figure 2B); or the carboxyl terminus of both the heavy and light chains (Figure 2C). Figure 2D is a diagram of a single gene construct for expression of a multispecific antibody fusion. [Figure 3A] FIG. 3A is a series of illustrations showing various multispecific activatable antibody formats suitable for use in the compositions and methods of the present disclosure. [Figure 3B] FIG. 3B is a diagram of the gene construct for expression of a multispecific activatable antibody fusion. [Figure 4]Panels AJ of Figure 4 are schematic diagrams of a series of selected sets of possible permutations of multispecific antibodies of the present disclosure. The gray shaded boxes encompass the configurations shown in Figure 2 and are included here for completeness and comparison. [Figure 5] Panels AJ of Figure 5 are a series of schematic diagrams of a selected set of possible permutations of multispecific antibodies of the present disclosure. The gray shaded boxes encompass the configurations shown in Figure 3 and are included herein for completeness and comparison. In particular, this diagram shows a multispecific activatable antibody in which the primary antigen-binding site is masked (i.e., capable of activation) and the additional antigen-binding domains are unmasked. [Figure 6] Panels AJ of Figure 6 are schematic diagrams of a series of multispecific activatable antibody sequences in which all antigen-binding domains are masked. [Figure 7] Panels AJ of Figure 7 are schematic diagrams of a series of multispecific activatable antibody sequences in which the secondary antigen-binding domain is masked and the additional antigen-binding domain is unmasked. [Figure 8] Panels AJ of Figure 8 are schematic diagrams of a series of multispecific activatable antibody sequences in which most, but not all, of the antigen-binding domains are masked and at least one additional antigen-binding domain is unmasked. [Figure 9] Panels AD of Figure 9 are schematic diagrams of a series of multispecific activatable antibody sequences in which a primary antigen-binding domain and another antigen-binding domain are masked and the remaining antigen-binding domains are unmasked. [Figure 10] FIG. 10 is a graph showing PAGE analysis of multispecific and multispecific activatable antibodies showing the molecular weight of the scFv-fused heavy chains in samples 1-8 compared to the monospecific or activatable antibodies in samples 9-11, respectively. [Figure 11] FIG. 11 is a graph depicting the ability of multispecific antibodies to specifically bind to their cognate antigens. [Figure 12A]FIG. 12A is a series of graphs showing the ability of multispecific antibodies and multispecific activatable antibodies comprising anti-human CTLA-4 scFv to specifically bind to (A) mouse and (B) human CTLA-4. [Figure 12B] FIG. 12B is a series of graphs showing the ability of multispecific antibodies and multispecific activatable antibodies comprising anti-human CTLA-4 scFv to specifically bind to (A) mouse and (B) human CTLA-4. [Figure 13] Figure 13 is a graph showing the ability of OKT3-containing multispecific activatable antibodies, referred to herein as anti-EGFR multispecific activatable antibody 3954-1204-C225v5-OKT3 and anti-Jagged multispecific activatable antibody 5342-1204-4D11v2-OKT3, to specifically bind human CD3ε in an ELISA binding experiment. [Figure 14A] FIG. 14A is a series of photographs showing PAGE analysis of uPA digests demonstrating activation of multispecific activatable antibodies without cleavage of the H chain scFv fusion. [Figure 14B] FIG. 14B is a series of photographs showing PAGE analysis of uPA digests demonstrating activation of the multispecific activatable antibody without cleavage of the H chain scFv fusion. [Figure 15A] FIG. 15A is a series of graphs showing the ability of activated multispecific activatable antibodies to bind various targets. [Figure 15B] FIG. 15B is a series of graphs showing the ability of activated multispecific activatable antibodies to bind various targets. [Figure 15C] FIG. 15C is a series of graphs showing the ability of activated multispecific activatable antibodies to bind various targets. [Figure 15D] FIG. 15D is a series of graphs showing the ability of activated multispecific activatable antibodies to bind various targets. [Figure 16A] FIG. 16A is a graph showing binding of multispecific antibodies of the present disclosure to CD3ε-positive Jurkat T cells. [Figure 16B]FIG. 16B is a graph showing simultaneous binding of multispecific antibodies of the disclosure to CD3ε-positive Jurkat T cells and EGFR. [Figure 17] FIG. 17 is a series of graphs showing EGFR-dependent activation of Jurkat T cells by multispecific antibodies of the disclosure. [Figure 18A] FIG. 18A is a graph showing EGFR-dependent activation of primary CD8+ T cells by multispecific antibodies of the disclosure. [Figure 18B] FIG. 18B is a graph showing EGFR-dependent killing of target cells by multispecific antibodies of the disclosure. [Figure 18C] FIG. 18C is a series of graphs showing CD69 activation of primary T cells by multispecific antibody C225v5-OKT3m-HN in EGFR-expressing SW480 cells compared to minimal activation in EGFR-negative U266 cells. [Figure 18D] FIG. 18D is a series of graphs showing EGFR-dependent killing of target cells by multispecific antibody C225v5-OKT3m-HN in EGFR-expressing SW480 cells compared to negligible killing in EGFR-negative U266 cells. [Figure 19] FIG. 19 is a series of graphs showing primary T cell lysis of a panel of EGFR-expressing cells by multispecific antibody C225v5-OKT3m-HN compared to the absence of lysis of the EGFR-negative U266 cell line. [Figure 20A]Figure 20A is a series of graphs showing reduced binding of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN to EGFR-expressing cells compared to EGFR binding by multispecific antibody C225v5-OKT3m-HN. Furthermore, Figure 20A shows that protease activation of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN with matriptase (also referred to herein as MTSP1 or MT-SP1) restores EGFR binding to levels comparable to those exhibited by multispecific antibody C225v5-OKT3m-HN. Figure 20A also shows that such multispecific activatable antibodies and multispecific antibodies bind EGFR in a manner similar to that of the respective monospecific activatable and monospecific antibodies. [Figure 20B] Figure 20B is a series of graphs showing reduced binding of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN to EGFR-expressing cells compared to EGFR binding by multispecific antibody C225v5-OKT3m-HN. Furthermore, Figure 20B shows that protease activation of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN with matriptase (also referred to herein as MTSP1 or MT-SP1) restores EGFR binding to levels comparable to those exhibited by multispecific antibody C225v5-OKT3m-HN. Figure 20B also shows that such multispecific activatable antibodies and multispecific antibodies bind EGFR in a manner similar to that of the respective monospecific activatable and monospecific antibodies. [Figure 21A] Figure 21A is a series of graphs showing binding of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN, activated multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN, and multispecific antibody C225v5-OKT3m-HN to CD3ε-positive Jurkat T cells. [Figure 21B]Figure 21B is a series of graphs showing binding of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN, activated multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN, and multispecific antibody C225v5-OKT3m-HN to CD3ε-positive Jurkat T cells. [Figure 22] 22 is a graph showing reduced EGFR-dependent activation of Jurkat T cells by multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN compared to activation by multispecific antibody C225v5-OKT3m-HN. Furthermore, this figure shows that protease activation of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN by matriptase restores EGFR-dependent activation to levels equivalent to those displayed by multispecific antibody C225v5-OKT3m-HN. [Figure 23A] Figure 23A is a graph showing reduced EGFR-dependent activation of primary CD8+ T cells by multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN compared to activation by multispecific antibody C225v5-OKT3m-HN. Furthermore, this figure shows that protease activation of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN by matriptase restores EGFR-dependent activation to levels equivalent to those displayed by multispecific antibody C225v5-OKT3m-HN. [Figure 23B] Figure 23B is a graph showing reduced EGFR-dependent killing of target cells by multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN compared to killing by multispecific antibody C225v5-OKT3m-HN. Furthermore, this figure shows that protease activation of multispecific activatable antibody 3954-1204-C225v5-OKT3m-HN by matriptase restores EGFR-dependent killing to levels equivalent to those exhibited by multispecific antibody C225v5-OKT3m-HN. DETAILED DESCRIPTION OF THE INVENTION

[0127] The present disclosure provides multispecific and / or multispecific activatable antibodies. As used herein, a multispecific antibody is an antibody that recognizes multiple different antigens or epitopes, and a multispecific activatable antibody is a multispecific antibody that includes at least one masking moiety (MM) attached to at least one antigen- or epitope-binding domain of the multispecific antibody, such that the coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. The activatable multispecific antibodies provided herein are stable in the circulation, are activated at intended sites of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the target that is at least equivalent to that of the corresponding, unmodified multispecific antibody.

[0128] Non-limiting examples of multispecific antibodies include bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and other multispecific antibodies. The multispecific antibodies provided herein are also multivalent; as used herein, multivalent refers to the total number of binding sites on the antibody, regardless of whether the binding sites recognize the same or different antigens or epitopes. Non-limiting examples of multispecific activatable antibodies include bispecific activatable antibodies, trispecific activatable antibodies, tetraspecific activatable antibodies, and other multispecific activatable antibodies. The multispecific activatable antibodies provided herein are also multivalent.

[0129] According to some embodiments, the multispecific antibodies or fragments thereof and / or multispecific activatable antibodies or fragments thereof are designed to recruit T cells and / or other immune effector cells. Multispecific activatable antibodies or fragments thereof that recruit T cells are also referred to herein as T-cell-recruiting multispecific antibodies or fragments thereof and / or T-cell-recruiting multispecific activatable antibodies or fragments thereof. Multispecific activatable antibodies or fragments thereof that recruit immune effector cells are also referred to herein as immune effector cell-recruiting multispecific antibodies or fragments thereof and / or immune effector cell-recruiting multispecific activatable antibodies or fragments thereof. According to some embodiments, the multispecific antibodies or fragments thereof and / or multispecific activatable antibodies or fragments thereof are designed to bind or otherwise interact with multiple targets and / or multiple epitopes, and are also referred to herein as multi-antigen-targeting antibodies or fragments thereof and / or multi-antigen-targeting activatable antibodies or fragments thereof.

[0130] 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 domain and a csFv domain. According to some embodiments, one antibody domain of the multispecific antibody or fragment thereof has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. According to some embodiments, one antibody domain of the multispecific antibody or fragment thereof has specificity for a target antigen and another antibody domain has specificity for a different target antigen. According to some embodiments, one antibody domain of the multispecific antibody or fragment thereof has specificity for a different epitope of the target antigen.

[0131] Various embodiments of the multispecific activatable antibody or fragment thereof of the present disclosure are shown in Figures 3A and 5-9. According to some embodiments, the multispecific activatable antibody or fragment thereof comprising an IgG has a masked IgG variable domain. According to some embodiments, the multispecific activatable antibody or fragment thereof comprising an scFv has a masked scFv domain. According to some embodiments, the multispecific activatable antibody or fragment thereof has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody or fragment thereof has both an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody or fragment thereof has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody or fragment thereof has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody or fragment thereof has both an IgG variable domain and an scFv domain, wherein each of the IgG variable domain and the scFv domain is coupled to its own masking moiety. According to some embodiments, one antibody domain of the multispecific activatable antibody 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 activatable 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 activatable antibody or fragment thereof has specificity for an epitope of a target antigen, and another antibody domain has specificity for a different epitope of the same target antigen.

[0132] According to some embodiments, the multispecific antibody or fragment thereof of the present disclosure comprises at least (i) a T-cell-engaging antibody or fragment thereof comprising a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, which is a surface antigen on a T cell, and (ii) a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target. According to some embodiments, the first and second targets are different antigens. According to some embodiments, the first and second targets are different epitopes on the same antigen. According to some embodiments, the T-cell-engaging antibody or fragment thereof is attached to the N-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell-engaging antibody or fragment thereof is attached to the C-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell-engaging antibody or fragment thereof is attached to the N-terminus of the light chain of the target-specific antibody. According to some embodiments, the T-cell-engaging antibody or fragment thereof is attached to the C-terminus of the light chain of the target-specific antibody. According to some embodiments, the multispecific antibody comprises a T-cell-engaging antibody or fragment thereof attached to a combination of one or more N-termini and / or one or more C-termini of the multispecific antibody. The present disclosure also includes multispecific antibodies comprising other immune effector cell-engaging antibodies or fragments thereof, such as those antibodies that bind surface antigens of natural killer (NK) cells, monocytes, e.g., bone marrow mononuclear cells, macrophages, and / or other immune effector cells.

[0133] According to some embodiments, the multispecific antibodies or fragments thereof of the present disclosure comprise at least two T-cell-engaging antibodies or fragments thereof that recruit one or more T-cell activating receptors, such as, but not limited to, T-cell-engaging scFv fragments, for example, OX40 / GITR, CD137 / GITR, CD137 / OX40, CD27 / NKG2D, and an additional combination of activating receptors, and one target-binding antibody such that the T-cell-engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the multispecific antibodies or fragments thereof of the present disclosure comprise two T-cell-engaging antibodies or fragments thereof that recruit one or more T-cell inhibitory receptors, and one target-binding antibody such that the T-cell-engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the two T-cell-engaging antibody fragments bind the same T-cell-engaging target. According to some embodiments, the two T-cell engaging antibody fragments bind different T-cell engaging targets. According to some embodiments, the two T-cell engaging antibody fragments bind different epitopes on the same T-cell engaging target.

[0134] According to some embodiments, the multispecific antibody or fragment thereof of the present disclosure comprises at least (i) a first arm comprising an antigen-binding fragment of a T-cell engaging antibody comprising a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, and (ii) a second arm comprising an antigen-binding fragment of a target-binding antibody comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target. According to some embodiments, the multispecific antibody or fragment thereof comprises at least (iii) a third antigen-binding fragment of a target-binding antibody comprising a third antibody or antigen-binding fragment thereof (AB3) that binds a third target. According to some embodiments, the second and third or more targets are the same antigen. According to some embodiments, the second and third or more targets are different antigens. According to some embodiments, the second and third or more targets are different epitopes on the same antigen.

[0135] According to some embodiments, the multispecific activatable antibody or fragment thereof of the present disclosure comprises at least: (i) a T-cell-engaging antibody or fragment thereof comprising a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, which is a surface antigen on a T cell, where AB1 is conjugated to a masking moiety (MM), such that the MM reduces the ability of AB1 to bind the first target; and (ii) a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target. According to some embodiments, the first and second targets are different antigens. According to some embodiments, the first and second targets are different epitopes on the same antigen. According to some embodiments, the T-cell-engaging antibody or fragment thereof is conjugated to the N-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell-engaging antibody or fragment thereof is conjugated to the C-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell engaging antibody or fragment thereof is attached to the N-terminus of the light chain of the target-specific antibody. According to some embodiments, the T-cell engaging antibody or fragment thereof is attached to the C-terminus of the light chain of the target-specific antibody. According to some embodiments, the multispecific activatable antibody comprises a T-cell engaging antibody or fragment thereof attached to a combination of one or more N-termini and / or one or more C-termini of the multispecific activatable antibody. The present disclosure also includes multispecific activatable antibodies, including other immune effector cell-engaging antibodies or fragments thereof, such as those antibodies that bind surface antigens on natural killer (NK) cells, monocytes, e.g., myelomonocytic cells, macrophages, and / or other immune effector cells.

[0136] According to some embodiments, the multispecific activatable antibodies or fragments thereof of the present disclosure comprise: (i) two T-cell-engaging antibodies or fragments thereof that engage one or more T-cell activating receptors, e.g., T-cell-engaging scFv fragments, e.g., but not limited to, OX40 / GITR, CD137 / GITR, CD137 / OX40, CD27 / NKG2D, and additional combinations of activating receptors, wherein the AB1 of one of the T-cell-engaging antibody fragments is conjugated to a masking moiety (MM), such that the coupling of the MM reduces the ability of the AB1 to bind its respective T-cell binding target; and (ii) at least one target-binding antibody, such that the T-cell-engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the multispecific antibodies or fragments thereof of the present disclosure comprise two T-cell engaging antibodies or fragments thereof that recruit one or more T cell inhibitory receptors and one target-binding antibody such that the T-cell engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the two T-cell engaging antibody fragments bind the same T-cell engaging target. According to some embodiments, the two T-cell engaging antibody fragments bind different T-cell engaging targets. According to some embodiments, the two T-cell engaging antibody fragments bind different epitopes on the same T-cell engaging target.

