Bispecific antibodies that bind CD3 and MUC1 and methods of use thereof

Abstract

The present disclosure is directed to antibodies, e.g., monoclonal antibodies, bispecific antibodies and bispecific activatable antibodies that bind MUC1 and / or CD3, and uses of such antibodies, in particular use thereof in the treatment of diseases, e.g., cancers.

Description

BISPECIFIC ANTIBODIES THAT BIND CD3 AND MUC1 AND METHODS OF USETHEREOFRELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 674,842, filed on July 24, 2024, and U.S. Provisional Application No. 63 / 812,390, filed on May 27, 2025. The entire contents of each of the foregoing applications are incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 20, 2025, is named 127206-11920. xml and is 140,032 bytes in size.BACKGROUND OF THE INVENTION

[0003] CD3 is an antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four different chains: epsilon, zeta, delta and gamma. The CD3 dimeric arrangements include gamma / epsilon, delta / epsilon and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes involving the activation of T cells.

[0004] Bispecific antibodies that are capable of binding CD3 and a target antigen have been proposed for therapeutic uses involving targeting T cell immune responses to tissues and cells expressing the target antigen. A bispecific antibody having a CD3-binding arm and a tumor antigen-binding arm may provide the necessary immunological synapse to elicit antitumor activity, and could be useful in therapeutic settings in which specific targeting and T cell- mediated killing of tumor cells that express the target antigen is desired. On-target off-tumor toxicities due to broad expression of the target antigen, however, have limited the therapeutic application of these antibodies. Accordingly, there is an ongoing need for therapeutic bispecific antibodies that overcome this, as well as other hurdles.SUMMARY OF THE INVENTION

[0005] The present invention is based on the discovery of bispecific antibodies that specifically bind to a T cell antigen, i.e., CD3, and a tumor cell antigen, i.e., MUC1. In particular, these bispecific antibodies are activatable antibodies that include a masking moiety coupled to each of the antigen-binding domains. The masking moiety is designed to limit the ability of the antibody to bind to its target in healthy tissues and, thus, minimize undesired toxicities. The masking moiety is coupled to the antigen-binding domain via a cleavable moiety (either directly or indirectly, e.g., via one or more linkers). The cleavable moiety is cleaved only under certain conditions, e.g., in a tumor environment where there is a high level of protease activity, to thereby release the masking moiety from the antigen-binding domain, and activate the bispecific antibodies to bind to the targets on both T cells and tumor cells, thus leading to T-cell activation, proliferation, and tumor cell death. In particular, the present inventors have successfully demonstrated that administration of the bispecific activatable antibodies of the invention lead to significant tumor regression in mice, including mice having a low-MUCl expressing cancer. Therefore, the bispecific activatable antibodies of the invention have the potential to treat a wider range of patients with different MUC1 expression levels.

[0006] Accordingly, in one aspect, the present invention provides a bispecific activatable antibody (BAA), wherein said BAA comprises a first binding component (FBC) and a second binding component (SBC), wherein the FBC comprises (a) an antigen binding domain (AB1) that specifically binds to CD3, wherein the AB1 comprises a heavy chain variable domain (AB1 VH) and a light chain variable domain (AB1 VL); and (b) a first masking moiety (MM1) linked to a first cleavable moiety (CM1), wherein the MM1 inhibits the binding of the AB1 to CD3, wherein the CM1 is a polypeptide that functions as a substrate for a first protease, wherein the MM1 is linked in an amino- (N-) to carboxyl- (C-) terminal direction to the CM1 to form an MM1-CM1 construct, and wherein the C-terminus of the MM1-CM1 construct is linked to the N-terminus of AB1; and wherein the SBC comprises (a) an antigen binding domain (AB2) that specifically binds to MUC1, wherein the AB2 comprises a heavy chain variable region (AB2 VH) and a light chain variable region (AB2 VL), and (b) a second masking moiety (MM2) linked to a second cleavable moiety (CM2), wherein the MM2 inhibits the binding of the AB2 to MUC1; wherein the CM2 is a polypeptide that functions as a substrate for a second protease, wherein the MM2 is linked in an N- to C- terminal direction to the CM2 to form an MM2-CM2 construct, and wherein the C-terminus of the MM2-CM2 construct is linked to the N-terminus of AB2.

[0007] In some embodiments, the AB1 VH comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO:3, and wherein the AB1 VL comprises a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6.

[0008] In some embodiments, the AB1 VH comprises a sequence of SEQ ID NO: 13 and the AB1 VL comprises a sequence of SEQ ID NO: 14.

[0009] In some embodiments, the AB1 is a single chain fragment variable (scFv) comprising the AB 1 VH and the AB 1 VL.

[0010] In some embodiments, the MM1 comprises an amino acid sequence of SEQ ID NO:30.

[0011] In some embodiments, the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.

[0012] In some embodiments, the CM1 comprises an amino acid sequence of SEQ ID NO:51. In some embodiments, the CM1 comprises an amino acid sequence of SEQ ID NO:52. In some embodiments, the CM1 comprises an amino acid sequence of SEQ ID NO:53. In some embodiments, the CM1 comprises an amino acid sequence of SEQ ID NO:54. In some embodiments, the CM1 comprises an amino acid sequence of SEQ ID NO:55.

[0013] In some embodiments, the AB2 VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and wherein the AB2 VL comprises a LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

[0014] In some embodiments, the AB2 VH comprises a sequence of SEQ ID NO: 15 and the AB2 VL comprises a sequence of SEQ ID NO: 16.

[0015] In some embodiments, the AB2 is a Fab fragment comprising the AB2 VH and the AB2 VL.

[0016] In some embodiments, the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.

[0017] In some embodiments, the MM2 comprises SEQ ID NO:36.

[0018] In some embodiments, the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55.

[0019] In some embodiments, the CM2 comprises an amino acid sequence of SEQ ID NO:51. In some embodiments, the CM2 comprises an amino acid sequence of SEQ ID NO: 52. In some embodiments, the CM2 comprises an amino acid sequence of SEQ ID NO:53. In some embodiments, the CM2 comprises an amino acid sequence of SEQ ID NO: 54. In some embodiments, the CM2 comprises an amino acid sequence of SEQ ID NO: 55.

[0020] In some embodiments, the BAA comprises a first antibody component (FAC) and a second antibody component (SAC). In some embodiments, the FAC comprises the FBC, a first hinge region and a first Fc region (Fcl), and the Fcl is linked in an N- to C-terminal direction to the FBC via the first hinge region. In some embodiments, the SAC comprises the SBC, a second hinge region and a second Fc region (Fc2), and the Fc2 is linked in an N- to C-terminal direction to the SBC via the second hinge region.

[0021] In some embodiments, the SAC comprises a heavy chain (HC) and a light chain (LC), the AB2 VH is present within the HC that further comprises a CHI constant domain, the second hinge region and / or the Fc2, and the AB2 VL is present within the LC that further comprises a light chain constant domain.

[0022] In some embodiments, the first hinge region and / or the second hinge region comprise an amino acid sequence encoding a human IgG hinge region or a variant thereof comprising from one to ten substitutions.

[0023] In some embodiments, the first hinge region and / or the second hinge region comprise an amino acid substitution in at least one of amino acid positions at S228, L234, and L235, as numbered by the EU index as set forth in Kabat.

[0024] In some embodiments, the Fcl and / or the Fc2 comprise an amino acid sequence encoding a human IgG Fc domain or a variant thereof comprising from one to ten substitutions.

[0025] In some embodiments, the Fcl and / or the Fc2 comprise an amino acid substitution in at least one of amino acid positions at S354, T366, L368, Y349 and Y407, as numbered by the EU index as set forth in Kabat.

[0026] In some embodiments, the BAA comprises at least one linker present between a pair of components selected from the group consisting of MM1 and CM1, CM1 and AB1 VH,AB1 VH and AB1 VL, AB1 VL and a first hinge region, MM2 and CM2, and CM2 and AB2 VH.

[0027] In some embodiments, the FAC comprises an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 17, (b) SEQ ID NO:20, (c) SEQ ID NO:22, and (d) an amino acid sequence at least 90% identical thereto.

[0028] In some embodiments, the SAC comprises an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 18 and SEQ ID NO: 19, (b) SEQ ID NO:21 and SEQ ID NO: 19, (c) SEQ ID NO:23 and SEQ ID NO: 19, and (d) an amino acid sequence at least 90% identical thereto.

[0029] In some embodiments, the BAA comprises a set of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; (b) SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO: 19; (c) SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO: 19; and (d) an amino acid sequence at least 90% identical thereto.

[0030] In some embodiments, the BAA comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0031] In some embodiments, the first protease and / or the second protease is a matrix metalloproteinase (MMP), a cysteine protease, and / or a serine protease.

[0032] In one aspect, the present invention provides an isolated nucleic acid molecule encoding the BAA of the present invention selected from the group of consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;.(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO: 18;(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO: 19;(h) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20;(i) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO:21;(j) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22; and(k) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO:23.

[0033] In another aspect, the present invention provides a vector comprising an isolated nucleic acid molecule of the invention.

[0034] In yet another aspect, the present invention provides a cell comprising an isolated nucleic acid molecule, or a vector of the invention.