[0137] According to some embodiments, the multispecific activatable antibodies or fragments thereof of the present disclosure comprise: (i) two T-cell-engaging antibodies or fragments thereof that engage one or more T-cell activating receptors, e.g., T-cell-engaging scFv fragments, e.g., OX40 / GITR, CD137 / GITR, CD137 / OX40, CD27 / NKG2D, and additional combinations of activating receptors, where each AB1 of the T-cell-engaging antibody fragments is linked to its own masking moiety (MM), such that coupling of each MM to its respective AB1 reduces the ability of the AB1 to bind its respective T-cell binding target; and (ii) at least one target-binding antibody, such that the T-cell-engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the multispecific antibodies or fragments thereof of the present disclosure comprise two T-cell engaging antibodies or fragments thereof that recruit one or more T cell inhibitory receptors and one target-binding antibody such that the T-cell engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the two T-cell engaging antibody fragments bind the same T-cell engaging target. According to some embodiments, the two T-cell engaging antibody fragments bind different T-cell engaging targets. According to some embodiments, the two T-cell engaging antibody fragments bind different epitopes on the same T-cell engaging target. According to some embodiments, the T-cell engaging antibody or fragments thereof recruit one or more T cell inhibitory receptors instead of one or more T cell activating receptors.

[0138] According to some embodiments, the multispecific activatable antibody or fragment thereof of the present disclosure comprises at least (i) a first arm comprising an antigen-binding fragment of a T-cell engaging antibody, comprising a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, where AB1 is conjugated to a masking moiety (MM), such that coupling of the MM reduces the ability of AB1 to bind the first target, and (ii) a second arm comprising an antigen-binding fragment of a target-binding antibody, comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target. According to some embodiments, the first and second targets are the same antigen. According to some embodiments, the first and second targets are different antigens. According to some embodiments, the first and second targets are different epitopes on the same antigen. According to some embodiments, the T-cell engaging antibody or fragment thereof engages one or more T-cell inhibitory receptors instead of one or more T-cell activating receptors.

[0139] According to some embodiments, the multispecific activatable antibody or fragment thereof of the present disclosure comprises at least: (i) a T-cell-engaging antibody or fragment thereof, comprising a first antibody or antigen-binding fragment thereof (AB1), that binds a first T-cell-engaging target, which is a surface antigen on a T cell; and (ii) a second antibody or fragment thereof, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein AB2 is conjugated to a masking moiety (MM), such that the MM reduces the ability of AB2 to bind the second target. According to some embodiments, the first and second targets are different antigens. According to some embodiments, the first and second targets are different epitopes on the same antigen. According to some embodiments, the T-cell-engaging antibody or fragment thereof is conjugated to the N-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell-engaging antibody or fragment thereof is conjugated to the C-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell engaging antibody or fragment thereof is attached to the N-terminus of the light chain of the target-specific antibody. According to some embodiments, the T-cell engaging antibody or fragment thereof is attached to the C-terminus of the light chain of the target-specific antibody. According to some embodiments, the multispecific activatable antibody comprises a T-cell engaging antibody or fragment thereof attached to a combination of one or more N-termini and / or one or more C-termini of the multispecific activatable antibody. The present disclosure also includes multispecific activatable antibodies, including other immune effector cell-engaging antibodies or fragments thereof, such as those antibodies that bind surface antigens on natural killer (NK) cells, monocytes, e.g., myelomonocytic cells, macrophages, and / or other immune effector cells.

[0140] According to some embodiments, the multispecific activatable antibodies or fragments thereof of the present disclosure comprise at least (i) two T-cell engaging antibodies or fragments thereof that recruit one or more T-cell activating receptors, e.g., T-cell engaging scFv fragments, such as, but not limited to, OX40 / GITR, CD137 / GITR, CD137 / OX40, CD27 / NKG2D, and additional combinations of activating receptors, and (ii) a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is conjugated to a masking moiety (MM), such that coupling of the MM reduces the ability of AB2 to bind the second target, wherein the T-cell scFv fragments are linked to both arms of the target-binding antibodies. According to some embodiments, the multispecific antibodies or fragments thereof of the present disclosure comprise two T-cell engaging antibodies or fragments thereof that recruit one or more T cell inhibitory receptors and one target-binding antibody such that the T-cell engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the two T-cell engaging antibody fragments bind the same T-cell engaging target. According to some embodiments, the two T-cell engaging antibody fragments bind different T-cell engaging targets. According to some embodiments, the two T-cell engaging antibody fragments bind different epitopes on the same T-cell engaging target. According to some embodiments, the T-cell engaging antibody or fragments thereof recruit one or more T cell inhibitory receptors instead of one or more T cell activating receptors.

[0141] According to some embodiments, the multispecific activatable antibody or fragment thereof of the present disclosure comprises at least (i) a first arm comprising an antigen-binding fragment of a T-cell engaging antibody, and (ii) a second arm comprising an antigen-binding fragment of a target-binding antibody comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is conjugated to a masking moiety (MM), such that coupling of the MM reduces the ability of AB2 to bind the second target. According to some embodiments, the first and second targets are the same antigen. According to some embodiments, the first and second targets are different antigens. According to some embodiments, the first and second targets are different epitopes on the same antigen. According to some embodiments, the T-cell engaging antibody or fragment thereof engages one or more T cell inhibitory receptors instead of one or more T cell activating receptors.

[0142] According to some embodiments, the multispecific activatable antibody or fragment thereof of the present disclosure comprises at least: (i) a T-cell-engaging antibody or fragment thereof comprising a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, which is a surface antigen on a T cell, wherein AB1 is conjugated to a masking moiety (MM1), such that MM1 reduces the ability of AB1 to bind the first target; and (ii) a second antibody or fragment thereof comprising a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is conjugated to a masking moiety (MM2), such that MM2 reduces the ability of AB2 to bind the second target. According to some embodiments, the first and second targets are different antigens. According to some embodiments, the first and second targets are different epitopes on the same antigen. According to some embodiments, the T-cell-engaging antibody or fragment thereof is conjugated to the N-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell engaging antibody or fragment thereof is attached to the C-terminus of the heavy chain of the target-specific antibody. According to some embodiments, the T-cell engaging antibody or fragment thereof is attached to the N-terminus of the light chain of the target-specific antibody. According to some embodiments, the T-cell engaging antibody or fragment thereof is attached to the C-terminus of the light chain of the target-specific antibody. According to some embodiments, the multispecific activatable antibody comprises a T-cell engaging antibody or fragment thereof attached to a combination of one or more N-termini and / or one or more C-termini of the multispecific activatable antibody. The present disclosure also includes multispecific activatable antibodies, including other immune effector cell-engaging antibodies or fragments thereof, such as those antibodies that bind surface antigens of natural killer (NK) cells, monocytes, e.g., bone marrow mononuclear cells, macrophages, and / or other immune effector cells.

[0143] According to some embodiments, the multispecific activatable antibodies or fragments thereof of the present disclosure comprise: (i) two T-cell-engaging antibodies or fragments thereof that engage one or more T-cell activating receptors, e.g., T-cell-engaging scFv fragments, e.g., OX40 / GITR, CD137 / GITR, CD137 / OX40, CD27 / NKG2D, and additional combinations of activating receptors, wherein AB1 of one of the T-cell-engaging antibody fragments is conjugated to a masking moiety (MM), such that the coupling of the MM reduces the ability of AB1 to bind its respective T-cell binding target; and (ii) at least a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is conjugated to a masking moiety (MM2), such that the coupling of the MM reduces the ability of AB2 to bind the second target, wherein the T-cell scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the multispecific antibodies or fragments thereof of the present disclosure comprise two T-cell engaging antibodies or fragments thereof that recruit one or more T cell inhibitory receptors and one target-binding antibody such that the T-cell engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the two T-cell engaging antibody fragments bind the same T-cell engaging target. According to some embodiments, the two T-cell engaging antibody fragments bind different T-cell engaging targets. According to some embodiments, the two T-cell engaging antibody fragments bind different epitopes on the same T-cell engaging target. According to some embodiments, the T-cell engaging antibody or fragments thereof recruit one or more T cell inhibitory receptors instead of one or more T cell activating receptors.

[0144] According to some embodiments, the multispecific activatable antibodies or fragments thereof of the present disclosure comprise: (i) two T-cell-engaging antibodies or fragments thereof that engage one or more T-cell activating receptors, e.g., T-cell-engaging scFv fragments, e.g., OX40 / GITR, CD137 / GITR, CD137 / OX40, CD27 / NKG2D, and additional combinations of activating receptors, wherein each AB1 of the T-cell-engaging antibody fragments is conjugated to a masking moiety (MM1), such that coupling of each MM1 to its respective AB1 reduces the ability of AB1 to bind its respective T-cell binding target; and (ii) at least a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is conjugated to a masking moiety (MM2), such that coupling of MM2 reduces the ability of AB2 to bind the second target, wherein the T-cell scFv fragments are linked to both arms of the target-binding antibodies. According to some embodiments, the multispecific antibodies or fragments thereof of the present disclosure comprise two T-cell engaging antibodies or fragments thereof that recruit one or more T cell inhibitory receptors and one target-binding antibody such that the T-cell engaging scFv fragments are linked to both arms of the target-binding antibody. According to some embodiments, the two T-cell engaging antibody fragments bind the same T-cell engaging target. According to some embodiments, the two T-cell engaging antibody fragments bind different T-cell engaging targets. According to some embodiments, the two T-cell engaging antibody fragments bind different epitopes on the same T-cell engaging target. According to some embodiments, the T-cell engaging antibody or fragments thereof recruit one or more T cell inhibitory receptors instead of one or more T cell activating receptors.

[0145] According to some embodiments, the multispecific activatable antibody or fragment thereof of the present disclosure comprises at least: (i) a first arm comprising an antigen-binding fragment of a T-cell engaging antibody, comprising a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell engaging target, wherein AB1 is conjugated to a masking moiety (MM1), such that coupling of the MM reduces the ability of AB1 to bind the first target; and (ii) a second arm comprising an antigen-binding fragment of a target-binding antibody, comprising a second antibody or antigen-binding fragment thereof (AB2), that binds a second target, wherein AB2 is conjugated to a masking moiety (MM2), such that coupling of the MM reduces the ability of AB2 to bind the first target. According to some embodiments, the first and second targets are the same antigen. According to some embodiments, the first and second targets are different antigens. According to some embodiments, the first and second targets are different epitopes on the same antigen. According to some embodiments, the T-cell engaging antibody or fragment thereof engages one or more T-cell inhibitory receptors instead of one or more T-cell activating receptors.

[0146] According to some embodiments, the target antigen is an antigen that is highly expressed on both normal, healthy tissue and diseased tissue. According to some embodiments, the target antigen is an antigen from Table 1 that is highly expressed on both normal, healthy tissue and diseased tissue.

[0147] According to some embodiments, the target antigen is an antigen that is highly expressed on diseased tissue but not highly expressed on normal healthy tissue. According to some embodiments, the target antigen is an antigen from Table 1 that is highly expressed on diseased tissue but not highly expressed on normal healthy tissue. The target antigen may be expressed on normal healthy tissue, but it is not highly expressed, or is otherwise overexpressed, on normal healthy tissue.

[0148] According to some embodiments, the masking moiety (MM) is coupled to the antibody or antigen-binding fragment thereof (AB) via a cleavable moiety (CM) that functions as a substrate for a protease. The appropriate protease for use with the multispecific activatable antibodies of the present disclosure is determined based on protease expression at the intended site of therapy and / or diagnosis. According to some embodiments, the protease is u-type plasminogen activator (uPA, also known as urokinase), legumain, and / or matriptase (also known as MT-SP1 or MTSP1). According to some embodiments, the protease is a matrix metalloproteinase (MMP).

[0149] According to some embodiments, a multispecific activatable antibody is constructed to include a masking moiety (MM) coupled to an antibody or antigen-binding fragment thereof (AB) via a non-cleavable linker. For example, according to some embodiments, the multispecific activatable antibody is a T-cell-engaging multispecific activatable antibody comprising a targeting antibody or antigen-binding fragment thereof and a T-cell-engaging antibody or antigen-binding portion thereof, wherein the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein AB1 is coupled to the masking moiety (MM1) via a non-cleavable linker, such that the coupling of the MM reduces the ability of AB1 to bind the first target and the targeting antibody or antigen-binding fragment thereof is unmasked.

[0150] According to some embodiments, the multispecific activatable antibody is a T-cell-engaging multispecific activatable antibody comprising a targeting antibody or antigen-binding fragment thereof and a T-cell-engaging antibody or antigen-binding portion thereof, wherein the T-cell-engaging antibody or antigen-binding fragment thereof comprises a first antibody or antigen-binding fragment thereof (AB1) that binds a first T-cell-engaging target, wherein AB1 is linked to a masking moiety (MM1) via a non-cleavable linker such that coupling of the MM reduces the ability of AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof comprises a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, wherein AB2 is linked to a masking moiety (MM2) via a cleavable linker such that coupling of the MM reduces the ability of AB2 to bind the second target.

[0151] The general concept of bispecific antibodies was first introduced at least 50 years ago (Nisonoff, A. and Mandy, WJ, Nature 194, 355-359 (1962), as cited in Chan, AC and Carter, PJ, Nature Reviews Immunol. 10, 301-316 (2010)).

[0152] A variety of bispecific platforms have been described (see, e.g., Figure 1; Liu, MA, et al., Proc. Natl. Acad. Sci. USA 82, 8648-8652 (1985); reviewed by Kroesen, BJ, et al. Adv. Drug Delivery Rev. 31, 105-129 (1998); Marvin, JS and Zhu, Z., Acta Pharm. Sinica 26, 649-658 (2005); Chan and Carter, Nat. Rev. Immunol. 2010; Fitzgerald and Lugovsky, MAbs. 3(3):299-309 (2011); Riethmuller, G., Cancer Immunity 12, 12-18 (2012)). The general concept for constructing bispecific antibodies is to link together protein-binding domains, usually based on multiple immunoglobulin domains, to construct molecules capable of binding to multiple target antigens and exhibit IgG-like physiological distribution, pharmacokinetics, and effector functions, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), T-cell recruitment (BiTEs®) (see, e.g., Baeuerle, PA and Reinhardt, C., Cancer Res. 69, 4941-4944 (2009)), and TandAbs® (see, e.g., Cochlovius et al., Cancer Res. 60, 4336-4341 (2000)), and / or delivery of cytotoxic payloads in the form of chemically conjugated moieties, such as microtubule inhibitors, DNA alkylating agents, or other toxins in the form of antibody-drug conjugates (ADCs). Many bispecific antibody formats have been investigated, with traditional heterodimeric IgG and BiTEs® representing the most clinically advanced properties, despite their suboptimal production and pharmacological properties (see Chan and Carter, Nat. Rev. Immunol. 2010).Bispecific antibodies have many potential uses, depending on the specific characteristics of the particular format and the target antigen selected; not all bispecific formats are suitable for all applications. For example, the two-in-one Fab format (Bostrom, J., et al., Science 323, 1610-1614 (2009)) consists of a single binding domain for binding antigens A and B in either manner; thus, this type of bispecific antibody can recruit two copies of antigen A, two copies of antigen B, or one copy of each antigen. On the other hand, the DVD format (Wu, C., et al., Nature Biotechnol. 25, 1290-1297 (2007)) provides for the recruitment of one copy of antigen A in addition to two copies of antigen B. The spacing and relative orientation of each binding domain is also important for recruitment of multiple antigens in a manner that confers the intended activity; for example, cross-linking receptors (Jackman, J., et al., J. Biol. Chem. 285, 20850-20859 (2010)) requires monovalent interactions with each of two different antigens, where recruitment of one copy of each receptor is required.

[0153] A mechanistically distinct area of ​​bispecific antibody application involves the recruitment of immune effector cells (e.g., cytotoxic T-lymphocytes, NK cells, and / or myeloid effector cells) to attack tumor cells by constructing bispecific antibodies that recruit both antigens on the surface of targeted cells (e.g., tumor cells) and antigens on the surface of cytotoxic immune cells (e.g., T-cells) for cytotoxicity. Catumaxomab is an example of a clinically validated bispecific antibody that targets the tumor antigen EpCAM on cancer cells and recruits cytotoxic T-cells via binding to CD3 on the T-cell surface (Linke, R., Klein, A., and Seimetz, D., mAbs 2, 129-136 (2010)); however, its use is limited, and it requires intraperitoneal administration. BiTE® antibodies are bispecific antibodies that achieve T-cell recruitment in different formats with distinct pharmacological properties (Baeuerle, PA and Reinhardt, C., Anti-Cancer Res. 69, 4941-4944 (2009)). In particular, BiTE antibodies are composed of two single-chain variable fragments (scFvs) and have very short pharmacological half-lives in vivo; therefore, they require delivery via sustained infusion. A similar format, called TandAb®, is a tetravalent bispecific diabody construct in which two variable domains bind to a target antigen and two variable domains bind to CD3 on the surface of T-cells (Cochlovius et al., Cancer Res. 60, 4336-4341 (2000)). This format demonstrates that bivalent recruitment of both a target antigen and a T-cell surface antigen can be used for T-cell recruitment and antigen-directed cytotoxicity. However, the TandAb format also has a very short half-life in vivo.

[0154] A common limitation of T-cell-engaging bispecific antibodies is related to the fact that T-cell cytotoxicity is very potent and can be activated by relatively low levels of target antigen on the cell surface. Even moderate levels of target antigen, such as EGFR, expression on healthy tissue can lead to significant toxicity, limiting or preventing clinical therapeutic use (Lutterbuese, R. et al., Proc. Natl. Acad. Sci. USA 107, 12605-12610 (2010)). Therefore, there remains a need for effective antibody-based therapies that exhibit improved safety, e.g., reduced toxicity.

[0155] The multispecific activatable antibodies of the present disclosure are safer than multispecific (e.g., bispecific) antibodies because the activity of the antibodies is not restricted to localized disease environments. According to some embodiments, the multispecific activatable antibodies are immune effector cell-recruiting multispecific activatable antibodies. According to some embodiments, the multispecific activatable antibodies are T-cell-recruiting multispecific activatable antibodies. According to some embodiments, the multispecific activatable antibodies recognize multiple targets. According to some embodiments, the multispecific activatable antibodies encompass an IgG-scFv format, which confers the long half-life properties of IgG. According to some embodiments, such multispecific activatable antibodies are further modulated through the use of Fc mutations in the FcRn binding site (Petkova, S. B. et al., Intl. Immunol. 18, 1759-1769 (2006)); Deng, R. et al., mAbs 4, 101-109 (2012)); Olafson, T. Methods Mol. Biol. 907, 537-556 (2012)). According to some embodiments, such multispecific activatable antibodies include mutations in the Fc domain, such as the N297A mutation, which reduces IgG effector function (ADCC and CDC) to reduce off-target toxicity (Lund, J. et al., Mol. Immunol. 29, 53-39 (1992)).

[0156] Multispecific activatable antibodies exploit the upregulation of protease activity, a widely recognized hallmark of multiple disease states, to achieve disease- and tissue-specific targeting of such therapies. Activatable antibodies are based on the use of IgG antibodies or fragments thereof, such as scFv regions, Fab regions, single VH, or VL domains, that have been modified to include a masking moiety (MM) linked to the antibody via a cleavable moiety (CM), e.g., a protease-cleavable moiety (see, e.g., PCT International Publication No. 2009 / 025846, published February 26, 2009; PCT International Publication No. 2010 / 081173, published July 15, 2010). Alternatively, non-antibody protein domains (see, e.g., PCT International Publication No. 2010 / 096838, published August 26, 2010; Boersma, YL et al., J. Biol. Chem. 286, 41273-41285 (2011)) can be used to achieve one or more binding specificities. Multispecific antibodies, such as conventional IgGs, can be used as a starting point to generate multispecific activatable antibodies. Such multispecific activatable antibodies allow highly affinity targeting of all antigens recognized by the parent multispecific antibody, but with tissue specificity determined by a selectively activated cleavable linker. Depending on the multispecific format used, the MM and CM can be positioned at the N- or C-terminus of the appropriate domain.

[0157] Multispecific activatable antibodies comprise at least one IgG-derived domain with specificity for a first antigen, synthetically or biosynthetically linked to another domain (which may be derived from IgG or another protein) with specificity for binding to a second antigen. Additional specificity can be conferred by linking such a multispecific activatable antibody to one or more additional domains that confer additional binding specificity for the first, second, or additional antigens. According to some embodiments, multispecific activatable antibodies have one or more of these domains masked with a suitable masking moiety (MM). Each of the multispecific formats described in Figure 1 can potentially be converted into a multispecific activatable antibody by masking one or more antigen-binding portions of one or more immunoglobulin domains. Examples of suitable multispecific activatable antibody formats are shown in Figures 3A and 5-9.

[0158] The use of scFv domains to confer specificity to a particular antigen allows for the modular construction of multispecific and multispecific activatable antibodies. The use of scFv domains fused to the termini of IgG heavy or light chains to construct bispecific antibodies has been previously described (see, e.g., Orcutt, KD et al., Prot. Eng. Design Select. 23, 221-228 (2010)); Dong et al., (2011)). This format ("IgG-scFv") allows the conversion of conventional IgG into bispecific antibodies, in which the first specificity is encoded in the IgG variable domain and the second specificity is encoded in an scFv domain linked via a flexible linker region. Variations of this format include the fusion of scFv domains at the N- or C-terminus of the H or L chain; the scFvs can have the same or different antigen-binding specificities (Pangler, JB et al., J. Mol. Biol. 422, 532-544 (2012)). Furthermore, through the use of H chain heterodimers (e.g., using knob-hole or similar constructs), scFvs of different specificities can be attached to the N- or C-terminus of each H chain.

[0159] According to some embodiments, the multispecific activatable antibody has a masked IgG variable domain. According to some embodiments, the multispecific activatable antibody has both masked scFv domains. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for an epitope of the target antigen and another antibody domain has specificity for another epitope of the target antigen.

[0160] The present disclosure provides examples of multispecific activatable antibody constructs, including, but not limited to, the following: (VL-CL)2:(VH-CH1-CH2-CH3-L4-VH * -L3-VL * -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-L2-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; (VL-CL-L4-VL * -L3-VH * )2:(MM-L1-CM-L2-VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2:(VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2:(VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2:(VL * -L3-VH * -L4-VH-CH1-CH2-CH3)2; or (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2: (VH * -L3-VL * -L4-VH-CH1-CH2-CH3)2, where VL and VH represent the light and heavy chain variable domains of the first specificity contained in IgG; VL * and VH *represents the variable domain of the second specificity contained in the scFv; L1 is a linker peptide connecting the masking moiety (MM) and the cleavable moiety (CM); L2 is a linker peptide connecting the cleavable moiety (CM) and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is a light chain constant domain; and CH1, CH2, and CH3 are heavy chain constant domains. The first and second specificities can be for any antigen or epitope. Additional structures include, but are not limited to: (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-CL-L4-VH * -L3-VL * )2:(VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL-CL--L4-VL * -L3-VH * )2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VH * -L3-VL * -L2-CM-L1-MM)2:(VH-CH1-CH2-CH3-L4-VH * -L3-VL * -L2-CM-L1-MM)2; (VL-CL-L4-VL * -L3-VH * -L2-CM-L1-MM)2:(VH-CH1-CH2-CH3-L4-VL * -L3-VH * -L2-CM-L1-MM)2; (MM-L1-CM-L2-VL-CL-L4-VH * -L3-VL * )2:(VH-CH1-CH2-CH3)2; or (MM-L1-CM-L2-VL-CL--L4-VL * -L3-VH* )2:(VH-CH1-CH2-CH3)2.

[0161] According to some embodiments, the multispecific activatable antibody comprising an IgG has a masked IgG variable domain. According to some embodiments, the multispecific activatable antibody comprising an scFv has a masked scFv domain. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. According to some embodiments, the multispecific activatable antibody has both an IgG variable domain and an scFv domain, wherein each of the IgG domain and the scFv domain is coupled to its own masking moiety. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for another target antigen. According to some embodiments, one antibody domain of the multispecific activatable antibody has specificity for an epitope of the target antigen and another antibody domain has specificity for another epitope of the target antigen.

[0162] According to some embodiments of immune effector cell-recruiting multispecific activatable antibodies, e.g., T-cell-recruiting multispecific activatable antibodies, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with the disease, e.g., any of the targets listed in Table 1, e.g., EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71. (transferrin receptor), and the other antigen is typically a stimulatory or inhibitory antigen present on T-cells, natural killer (NK) cells, myelomonocytic cells, macrophages, and / or other immune effector cells, such as B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain that confers specificity for a T-cell surface antigen may also be replaced with a ligand or ligand domain that binds to a T-cell receptor, an NK-cell receptor, a macrophage receptor, and / or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2, or TNFSF9. According to some embodiments of the multi-antigen targeting activatable antibody, one antigen is selected from the target groups listed in Table 1, and another antigen is selected from the target groups listed in Table 1.

[0163] According to some embodiments, a single-chain variable domain specific for binding a T-cell surface-displayed target is fused to the carboxyl terminus of a fully human IgG1 antibody (targeting antibody) against a cell surface antigen. The scFv fusion can be to the carboxyl terminus of the heavy chain, the carboxyl terminus of the light chain, or both chains (Figure 2). According to some embodiments, a single-chain variable domain specific for binding a T-cell surface-displayed target is fused to the amino terminus of a fully human IgG1 antibody (targeting antibody) against a cell surface antigen. The scFv fusion can be to the amino terminus of the heavy chain, the amino terminus of the light chain, or both chains. The fusion is constructed as a single gene construct and expressed in cells in culture. The targeting antibody can be specific for binding to one or more tumor surface antigens, or any cell targeted for depletion. The scFvs can be specific for the same or different antigens.

[0164] In some embodiments, the targeting antibody is an anti-EGFR antibody. In some embodiments, the targeting antibody is C225v5, which is specific for binding to EGFR. In some embodiments, the targeting antibody is c225, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v4, which is specific for binding to EGFR. In some embodiments, the targeting antibody is C225v6, which is specific for binding to EGFR. In some embodiments, the targeting antibody is an anti-Jagged antibody. In some embodiments, the targeting antibody is 4D11, which is specific for binding to human and mouse Jagged1 and Jagged2. In some embodiments, the targeting antibody is 4D11v2, which is specific for binding to human and mouse Jagged1 and Jagged2.

[0165] In some embodiments, the targeting antibody can be in the form of an activatable antibody. In some embodiments, the scFv can be in the form of a Pro-scFv (see, e.g., WO 2009 / 025846, WO 2010 / 081173).

[0166] In some embodiments, the scFv is specific for binding CD3ε, e.g., OKT3. In some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).

[0167] According to some embodiments, the scFv is specific for binding one or more T-cells, one or more NK-cells, and / or one or more macrophages. According to some embodiments, the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA, and combinations thereof.

[0168] definition Unless otherwise specified, scientific and technical terms used in connection with this disclosure will have the meaning commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms will include the plural and plural terms will include the singular. Generally, the nomenclature used in connection with and in connection with the techniques of cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein is that well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the nuclear art or as described herein. The foregoing techniques and procedures are generally performed according to conventional techniques well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0169] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0170] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with, or with a lower affinity (K) with, other polypeptides. d >10 -6 ) means to bind. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, fully human, domain antibodies, single chain, Fab and F(ab')2 fragments, scFv, and a Fab expression library.

[0171] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Generally, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chain present in the molecule. Specific classes have subclasses, e.g., IgG1, IgG2, IgG3, IgG4, and others. Furthermore, in humans, the light chains can be kappa or lambda chains.

[0172] The terms "monoclonal antibody" (mAb) or "monoclonal antibody composition," as used herein, refer to a population of antibody molecules containing only one species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0173] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions" or FRs. Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to be bound, and each of the three hypervariable regions of the H and L chains is referred to as the "complementarity-determining regions" or "CDRs." The amino acid assignment to each domain follows the definitions in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0174] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific conformational and charge characteristics. For example, antibodies are raised against N- or C-terminal peptides of polypeptides. An antibody is said to specifically bind an antigen when the dissociation constant is ≦1 μM, e.g., in some embodiments, ≦100 nM, and in some embodiments, ≦10 nM.

[0175] As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of noncovalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed by the dissociation constant (Kd) of the interaction, where a smaller Kd represents a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio Koff / Kon allows for the cancellation of all parameters not related to affinity and gives the dissociation constant K d(See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) An antibody of the present disclosure is said to specifically bind to EGFR if the equilibrium binding constant (Kd), as measured by an assay known to one of skill in the art, e.g., a radioligand binding assay or similar assay, is ≦1 μM, e.g., in some embodiments, ≦100 nM, in some embodiments, ≦10 nM, and in some embodiments, ≦100 pM to about 1 pM.

[0176] The term "isolated polynucleotide," as used herein, means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an "isolated polynucleotide" is one that: (1) is not associated with all or part of a polynucleotide with which the "isolated polynucleotide" is found in nature; (2) is operably linked to a polynucleotide with which it is not linked in nature; or (3) is not found in nature as part of a larger sequence. Polynucleotides of the present disclosure include nucleic acid molecules encoding the heavy chain immunoglobulin molecules set forth herein and nucleic acid molecules encoding the light chain immunoglobulin molecules set forth herein.

[0177] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; by its origin or source of derivation, an "isolated protein" is (1) not associated with a protein found in nature, (2) free from other proteins from the same source, e.g., murine proteins, (3) expressed by cells from a different species, or (4) not naturally occurring.

[0178] The term "polypeptide" is used herein as a genetic term to refer to a naturally occurring protein fragment or analog of a polypeptide sequence. Naturally occurring protein fragments and analogs are therefore species of the polypeptide genus. Polypeptides of the present disclosure include heavy chain immunoglobulin molecules as set forth herein, and light chain immunoglobulin molecules as set forth herein, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules together with light chain immunoglobulin molecules, e.g., kappa light chain immunoglobulins, and vice versa, and fragments and analogs thereof.

[0179] As used herein, the term "naturally occurring," as applied to an object, refers to the fact that the object can be found in nature. For example, it can refer to a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in the laboratory, or that otherwise occurs in nature.

[0180] The term "operably linked," as used herein, means that the positions of the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0181] The term "control sequence," as used herein, refers to polynucleotide sequences necessary to affect the expression and processing of linked coding sequences. The nature of such control sequences varies depending on the host organism in prokaryotes; in eukaryotes, such control sequences generally include a promoter, ribosomal binding site, 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 leader sequences and fusion partner sequences. The term "polynucleotide," as used herein, refers to a sequence of nucleotides, ribonucleotides or deoxynucleotides, of at least 10 bases in length, or modified forms of either type of nucleotide. The term encompasses single- and double-stranded forms of DNA.