[0035] In one aspect, the present invention provides a cell comprising an isolated nucleic acid molecule encoding the BAA of the present invention selected from the group consisting of:(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 comprising an amino acid sequence of SEQ ID NO: 13 and an amino acid sequence of SEQ ID NO: 14; and a nucleotide sequence encoding an AB2 comprising an amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO: 16;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO: 18 and an amino acid sequence of SEQ ID NO: 19;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO:21 and an amino acid sequence of SEQ ID NO: 19; and(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO:23 and an amino acid sequence of SEQ ID NO: 19.

[0036] In one aspect, the present invention provides a pharmaceutical composition comprising a BAA and a pharmaceutically acceptable excipient.

[0037] In some embodiments, the pharmaceutical composition is for use in combination of another therapeutic agent for treating cancer.

[0038] In another aspect, the present invention provides a kit comprising a BAA, or a pharmaceutical composition, and instructions for use.

[0039] In one aspect, the present invention provides a method of producing a BAA by culturing a cell under conditions that lead to expression of the BAA, wherein the cell comprises the nucleic acid molecule or the vector as described herein.

[0040] In one aspect, the present invention provides a method of treating cancer comprising administering a therapeutically effective amount of a BAA, or a pharmaceutical composition as described herein, to a subject in need thereof.

[0041] In one aspect, the present invention provides a method of treating cancer comprising administering a therapeutically effective amount of a BAA, or a pharmaceutical composition, as described herein, in combination with another therapeutic agent for treating cancer to a subject in need thereof.

[0042] In another aspect, the present invention provides use of a BAA in production of a pharmaceutical composition for treating cancer.

[0043] In one aspect, the present invention provides a bispecific antibody that binds to CD3 and MUC1, comprising: (a) a first antigen binding domain (AB1) that specifically binds to a CD3, wherein the AB1 comprises a AB1 VH and a AB1 VL; wherein the AB1 VH comprises a HCDR1 of SEQ ID NO:1, a HCDR2 of SEQ ID NO:2, and a HCDR3 of SEQ ID NO:3, and wherein the AB1 VL comprises a LCDR1 of SEQ ID NO:4, a LCDR2 of SEQ ID NO:5, and a LCDR3 of SEQ ID NO:6, and (b) a second antigen binding domain (AB2) that specifically binds to a MUC1, wherein the AB2 comprises an AB2 VH and an AB2 VL.

[0044] In some embodiments, the AB1 VH comprises a sequence of SEQ ID NO: 13 and the AB1 VL comprises a sequence of SEQ ID NO: 14.

[0045] In some embodiments, the AB1 is a scFv comprising the AB1 VH and the AB1 VL.

[0046] In some embodiments, the AB2 VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and the AB2 VL comprises a LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

[0047] In some embodiments, the AB2 VH comprises a sequence of SEQ ID NO: 15 and the AB2 VL comprises a sequence of SEQ ID NO: 16.

[0048] In some embodiments, the AB2 is a Fab fragment comprising the AB2 VH and the AB2 VL.

[0049] In some embodiments, the bispecific antibody further comprises a first hinge region and a Fcl, and a second hinge region and a Fc2.

[0050] In some embodiments, the first hinge region and the second hinge region comprise an amino acid sequence encoding a human IgG hinge region or a variant thereof comprising from one to ten substitutions.

[0051] In some embodiments, the first hinge region and the second hinge region comprise an amino acid substitution in at least one of amino acid positions at S228, L234, and L235, as numbered by the EU index as set forth in Kabat.

[0052] In some embodiments, the Fcl and the Fc2 comprise an amino acid sequence encoding a human IgG Fc domain or a variant thereof comprising from one to ten substitutions.

[0053] In some embodiments, the Fcl and the Fc2 comprise an amino acid substitution in at least one of amino acid positions at S354, T366, E368, Y349 and Y407, as numbered by the EU index as set forth in Kabat.

[0054] In some embodiments, the bispecific antibody comprises an AB 1 Fc fusion, an AB2 HC and an AB2 EC. In some embodiments, the AB1 Fc fusion comprises the AB1, the first hinge region and the Fcl. In some embodiments, the AB2 HC comprises the AB2 VH, a CHI constant domain, the second hinge region and the Fc2. In some embodiments, the AB2 EC comprises the AB2 VE and a light chain constant domain.

[0055] In some embodiments, the AB1 Fc fusion comprises an amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 90% identical thereto; the AB2 HC comprises an amino acid sequence of SEQ ID NO:25, an amino acid sequence at least 90% identical thereto; and the AB2 EC comprises an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 90% identical thereto.

[0056] In one aspect, the present invention provides an isolated nucleic acid molecule encoding the bispecific antibody of the present invention selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;.(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 Fc fusion consisting of an amino acid sequence of SEQ ID NO:24;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 HC consisting of an amino acid sequence of SEQ ID NO:25; or(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 LC consisting of an amino acid sequence of SEQ ID NO: 19.

[0057] In another aspect, the present invention provides a vector comprising an isolated nucleic acid molecule of the invention.

[0058] In yet another aspect, the present invention provides a cell comprising an isolated nucleic acid molecule, or a vector of the invention.

[0059] In one aspect, the present invention provides a cell comprising an isolated nucleic acid molecule encoding the bispecific antibody of the present invention selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 comprising an amino acid sequence of SEQ ID NO: 13 and an amino acid sequence of SEQ ID NO: 14, and a nucleotide sequence encoding an AB2 comprising an amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO: 16; and(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 Fc fusion consisting of an amino acid sequence of SEQ ID NO:24, and a nucleotide sequence encoding an AB2 HC consisting of an amino acid sequence of SEQ ID NO:25 and an AB2 LC consisting of an amino acid sequence of SEQ ID NO: 19.

[0060] In one aspect, the present invention provides a monoclonal antibody comprising an antigen binding domain that specifically binds to MUC1, wherein the antigen binding domain comprises a VH and a VL; wherein the VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and wherein the VL comprises a LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

[0061] In some embodiments, the VH comprises a sequence of SEQ ID NO: 15 and the VL comprises a sequence of SEQ ID NO: 16.

[0062] In another aspect, the present invention provides a monoclonal antibody comprising an antigen binding domain that specifically binds to MUC1, wherein the antigen binding domain comprises a VH and a VL; and wherein the VH comprises a HCDR1 of SEQ ID NO:45, a HCDR2 of SEQ ID NO:46, and a HCDR3 of SEQ ID NO:47, and wherein the VL comprises a LCDR1 of SEQ ID NO:48, a LCDR2 of SEQ ID NO:49, and a LCDR3 of SEQ ID NO:50.

[0063] In some embodiments, the VH comprises a sequence of SEQ ID NO:28 and the VL comprises a sequence of SEQ ID NO:29.

[0064] In one aspect, the present invention provides an isolated nucleic acid molecule encoding the monoclonal antibody of the present invention selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 15;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO: 16;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 28; or(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO:29.

[0065] In another aspect, the present invention provides a vector comprising an isolated nucleic acid molecule of the invention.

[0066] In yet another aspect, the present invention provides a cell comprising an isolated nucleic acid molecule, or a vector of the invention.

[0067] In one aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody of the present invention, or a monoclonal antibody of the present invention, and a pharmaceutically acceptable excipient.

[0068] In some embodiments, the pharmaceutical composition is for use in combination of another therapeutic agent for treating cancer.

[0069] In another aspect, the present invention provides a kit comprising a bispecific antibody of the present invention, or a monoclonal antibody of the present invention, or a pharmaceutical composition, and instructions for use.

[0070] In one aspect, the present invention provides a method of producing a bispecific antibody or a monoclonal antibody of the present invention by culturing a cell under conditions that lead to expression of the bispecific antibody or monoclonal antibody, wherein the cell comprises the nucleic acid molecule or the vector as described herein.

[0071] In one aspect, the present invention provides a method of treating cancer comprising administering a therapeutically effective amount of a bispecific antibody or a monoclonal antibody of the present invention, or a pharmaceutical composition as described herein, to a subject in need thereof.

[0072] In one aspect, the present invention provides a method of treating cancer comprising administering a therapeutically effective amount of a bispecific antibody or a monoclonal antibody of the present invention, or a pharmaceutical composition, as described herein, in combination with another therapeutic agent for treating cancer to a subject in need thereof.

[0073] In one aspect, the present invention provides a method of treating a low-MUCl expressing cancer, the method comprising administering a therapeutically effective amount of a bispecific antibody or a monoclonal antibody of the present invention, or the pharmaceutical composition, as described herein, to a subject in need thereof.

[0074] In one aspect, the present invention provides a method of treating a low-MUCl expressing cancer, the method comprising selecting a subject having a low-MUCl expressing cancer, and administering a therapeutically effective amount of a bispecific antibody or a monoclonal antibody of the present invention, or the pharmaceutical composition, as described herein, to a subject in need thereof.

[0075] In some embodiments, the low-MUCl expressing cancer expresses MUC1 at a level of less than about 170, e.g., less than about 160, less than about 150, less than about 140, less than about 130, less than about 120, less than about 110, less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, or less than about 55, as measured by RFI (relative fluorescence intensity).

[0076] In some embodiments, the low-MUCl expressing cancer expresses MUC1 at a level of about 1 to 170, or about 1 to 150, or about 1 to 130, or about 1 to 100, or about 1 to 80, or about 1 to 60, or about 1 to 55, as measured by RFI (relative fluorescence intensity).

[0077] In another aspect, the present invention provides use of a bispecific antibody or a monoclonal antibody of the present invention in production of a pharmaceutical composition for treating cancer.BRIEF DESCRIPTION OF DRAWINGS

[0078] FIG. 1A depicts the structure of a bispecific antibody that binds MUC1 and CD3.

[0079] FIG. IB and FIG. 1C depict the structure of a dually masked bispecific activatable antibody that binds MUC1 and CD3.

[0080] FIGS. 2A and 2B depict the sequence alignments of the humanized sequences with chimeric 74G5 and 21D3. FIG. 2A depicts SEQ ID NOs: 101-116. FIG. 2B decpits SEQ ID NOs: 117-118, 135-136, and 119-132.

[0081] FIG. 3A depicts the MUC1 binding activity of chimeric 74G5 bispecific scFv-Fab-Fc (ProC1465) and humanized MUC1 bispecific scFv-Fab-Fc (ProC2231) with human HP AC and cyno small intestine epithelial cells.

[0082] FIG. 3B depicts the cytotoxic activity of chimeric 74G5 bispecific scFv-Fab-Fc (ProC1465) and humanized MUC1 bispecific scFv-Fab-Fc (ProC2231) to human HP AC and cyno small intestine epithelial cells.

[0083] FIG. 4A depicts the MUC1 binding activity of the humanized MUC1 bispecific scFv- Fab-Fc (ProC2231) and the deglycosylated variants (ProC2689 and ProC2690) with HCT116-huMUCl, human HP AC and cyno small intestine epithelial cells.

[0084] FIGS. 5B depicts the cytotoxic activity of the humanized MUC1 bispecific scFv-Fab- Fc (ProC2231) and the deglycosylated variants (ProC2689 and ProC2690) with HCT116- huMUCl and human HP AC cells.

[0085] FIG. 5 depicts the on-cell binding of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 to MUC1 on HCT116-huMUCl cells.

[0086] FIG. 6 depicts the on-cell binding of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 to MUC1 on HP AC cells.

[0087] FIG. 7 depicts the on-cell binding of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 to MUC1 on cyno primary small intestine epithelial cells.

[0088] FIG. 8 depicts the on-cell binding of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 to CD3 on Jurkat cells.

[0089] FIG. 9 depicts the cytotoxic activity of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 in HCT116-huMUCl cells.

[0090] FIG. 10 depicts the cytotoxic activity of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 in HP AC cells.

[0091] FIG. 11 depicts the cytotoxic activity of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 in cyno primary small intestine epithelial cells.

[0092] FIG. 12 depicts the on-cell binding of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 to MUC1 on HCT116-huMUCl cells upon protease activation.

[0093] FIG. 13 depicts the on-cell binding of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 to MUC1 on HP AC cells upon protease activation.

[0094] FIG. 14 depicts the on-cell binding of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 to CD3 on Jurkat cells upon protease activation.

[0095] FIG. 15 depicts the cytotoxic activity of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 in HCT116-huMUCl cells upon protease activation.

[0096] FIG. 16 depicts the cytotoxic activity of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 against HPAC cells upon protease activation.

[0097] FIG. 17 depicts the anti-tumor efficacy of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 in HCT116-huMUCl tumor model (high MUC-1 expressing cells).

[0098] FIG. 18 depicts the anti-tumor efficacy of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 in HPAC tumor model (moderate MUC-1 expressing cells).

[0099] FIG. 19 depicts the anti-tumor efficacy of the dually masked, activatable bispecific antibody ProC3405, ProC3699, and ProC3687 in NCI-H1650 tumor model (low MUC-1 expressing cells).DETAILED DESCRIPTION OF THE INVENTION

[0100] The present invention is based on the discovery of bispecific antibodies that specifically bind to a T cell antigen, i.e., CD3, and a tumor cell antigen, i.e., MUC1. In particular, these bispecific antibodies are activatable antibodies that include a masking moiety coupled to an antigen-binding domain. The masking moiety is designed to limit the ability of the antibody to bind to its target in healthy tissues and, thus, minimize undesired toxicities. The masking moiety is coupled to the antigen-binding domain via a cleavable moiety (either directly or indirectly, e.g., via one or more linkers). The cleavable moiety is cleaved only under certain conditions, e.g., in a tumor environment where there is a high level of protease activity, to thereby release the masking moiety from the antigen-binding domain, and activate the antibodies to bind to the targets on both T cells and tumor cells, leading to T-cell activation, proliferation, and tumor cell death.

[0101] Also provided herein are related compositions, kits, nucleic acids, vectors, and recombinant cells, as well as related methods, including methods of using and of producing any of the bispecific antibodies described herein.I. Definitions

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0103] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

[0104] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art. For example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value where appropriate. Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 0.01 to 2.0” should be interpreted to include not only the explicitly recited values of about 0.01 to about 2.0, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from 0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc.Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. Additionally, it is noted that all percentages are in weight, unless specified otherwise.

[0105] In understanding the scope of the present disclosure, the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of thestated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of,” as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transition terms (i.e. “comprising,” “consisting,” or “consisting essentially”) provides direct support for replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of’ or “consisting of’ would find direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0106] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[0107] The term “exemplary” is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more concrete fashion.

[0108] Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, disorders, conditions, properties, steps, or the like may be discussed in the context of one specific embodiment or aspect or in a separate paragraph or section of this disclosure. It is understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other embodiments or aspects found anywhere in the present disclosure and claims, which all form the application and claimed invention at the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, composition, or method is intended to and does find direct support for embodiments related to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0109] The term “antibody”, or “antigen-binding domain” as used herein, in its broadest sense and includes an antibody or antigen-binding fragment thereof that specifically binds to an antigen or epitope. Examples of antibodies include intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multi- specific antibodies.

[0110] The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains and two light chains inter-connected by disulfide bonds. The five major classes of immunoglobulin are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is by far the most abundant immunoglobulin and has several subclasses (IgGl, IgG2, IgG3, and IgG4 in humans). A “light chain” includes one light chain variable region or variable domain (VL) and one constant domain (CL). There are two different light chains termed kappa or lambda.

[0111] A “heavy chain” consists of one heavy chain variable region or variable domain (VH) and three constant region domains (CHI, CH2, CH3). There are five main heavy-chain classes or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule.

[0112] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl- terminus in the following order: FR1, CDR1, FR1, CDR2, FR3, CDR3, FR4. An amino acid sequence of three complementarity determining regions (CDRs) present in the VH and VL is largely varied, and contributes to variability of the variable regions. Each CDR is a region consisting of 5 to 10 amino acid residues, which is present in the order of CDR1, CDR2 and CDR3 in the N- terminal of each of the heavy chain and the light chain, and comes into contact with an antigen to form an antibody binding site. It is known that the CDRs of the heavy chain make a larger contribution to antigen binding than the CDRs of the light chain, and that the CDR3 makes a largest contribution among the CDR1 to CDR3. On the other hand, a portion excluding the CDRs of the variable region is designated as a framework region (FR), consists of FR1 to FR4, and is comparatively little varied in the amino acid sequence.

[0113] The term “antibody”, as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, “antigen-binding domain” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0114] Non-limiting examples of antigen-binding fragments include: Fab fragments; Fab’ fragments, F(ab')2 fragments; Fv fragments; single-chain Fv (scFv) molecules; dAb fragments; and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain- specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.

[0115] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0116] A “fragment antigen binding” (Fab) includes a complete light chain paired with the VH domain and the CHI domain of a heavy chain. A F(ab')2 fragment is formed when an antibody is cleaved by pepsin (or otherwise truncated) below the hinge region, in which case the two fragment target-binding domains (Fabs) of the antibody molecule remain linked. A F(ab')2 fragment contains two complete light chains paired with the two VH and CHI domains of the heavy chains joined together by the hinge region.

[0117] A “fragment crystallizable” (Fc) fragment (also referred to herein as FC domain) corresponds to the paired CH2 and CH3 domains and is the part of the antibody molecule that interacts with effector molecules and cells. The functional differences between heavy-chain isotypes lie mainly in the Fc fragment.

[0118] An “Fv” fragment includes a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[0119] A “single chain fragment variable” (scFv) contains only the variable domain of a light chain (VL) linked by a stretch of peptide to a variable domain of a heavy chain (VH).

[0120] A “hinge region” or “interdomain” is flexible amino acid stretch that joins or links the Fab fragment to the Fc domain. A “synthetic hinge region” is an amino acid sequence that joins or links a Fab fragment to an Fc domain.

[0121] In some embodiments, the antibodies of the invention may be a mouse, rat, rabbit, goat, camel, donkey, primate, chimeric, human, or humanized antibody. In one example, the antibody may be a human antibody. In one example, the antibody may be a humanized (e.g., fully humanized) antibody.

[0122] The term “human antibody” is intended to include antibodies having variable and constant regions generated, assembled, or derived from human immunoglobulin sequences. In some embodiments, antibodies may be considered to be “human” even though their amino acid sequences include residues or elements not encoded by human germline immunoglobulin sequences (e.g., include sequence variations, for example that may (originally) have been introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in one or more CDRs.