[0182] The term "polynucleotide," as used herein, encompasses naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides generally containing 200 or fewer bases in length. In some embodiments, oligonucleotides are 10-60 bases in length, e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length in some embodiments. Oligonucleotides are typically single-stranded for probes, although oligonucleotides can be double-stranded for use in constructing gene variants. Oligonucleotides of the present disclosure can be either sense or antisense oligonucleotides.

[0183] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkages" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoronmidate, and the like. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anticancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). Oligonucleotides can contain a label for detection, if desired.

[0184] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers of the 20 conventional amino acids, unnatural amino acids, such as α-substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids (e.g., D-amino acids), may also be suitable components for the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, δ-N-methylarginine, and other similar amino acids, and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0185] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5'-end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The 5' to 3' addition direction of the nascent RNA transcript is referred to as the transcription direction sequence with the DNA strand having the same sequence as the RNA, and the 5' to 5' end direction of the RNA transcript is referred to as the "upstream sequences," i.e., the region of sequences on the DNA strand having the same sequence as the RNA, and the 3' to 3' end direction of the RNA transcript is referred to as the "downstream sequences."

[0186] The term "substantial identity," as applied to polypeptides, means that two peptide sequences, when optimally aligned with the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity; for example, according to some embodiments, at least 90% sequence identity, according to some embodiments, at least 95% sequence identity, and according to some embodiments, at least 99% sequence identity.

[0187] According to some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0188] As discussed herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are intended to be encompassed by the present disclosure, provided that the variation in amino acid sequence is maintained at least 75%, e.g., according to some embodiments, at least 80%, 90%, 95%, and according to some embodiments, 99%. Conservative amino acid substitutions are particularly contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic-hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. It is reasonable to predict that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the backbone. Whether an amino acid change results in a functional peptide can be easily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by one of skill in the art. According to some embodiments, the amino- and carboxy-termini of the fragment or analog reside near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequences to public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Methods for identifying proteins that fold into known three-dimensional structures are known (Bowie et al. Science 253:164 (1991)). Thus, the foregoing examples demonstrate that one of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present disclosure.

[0189] According to some embodiments, amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence (in portions of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., amino acid substitutions should not tend to disrupt helices present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Principles of Proteins, Structure and Molecules (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0190] The term "polypeptide fragment," as used herein, refers to a polypeptide having an amino- and / or carboxy-terminal deletion and / or one or more internal deletions, but in which the remaining amino acid sequence is identical to the corresponding positions in a naturally occurring sequence, e.g., as deduced from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, e.g., at least 14 amino acids in length, in some embodiments, at least 20 amino acids in length, usually at least 50 amino acids in length, and in some embodiments, at least 70 amino acids in length. The term "analog," as used herein, refers to a polypeptide comprised of a segment of at least 25 amino acids that has substantial identity to a portion of the deduced amino acid sequence and has specific binding to EGFR under appropriate binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids in length, e.g., in some embodiments at least 50 or more amino acids in length, and can often be as long as a full-length naturally occurring polypeptide.

[0191] The term "agent" is used herein to denote a compound, a mixture of compounds, a biopolymer, or an extract made from biomaterials.

[0192] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment to the polypeptide of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected optically or colorimetrically). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., fluorophores, rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug," as used herein, refers to a compound or composition capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0193] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and the substantially purified fraction is a composition in which the target species accounts for at least about 50% (on a molar basis) of all macromolecular species present.

[0194] Generally, a substantially pure composition will comprise greater than about 80%, and in some embodiments, greater than about 85%, 90%, and 99% of all macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity (contaminating species in the composition cannot be detected by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0195] The term "patient" includes human and veterinary subjects.

[0196] Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0197] Multispecific antibodies and multispecific activatable antibodies Exemplary multispecific and / or multispecific activatable antibodies of the present disclosure include, for example, those shown in the Examples provided herein, and variants thereof.

[0198] According to some non-limiting embodiments, at least one of the ABs in the multispecific antibody is a binding partner for any of the targets listed in Table 1.

[0199] [Table 1]

[0200] [Table 2]

[0201] According to some non-limiting embodiments, at least one of the ABs of the multispecific antibody is derived from a sequence shown in Table 7 in the Examples provided herein.

[0202] According to some non-limiting embodiments, at least one of the ABs of the multispecific antibody is derived from an antibody listed in Table 2.

[0203] [Table 3]

[0204] [Table 4]

[0205] Also included in the present disclosure are multispecific and / or multispecific activatable antibodies that bind to the same epitope as the multispecific and / or multispecific activatable antibodies described herein.

[0206] Those skilled in the art will recognize that it is possible to determine without undue experimentation if a multispecific antibody and / or multispecific activatable antibody has the same or similar specificity as a multispecific antibody and / or multispecific activatable antibody of the present disclosure by determining whether the former prevents binding to the target by the latter. If the multispecific antibody and / or multispecific activatable antibody being tested competes with the multispecific antibody and / or multispecific activatable antibody of the present disclosure, as indicated by reduced binding by the multispecific antibody and / or multispecific activatable antibody of the present disclosure, then the two multispecific antibodies and / or multispecific activatable antibodies bind to the same or closely related epitopes.

[0207] One embodiment for determining whether a multispecific and / or multispecific activatable antibody has the same or similar specificity as a multispecific and / or multispecific activatable antibody of the present disclosure is to pre-incubate the multispecific and / or multispecific activatable antibody of the present disclosure with the soluble target it is normally reactive with, and then add the multispecific and / or multispecific activatable antibody to be tested to determine whether the multispecific and / or multispecific activatable antibody to be tested inhibits its ability to bind the target. If the multispecific and / or multispecific activatable antibody to be tested is inhibited, then it likely has the same or functionally equivalent epitope specificity as a multispecific and / or multispecific activatable antibody of the present disclosure.

[0208] Multispecific and / or multispecific activatable antibodies are produced, for example, using the procedures described in the Examples provided below. Multispecific and / or multispecific activatable antibodies can also be produced using any of a number of art-recognized techniques for antibody production and / or purification.

[0209] Antibody fragments, such as Fv, F(ab')2, and Fab, for use in multispecific and / or multispecific activatable antibodies can be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage. Alternatively, truncated genes are contemplated. For example, a chimeric gene encoding a portion of the F(ab')2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the heavy chain followed by a translation stop codon to generate a truncated molecule.

[0210] Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. Convenient vectors are those encoding functionally complete human CH or CL immunoglobulin sequences with appropriate restriction sites engineered to allow for easy insertion and expression of any VH or VL sequence. In such vectors, splicing occurs between the splice donor site in the inserted J region and the splice acceptor site following the human C region, and also at splice regions present within the human CH exon. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. The resulting antibody can be linked to any strong promoter, such as a retroviral LTR, such as the SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. PNAS 79:6777 (1982)), and Moloney Mouse Leukemia Virus LTR (Grosschedl et al. Cell 41:885 (1985)). It will be appreciated that native Ig promoters and the like can also be used.

[0211] Furthermore, multispecific and / or multispecific activatable antibodies can be generated via display-type technologies, including but not limited to, phage display, retroviral display, ribosome display and other techniques, using techniques well known in the art, and the resulting molecules can be subjected to further maturation, such as affinity maturation, for example, techniques well known in the art. Wright et al. Crit. Reviews in Immunol. 12125-168 (1992), Hanes and Pluckthun PNAS USA 94:4937-4942 (1997) (ribosome display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol. 17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russell et al. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A (1992), and U.S. Patent No. No. 5,733,743.

[0212] It may be desirable to modify the multispecific and / or multispecific activatable antibodies of the present disclosure with respect to effector function to enhance or decrease such function to improve the effectiveness of the antibody in treating diseases and disorders. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The heterodimeric antibody thus generated has improved internalization capability and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, antibodies can be constructed which have dual Fc regions and thereby can have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)). According to some embodiments, Fc maturation is performed to remove glycosylation sites, thereby reducing Fc function.

[0213] Multispecific activatable antibodies The multispecific activatable antibodies and multispecific activatable antibody compositions provided herein comprise at least a first antibody or antibody fragment thereof (collectively referred to throughout this disclosure as AB1) that specifically binds a first target and / or a first epitope, and a second antibody or antibody fragment thereof (collectively referred to throughout this disclosure as AB2) that specifically binds a second target and / or a second epitope, wherein at least one of the ABs is modified with a masking moiety (MM). According to some embodiments, each AB in the multispecific activatable antibody is modified with its own masking moiety.

[0214] When at least one of the ABs in a multispecific activatable antibody is modified with a MM and in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to its target.

[0215] K of MM-modified AB against target d is the K of the AB unmodified by MM or the parent AB against the target dor more than at least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of an AB modified with a MM to a target is at least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times lower.

[0216] The dissociation constant (K) of MM for at least one of the ABs in the multispecific activatable antibody d ) is generally the K of AB against the target d Higher than AB. d is the K of AB against the target d The binding affinity of the MM to the AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times higher than the binding affinity of the MM to the AB. Conversely, the binding affinity of the MM to the AB is generally lower than the binding affinity of the AB to the target. The binding affinity of the MM to the AB is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times lower than the binding affinity of the MM to the target.

[0217] When at least one of the ABs in a multispecific activatable antibody is modified with a MM and in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to its target. The ability of the AB to bind a target when modified with a MM, when compared to the target binding of an AB not modified with a MM or the target binding of the parent AB, is at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, as measured in an in vivo or in vitro assay. It can be reduced to 99% and even 100%.

[0218] The MM inhibits the binding of AB in a multispecific activatable antibody to at least one of its targets. The MM binds the antigen-binding domain of AB and inhibits the binding of AB to its target. The MM sterically inhibits the binding of AB to its target. The MM can allosterically inhibit the binding of AB to its target. According to these embodiments, when the AB is modified or coupled to a MM and in the presence of a target, there is no, substantially no, or only 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, or more than 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more of binding of the AB to the target when measured in an in vivo or in vitro assay compared to binding of the AB unmodified with the MM, the parent AB, or the AB not coupled to the MM. There is 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% binding of AB to the target.

[0219] When at least one of the ABs in a multispecific activatable antibody is coupled to or modified with a MM, the MM "masks" or reduces or otherwise inhibits the specific binding of the AB to its target. When at least one of the ABs in a multispecific activatable antibody is coupled to or modified with a MM, such coupling or modification can affect a conformational change that reduces or inhibits the ability of the AB to specifically bind to its target.

[0220] In a multispecific activatable antibody, when at least one AB is coupled to or modified with a MM, at least a portion of the multispecific activatable antibody can be represented, in order from the amino (N)-terminal region to the carboxyl (C)-terminal region, by the formula: (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM)

[0221] where MM is a masking moiety, AB is an antibody or antibody fragment thereof, and L is a linker. In many embodiments, it is desirable to include one or more linkers, e.g., flexible linkers, in the composition to provide flexibility.

[0222] According to certain embodiments, the MM is not a natural binding partner of AB. According to some embodiments, the MM does not include or contain homology to any natural binding partner of AB. According to other embodiments, the MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0223] According to some embodiments, a multispecific activatable antibody comprises an AB modified with a MM and also comprising one or more cleavable moieties (CM). Such a multispecific activatable antibody exhibits activatable / switchable binding of the AB to a target. A 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 a protease of interest.

[0224] The elements in a multispecific activatable antibody are positioned such that each MM and CM is positioned such that, under cleaved (or relatively active) conditions and in the presence of a target, its corresponding AB binds the target, whereas under uncleaved (or relatively inactive) conditions in the presence of a target, specific binding of the AB to its target is reduced or inhibited. Specific binding of the AB to its target can be reduced due to inhibition or masking of the AB's ability to specifically bind its target by the MM.

[0225] K of each AB modified by MM and CM against the target d is the K of the AB unmodified by MM and CM, or the parent AB, against the target. dor more than at least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of each AB modified with MM and CM for the target is at least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times lower.

[0226] When at least one AB is modified with a MM and a CM and in the presence of its target, but not in the presence of a modifying agent (e.g., a protease), the specific binding of the AB to its target is reduced or inhibited compared to the specific binding of an AB that is not modified with a MM and a CM or the specific binding of the parent AB to its target. The ability of the AB when modified with MM and CM to bind a target when compared to the binding of the parent AB or the binding of an AB not modified with MM and CM to a target is at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 11109, 11209, 112109, 11309, 11409, 11509, 11609, 11709, 11809, 11909, 12009, 12109, 12209, 12309, 12409, 12509, 12609, 12709, 12809, 12909, 13009, 13109, 13209, 13309, 13409, 13509, 13609, 13709, 13809, 13909, 14009, 14109, 14209, 14309, 14409, 14509, 14609, 14709, 14809, 14909, 14910, 149209, 149309, 149409, 149509, 149609, 149709, 149809, 149809, 1 It can be reduced to 98%, 99% and even 100%.

[0227] As used herein, the term cleaved state refers to the state of a multispecific activatable antibody following modification of the CM by a protease. The term uncleaved state, as used herein, refers to the state of a multispecific activatable antibody in the absence of cleavage of the CM by a protease. As discussed above, the term "multispecific activatable antibodies" is used herein to refer to a multispecific activatable antibody in both its uncleaved (native) state and its cleaved state. It will be apparent to those skilled in the art that, according to some embodiments, a cleaved multispecific 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 attached to the multispecific activatable antibody by a covalent bond (e.g., a disulfite bond between cysteine ​​residues)).

[0228] Activatable or switchable means that the multispecific activatable antibody exhibits a first level of target binding (first conformation) under an inhibited, masked, or uncleaved state, and a second level of target binding (i.e., second conformation) under an uninhibited, unmasked, and / or cleaved state, where the second level of target binding is greater than the first level of binding. Generally, target access to its corresponding AB of a multispecific activatable antibody is higher in the presence of a cleaving agent capable of cleaving the CM than in the absence of such a cleaving agent. Thus, when the multispecific activatable antibody is under an uncleaved state, at least one AB can be inhibited from target binding and masked from target binding (i.e., the first conformation is such that the AB cannot bind the target), and under a cleaved state, the AB is not inhibited or masked from target binding.

[0229] The CM and AB of the multispecific activatable antibody are selected such that the first AB represents a binding moiety for a first target and / or epitope, and the CM represents a substrate for a protease that is co-localized with the target at a therapeutic or diagnostic site in a subject. The multispecific activatable antibodies disclosed herein are particularly useful, for example, when a protease capable of cleaving a site in the CM is present at relatively higher levels in target-containing tissue at a therapeutic or diagnostic site than in tissue at a non-therapeutic site (e.g., than in healthy tissue).

[0230] According to some embodiments, the multispecific activatable antibody provides reduced toxicity and / or adverse side effects resulting from binding of the first AB at a non-therapeutic site when the AB is unmasked or otherwise inhibited from binding its target.

[0231] In general, multispecific activatable antibodies can be designed by selecting for a first AB of interest and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides masking of the AB or reduced binding of the AB to its target. Architectural design criteria should be considered to provide this functional characteristic.

[0232] Multispecific activatable antibodies are provided that exhibit a desired dynamic range of switchable phenotypes for target binding in inhibited versus uninhibited conformations. Dynamic range generally refers to the ratio of the maximum detectable level of a parameter under (a) a first set of conditions to the minimum detectable level of that parameter under (b) a second set of conditions. For example, in the case of a multispecific activatable antibody, dynamic range refers to the ratio of the maximum detectable level of target protein binding to the multispecific activatable antibody in the presence of a protease capable of cleaving the CM of the activatable antibody to the minimum detectable level of target protein binding to the multispecific activatable antibody in the absence of a protease. The dynamic range of a multispecific activatable antibody can be calculated as the ratio of the equilibrium dissociation constant of the multispecific activatable antibody cleaving agent (e.g., an enzyme) treatment to the equilibrium dissociation constant of the activatable antibody cleaving agent treatment. The greater the dynamic range of the multispecific activatable antibody, the better the switchable phenotype of the activatable antibody. Activatable antibodies with a relatively high dynamic range value (e.g., higher than 1) exhibit a more desirable switching phenotype, such that target protein binding by the activatable antibody occurs to a greater extent (e.g., occurs primarily) in the presence of a cleaving agent (e.g., an enzyme) capable of cleaving the CM of the activatable antibody than in the absence of the cleaving agent.

[0233] Multispecific activatable antibodies can be provided in a variety of structural configurations. Exemplary formulas for at least a portion of a multispecific activatable antibody are provided below. It is specifically contemplated that the N- to C-terminal order of a first AB and its corresponding MM and CM can be reversed within the activatable antibody. It is also specifically contemplated that the CM and MM can overlap in amino acid sequence, e.g., such that a CM is contained within a MM.

[0234] For example, at least a portion of the multispecific activatable antibody can be represented, from amino (N) terminal region to carboxyl (C) terminal region, by the following formula: (MM)-(CM)-(AB) (AB)-(CM)-(MM)

[0235] where MM is a masking moiety, CM is a cleavable moiety, and AB is a first antibody or fragment thereof. Although MM and CM are shown as distinct moieties in the above formula, it is contemplated that, according to all exemplary embodiments (including formulas) disclosed herein, the amino acid sequences of MM and CM can overlap, such that the CM is fully or partially contained within the MM. Additionally, the above formula provides for additional amino acid sequences that can be located N-terminal or C-terminal to the activatable antibody element.

[0236] According to certain embodiments, the MM is not a natural binding partner of AB. According to some embodiments, the MM does not include or contain homology to any natural binding partner of AB. According to other embodiments, the MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 50% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 25% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 20% identical to any natural binding partner of AB. According to some embodiments, the MM is no more than 10% identical to any natural binding partner of AB.

[0237] According to many embodiments, it may be desirable to insert one or more linkers, e.g., flexible linkers, into a multispecific activatable antibody construct to provide flexibility at one or more MM-CM junctions, CM-AB junctions, or both. For example, the AB, MM, and / or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, especially Gly) to provide the desired flexibility. The switchable phenotype of such a multispecific activatable antibody construct may benefit from the introduction of one or more amino acids to provide a flexible linker. Furthermore, as described below, when a multispecific activatable antibody is provided as a conformationally constrained construct, a flexible linker may be operatively introduced to promote the formation and maintenance of a cyclic structure in the uncleaved multispecific activatable antibody.

[0238] For example, according to certain embodiments, the multispecific activatable antibody comprises one of the following formulas (wherein the formulas represent, from N- to C-terminal or C- to N-terminal, the amino acid sequence): (MM)-L1-(CM)-(AB) (MM)-(CM)-L2-(AB) (MM)-L1-(CM)-L2-(AB)

[0239] wherein MM, CM, and AB are as defined above; and L1 and L2 are each independently, and optionally, the same or different, flexible linkers, which may be present or absent and comprise at least one flexible amino acid (e.g., Gly). Furthermore, the above formula provides additional amino acid sequences that may be located N-terminally or C-terminally to the multispecific activatable antibody element. Examples include, but are not limited to, targeting moieties (e.g., ligands for receptors on cells present in target tissues) and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulins (i.e., IgG) or serum albumin (e.g., human serum albumin (HAS))).

[0240] According to some non-limiting embodiments, at least one of the ABs in the multispecific activatable antibody is a binding partner for any of the targets listed in Table 1.

[0241] According to some non-limiting embodiments, at least one of the ABs in the multispecific activatable antibody comprises, is, or is derived from a sequence shown in Table 7 in the Examples provided herein.

[0242] According to some non-limiting embodiments, at least one of the ABs in the multispecific activatable antibody comprises, is, or is derived from the sequence shown in Example 5 of the Examples provided herein. According to some non-limiting embodiments, at least one of the ABs in the multispecific activatable antibody comprises, is, or is derived from the sequence shown in Table 11 of the Examples provided herein.

[0243] According to some non-limiting embodiments, at least one of the ABs in the multispecific activatable antibody is or is derived from an antibody listed in Table 2.

[0244] According to some embodiments, a masking moiety is selected for use with a specific antibody or antibody fragment. For example, a suitable masking moiety for use with an antibody that binds EGFR includes a MM comprising the sequence CISPRG (SEQ ID NO: 75). As a non-limiting example, a MM can include sequences such as CISPRGC (SEQ ID NO: 339), CISPRGCG (SEQ ID NO: 76); CISPRGCPDGPYVMY (SEQ ID NO: 77); CISPRGCPDGPYVM (SEQ ID NO: 78), CISPRGCEPGTYVPT (SEQ ID NO: 79), and CISPRGCPGQIWHPP (SEQ ID NO: 80).Other suitable masking moieties include any of the EGFR-specific masks disclosed in PCT Publication No. WO 2010 / 081173, for example, by way of non-limiting example, the following sequences: GSHCLIPINMGAPSC (SEQ ID NO: 81); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 82); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 83); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 84); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 85); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 86); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 87); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 88); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 89); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 90); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 91); CHHFTLTTCGCISPRGCPG (SEQ ID NO: 92); YNPCATPMCCISPRGCPG (SEQ ID NO: 92)); CNHHYFYTCGCISPRGCG (SEQ ID NO: 94); CNHHYHYYCGCISPRGCG (SEQ ID NO: 95); CNHVYFGTCGCISPRGCG (SEQ ID NO: 96); CHHVYWGHCGCISPRGCG (SEQ ID NO: 97); CPHFTTTSCGCISPRGCG (SEQ ID NO: 98); CNHFTLTTCGCISPRGCG (SEQ ID NO: 99); CHHFTLTTCGCISPRGCG (SEQ ID NO: 100); CPYYTLSYCGCISPRGCG (SEQ ID NO: 101); CPHVSFGSCGCISPRGCG (SEQ ID NO: 102); ADHVFWGSYGCISPRGCG (SEQ ID NO: 103); YNPCATPMCCISPRGCG (SEQ ID NO: 104); CHHVYWGHCGCISPRGCG (SEQ ID NO: 105); C(N / P)H(H / V / F)(Y / T)(F / W / T / L)(Y / G / T / S)(T / S / Y / H)CGCISPRGCG (SEQ ID NO: 106); CISPRGCGQPIPSVK ​​(SEQ ID NO: 107); CISPRGCTQPYHVSR (SEQ ID NO: 108); and / or CISPRGCNAVSGLGS (SEQ ID NO: 109).

[0245] Suitable masking moieties for use with antibodies that bind Jagged targets, such as Jagged1 and / or Jagged2, include, by non-limiting example, masking moieties comprising the following sequences: QGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 338); QGQSGQGQQQWCNIWINGGDCRGWNG (SEQ ID NO: 110); PWCMQRQDFLRCPQP (SEQ ID NO: 111); QLGLPAYMCTFECLR (SEQ ID NO: 112); CNLWVSGGDCGGLQG (SEQ ID NO: 113); SCSLWTSGSCLPHSP (SEQ ID NO: 113)); YCLQLPHYMQAMCGR (SEQ ID NO: 115); CFLYSCTDVSYWNNT (SEQ ID NO: 116); PWCMQRQDYLRCPQP (SEQ ID NO: 117); CNLWISGGDCRGLAG (SEQ ID NO: 118); CNLWVSGGDCRGVQG (SEQ ID NO: 119); CNLWVSGGDCRGLRG (SEQ ID NO: 120); CNLWISGGDCRGLPG (SEQ ID NO: 121); CNLWVSGGDCRDAPW (SEQ ID NO: 122); CNLWVSGGDCRDLLG (SEQ ID NO: 123); CNLWVSGGDCRGLQG (SEQ ID NO: 124); CNLWLHGGDCRGWQG (SEQ ID NO: 124)); CNIWLVGGDCRGWQG (SEQ ID NO: 126); CTTWFCGGDCGVMRG (SEQ ID NO: 127); CNIWGPSVDCGALLG (SEQ ID NO: 128); CNIWVNGGDCRSFEG (SEQ ID NO: 129); YCLNLPRYMQDMCWA (SEQ ID NO: 130); YCLALPHYMQADCAR (SEQ ID NO: 131); CFLYSCGDVSYWGSA (SEQ ID NO: 132); CYLYSCTDSAFWNNR (SEQ ID NO: 133); CYLYSCNDVSYWSNT (SEQ ID NO: 134); CFLYSCTDVSYW (SEQ ID NO: 135); CFLYSCTDVAYWNSA (SEQ ID NO: 136); CFLYSCTDVSYWGDT (SEQ ID NO: 137); CFLYSCTDVSYWGNS (SEQ ID NO: 137)); CFLYSCTDVAYWNNT (SEQ ID NO: 139); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 140); CFLYSCTDVAYWSGL (SEQ ID NO: 141);CYLYSCTDGSYWNST (SEQ ID NO: 142); CFLYSCSDVSYWGNI (SEQ ID NO: 143); CFLYSCTDVAYW (SEQ ID NO: 144); CFLYSCTDVSYWGST (SEQ ID NO: 145); CFLYSCTDVAYWGDT (SEQ ID NO: 146); GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 147); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 148); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 149); GCNIWANGGDCRGWIDNIDG (SEQ ID NO: 150); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 151); GCNIWLVGGDCRGWLEEAVG (SEQ ID NO: 152); GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 153); GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 154); GQQQWCNIWINGGDCRGWNG (SEQ ID NO: 155); GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 156); GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 157); GASQYCNLWINGGDCRGWRG (SEQ ID NO: 158); GCNIWLVGGDCRPWVEGG (SEQ ID NO: 159); GCNIWAVGGDCRPFVDGG (SEQ ID NO: 160); GCNIWLNGGDCRAWVDTG (SEQ ID NO: 161); GCNIWIVGGDCRPFINDG (SEQ ID NO: 162); GCNIWLNGGDCRPVVFGG (SEQ ID NO: 163); GCNIWLSGGDCRMFMNEG (SEQ ID NO: 164); GCNIWVNGGDCRSFVYSG (SEQ ID NO: 165); GCNIWLNGGDCRGWEASG (SEQ ID NO: 166); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 167); GCNIWLNGGDCRTFVASG (SEQ ID NO: 168); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 169); GFLENCNIWLNGGDCRTG (SEQ ID NO: 170); GIYENCNIWLNGGDCRMG (SEQ ID NO: 170)); and / or GIPDNCNIWINGGDCRYG (SEQ ID NO: 172).

[0246] Suitable masking moieties for use with antibodies that bind an interleukin-6 target, such as the interleukin-6 receptor (IL-6R), include, by non-limiting example, masking moieties comprising the following sequences: QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 174); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 175); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 175)); YRSCNWNYVSIFLDC (SEQ ID NO: 177); PGAFDIPFPAHWVPNT (SEQ ID NO: 178); ESSCVWNYVHIYMDC (SEQ ID NO: 179); YPGCKWNYDRIFLDC (SEQ ID NO: 180); YRTCSWNYVGIFLDC (SEQ ID NO: 181); YGSCSWNYVHIFMDC (SEQ ID NO: 182); YGSCSWNYVHIFLDC (SEQ ID NO: 183); YGSCNWNYVHIFLDC (SEQ ID NO: 184); YTSCNWNYVHIFMDC (SEQ ID NO: 185); YPGCKWNYDRIFLDC (SEQ ID NO: 186); WRSCNWNYAHIFLDC (SEQ ID NO: 187); WSNCHWNYVHIFLDC (SEQ ID NO: 188); DRSCTWNYVRISYDC (SEQ ID NO: 189); SGSCKWDYVHIFLDC (SEQ ID NO: 190); SRSCIWNYAHIHLDC (SEQ ID NO: 191); SMSCYWQYERIFLDC (SEQ ID NO: 192); YRSCNWNYVSIFLDC (SEQ ID NO: 193); YGSCSWNYVHIFMDC (SEQ ID NO: 194); SGSCKWDYVHIFLDC (SEQ ID NO: 195); YKSCHWDYVHIFLDC (SEQ ID NO: 196); YGSCTWNYVHIFMEC (SEQ ID NO: 197); FSSCNWNYVHIFLDC (SEQ ID NO: 198); WRSCNWNYAHIFLDC (SEQ ID NO: 199); YGSCQWNYVHIFLDC (SEQ ID NO: 200); YRSCNWNYVHIFLDC (SEQ ID NO: 201); NMSCHWDYVHIFLDC (SEQ ID NO: 202); FGPCTWNYARISWDC (SEQ ID NO: 203); XXsCXWXYvhIfXdC (SEQ ID NO: 204); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 205);RDTGGQCRWDYVHIFMDC (SEQ ID NO:206); AGVPAGCTWNYVHIFMEC (SEQ ID NO:207); VGVPNGCVWNYAHIFMEC (SEQ ID NO:208); DGGPAGCSWNYVHIFMEC (SEQ ID NO:209); AVGPAGCWWNYVHIFMEC (SEQ ID NO:209)); CTWNYVHIFMDCGEGEGP (SEQ ID NO:211); GGVPEGCTWNYAHIFMEC (SEQ ID NO:212); AEVPAGCWWNYVHIFMEC (SEQ ID NO:213); AGVPAGCTWNYVHIFMEC (SEQ ID NO:214); SGASGGCKWNYVHIFMDC (SEQ ID NO:215); MGVPAGCVWNYAHIFMDC (SEQ ID NO:216); TPGCRWNYVHIFMECEAL (SEQ ID NO:217); VGVPNGCVWNYAHIFMEC (SEQ ID NO:218); PGAFDIPFPAHWVPNT (SEQ ID NO:219); RGACDIPFPAHWIPNT (SEQ ID NO: 220); QGDFDIPFPAHWVPIT (SEQ ID NO: 221); XGafDIPFPAHWvPnT (SEQ ID NO: 222); RGDGNDSDIPFPAHWVPRT (SEQ ID NO: 223); SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 224); WAGGNDCDIPFPAHWIPNT (SEQ ID NO: 225); WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 226); AGSGNDSDIPFPAHWVPRT (SEQ ID NO: 227); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 228); and / or RECGRCGDIPFPAHWVPRT (SEQ ID NO: 173).

[0247] According to some embodiments, the cleavable moiety (CM) of the multispecific activatable antibody comprises amino acids that can act as a substrate for a protease, typically an extracellular protease. The CM can be selected based on the protease co-localizing in a tissue with the desired target of at least one AB in the multispecific activatable antibody. A variety of different situations in which a target of interest is co-localized with a protease are known, where the substrate of the protease is known in the art. In the example of cancer, the target tissue can be cancerous tissue, particularly cancerous tissue of a solid tumor. In many cancers, e.g., solid tumors, elevated levels of proteases with known substrates have been reported in the literature. See, e.g., La Rocca et al., (2004) British Journal of Cancer 90(7): 1414-1421. Non-limiting examples of diseases include: all types of cancer (breast cancer, lung cancer, colorectal cancer, prostate cancer, melanoma, head and neck, pancreatic, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular disorders, ischemia, etc. For example, indications include leukemia, such as T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases, such as multiple myeloma, and solid tumors, such as lung, colorectal, prostate, pancreatic, and breast tumors, such as triple-negative breast cancer. For example, indications include bone disease or metastasis in cancer, regardless of the initial tumor origin; breast cancer, for example, by way of non-limiting example, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, for example, by way of non-limiting example, non-small cell lung cancer. Cancers include, but are not limited to, multiple myeloma, ovarian cancer; pancreatic cancer; prostate cancer; sarcomas, such as osteosarcoma; kidney cancer, such as, by way of non-limiting example, renal cell carcinoma, and / or skin cancer, such as, by way of non-limiting example, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0248] CM is approximately 0.001-1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250 or 1500 x 10 4 M -1 S -1 It is specifically cleaved by the enzyme at a rate of

[0249] For specific cleavage by an enzyme, contact between the enzyme and the CM is required. When a multispecific activatable antibody comprising at least a first AB coupled to a MM and a CM is in the presence of the target and sufficient enzymatic activity, the CM can be cleaved. Sufficient enzymatic activity refers to the ability of the enzyme to contact the CM and effect cleavage. It is readily envisioned that the enzyme may be in proximity to the CM but unable to cleave it due to protein modifications by other cellular factors or enzymes.