[0123] The term “humanized” refer to an antibody having an amino acid sequence that includes VH and VL region sequences from a reference protein raised in a non-human species (e.g., a mouse), but also includes modifications in those sequences relative to the reference protein intended to render them more “human-like,” i.e., more similar to human germline variable sequences. In some embodiments, a “humanized” antibody is one that immunospecifically binds to an antigen of interest and that has a framework (FR) region having substantially the amino acid sequence as that of a human antibody, and a complementary determining region (CDR) having substantially the amino acid sequence as that of a non-human antibody contains humanized VH and VL regions.

[0124] The antibodies of the invention may be isolated antibodies. An “isolated antibody,” as used herein, means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody” for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0125] The term “bispecific”, as used herein, refers to the ability of an antibody to specifically bind to two distinct targets. Typically, a bispecific antibody comprises two antigen-binding domains, each of which comprises at least one CDR that alone, or in combination with one or more additional CDRs and / or FRs, and is capable of specifically binding to a different target. In some embodiments, the bispecific antibody may be capable of simultaneously binding two targets, e.g., two target proteins expressed on two distinct cells. In the context of the present invention, the first antigen-binding domain specifically binds a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds a second, distinct antigen (e.g., MUC1).

[0126] As used herein, the term “CD3” refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3-epsilon, CD3- delta, CD3-zeta, and CD3-gamma. The amino acid sequence of a human CD3-epsilon can be found at, for example, NP_000724.1 (SEQ ID NO: 94).

[0127] As used herein, “CD3-binding domain” includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments of the present invention may bind soluble CD3 and / or cell surface expressed CD3. Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants such as, e.g., monomeric and dimeric CD3 constructs, that lack a transmembrane domain or are otherwise unassociated with a cell membrane.

[0128] As used herein, the term “mucin 1” or “MUC1” (also known as KL-6, PEM, EMA, ADMCKD1, Cal5-3, CD227, MCKD, MCD, PUM, Tumor-Associated Epithelial Membrane Antigen, Breast Carcinoma-Associated Antigen DF3, Peanut-Reactive Urinary Mucin, Polymorphic Epithelial Mucin, Carcinoma- Associated Mucin, Krebs Von Den Lungen-6, or Cancer Antigen 15-3) refers to a member of the mucin family. Mucins are O-glycosylatedproteins that play an essential role in forming protective mucous barriers on epithelial surfaces. These proteins also play a role in intracellular signaling. This protein is expressed on the apical surface of epithelial cells that line the mucosal surfaces of many different tissues including lung, breast stomach and pancreas. This protein is proteolytically cleaved into alpha and beta subunits that form a heterodimeric complex. The N-terminal alpha subunit functions in cell-adhesion and the C-terminal beta subunit is involved in cell signaling. Overexpression, aberrant intracellular localization, and changes in glycosylation of this protein have been associated with carcinomas. The amino acid sequence of a human MUC1 can be found at, for example, NP_002447.4 (SEQ ID NO: 95).

[0129] As used herein, the term “activatable antibody” refers to an antibody in its inactive (uncleaved or native) state. Activatable antibodies include a masking moiety (MM) coupled to an antigen-binding domain via a cleavable moiety (CM) (either directly or indirectly, e.g., via one or more linkers). The masking moiety is designed to limit the ability of the antibodies to bind to its antigen. The cleavable moiety is cleaved only under certain conditions, e.g., in a tumor environment where there is a high level of proteases, to thereby release the masking moiety from the antigen-binding domain, and activate the antibodies to bind to the targets. It will be apparent to the ordinarily skilled artisan that, in some embodiments, a cleaved activatable antibody may be connected to a MM that is not reducing, inhibiting, or interfering with binding between the antigen-binding domain and its target. In some embodiments, a cleaved activatable antibody may lack a MM due to cleavage of the CM (e.g., by a protease), resulting in release of the MM. As used herein, the term “cleaved state” or “active state” refers to the condition of the activatable antibody following cleavage of the CM by at least one protease. The term “uncleaved state” or “inactive state” refers to the condition of the activatable antibody in the absence of cleavage of the CM by a protease.

[0130] By activatable is meant that the activatable antibody exhibits a first level of binding to an antigen when the activatable antibody is in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the antigen in the uninhibited, unmasked and / or cleaved state (i.e., a second conformation), where the second level of antigen binding is greater than the first level of binding. In general, the access of antigen to the antigenbinding domain of the activatable antibody is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleaving agent. Thus, when the activatable antibody is in the uncleaved state, the antigen-binding domain is inhibited from antigen binding and can be masked from antigen binding (i.e., the first conformation is such that the antigen-binding domain cannot bind the antigen or is inhibitedin binding the antigen), and in the cleaved state the antigen-binding domain is not inhibited or is unmasked to antigen binding.

[0131] The term “binding component” (e.g., “first binding component (FBC) or second binding component (SBC)), as used herein, refers to a component of an antibody, wherein the binding component comprises an antigen-binding domain, e.g., a heavy chain variable region (VH) and a light chain variable region (VL). The binding component may further comprise a masking moiety and / or a cleavable moiety. In some embodiments, the binding component may further comprise a constant region (e.g., a CHI and CL region). In some embodiments, the binding component comprises one polypeptide that specifically binds to an antigen. In some embodiments, the binding component comprises two polypeptides that specifically bind to an antigen. In some embodiments, the FBC comprises an AB1 VH, an AB1 VL, a masking moiety (MM1), and a cleavable moiety (CM1). In some embodiments, the SBC comprises an AB2 VH, an AB2 VL, a CHI, a CL, a masking moiety (MM2), and a cleavable moiety (CM2).

[0132] The term “antibody component” (e.g., “first antibody component (FAC) or second antibody component (SAC)), as used herein, refers to a component of an antibody, wherein the antibody component comprises a binding component as described herein. In some embodiments, the antibody component further comprises a hinge region, and / or a Fc region. In some embodiments, the antibody component comprises one polypeptide that specifically binds to an antigen. In some embodiments, the antibody component comprises two polypeptides that specifically bind to an antigen. In some embodiments, the FAC comprises an FBC, a hinge region, and a Fc region. In some embodiments, the SAC comprises an SBC, a hinge region and a Fc region.

[0133] The term "post-translational modification" refers to that an antibody expressed in a cell is modified after translation. Examples of the post-translational modification include modification such as pyroglutamylation, glycosylation, oxidation, deamidation or glycation of glutamine or glutamic acid at the heavy chain N-terminal, and lysine deletion by cutting lysine at the heavy chain C-terminal with carboxypeptidase. It is known that such post- translational modification is caused in various antibodies (J. Pharm. Sci., 2008, Vol. 97, p. 2426-2447).

[0134] The term “subject” is used herein to refer to an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, a gorilla, an orangutan, a gibbon, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and a whale), abird (e.g., a duck or a goose), and a shark. In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and / or human being treated for a disease, disorder or condition as described herein. In one embodiment, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age. In another embodiment, the subject is about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100 years of age. The terms “subject” and “patient” are used interchangeably herein. Values and ranges intermediate to the above recited ranges are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above-recited values as upper and / or lower limits are intended to be included.

[0135] As used herein, the term “a low-MUCl expressing cancer” refers to a cancer having a MUC1 expresssion level that is higher than a normal healthy cell, but relatively lower than the MUC1 expression level of other cancer cells. The MUC1 expression level can be determined by any methods known in the art, for example, by flow cytometry. In some embodiments, a low-MUCl expressing cancer may express MUC1 at a level of less than about 170, e.g., less than about 160, less than about 150, less than about 140, less than about 130, less than about 120, less than about 110, less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, or less than about 55, as measured by RFT (relative fluorescence intensity). Relative fluorescence intensity (RFI), as used herein, is calculated by the following formula: (mean fluorescence intensity (MFI) of a sample incubated with anti-MUCl antibody) / (MFI of a sample incubated with a negative control). In some embodiments, a low-MUCl expressing cancer expresses MUC1 at a level of about 1 to 170, or about 1 to 150, or about 1 to 130, or about 1 to 100, or about 1 to 80, or about 1 to 60, or about 1 to 55, as measured by RFI (relative fluorescence intensity).

[0136] As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms, diminishing the extent of a disorder, stabilized (i.e., not worsening) state of a disorder, delaying progression of a disorder, amelioration or palliation of the disorder, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0137] As used herein, the term "effective amount" refers to the amount of a therapy, which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, inhibit or prevent the advancement of a disorder, cause regression of adisorder, inhibit or prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). An effective amount can require more than one dose.II. Monoclonal Antibodies, Bispecific Antibodies, and Bispecific Activatable Antibodies

[0138] The present invention provides bispecific antibodies comprising two antigen-binding domains, wherein one arm of the antibody comprises an antigen-binding domain (AB1) that specifically binds a T cell antigen, i.e., CD3, and the other arm of the antibody comprises an antigen-binding domain (AB2) that specifically binds a tumor cell antigen, i.e., MUC1. The bispecific antibody can bind to CD3 on an immune cell, e.g., a T cell, and MUC1 on a tumor cell, thus activating the immune cell and crosslinking the activated immune cell to the tumor cell.