[0250] Exemplary substrates include, but are not limited to, substrates that can be cleaved by the following enzymes or proteases in Table 3:

[0251] [Table 5]

[0252] For example, in some embodiments, the substrate can be cleaved by one or more of the following enzymes or proteases: uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, and / or MMP-14. In some embodiments, the protease is selected from the group of uPA, legumain, and MT-SP1. In some embodiments, the protease is a matrix metalloproteinase. In some embodiments, the protease comprises uPA. In some embodiments, the protease comprises legumain. In some embodiments, the protease comprises MT-SP1.

[0253] In some embodiments, the CM is selected for use with a particular protease. In some embodiments, the CM is a substrate for at least one protease selected from the group consisting of ADAM17, BMP-1, a cysteine ​​protease such as a cathepsin, HTRA1, legumain, matriptase (MT-SP1), a matrix metalloproteinase (MMP), neutrophil elastase, a TMPRSS such as MPRSS3 or TMPRSS4, thrombin, and u-type plasminogen activator (uPA, also known as urokinase).

[0254] According to some embodiments, CM is a substrate for ADAM17. According to some embodiments, CM is a substrate for BMP-1. According to some embodiments, CM is a substrate for cathepsin. According to some embodiments, CM is a substrate for cysteine. According to some embodiments, CM is a substrate for HtrA1. According to some embodiments, CM is a substrate for legumain. According to some embodiments, CM is a substrate for MT-SP1. According to some embodiments, CM is a substrate for MMP. According to some embodiments, CM is a substrate for neutrophil elastase. According to some embodiments, CM is a substrate for thrombin. According to some embodiments, CM is a substrate for TMPRSS. According to some embodiments, CM is a substrate for TMPRSS3. According to some embodiments, CM is a substrate for TMPRSS4. According to some embodiments, CM is a substrate for uPA.

[0255] According to some embodiments, the cleavable moiety is selected for use with a particular protease, for example, a protease known to co-localize with the target of the activatable antibody. For example, suitable cleavable moieties for use in the activatable antibodies of the present disclosure include the sequences TGRGPSWV (SEQ ID NO:27); SARGPSRW (SEQ ID NO:28); TARGPSFK (SEQ ID NO:29); LSGRSDNH (SEQ ID NO:26); GGWHTGRN (SEQ ID NO:30); HTGRSGAL (SEQ ID NO:31); PLTGRSGG (SEQ ID NO:32); AARGPAIH (SEQ ID NO:33); RGPAFNPM (SEQ ID NO:37)); SSRGPAYL (SEQ ID NO:35); RGPATPIM (SEQ ID NO:36); RGPA (SEQ ID NO:37); GGQPSGMWGW (SEQ ID NO:38); FPRPLGITGL (SEQ ID NO:39); VHMPLGFLGP (SEQ ID NO:40); SPLTGRSG (SEQ ID NO:41); SAGFSLPA (SEQ ID NO:45)); LAPLGLQRR (SEQ ID NO:43); SGGPLGVR (SEQ ID NO:44); and / or PLGL (SEQ ID NO:45).

[0256] According to some embodiments, the CM is a substrate for at least one matrix metalloproteinase (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. According to some embodiments, the CM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. According to some embodiments, the CM is a substrate for MMP7. According to some embodiments, the CM is a substrate for MMP9. According to some embodiments, the CM is a substrate for MMP14. According to some embodiments, the CM is a substrate for multiple MMPs. According to some embodiments, the CM is a substrate for multiple MMPs, at least MMP9 and MMP14. According to some embodiments, the CM1 comprises multiple substrates for the same MMP. According to some embodiments, the CM comprises at least multiple MMP9 substrates. According to some embodiments, the CM comprises at least multiple MMP14 substrates.

[0257] According to some embodiments, the CM is a substrate for MMPs and comprises the sequences ISSGLLSS (SEQ ID NO:316); QNQALRMA (SEQ ID NO:317); AQNLLGMV (SEQ ID NO:318); STFPFGMF (SEQ ID NO:319); PVGYTSSL (SEQ ID NO:320); DWLYWPGI (SEQ ID NO:321); MIAPVAYR (SEQ ID NO:322); RPSPMWAY (SEQ ID NO:21); WATPRPMR (SEQ ID NO:323); FRLLDWQW (SEQ ID NO:324); LKAAPRWA (SEQ ID NO:325); GPSHLVLT (SEQ ID NO:326); LPGGLSPW (SEQ ID NO:327); MGLFSEAG (SEQ ID NO:328); SPLPLRVP (SEQ ID NO:329); RMHLRSLG (SEQ ID NO:330); LAAPLGLL (SEQ ID NO:331); AVGLLAPP (SEQ ID NO:332); LLAPSHRA (SEQ ID NO:333); PAGLWLDP (SEQ ID NO:334); and / or ISSGLSS (SEQ ID NO:335).

[0258] According to some embodiments, the multispecific and / or multispecific activatable antibodies of the present disclosure can be biosynthetically produced using recombinant DNA techniques and expression in eukaryotic or prokaryotic species. For multispecific activatable antibodies, cDNAs encoding a masking moiety, a linker sequence (which may include a cleavable moiety (CM) and an antibody chain (H or L)) are linked in 5' to 3' order (N- to C-terminus in the translated product) to create a nucleic acid construct, which is expressed as a multispecific activatable antibody protein following conventional antibody expression processes. According to some embodiments, multispecific activatable antibodies can be produced semisynthetically by expressing a CM-antibody and then chemically coupling a mask at or near the N-terminus of the protein. According to some embodiments, multispecific activatable antibodies can be generated by expressing an antibody and chemically coupling a mask and a CM at or near the N-terminus of the protein, such that the multispecific activatable antibody in its uncleaved state has the following structural arrangement from N- to C-terminus: MM-CM-AB or AB-CM-MM.

[0259] Linkers suitable for use in the compositions described herein are generally those that provide flexibility for the modified AB or multispecific activatable antibody to facilitate inhibition of binding of at least the first AB to a target. Suitable linkers are readily selected and can be of any suitable different length, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0260] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 18), and (GGGS)n (SEQ ID NO: 19), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore act as neutral tethers between components. Glycine has significantly more access to phi-psi space than alanine, even though it is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, the following sequences: Gly-Gly-Ser-Gly (SEQ ID NO: 20), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 21), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 22), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 23), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 24), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 25), and the like. Those skilled in the art will recognize that the design of an activatable antibody can include a linker that is fully or partially flexible, and consequently, the linker can include a flexible linker and one or more moieties that confer a less flexible structure to provide the desired multispecific activatable antibody structure.

[0261] In addition to the elements described above, the multispecific activatable antibody can include additional elements, such as the N- or C-terminal amino acid sequence of the multispecific activatable antibody. For example, the multispecific activatable antibody can include a targeting moiety that facilitates delivery to a cell or tissue of interest. The multispecific activatable antibody can be conjugated to an agent, such as a therapeutic agent, an anti-tumor agent, a toxin or fragment thereof, a detectable moiety, or a diagnostic agent. Examples of agents are disclosed herein.

[0262] The multispecific activatable antibody can also include any of the conjugated agents, linkers, and other components described herein in connection with the multispecific activatable antibodies of the present disclosure, such as, by non-limiting example, any of the agents listed in Table 4, and / or any of the linkers listed in Table 5 and / or Table 6.

[0263] Conjugated multispecific antibodies and conjugated multispecific activatable antibodies The present disclosure also relates to immunoconjugates comprising multispecific antibodies and / or multispecific activatable antibodies conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragment thereof), or a radioactive isotope (i.e., a radioconjugate). Suitable cytotoxic agents include, for example, dolastatins and their derivatives (e.g., auristatin E, AFP, MMAD, MMAF, MMAE). For example, the cytotoxic agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is an agent selected from the group listed in Table 4. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is maytansinoid or a derivative thereof. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0264] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, croton, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 212 Bi, 64 Cu, 125 I, 131 I, 131 In, 99m Tc, 90 Y, 186 Re and 89 Zr can be mentioned.

[0265] Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). 14C-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionuclides to antibodies (see WO 94 / 11026).

[0266] Table 4 lists some exemplary agents that may be used in the disclosure described herein, but is by no means meant to be an exhaustive list.

[0267] [Table 6]

[0268] [Table 7]

[0269] Those skilled in the art will recognize that a wide variety of possible moieties can be coupled to the resulting multispecific and / or multispecific activatable antibodies of the present disclosure (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0270] Coupling can be achieved by any chemical reaction that will link two molecules together so that the antibody and other moiety retain their respective activities for as long as possible. This binding can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. However, according to some embodiments, covalent bonding is preferred. Covalent bonding can be achieved by direct condensation of existing side chains or by the use of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various types of coupling agents known in the art, but rather is illustrative of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0271] In addition to the compositions and methods provided herein, according to some embodiments, conjugated activatable antibodies can also be modified for site-specific conjugation through modified amino acid sequences inserted into or otherwise included in the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled substitution and / or administration of conjugated agents within the conjugated activatable antibody. For example, activatable antibodies can be engineered to contain cysteine ​​substitutions at positions on the light and heavy chains that provide reactive thiol groups and do not negatively affect protein folding and assembly or alter antigen binding. According to some embodiments, activatable antibodies can be engineered to contain or otherwise incorporate one or more non-naturally occurring amino acid residues within the activatable antibody to provide suitable sites for conjugation. According to some embodiments, activatable antibodies can be engineered to contain or otherwise incorporate enzymatically activatable peptide sequences within their activatable antibody sequence.

[0272] Suitable linkers are described in the literature (see, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies by oligopeptide linkers. Particularly suitable linkers include: (i) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (21558G)); (ii) SPDP (succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co. catalog number 21651G); and (iii) sulfo-LC-SPDP (sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co. catalog number 2165-G). Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

[0273] The linkers described above contain components with different attributes and therefore lead to conjugates with different physico-chemical properties. For example, the linker SMPT contains a sterically hindered disulfide bond and can form conjugates with enhanced stability. Disulfide bonds are generally less stable than other linkages because the disulfide bond is cleaved in vitro, resulting in less available conjugate.

[0274] The reagent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) is useful for creating carboxamides starting with a carboxylic acid and a primary or secondary amine. Thus, EDC can be used to link a carboxylic acid in a linker or toxin to a lysine residue in an antibody, or to link an amine in a linker or toxin to an aspartic acid or glutamic acid residue in an antibody. Such conjugation reactions using EDC can be enhanced by the addition of NHS (N-hydroxysuccinimide) or sulfo-NHS (N-hydroxy-3-oxysulfonylsuccinimide). The addition of NHS or sulfo-NHS to such conjugation reactions can enhance the rate, completeness, selectivity, and reproducibility of the conjugation reaction.

[0275] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. In some embodiments, there are multiple linkers. The multiple linkers can all be the same, e.g., cleavable or non-cleavable, or the multiple linkers can be different, e.g., at least one cleavable and at least one non-cleavable.

[0276] The present disclosure utilizes several methods for conjugating agents to the ABs of multispecific antibodies and / or multispecific activatable antibodies, including: (a) attachment to the carbohydrate moiety of the ABs, or (b) attachment to the sulfhydryl group of the ABs, or (c) attachment to the amino group of the ABs, or (d) attachment to the carboxyl group of the ABs. According to the present disclosure, the ABs can be covalently attached to the agent through an intermediate linker having at least two reactive groups, where one reactive group reacts with the ABs and one group reacts with the agent. The linker, which can comprise any compatible organic compound, is selected so that reaction with the ABs (or agent) does not adversely affect the AB reactivity and selectivity. Furthermore, attachment of the linker to the agent does not destroy the activity of the agent. Suitable linkers for reaction with oxidized antibodies or oxidized antibody fragments are those linkers containing an amine selected from the group consisting of primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and thiosemifulvazides. Such reactive functional groups may be present as part of the structure of the linker or may be introduced by appropriate chemical modification of a linker that does not contain such groups.

[0277] According to the present disclosure, suitable linkers for attachment of multispecific and / or multispecific activatable antibodies to reduced ABs include those linkers having specific reactive groups capable of reacting with sulfhydryl groups on reduced antibodies or fragments. Such reactive groups include, but are not limited to, reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercurybenzoic acid groups, and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

[0278] According to the present disclosure, suitable linkers for attachment of multispecific and / or multispecific activatable antibodies to unoxidized or unreduced ABs are those linkers that have specific functional groups that can react with primary amines present in unmodified lysine residues in ABs. Such reactive groups include, but are not limited to: NHS carboxylic acid or carbonate, sulfo-NHS carboxylic acid or carbonate, 4-nitrophenyl carboxylic acid or carbonate, pentafluorophenyl carboxylic acid or carbonate, acylimidazole, isocyanate, and isothiocyanate.

[0279] According to the present disclosure, suitable linkers for attachment to unoxidized and unreduced AB include those linkers having specific functional groups activated by an appropriate reagent that can react with carboxylic acid groups present in aspartic acid or glutamic acid residues in AB. Suitable activation reagents include EDC with or without an added NHS or sulfo-NHS, and other dehydrating agents utilized for carboxamide formation. In these cases, functional groups present on suitable linkers include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0280] The agent can be attached to the linker before or after the linker is attached to the AB. Depending on the particular application, it may be desirable to first generate an Ab-linker intermediate in which the linker does not have an associated agent. Depending on the particular application, a particular agent can be covalently attached to the linker. In other embodiments, the AB is first attached to the MM, CM, and associated linker, and then attached to the linker for conjugation purposes.

[0281] Branched Linkers: According to certain embodiments, branched linkers are used that have multiple sites for attachment of agents. For multi-site linkers, a single covalent bond to AB results in an AB-linker intermediate that can attach agents at multiple sites. The sites can be aldehydes or sulfhydryl groups, or any chemical site to which an agent can be attached.

[0282] On the other hand, higher specific activity (or a higher ratio of agent to AB) can be achieved by attaching a single-site linker at multiple sites on AB. These multiple sites can be introduced into AB by either of two methods. First, one method can generate multiple aldehyde and / or sulfhydryl groups on the same AB. Second, one method can attach a "branched linker" with multiple functional sites to the aldehydes or sulfhydryls of AB for subsequent attachment to a linker. The functional sites of the branched or multi-site linker can be aldehyde or sulfhydryl groups, or any chemical site to which a linker can be attached. Even higher specific activity can be obtained by combining these two approaches, i.e., by attaching a multi-site linker at several sites on AB.

[0283] Cleavable linker: A peptide linker that is susceptible to cleavage by an enzyme of the complement system, such as, but not limited to, urokinase, tissue plasminogen activator, trypsin, plasmin, or another enzyme with proteolytic activity, can be used in one embodiment of the present disclosure. According to one method of the present disclosure, an agent is attached via a linker that is susceptible to cleavage by complement. The antibody is selected from a species that can activate complement. The antibody-agent conjugate thus activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, an agent is attached via a linker that is susceptible to cleavage by an enzyme with proteolytic activity, such as urokinase, tissue plasminogen activator, plasmin, or trypsin. These cleavable linkers are useful in conjugated activatable antibodies that contain exotoxins, such as, by way of non-limiting example, any of the exotoxins listed in Table 4.

[0284] Non-limiting examples of cleavable linker sequences are provided in Table 5.

[0285] [Table 8]

[0286] Additionally, the agent can be conjugated to the AB via a disulfide bond (e.g., a disulfide bond on a cysteine ​​residue). Many tumors naturally release high levels of glutathione (a reducing agent), which reduces the disulfide bond and causes subsequent release of the agent at the site of delivery. According to certain embodiments, the reducing agent that modifies the CM will also modify the linker of the conjugated activatable antibody.

[0287] Spacers and Cleavable Elements: According to yet another embodiment, the linker should be configured in such a way as to optimize the spacing between the agent and the AB of the activatable antibody. This can be achieved by using linkers of the following general structure: W-(CH2)nQ where W is either -N-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer from 0 to 20.

[0288] According to yet other embodiments, the linker can comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the core of the AB so that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain branched linkers described above can act as spacer elements.

[0289] It should be understood throughout this discussion that the attachment of a linker to an agent (or of a spacer element to a cleavable element, or of a cleavable element to an agent) does not require a particular mode of attachment or reaction: any reaction that provides a product of suitable stability and biological compatibility is acceptable.

[0290] Selection of serum complement and linker: According to one method of the present disclosure, if release of an agent is desired, an antibody of a type capable of activating complement, an AB, is used. The resulting conjugate retains both the ability to conjugate antigen and activate the cascade. Thus, according to this embodiment of the present disclosure, the agent is linked to one end of a cleavable linker or cleavable element, and the other end of the linker group is attached to a specific site on the AB. For example, if the agent has a hydroxy or amino group, it can be attached to the carboxy terminus of a peptide, amino acid, or other appropriately selected linker via an ester or amide bond, respectively. For example, such agents can be attached to the linker peptide via a carbodiimide reaction. If the agent contains functional groups that prevent binding to the linker, those interfering functional groups are blocked prior to conjugation and deblocked once the product conjugate or intermediate is prepared. The opposite or amino terminus of the linker is then used, either directly or after further modification, for conjugation to an AB capable of activating complement.

[0291] The linker (or spacer element of the linker) can be of any desired length, and one end is covalently attached to a specific site on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker.

[0292] Thus, when the conjugates bind to an antigen in the presence of complement, the amide or ester bond connecting the agent to the linker is cleaved, resulting in release of the agent in its active form. These conjugates, when administered to a subject, achieve delivery or release of the agent at the target site and are particularly effective for in vivo delivery of drugs, antibiotics, antimetabolites, antiproliferative agents, and the like, as presented (but not limited to) in Table 4.

[0293] Linkers for release without complement activation: According to yet another application of targeted delivery, release of an agent without complement activation is desirable because activation of the complement cascade ultimately lyses target cells. Therefore, this approach is useful when delivery and release of an agent must be achieved without killing the target cells. This is the case when delivery of cellular mediators, such as hormones, enzymes, corticosteroids, neurotransmitters, genes, or enzymes to target cells is desired. These conjugates can be prepared by linking the agent to an AB that cannot activate complement via a linker that is mildly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complement forms quickly, but cleavage of the agent occurs slowly, thereby resulting in release of the compound at the target site.

[0294] Biochemical Crosslinkers: According to other embodiments, activatable antibodies can be conjugated to one or more therapeutic agents using specific biochemical crosslinkers. Crosslinking reagents form molecular bridges that link together functional groups of two different molecules. To link two different proteins in a stepwise fashion, hetero-bifunctional crosslinkers can be used, which eliminates undesired homopolymer formation.

[0295] Peptidyl linkers cleavable by lysosomal proteases, such as Val-Cit, Val-Ala, or other dipeptides, are also useful. Additionally, acid-labile linkers cleavable in the low pH environment of the lysosome, such as bis-sialyl ether, can be used. Other suitable linkers include cathepsin-labile substrates, particularly those that function optimally at acidic pH.

[0296] Exemplary hetero-bifunctional cross-linkers are referenced in Table 6. [Table 9]

[0297] Non-cleavable linker or direct bond: According to yet another embodiment of the present disclosure, the conjugate can be designed so that the agent is delivered to the target but not released. This can be achieved by attaching the agent to the AB directly or via a non-cleavable linker.

[0298] These non-cleavable linkers can include amino acids, peptides, D-amino acids, or other organic compounds that can be subsequently modified to include functional groups that can be utilized for attachment to AB by the methods described herein. A general formula for such organic linkers can be: W-(CH2)nQ where W is either -N-CH2- or -CH2-; Q is an amino acid, a peptide; and n is an integer from 0 to 20.

[0299] Non-cleavable conjugates: Alternatively, a compound can be conjugated to an AB that is incapable of activating complement. When using an AB that is incapable of activating complement, this conjugation can be achieved using a linker that is susceptible to cleavage by activated complement, or using a linker that is not susceptible to cleavage by activated complement.

[0300] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0301] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be coupled to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-exchange reaction.

[0302] Multispecific activatable antibodies having non-binding steric moieties or binding partners for non-binding steric moieties The present disclosure also provides multispecific activatable antibodies comprising a non-binding steric moiety (NB), or a binding partner (BP) for the non-binding steric moiety, where the BP recruits or otherwise attracts the NB to the multispecific activatable antibody. Multispecific activatable antibodies provided herein include, for example, multispecific activatable antibodies comprising a non-binding steric moiety (AB), a cleavable gastric linker (CL), and at least a first antibody or antibody fragment (AB1) that binds a first target or epitope; multispecific activatable antibodies comprising a binding partner for the non-binding steric moiety (BP), the CL, and AB1; and multispecific activatable antibodies comprising a BP to which the NB is recruited, the CL, and AB1 that binds the first target or epitope. Multispecific activatable antibodies in which the NB is covalently linked to the CL and AB1, or which are associated by interaction with a BP that is covalently linked to the CL and AB1, are referred to herein as "NB-containing multispecific activatable antibodies." Activatable or switchable means that the activatable antibody exhibits a first level of target binding (first conformation) when under inhibited, masked, or uncleaved conditions, and a second level of target binding (i.e., second conformation) when under uninhibited, unmasked, and / or cleaved conditions, where the second level of target binding is greater than the first level of binding. Multispecific activatable antibody compositions can exhibit enhanced bioavailability and better biodistribution compared to conventional antibody therapies.

[0303] According to some embodiments, the multispecific activatable antibody provides reduced toxicity and / or adverse side effects resulting from binding of the multispecific activatable antibody at non-therapeutic and / or non-diagnostic sites when the multispecific activatable antibody is not masked or otherwise inhibited from binding to such sites.

[0304] According to one embodiment, the multispecific activatable antibody comprises a non-binding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (AB1) that specifically binds to a first target or epitope, where NB is a polypeptide that does not specifically bind to AB1; CL is a polypeptide that comprises a substrate (S) for an enzyme; CL is positioned such that, in its uncleaved state, NB interferes with the binding of AB1 to its target and, in its cleaved state, NB does not interfere with the binding of AB1 to its target; and NB does not inhibit cleavage of CL by the enzyme. As used herein and throughout, the term polypeptide refers to any polypeptide comprising at least two amino acid residues, e.g., larger polypeptides, full-length proteins and fragments thereof, and the term polypeptide is not limited to single-chain polypeptides but can encompass multiple units, e.g., multi-chain polypeptides. The terms peptide and polypeptide are used interchangeably herein when the polypeptide is of short length, e.g., a total of 50 amino acids or less, and the terms polypeptide and protein are used interchangeably herein when the polypeptide is of longer length, e.g., 50 or more amino acids.

[0305] According to one embodiment, the multispecific activatable antibody comprises a non-binding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (AB1) that specifically binds to a first target or epitope, where (i) NB is a polypeptide that does not specifically bind to AB1; (ii) CL is a polypeptide up to 50 amino acids in length that comprises a substrate (S) for an enzyme; (iii) CL is positioned such that, in the uncleaved state, NB interferes with binding of AB1 to its target, and in the cleaved state, NB does not interfere with binding of AB1 to its target; and (iv) NB does not inhibit cleavage of CL by the enzyme. For example, the CLs have the following lengths of amino acids: up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, up to 30 amino acids, up to 35 amino acids, up to 40 amino acids, up to 45 amino acids, up to 50 amino acids, lengths in the range of 10-50 amino acids, 20-50 amino acids, 25-50 amino acids, 30-50 amino acids, 35-50 amino acids, 40-50 amino acids. Amino acids of length, , amino acids of length in the range of 45-50, amino acids of length in the range of 10-40, amino acids of length in the range of 15-40, amino acids of length in the range of 20-40, amino acids of length in the range of 25-40, amino acids of length in the range of 30-40, amino acids of length in the range of 35-40, amino acids of length in the range of 10-30, amino acids of length in the range of 15-30, amino acids of length in the range of 20-30, amino acids of length in the range of 25-30, amino acids of length in the range of 10-20, or amino acids of length in the range of 10-15.

[0306] According to one embodiment, the multispecific activatable antibody comprises a non-binding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (AB1) that specifically binds to a first target or epitope, where (i) NB is a polypeptide that does not specifically bind to AB1; (ii) CL is a polypeptide that comprises a substrate (S) for an enzyme; (iii) CL is positioned such that, in the uncleaved state, NB interferes with binding of AB1 to its target, and in the cleaved state, NB does not interfere with binding of AB1 to its target; (iv) NB does not inhibit cleavage of CL by the enzyme; and (v) at least a portion of the multispecific activatable antibody, in the uncleaved state, has the following structural arrangement from N-terminal to C-terminal: NB-CL-AB1 or AB1-CL-NB.

[0307] According to one embodiment, the multispecific activatable antibody comprises a non-binding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (AB1) that specifically binds to a first target or epitope, wherein (i) NB is a polypeptide that does not specifically bind to AB1; (ii) CL is a polypeptide that comprises a substrate (S) for an enzyme; and (iii) CL is such that, in its uncleaved state, NB prevents AB1 from binding to its target, and in its cleaved state, NB prevents AB1 from binding to its target. and (iv) the NB does not inhibit enzymatic cleavage of the CL. The reduction in the ability of the AB to bind its target can be determined, for example, using an assay such as those described herein or an in vitro target displacement assay, such as those described in WO 2009 / 025846 and WO 2010 / 081173.

[0308] According to one embodiment, a multispecific activatable antibody comprises a binding partner (BP) for a non-binding steric moiety (NB); a cleavable linker (CL); and at least a first antibody or antibody fragment (AB1) that specifically binds to a first target and / or epitope, where BP is a polypeptide that binds to NB when exposed to NB; NB is a polypeptide that does not specifically bind to AB1; CL is a polypeptide that includes a substrate (S) for an enzyme; CL is positioned such that, in the presence of NB, in its uncleaved state, NB prevents AB1 from binding to its target, and in its cleaved state, NB does not prevent AB1 from binding to its target, and BP does not prevent AB from binding to its target; and NB and BO do not inhibit cleavage of CL by the enzyme. According to some examples of this embodiment, the BP of the activatable antibody is optionally bound to the NB. According to one embodiment, the NB is recruited by the BP of the activatable antibody in vivo.

[0309] According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody is formulated as a composition. According to some of these embodiments, the composition also includes a NB, where the NB is co-formulated with the multispecific activatable antibody, including the BP, the CL, and the AB. According to some examples of this embodiment, the BP is selected from the group consisting of an albumin-binding peptide, a fibrinogen-binding peptide, a fibronectin-binding peptide, a hemoglobin-binding peptide, a transferrin-binding peptide, an immunoglobulin domain-binding peptide, and another serum protein-binding peptide.

[0310] According to some examples of any of these multispecific activatable antibody embodiments, the NB is a soluble globular protein. According to some examples of any of these multispecific activatable antibody embodiments, the NB is a protein that circulates in the bloodstream. According to some examples of any of these multispecific activatable antibody embodiments, the NB is selected from the group consisting of albumin, fibrinogen, fibronectin, hemoglobin, transferrin, immunoglobulin domains, and other serum proteins.

[0311] According to some examples of any of these multispecific activatable antibody embodiments, the CL is a polypeptide comprising a substrate for a protease. According to some examples of any of these multispecific activatable antibody embodiments, the protease co-localizes with its target in a tissue, and the protease cleaves the CL of the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease. According to some examples of any of these multispecific activatable antibody embodiments, the CL is a polypeptide up to 50 amino acids in length. According to some examples of any of these multispecific activatable antibody embodiments, the CL is a polypeptide comprising a substrate (S) up to 15 amino acids in length, e.g., 3 amino acids in length, 4 amino acids in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, or 15 amino acids in length.

[0312] According to some examples of any of these multispecific activatable antibody embodiments, at least a portion of the multispecific activatable antibody, in its uncleaved state, has the following N-terminal to C-terminal structural arrangement: NB-CL-AB, AB-CL-NB, BP-CL-AB, or AB-CL-BP. According to embodiments in which the multispecific activatable antibody comprises a BP and is in the presence of its corresponding NB, at least a portion of the multispecific activatable antibody, in its uncleaved state, has the following N-terminal to C-terminal structural arrangement: NB:BP-CM-AB, or AB-CM-BP:NB, where ":" represents an interaction, e.g., binding, between the NB and the BP.

[0313] According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody specifically binds its target and includes antibodies or antigen-binding fragments thereof that are monoclonal antibodies, domain antibodies, single chains, Fab fragments, F(ab')2 fragments, scFvs, scabs, dAbs, single domain heavy chain antibodies, and single domain light chain antibodies. According to some embodiments, such antibodies or immunologically active fragments thereof that bind its target are murine, chimeric, humanized, or fully human monoclonal antibodies.

[0314] According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody also includes an agent conjugated to AB. According to some embodiments, the agent is a therapeutic agent. According to some embodiments, the agent is an anti-tumor agent. According to some embodiments, the agent is a toxin or a fragment thereof. According to some embodiments, the agent is conjugated to AB via a linker. According to some embodiments, the linker is a cleavable linker. According to some embodiments, the agent is an agent selected from the group listed in Table 4. According to some embodiments, the agent is a dolastatin. According to some embodiments, the agent is an auristatin or a derivative thereof. According to some embodiments, the agent is auristatin E or a derivative thereof. According to some embodiments, the agent is monomethyl auristatin E (MMAE). According to some embodiments, the agent is monomethyl auristatin D (MMAD). According to some embodiments, the agent is a maytansinoid or a derivative thereof. According to some embodiments, the agent is DM1 or DM4. According to some embodiments, the agent is a duocarmycin or a derivative thereof. According to some embodiments, the agent is a calicheamicin or a derivative thereof. According to some embodiments, the agent is a pyrrolobenzodiazepine.

[0315] According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody also comprises a detectable moiety. According to some embodiments, the detectable moiety is a diagnostic agent.

[0316] According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody also comprises a spacer. According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody also comprises a signal peptide. According to some embodiments, the signal peptide is conjugated to the multispecific activatable antibody via a spacer. According to some examples of any of these multispecific activatable antibody embodiments, the spacer is attached directly to the MM of the multispecific activatable antibody.