[0139] In particular, these bispecific antibodies are activatable antibodies that include a masking moiety coupled to each of the antigen-binding domains. The masking moiety is designed to limit the ability of the antibody to bind to its target in healthy tissues and, thus, minimize undesired toxicities. The masking moiety is coupled to the antigen-binding domain via a cleavable moiety. The cleavable moiety is cleaved only under certain conditions, e.g., in a tumor environment where there is a high level of protease activity, to thereby release the masking moiety from the antigen-binding domain, and activate the antibodies to bind to the targets on both T cells and tumor cells, leading to T-cell activation, proliferation, and tumor cell death.

[0140] Each antigen-binding domain within the bispecific antibody comprises at least one complementarity determining region (CDR) that alone, or in combination with one or more additional CDRs, specifically binds to a particular antigen. In some embodiments, each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

[0141] Accordingly, in one aspect, the present invention provides a bispecific activatable antibody comprising a first binding component (FBC) and a second binding component (SBC), wherein the FBC comprises (a) an antigen binding domain (AB1) that specifically binds to CD3, wherein the AB1 comprises a heavy chain variable domain (AB1 VH) and a light chain variable domain (AB1 VE); and (b) a first masking moiety (MM1) linked to a first cleavable moiety (CM1), wherein the MM1 inhibits the binding of the AB1 to CD3, whereinthe CM1 is a polypeptide that functions as a substrate for a first protease, wherein the MM1 is linked in an N- to C- terminal direction to the CM1 to form an MM1-CM1 construct, and wherein the C-terminus of the MM1-CM1 construct is linked to the N-terminus of AB1; and wherein the SBC comprises (a) an antigen binding domain (AB2) that specifically binds to MUC1, wherein the AB2 comprises a heavy chain variable region (AB2 VH) and a light chain variable region (AB2 VL), and (b) a second masking moiety (MM2) linked to a second cleavable moiety (CM2), wherein the MM2 inhibits the binding of the AB2 to MUC1; wherein the CM2 is a polypeptide that functions as a substrate for a second protease, wherein the MM2 is linked in an N- to C- terminal direction to the CM2 to form an MM2-CM2 construct, and wherein the C-terminus of the MM2-CM2 construct is linked to the N- terminus of AB2.

[0142] In another aspect, the present invention provides a bispecific antibody comprising (a) a first antigen binding domain that specifically binds to a CD3 (AB1) and (b) a second antigen binding domain that specifically binds to a MUC1 (AB2).

[0143] In yet another aspect, the present invention provides a monoclonal antibody comprising an antigen binding domain that specifically binds to MUC1.CD3-Binding Domain

[0144] In some embodiments, the CD3-binding domain (AB1) comprises a heavy chain variable domain (AB1 VH) comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain (AB1 VL) comprising LCDR1, LCDR2, and LCDR3.

[0145] In some embodiments, the HCDR1 comprises a sequence of SEQ ID NO: 1; the HCDR2 comprises a sequence of SEQ ID NO: 2; the HCDR3 comprises a sequence of SEQ ID NO: 3; the LCDR1 comprises a sequence of SEQ ID NO: 4; the LCDR2 comprises a sequence of SEQ ID NO: 5; and the LCDR3 comprises a sequence of SEQ ID NO: 6.

[0146] In some embodiments, the heavy chain variable domain (AB 1 VH) comprises a sequence of SEQ ID NO: 13, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 13.

[0147] In some embodiments, the light chain variable domain (AB1 VL) comprises a sequence of SEQ ID NO: 14, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 14.

[0148] In some embodiments, the AB1 VH comprises a sequence of SEQ ID NO: 13, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) to SEQ ID NO: 13, and the AB1 VL comprises a sequence of SEQ ID NO: 14, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) to SEQ ID NO: 14.

[0149] In some embodiments, the AB1 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 13. In some embodiments, the AB1 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the AB1 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 13 and the AB1 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 14.

[0150] In some embodiments, the AB1 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the AB1 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the AB1 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 13 and the AB1 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 14.

[0151] In some embodiments, the AB1 VH comprises a sequence of SEQ ID NO: 13 and the AB1 VL comprises a sequence of SEQ ID NO: 14.

[0152] In some embodiments, the AB1 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 1; a HCDR2 comprising a sequence of SEQ ID NO: 2; a HCDR3 comprising a sequence of SEQ ID NO: 3; and the AB1 VH comprises a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 13; and the AB1 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 4; a LCDR2 comprising a sequence of SEQ ID NO: 5; and a LCDR3 comprising a sequence of SEQ ID NO: 6, and the AB1 VL comprises a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 14.

[0153] In some embodiments, the AB1 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 1; a HCDR2 comprising a sequence of SEQ ID NO: 2; a HCDR3 comprising a sequence of SEQ ID NO: 3; and the AB1 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 13; and the AB1 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 4; a LCDR2 comprising a sequence of SEQ ID NO: 5; and a LCDR3 comprising a sequence of SEQ ID NO: 6, and the AB1 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 14.

[0154] In some embodiments, the AB1 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 1; a HCDR2 comprising a sequence of SEQ ID NO: 2; a HCDR3 comprising a sequence of SEQ ID NO: 3; and the AB1 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 13; and the AB1 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 4; a LCDR2 comprising a sequence of SEQ ID NO: 5; and a LCDR3 comprising a sequence of SEQ ID NO: 6, and the AB1 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 14.MUC1 -Binding Domain

[0155] In some embodiments, the MUC1 -binding domain (AB2) comprises a heavy chain variable domain (AB2 VH) comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain (AB2 VL) comprising LCDR1, LCDR2, and LCDR3.

[0156] In some embodiments, the HCDR1 comprises a sequence of SEQ ID NO: 7; the HCDR2 comprises a sequence of SEQ ID NO: 8; the HCDR3 comprises a sequence of SEQ ID NO: 9; the LCDR1 comprises a sequence of SEQ ID NO: 10; the LCDR2 comprises a sequence of SEQ ID NO: 11; and the LCDR3 comprises a sequence of SEQ ID NO: 12.

[0157] In some embodiments, the heavy chain variable domain (AB2 VH) comprises a sequence of SEQ ID NO: 15, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 15.

[0158] In some embodiments, the light chain variable domain (AB2 VL) comprises a sequence of SEQ ID NO: 16, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 16.

[0159] In some embodiments, the AB2 VH comprises a sequence of SEQ ID NO: 15, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) to SEQ ID NO: 15, and the AB2 VL comprises a sequence of SEQ ID NO: 16, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) to SEQ ID NO: 16.

[0160] In some embodiments, the AB2 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 15. In some embodiments, the AB2 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 16. In some embodiments, the AB2 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 15 and the AB2 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 16.

[0161] In some embodiments, the AB2 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the AB2 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the AB2 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 15 and the AB2 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 16.

[0162] In some embodiments, the AB2 VH comprises a sequence of SEQ ID NO: 15 and the AB2 VL comprises a sequence of SEQ ID NO: 16.

[0163] In some embodiments, the AB2 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 7; a HCDR2 comprising a sequence of SEQ ID NO: 8; a HCDR3 comprising a sequence of SEQ ID NO: 9; and the AB2 VH comprises a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 15; and the AB2 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 10; a LCDR2 comprising a sequence of SEQ ID NO: 11; and a LCDR3 comprising a sequence of SEQ ID NO: 12, and the AB2 VL comprises a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 16.

[0164] In some embodiments, the AB2 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 7; a HCDR2 comprising a sequence of SEQ ID NO: 8; a HCDR3 comprising a sequence of SEQ ID NO: 9; and the AB2 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 15; and the AB2 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 10; a LCDR2 comprising a sequence of SEQ ID NO: 11; and a LCDR3 comprising a sequence of SEQ ID NO: 12, and the AB2 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 16.

[0165] In some embodiments, the AB2 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 7; a HCDR2 comprising a sequence of SEQ ID NO: 8; a HCDR3 comprising a sequence of SEQ ID NO: 9; and the AB2 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 15; and the AB2 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 10; a LCDR2 comprising a sequence of SEQ ID NO: 11; and a LCDR3 comprising a sequence of SEQ ID NO: 12, and the AB2 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 16.

[0166] In some embodiments, the HCDR1 comprises a sequence of SEQ ID NO: 45; the HCDR2 comprises a sequence of SEQ ID NO: 46; the HCDR3 comprises a sequence of SEQ ID NO: 47; the LCDR1 comprises a sequence of SEQ ID NO: 48; the LCDR2 comprises a sequence of SEQ ID NO: 49; and the LCDR3 comprises a sequence of SEQ ID NO: 50.

[0167] In some embodiments, the heavy chain variable domain (AB2 VH) comprises a sequence of SEQ ID NO: 28, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 28.

[0168] In some embodiments, the light chain variable domain (AB2 VL) comprises a sequence of SEQ ID NO: 29, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 29.

[0169] In some embodiments, the AB2 VH comprises a sequence of SEQ ID NO: 28, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) to SEQ ID NO: 28, and the AB2 VL comprises a sequence of SEQ ID NO: 29, or a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 29.

[0170] In some embodiments, the AB2 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 28. In some embodiments, the AB2 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 29. In some embodiments, the AB2 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 28 and the AB2 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 29.

[0171] In some embodiments, the AB2 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 28. In some embodiments, the AB2 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 29. In some embodiments, the AB2 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 28 and the AB2 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 29.

[0172] In some embodiments, the AB2 VH comprises a sequence of SEQ ID NO: 28 and the AB2 VL comprises a sequence of SEQ ID NO: 29.

[0173] In some embodiments, the AB2 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 45; a HCDR2 comprising a sequence of SEQ ID NO: 46; a HCDR3 comprising a sequence of SEQ ID NO: 47; and the AB2 VH comprises a sequence that is at least 80% identical (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 28; and the AB2 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 48; a LCDR2 comprising a sequence of SEQ ID NO: 49; and a LCDR3 comprising a sequence of SEQ ID NO: 50, and the AB2 VL comprises a sequence that is at least 80% identical e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% ,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 29.

[0174] In some embodiments, the AB2 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 45; a HCDR2 comprising a sequence of SEQ ID NO: 46; a HCDR3 comprising a sequence of SEQ ID NO: 47; and the AB2 VH comprises a sequence that is at least 90% identical to SEQ ID NO: 28; and the AB2 VL comprises a LCDR1 comprising a sequence ofSEQ ID NO: 48; a LCDR2 comprising a sequence of SEQ ID NO: 49; and a LCDR3 comprising a sequence of SEQ ID NO: 50, and the AB2 VL comprises a sequence that is at least 90% identical to SEQ ID NO: 29.

[0175] In some embodiments, the AB2 VH comprises a HCDR1 comprising a sequence of SEQ ID NO: 45; a HCDR2 comprising a sequence of SEQ ID NO: 46; a HCDR3 comprising a sequence of SEQ ID NO: 47; and the AB2 VH comprises a sequence that is at least 95% identical to SEQ ID NO: 28; and the AB2 VL comprises a LCDR1 comprising a sequence of SEQ ID NO: 48; a LCDR2 comprising a sequence of SEQ ID NO: 49; and a LCDR3 comprising a sequence of SEQ ID NO: 50, and the AB2 VL comprises a sequence that is at least 95% identical to SEQ ID NO: 29.Construct format

[0176] The CD3-binding domain and the MUC1 -binding domain may reside within any construct formats known in the art, including an antibody or a fragment thereof, a single chain fragment variable (scFv), a Fab-scFv, a scFv-Fab-Fc, a Fab-scFv-Fc, a F(ab’)2-scFv2, a (scFv)2, a F(ab’)2, a scFv-KfH, BiTE or a component thereof, a NANOBODY®, a nanobody-HSA, VHH-scAb, a VHH-Fab, a Dual scFab, a diabody, a CROSSMAB®, a DAF (two-in-one), a DAE (four- in-one), a DUTAMAB®, a DT- IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a FcAb, a kl-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, ZYBODYTM, DVI-IgG, Diabody- CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, a VHH-Fc, a tandem VHH-Fc, a L'HH-Fc KiH, a Fab- VHH-Fc, an Intrabody, a dock and lock, an ImmTAC® (immune- mobilizing monoclonal TCRs (T cell receptors) against cancer), an IgG-IgG conjugate, a Cov-X-Body, a scFvl- PEG-scFv2, an Adnectin, a DARPin®, a fibronectin, an IgG, an IgM, an IgA, an IgE, an IgD, a DEP conjugate, TMEAbodyTM, SAFEbody®, TRITAC®, or SHIELD antibody. A VHH domain is a single monomeric variable antibody domain that can be found in camelids. A VNAR domain is a single monomeric variable antibody domain that can be found in cartilaginous fish.

[0177] In some embodiments, the CD3-binding domain and the MUC1 -binding domain may be both Fab fragments, or at least one may be a Fab fragment. In some embodiments, theCD3-binding domain and the MUC1 -binding domain may be both Fv fragments, or at least one may be a Fv fragment.

[0178] In some embodiment, the AB1 comprises a scFv. In some embodiments, the AB1 is a scFv comprising the AB1 VH and the AB1 VL. In some embodiments, the AB1 is a scFv that comprises AB1 VH comprising a HCDR1 comprising a sequence of SEQ ID NO: 1; a HCDR2 comprising a sequence of SEQ ID NO: 2; a HCDR3 comprising a sequence of SEQ ID NO: 3; and a light chain variable domain (AB1 VL) comprising a LCDR1 comprising a sequence of SEQ ID NO: 4; a LCDR2 comprising a sequence of SEQ ID NO: 5; and a LCDR3 comprising a sequence of SEQ ID NO: 6. In some embodiments, the scFv comprises a AB1 VH comprising a sequence of SEQ ID NO: 13, and a AB1 VL comprising a sequence of SEQ ID NO: 14.

[0179] In some embodiments, the AB2 comprises a Fab fragment. In some embodiments, the AB2 is a Fab fragment comprises a AB2 VH and a AB2 VL. In some embodiments, the AB2 is a Fab fragment that comprises a heavy chain variable domain (AB2 VH) comprising a HCDR1 comprising a sequence of SEQ ID NO: 7; a HCDR2 comprising a sequence of SEQ ID NO: 8; a HCDR3 comprising a sequence of SEQ ID NO: 9, and a light chain variable domain (AB2 VL) comprising a LCDR1 comprising a sequence of SEQ ID NO: 10; a LCDR2 comprising a sequence of SEQ ID NO: 11; and a LCDR3 comprising a sequence of SEQ ID NO: 12. In some embodiments, the Fab comprises a AB2 VH comprising a sequence of SEQ ID NO: 15, and a AB2 VL comprising a sequence of SEQ ID NO: 16.

[0180] In some embodiments, the antibodies further comprise a fragment crystallizable region (Fc domain). Fc domains that are known in the art are suitable for use in the antibodies of the present application. The Fc domain may be an Fc domain of an IgG (e.g., IgGl, IgG2, IgG3, or IgG4). In some embodiments, the antibodies comprise a dimer formed by two Fc domains. The Fc domain may be a wild type peptide or a mutant. For example, the antibodies may comprise a dimer formed by two Fc domain mutants.

[0181] In some embodiments, the heavy chain variable domain of the antigen-binding domain is present in a chain that further comprises a Fc region. In some embodiments, the heavy chain variable domain of the CD3-binding domain (AB 1 VH) is present within a FAC that further comprises a first Fc region (Fcl), and wherein the heavy chain variable domain of the MUC1 -binding domain (AB2 VH) is present within an AB2 heavy chain (AB2 HC) that further comprises a second Fc region (Fc2).

[0182] In some embodiments, the Fcl and / or the Fc2 comprise an amino acid sequence encoding a human IgG Fc domain (e.g., a human IgGl Fc domain, or a human IgG4 Fcdomain), or a variant thereof comprising from one to ten substitutions, e.g., one, two, three, four, five, six, seven, eight, nine or ten substitutions.

[0183] The mutations may confer a desired beneficial property to the antibodies. For example, certain mutations in the FcRn binding site are known to modulate effector function, (see, e.g., Petkova et al., Inti. Immunol. 18: 1759-1769, 2006; Deng et al., MAbs 4: 101-109, 2012; and Olafson et al., Methods Mol. Biol. 907:537-556, 2012.) The inclusion of any known mutations in an Fc domain that can modulate effector function are suitable. For example, a N297A or N297G mutation in the Fc amino acid sequence may be employed to reduce IgG effector functions (e.g., ADCC and CDC) which may reduce target independent toxicities (see, e.g., Lund et al., Mol. Immunol. 29:35-39, 1992). The Fc domains suitable for use in context with the present disclosure include any Fc domain known in the art, including but not limited to any known heterodimeric Fc (e.g., knob-into-holes, and the like). For example, the Fc domains may be a Fc domain hole mutant and a Fc domain knob mutant (also referred to as "knobs-into-holes"). The knob and hole mutants may interact with each other to facilitate the dimerization of the Fc domains. In some embodiments, the knob and hole mutants may comprise one or more amino acid modifications within the interface between the Fc domains (e.g., in the CH3 domain). The term “knob-into-hole” or “KnH” technology as mentioned herein refers to the technology directing the pairing of two polypeptides together in vitro or in vivo by introducing a pertuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact. For example, KnHs have been introduced in the Fc:Fc binding interfaces, CL:CH1 interfaces or VH / VL interfaces of antibodies (e.g., US2007 / 0178552, WO 96 / 027011, WO 98 / 050431 and Zhu et al. (1997) Protein Science 6:781-788). This is especially useful in driving the pairing of two different heavy chains together during the manufacture of multispecific antibodies. For example, multispecific antibodies having KnH in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains. KnH technology can also be used to pair two different receptor extracellular domains together or any other polypeptide sequences that comprises different target recognition sequences (e.g., including affibodies, peptibodies and other Fc fusions). In one embodiment, the knob-into-hole mutations comprise an amino acid substitution in at least one of amino acid positions at S354, T366, L368, Y349 and Y407, as numbered by the EU index as set forth in Kabat.