[0317] According to some embodiments, the serum half-life of the multispecific activatable antibody is longer than the half-life of its corresponding multispecific antibody; for example, the pK of the multispecific antibody is longer than the pK of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is similar to the half-life of its corresponding multispecific antibody. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 15 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 11 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 9 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 7 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 5 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 2 days when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 24 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 20 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 18 hours when administered to an organism.According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 16 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 14 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 12 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 10 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 8 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 7 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 6 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 4 hours when administered to an organism. According to some embodiments, the serum half-life of the multispecific activatable antibody is at least 3 hours when administered to an organism.

[0318] The present disclosure also provides isolated nucleic acid molecules encoding any of these multispecific activatable antibodies, and vectors comprising these isolated nucleic acid sequences. The present disclosure provides methods for producing multispecific activatable antibodies by culturing cells comprising such nucleic acid sequences under conditions conducive to expression of the multispecific activatable antibodies. According to some embodiments, the cells comprise such vectors.

[0319] The dissociation constant (Kd) of the NB-containing multispecific activatable antibody for the target is higher than the Kd of the AB for the target when the AB is not associated with the NB or NB:BP. The dissociation constant of the NB-containing multispecific activatable antibody for the target is higher than the Kd of the parent AB for the target. For example, the Kd of the NB-containing multispecific activatable antibody for a target may be at least 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times higher. Conversely, the binding affinity of the NB-containing multispecific activatable antibody for the target is lower than the binding affinity of the AB when the AB is not associated with the NB or NB:BP, or is lower than the binding affinity of the parent AB for the target.For example, the binding affinity of the NB-containing multispecific activatable antibody for a target may be at least 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times lower.

[0320] When the NB-containing multispecific activatable antibody is in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB when the AB is not associated with the NB or NB:BP. When the NB-containing multispecific activatable antibody is in the presence of its target, specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the parent AB to its target. The ability of the NB-containing multispecific activatable antibody to bind its target, as measured, e.g., in vitro and / or in vivo, when compared to the binding of an AB unrelated to the NB or NB:BP, or the binding of the parent AB, to its target, is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 300%, 360%, 400%, 420%, 440%, 460%, 500%, 520%, 540%, 560%, 580%, 600%, 620%, 640%, 660%, 700%, 720%, 740%, 760%, 780%, 840%, 860%, 880%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 2000%, 2100%, 2200%, 2300%, 2400%, 2500%, 3000%, 3600%, 4000%, 5200%, 5400%, 6600%, 7800%, 8000%, 9000%, 1000%, 1100%, 1200%, 1400%, 1500%, 1600%, 1700%, 1800%, 2000%, 2200%, 230 It can be reduced to 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100%.

[0321] When the NB-containing multispecific activatable antibody is in the presence of its target but not in the presence of a modifying agent (e.g., a protease or other enzyme), specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the AB when the AB is not associated with the NB or NB:BP. When the NB-containing multispecific activatable antibody is in the presence of its target but not in the presence of a modifying agent (e.g., a protease, other enzyme, reducing agent, or light), specific binding of the AB to its target is reduced or inhibited compared to the specific binding of the parent AB to its target. The ability of the NB-containing multispecific activatable antibody to bind its target, as measured, e.g., in vitro and / or in vivo, when compared to the binding of an AB unrelated to the NB or NB:BP, or the binding of the parent AB, to its target, is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 300%, 360%, 400%, 420%, 440%, 460%, 500%, 520%, 540%, 560%, 580%, 600%, 620%, 640%, 660%, 700%, 720%, 740%, 760%, 780%, 840%, 860%, 880%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 2000%, 2100%, 2200%, 2300%, 2400%, 2500%, 3000%, 3600%, 4000%, 5200%, 5400%, 6600%, 7800%, 8000%, 9000%, 1000%, 1100%, 1200%, 1400%, 1500%, 1600%, 1700%, 1800%, 2000%, 2200%, 230 It can be reduced to 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100%.

[0322] According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody also includes an agent conjugated to the AB to generate the multispecific activatable antibody conjugate. According to some embodiments of the multispecific activatable antibody conjugate, the agent is a therapeutic agent. According to some embodiments, the agent is a diagnostic agent. According to some embodiments, the agent is a detectable marker. According to some embodiments of the multispecific activatable antibody conjugate, the agent is an anti-tumor agent. According to some embodiments of the multispecific activatable antibody conjugate, the agent is a toxin or a fragment thereof. According to some embodiments of the multispecific activatable antibody conjugate, the agent is conjugated to the AB via a linker. According to some embodiments of the multispecific activatable antibody conjugate, the linker is a cleavable linker. According to some embodiments, the agent is an agent selected from the group listed in Table 4. According to some embodiments, the agent is a dolastatin. According to some embodiments, the agent is an auristatin or a derivative thereof. According to some embodiments, the agent is auristatin E or a derivative thereof. According to some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid or a derivative thereof. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or a derivative thereof. In some embodiments, the agent is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.

[0323] According to some examples of any of these multispecific activatable antibody embodiments, the multispecific activatable antibody is a dual-target binding multispecific activatable antibody. Such dual-target binding multispecific activatable antibodies comprise two Abs capable of binding to the same or different targets. According to particular embodiments, the dual-targeting multispecific activatable antibody comprises a bispecific antibody or fragment thereof.

[0324] A dual-target-binding multispecific activatable antibody is designed to have a CL that can be cleaved by a cleaving agent co-localized in a target tissue with one or both targets that can bind to the AB of the multispecific activatable antibody. A dual-target-binding multispecific activatable antibody with multiple ABs for the same or different targets can be designed to have multiple CLs, where a first CL can be cleaved by a cleaving agent in a first target tissue and a second CL can be cleaved by a cleaving agent in a second target tissue, and one or more targets bind to the AB of the multispecific activatable antibody. According to one embodiment, the first and second target tissues are spatially separated, for example, at different sites in an organism. According to one embodiment, the first and second target tissues are the same tissue but temporally separated, for example, the same tissue at two different time points, for example, the first time point is when the tissue is an early-stage tumor and the second time point is when the tissue is a later-stage tumor.

[0325] The present disclosure also provides nucleic acid molecules encoding the multispecific activatable antibodies described herein. The disclosure also provides vectors containing those nucleic acids. The multispecific activatable antibodies described herein can be produced by culturing cells containing those nucleic acid molecules or vectors under conditions conducive to expression of the multispecific activatable antibodies.

[0326] The present disclosure also provides a method for producing a multispecific activatable antibody. According to one embodiment, the method includes the steps of: (a) culturing cells containing a nucleic acid construct encoding the multispecific activatable antibody under conditions conducive to expression of the multispecific activatable antibody, where the multispecific activatable antibody comprises (i) a non-binding steric moiety (NB); (ii) a cleavable linker (CL); and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, where (1) the NB does not specifically bind to the AB; (2) the CL is a polypeptide that comprises a substrate (S) for an enzyme; (3) the CL is positioned such that, in its uncleaved state, the NB interferes with binding of the AB to its target and, in its cleaved state, the NB does not interfere with binding of the AB to its target; and (4) the NB does not inhibit cleavage of the CL by the enzyme; and (b) recovering the multispecific activatable antibody.

[0327] According to another embodiment, the method includes the steps of (a) culturing cells containing a nucleic acid construct encoding the multispecific activatable antibody under conditions conducive to expression of the multispecific activatable antibody, wherein the multispecific activatable antibody comprises (i) a binding partner (BP) for a non-binding steric moiety (NB); (ii) a cleavable linker (CL); and (iii) an antibody or antigen-binding fragment thereof (AB) that specifically binds a target, wherein (1) the NB does not specifically bind to the AB; (2) the CL is a polypeptide that comprises a substrate (S) for an enzyme; (3) the CL is positioned such that, in an uncleaved state and in the presence of the NB, the NB interferes with binding of the AB to the target, and in a cleaved state, the NB does not interfere with binding of the AB to the target, and the BP does not interfere with binding of the AB to the target; and (4) the NB does not inhibit cleavage of the CL by the enzyme; and (b) recovering the multispecific activatable antibody. According to some examples of this embodiment, the BP of the multispecific activatable antibody is conjugated to the NB.

[0328] Uses of multispecific and multispecific activatable antibodies It will be understood that administration of therapeutic entities according to the present disclosure will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and the like. Numerous suitable formulations can be found in formularies known to all pharmacists: Blaug, Seymour, Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), especially Chapter 87. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., Lipofectin®), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures are suitable in treatment and therapy in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation, and the formulation is physiologically compatible and acceptable to the route of administration.For additional information related to formulations, excipients, and carriers familiar to pharmacists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance," Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals," Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts," J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations," PDA J Pharm Sci Technol. 52:238-311 (1998), and references cited therein.

[0329] According to one embodiment, the multispecific antibodies and / or multispecific activatable antibodies of the present disclosure can be used as therapeutic agents. Such agents will generally be used to diagnose, prognose, monitor, treat, alleviate, and / or prevent a disease or condition in a subject. A treatment regimen is implemented by identifying a subject, e.g., a human patient or other mammal, suffering from (or at risk of developing) a disorder using standard methods. A multispecific antibody and / or multispecific activatable antibody formulation, e.g., according to some embodiments, a formulation with high specificity and high affinity for its multiple target antigens, is administered to a subject and will generally have an effect resulting from its binding to the targets. Administration of the multispecific antibody and / or multispecific activatable antibody can inhibit, block, or interfere with the signaling function of one or more targets. Administration of the multispecific antibody and / or multispecific activatable antibody can inhibit, block, or interfere with the binding of one or more targets to the endogenous ligands to which it naturally binds.

[0330] Generally, alleviation or treatment of a disease or disorder encompasses the alleviation of one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, a therapeutically effective amount of an agent can achieve one or a combination of the following phenomena: reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., reducing and / or stopping to some extent) cancer cell invasion into peripheral organs; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with cancer. According to some embodiments, the compositions of the present disclosure can be used to prevent the onset or recurrence of a disease or disorder in a subject, such as a human or other mammal, such as a non-human primate, a companion animal (e.g., a cat, dog, horse), livestock, a working animal, or a zoo animal. The terms subject and patient are used interchangeably herein.

[0331] A therapeutically effective amount of a multispecific antibody and / or multispecific activatable antibody of the present disclosure generally refers to the amount necessary to achieve a therapeutic goal. As noted above, this may be a binding interaction between the multispecific antibody and / or multispecific activatable antibody and its target antigen, which, in certain cases, interferes with the function of the target. The amount that needs to be administered will further depend on the binding affinity of the multispecific antibody and / or multispecific activatable antibody for its specific antigen, and also on the rate at which the administered multispecific antibody and / or multispecific activatable antibody is depleted from the free volume of the other subject to which it is administered. A typical range for a therapeutically effective dose of a multispecific antibody and / or multispecific activatable antibody of the present disclosure, by way of non-limiting example, may be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. A typical administration frequency may be, for example, twice a week to once a week.

[0332] The effectiveness of treatment is determined according to any known method for diagnosing or treating the particular disorder. Methods for screening for multispecific and / or multispecific activatable antibodies with the desired specificities include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immunologically mediated techniques known in the art.

[0333] According to further embodiments, the multispecific antibodies and / or multispecific activatable antibodies di...

Claims

1. A multispecific activatable antibody, comprising: at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or epitope; a second antibody or antigen-binding fragment thereof (AB2), which specifically binds a second target or epitope; at least a first masking moiety (MM1) that inhibits binding of said AB1 to said first target or epitope; and at least a first cleavage moiety (CM1) coupled to said AB1 and said MM1, said CM1 being a substrate for a protease; The CM1 is present between the AB1 and the MM1, The AB2 is linked to the N-terminus or C-terminus of the H chain or L chain of the AB1; said AB1 is a targeting antibody or antigen-binding fragment thereof that specifically binds to said first target or epitope present on the surface of a tumor cell or other cell type associated with a disease, and said AB2 is an immune cell-engaging antibody or antigen-binding fragment thereof that specifically binds to said second target or epitope present on the surface of an immune effector cell; and A multispecific activatable antibody, wherein the protease cleaves the CM1 in the multispecific activatable antibody when the multispecific activatable antibody is exposed to the protease.

2. The AB2 is (i) a T-cell engaging antibody or antigen-binding fragment thereof; (ii) an NK-cell recruiting antibody or antigen-binding fragment thereof; (iii) a bone marrow mononuclear cell recruiting antibody or an antigen-binding fragment thereof; or (iv) The multispecific activatable antibody of claim 1 which is a macrophage-recruiting antibody or an antigen-binding fragment thereof. (i) the first target or epitope present on the surface of a tumor cell or other cell type associated with a disease is EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrin receptor); or (ii) the second target or epitope present on the surface of an immune effector cell is CD3, CD4, CD8, CD16a, CD25, CD28, CD32, CD56, CD137, CTLA-4, PD-1, or CD137 (TNFRSF9).

4. The multispecific activatable antibody of claim 1, comprising: (a) a targeting antibody, or antigen-binding fragment thereof, that specifically binds to a first target or epitope present on the surface of a tumor cell or other cell type associated with a disease, and (b) a T-cell recruiting antibody, or antigen-binding fragment thereof.

5. The multispecific activatable antibody of claim 1, wherein at least one of AB1 and AB2 is an scFv.

6. The multispecific activatable antibody of claim 5, wherein the targeting antibody is an IgG and the immune effector cell recruiting antibody is an scFv.

7. The multispecific activatable antibody of claim 6, wherein the targeting antibody is an IgG and the T-cell recruiting antibody is an scFv.

8. The multispecific activatable antibody of claim 1, wherein AB2 is an anti-CD3 epsilon scFv.

9. The multispecific activatable antibody of claim 8, wherein the anti-CD3 epsilon scFv is derived from OKT3.

10. (i) the AB1 is an anti-Jagged antibody or an anti-EGFR antibody; (ii) the AB1 is an anti-EGFR antibody or an antigen-binding fragment thereof; (iii) the AB1 is an anti-Jagged antibody or an antigen-binding fragment thereof; (iv) said AB1 is an anti-Jagged antibody or an anti-EGFR antibody and said AB2 is an anti-CD3 epsilon scFv; (v) said AB1 is an anti-EGFR antibody and said AB2 is an anti-CD3 epsilon scFv; or (vi) The multispecific activatable antibody of claim 1, wherein said AB1 is an anti-Jagged antibody and said AB2 is an anti-CD3 epsilon scFv.

11. The multispecific activatable antibody of claim 1, (i) a linking peptide between said MM1 and said CM1: (ii) a connecting peptide between the CM1 and the AB1; or 2. The multispecific activatable antibody of claim 1, comprising (iii) a first connecting peptide (LP1) and a second connecting peptide (LP2), and wherein the multispecific activatable antibody has the following structural arrangement from N-terminus to C-terminus: MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. (a) LP1 and LP2 are not identical to each other; or (b) the multispecific activatable antibody of claim 11, wherein each of LP1 and LP2 is a peptide of 1-20 amino acids in length.

13. The multispecific activatable antibody of claim 1, wherein CM1 is a polypeptide up to 15 amino acids in length.

14. The multispecific activatable antibody of claim 1, wherein at least a portion of the multispecific activatable antibody has the following N-terminal to C-terminal structural arrangement: MM1-CM1-AB1 or AB1-CM1-MM1.

15. The multispecific activatable antibody of claim 1, wherein the antigen-binding fragment of AB1 and / or AB2 is selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. (i) at least a portion of the multispecific activatable antibody has the following N-terminal to C-terminal structural configuration: MM1-CM1-AB1 or AB1-CM1-MM1; and (ii) The multispecific activatable antibody of claim 1, wherein the antigen-binding fragment of AB1 and / or AB2 is selected from the group consisting of a Fab fragment, an F(ab')2 fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

17. The multispecific activatable antibody of claim 1, wherein CM1 is selected from SEQ ID NOs: 77-80 or 339.

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