[0184] In some embodiments, the Fcl and / or the Fc2 comprise an amino acid sequence encoding a human IgGl Fc domain or a variant thereof comprising from one to ten substitutions. In some embodiments, an IgGl Fc may comprise one or more amino acid substitutions. Such substitutions may include, for example, N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236-deleted, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y, M428L, N434S, L328F, M252Y, S254T, T256E, and / or any combination thereof (the residue numbering is according to the EU index as in Kabat) (Dall'Acqua W. F. et al., J Biol Chem. 2006 Aug 18;281(33):23514-24; Wang X. et al., Protein Cell. 2018 Jan; 9(1): 63-73), or for example, N434A, Q438R, S440E, L432D, N434L, and / or any combination thereof (the residue numbering according to EU numbering). Specific exemplary substitution combinations for an IgGl-type Fc include, but not limited to: M252Y, S254T, and T256E (“YTE” variant); M428L and N434A (“LA” variant), M428L and N434S (“LS” variant); M428L, N434A, Q438R, and S440E (“LA-RE” variant); L432D and N434L (“DEL” variant); and L234A, L235A, L432D, and N434L (“LALA-DEL” variant) (the residue numbering is according to the EU index as in Kabat).

[0185] In some embodiments, the Fcl and / or the Fc2 comprise an amino acid sequence encoding a human IgG4 Fc domain or a variant thereof comprising from one to ten substitutions. In some embodiments, an IgG4 Fc region may comprise one or more amino acid substitutions. Such substitutions may include but are not limited to, E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, S354C, T366S, T366W, L368A, Y349C, Y407V and / or any combination thereof (the residue numbering is according to the EU index as in Kabat).

[0186] In some embodiments, the Fcl and / or the Fc2 comprises an IgG4 Fc domain and comprises a knob-into-hole mutation, i.e., an amino acid substitution in at least one of amino acid positions at S354, T366, L368, Y349 and Y407, as numbered by the EU index as set forth in Kabat.

[0187] In some embodiments, the Fcl and / or Fc2 comprise an IgG4 Fc domain with at least one amino acid substitution selected from the group consisting of T366S, L368A, Y349C and Y407V. In some embodiments, the Fcl and / or Fc2 comprise an IgG4 Fc domain with T366S, L368A, Y349C and Y407V substitutions. In some embodiments, the Fcl and / or Fc2 comprise an amino acid sequence of SEQ ID NO:97, or an amino acid sequence at least 90%identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0188] In some embodiments, the Fcl and / or Fc2 comprise an IgG4 Fc domain with at least one amino acid substitution selected from the group consisting of T366W, and S354C. In some embodiments, the Fcl and / or Fc2 comprise an IgG4 Fc domain with T366W and S354C substitutions. In some embodiments, the Fcl and / or Fc2 comprise an amino acid sequence of SEQ ID NO:99, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0189] In some embodiments, the Fcl and / or the Fc2 comprise an amino acid substitution in at least one of amino acid positions at S354, T366, L368, Y349 and Y407.

[0190] In some embodiments, the Fcl comprises an IgG4 Fc domain with T366S, L368A, Y349C and Y407V substitutions and the Fc2 comprises an IgG4 Fc domain with T366W, and S354C substitutions. In some embodiments, the Fcl comprises an IgG4 Fc domain with T366W, and S354C substitutions and the Fc2 comprises an IgG4 Fc domain T366S, L368A, Y349C and Y407V substitutions.

[0191] In some embodiments, the antibodies disclosed herein further comprise an immunoglobulin hinge region. Suitable hinge regions include any hinge regions known in the art. For example, a hinge region from any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) are suitable for use in the present disclosure. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. In some embodiments, hinge region from any the five major classes of immunoglobulin may comprises from one to ten substitutions.

[0192] In some embodiments, the first hinge region and / or the second hinge region further comprise an amino acid sequence encoding a human IgG hinge region or a variant thereof comprising from one to ten substitutions.

[0193] In some embodiments, the first hinge region and / or the second hinge region comprise an amino acid sequence encoding a human IgG hinge region or a variant thereof comprising from one to ten substitutions. In some embodiments, the first hinge region and / or the second hinge region comprise an IgG hinge region or a variant thereof comprising from one to ten substitutions. In some embodiments, the first hinge region and / or the second hinge region comprise an IgG hinge region and comprise an amino acid substitution in at least one of amino acid positions at S228, L234, L235, as numbered by the EU index as set forth inKabat. In some embodiments, the first hinge region and / or the second hinge region comprise an IgG hinge region with S228P and / or L235E. In some embodiments, the first hinge region comprises an amino acid sequence of SEQ ID NO: 96, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto). In some embodiments, the second hinge region comprises an amino acid sequence of SEQ ID NO: 96, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0194] In some embodiments, the AB2 VH is present within an AB2 heavy chain (AB2 HC) that further comprises a CHI constant domain. In some embodiments, the AB2 VH is present within an AB2 heavy chain (AB2 HC) that further comprises a CHI constant domain, a second hinge region, and a Fc2. In some embodiments, CHI constant domain comprises an amino acid sequence of SEQ ID NO:98, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0195] In some embodiments, the light chain variable domain of the antigen-binding domain is present in a light chain that further comprises a light chain constant domain. In some embodiments, the light chain variable domain of the MUC1 -binding domain (AB2 VL) is present within a light chain (LC) that further comprises a light chain constant domain. In some embodiments, the light chain constant domain comprises an amino acid sequence of SEQ ID NO: 100, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0196] In some embodiments, the antibody may be a mouse, rat, rabbit, goat, camel, donkey, primate, chimeric, human, or humanized antibody. In one example, the antibody may be a human antibody. In one example, the antibody may be a humanized (e.g., fully humanized) antibody.

[0197] As used herein, the terms “specific binding” and “specifically binds” refer to the non- covalent interactions of the type that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of binding interactions can be expressed in terms of the dissociation constant (Kd or KD) of the interaction, wherein a smaller Kd represents a greater affinity. The strength or affinity of binding interactions can be expressed in terms of the rate of association (Kon) or the rate ofdissociation (Koff). The strength or affinity of binding interaction refers to the strength of the sum total of non-covalent interactions between a target-binding domain and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a target-binding domain and an antigen or epitope. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity for a target-binding domain and its corresponding antigen or epitope are known in the art.

[0198] As used herein, a statement that an antigen-binding domain “specifically binds” to CD3 or MUC1 refers to an antibody or antigen binding fragment thereof that binds to CD3 or MUC1 with a dissociation constant (Kd) of less than 100 pM (e.g., less than 5 pM or 10 pM). The antibody or antigen binding fragment thereof may specifically bind to CD3 or MUC1 with a Kd of about 0.01 nM to about 10 pM, about 0.01 nM to about 1 pM, about 0.01 nM to about 500 nM, about 0.01 nM to about 400 nM, about 0.01 nM to about 300 nM, about 0.01 nM to about 200 nM, or about 0.01 nM to about 100 nM.

[0199] In one aspect, the present invention provides a bispecific antibody comprising (a) a first antigen binding domain that specifically binds to a CD3 (AB1), wherein the AB1 comprises a AB1 VH and a AB1 VL; wherein the AB1 VH comprises a HCDR1 of SEQ ID NO: 1, a HCDR2 of SEQ ID NO:2, and a HCDR3 of SEQ ID NO:3, and wherein the AB 1 VL comprises a LCDR1 of SEQ ID NO:4, a LCDR2 of SEQ ID NO:5, and a LCDR3 of SEQ ID NO:6, and (b) a second antigen binding domain that specifically binds to a MUC1 (AB2), wherein the AB2 comprises an AB2 VH and an AB2 VL.

[0200] In some embodiments, the AB1 VH comprises a sequence of SEQ ID NO: 13 and the AB1 VL comprises a sequence of SEQ ID NO: 14. In some embodiments, the AB1 is a scFv comprising the AB 1 VH and the AB 1 VL.

[0201] In some embodiments, the AB2 VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and the AB2 VL comprises a LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

[0202] In some embodiments, the AB2 VH comprises a sequence of SEQ ID NO: 15 and the AB2 VL comprises a sequence of SEQ ID NO: 16. In some embodiments, the AB2 is a Fab fragment comprising the AB2 VH and the AB2 VL.

[0203] In some embodiments, the bispecific antibody comprises an AB 1 Fc fusion, an AB2 HC and an AB2 LC. In some embodiments, the AB1 Fc fusion comprises the AB1, the firsthinge region and the Fcl. In some embodiments, the AB2 HC comprises the AB2 VH, a CHI constant domain, the second hinge region and the Fc2, and the AB2 LC comprises the AB2 VL and a light chain constant domain.

[0204] In some embodiments, the AB1 Fc fusion comprises an amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 90% identical thereto. In some embodiments, the AB2 HC comprises an amino acid sequence of SEQ ID NO:25, an amino acid sequence at least 90% identical thereto; and the AB2 LC comprises an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 90% identical thereto.

[0205] In one aspect, the bispecific antibodies of the present invention are activatable. The activatable antibodies selectively activates in conditions that are more prevalent in a tumor microenvironment. Until such activation occurs, however, the capacity to bind its targets is impaired. The bispecific activatable antibodies of the present invention thus have the potential to reduce target-related toxicities by minimizing off-target binding.

[0206] In general, an activatable antibody may comprise a prodomain, which refers to a polypeptide that, when linked to an antigen-binding domain, functions to inhibit antigen binding and includes an amino acid sequence that form a protease cleavable substrate. The portion of the prodomain that inhibits antigen binding is referred to as a masking moiety (MM) and the amino acid sequence that is a protease cleavable substrate is referred to as a cleavable moiety (CM). The prodomain may include a linker (L) between the MM and the CM and / or at the prodomain’s terminus (e.g., carboxyl and / or amino terminus to facilitate the linkage of the prodomain to the antibody). In certain embodiments, a prodomain may comprise one of the following formulae (representing an amino acid sequence in an N- to C- terminal direction): MM-CM, MM-L-CM, MM-CM-L, MM-L-CM-L, CM-MM, CM-L-MM, L-CM-MM, or L-CM-L-MM, wherein each represents a direct or indirect (e.g., via a linker) linkage.

[0207] As mentioned above, an activatable antibody exhibits a first level of binding to a target when the activatable antibody is in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target in the uninhibited, unmasked and / or cleaved state (i.e., a second conformation), where the second level of antigen binding is greater than the first level of binding. In general, the access of antigen to the antigenbinding domain of the activatable antibody is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleaving agent. Thus, when the activatable antibody is in the uncleaved state, the antigen-binding domain is inhibited from antigen binding and can be masked from antigen binding (i.e., the firstconformation is such that the antigen-binding domain cannot bind the target or is inhibited in binding the antigen), and in the cleaved state the antigen-binding domain is not inhibited or is unmasked to antigen binding.

[0208] In some embodiments, an activatable antibody may be designed by selecting an antigen-binding domain of interest and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides for masking of the antigenbinding domain or reduction of binding of the antigen-binding domain to its antigen. Structural design criteria can be to be taken into account to provide for this functional feature.

[0209] Activatable antibodies herein may exhibit an activatable phenotype of a desired dynamic range for antigen binding in an inhibited versus an uninhibited conformation. Dynamic range generally refers to a ratio of (a) a maximum detected level of a parameter under a first set of conditions to (b) a minimum detected value of that parameter under a second set of conditions. For example, in the context of an activatable antibody, the dynamic range refers to the ratio of (a) a maximum detected level of antigen binding to an activatable antibody in the presence of a protease capable of cleaving a CM in the activatable antibody to (b) a minimum detected level of antigen binding to an activatable antibody in the absence of the protease. The greater the dynamic range of an activatable antibody, the better the activatable phenotype of the activatable antibody. Activatable antibodies having relatively higher dynamic range values (e.g., greater than 1) exhibit more desirable activatable phenotypes such that antigen binding by the activatable antibodies occurs to a greater extent (e.g., predominantly occurs) in the presence of a cleaving agent (e.g., enzyme) capable of cleaving the CM of the activatable antibodies than in the absence of a cleaving agent.

[0210] The bispecific activatable antibody of the present invention comprise two antigenbinding domains, two masking moieties (MMs) reducing, inhibiting, or interfering with antigen binding to each of the antigen-binding domains, two cleavable moieties (CMs) that couple each of the MMs to each of the antigen-binding domains.

[0211] In some embodiments, the bispecific activatable antibody comprises a first binding component (FBC) and a second binding component (SBC). FIG. IB and FIG. 1C are illustrative schematics depicting the dually masked bispecific activatable antibody.

[0212] The FBC comprises a CD3-binding domain (AB1) comprising a heavy chain variable domain (AB1 VH) and a light chain variable domain (AB1 VL), and a masking moiety (MM1) inhibiting or diminishing the ability of the antibody to specifically bind to CD3, wherein the MM1 is coupled to the CD3-binding domain via a cleavable moiety (CM1) (either directly or indirectly, e.g., via one or more linkers).

[0213] The SBC comprises a MUC1 -binding domain (AB2) comprising a heavy chain variable domain (AB2 VH) and a light chain variable domain (AB2 VL), and a masking moiety (MM2) inhibiting or diminishing the ability of the antibody to specifically bind to MUC1, wherein the MM2 is coupled to the MUC1 -binding domain via a cleavable moiety (CM2) (either directly or indirectly, e.g., via one or more linkers).

[0214] In some embodiments, the bispecific activatable antibody comprises a first antibody component (FAC) and a second antibody component (SAC).

[0215] In some embodiments, the FAC comprises the FBC, a first hinge region and a first Fc region (Fcl). In some embodiments, the Fcl is linked in an N- to C-terminal direction to the FBC via the first hinge region.

[0216] In some embodiments, the SAC comprises the SBC, a second hinge region and a second Fc region (Fc2). In some embodiments, the Fc2 is linked in an N- to C-terminal direction to the SBC via the second hinge region.

[0217] In some embodiments, the SAC comprises a heavy chain (HC) and a light chain (LC), wherein the AB2 VH is present within the HC that further comprises a CHI constant domain, the second hinge region and / or the Fc2, and wherein the AB2 VL is present within the LC that further comprises a light chain constant domain.

[0218] As used herein and unless otherwise stated, components of the activatable antibody that are “coupled” may be coupled either via a direct covalent linkage or indirect covalent linkage, e.g., via one or more linking peptides (also referred to as “linkers”), cleavable moieties, or other components of the activatable antibody.

[0219] The present invention also provides a monoclonal antibody comprising an antigen binding domain that specifically binds to MUC1, wherein the antigen binding domain comprises a VH and a VL; wherein the VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and wherein the VL comprises a 1LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

[0220] In some embodiments, the VH comprises a sequence of SEQ ID NO: 15 and the VL comprises a sequence of SEQ ID NO: 16.

[0221] In another aspect, the present invention provides a monoclonal antibody comprising an antigen binding domain that specifically binds to MUC1, wherein the antigen binding domain comprises a VH and a VL; wherein the VH comprises a HCDR1 of SEQ ID NO:45, a HCDR2 of SEQ ID NO:46, and a HCDR3 of SEQ ID NO:47, and wherein the VL comprises a LCDR1 of SEQ ID NO:48, a LCDR2 of SEQ ID NO:49, and a LCDR3 of SEQ ID NO:50.

[0222] In some embodiments, the VH comprises a sequence of SEQ ID NO:28 and the VL comprises a sequence of SEQ ID NO:29.Masking moieties (MMs)

[0223] The bispecific activatable antibodies of the present invention comprise a first masking moiety (MM1) capable of interfering with the binding of CD3-binding domain to CD3, and a second masking moiety (MM2) capable of interfering with the binding of MUC1 -binding domain to MUC1.

[0224] A masking moiety (MM) in an activatable molecule (that is not yet activated) “masks” or reduces or otherwise inhibits the binding of the antigen-binding domain to its target. In some embodiments, the coupling or modifying of an antibody with a MM may inhibit the ability of the antibody to specifically bind its target by means of inhibition known in the art (e.g., structural change and competition for antigen-binding domain). In some embodiments, the coupling or modifying of an antibody with a MM may effect a structural change that reduces or inhibits the ability of the antibody to specifically bind its target. In some embodiments, the coupling or modifying of an antibody with a MM sterically blocks, reduces or inhibits the ability of the antigen-binding domain to specifically bind its target.

[0225] A MM may be coupled to an antigen-binding domain by a CM and optionally one or more linkers described herein. In some embodiments, when an activatable antibody is not activated, the MM prevents the antigen-binding domain from antigen binding; but when the activatable antibody is activated (when the CM is cleaved by a protease), the MMs does not substantially or significantly interfere with the antigen-binding domain’s binding to the target.

[0226] The structural properties of the MMs may be selected according to factors such as the minimum amino acid sequence required for interference with antibody binding to antigen, the antigen / antibody binding pair of interest, the size of the antigen-binding domain, the presence or absence of linkers, and the like.

[0227] As used herein, the term “masking efficiency” refers to the activity (e.g., EC50) of the activatable antibody in the inactivated state divided by the activity of a control antibody, wherein the control antibody may be either cleavage product of the activatable antibody or the antibody or fragment thereof used as the antigen-binding domain of the activatable antibody. In some embodiments, the masking efficiency (ME) is calculated as the ratio of the Kd of the masked activatable antibody over the Kd of the unmasked antibody. An activatable antibody having a reduced level of an antigen-binding domain activity may have a maskingefficiency that is greater than 10. In some embodiments, the activatable antibodies described herein may have a masking efficiency that is greater than 10, 100, 1000, or 5000.

[0228] In some embodiments, the MM may be a polypeptide of about 2 to 50 amino acids in length. For example, the MM may be a polypeptide of from 2 to 40, from 2 to 30, from 2 to 20, from 2 to 10, from 5 to 15, from 10 to 20, from 15 to 25, from 20 to 30, from 25 to 35, from 30 to 40, from 35 to 45, from 40 to 50 amino acids in length. For example, the MM may be a polypeptide with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 amino acids in length. In some examples, the MM may be a polypeptide of more than 50 amino acids in length, e.g., 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0229] In some embodiments, the MMs may contain genetically encoded or genetically nonencoded amino acids. Examples of genetically non-encoded amino acids include but are not limited to D-amino acids, P-amino acids, and y-amino acids. In specific embodiments, the MMs contain no more than 50%, 40%, 30%, 20%, 15%, 10%, 5% or 1% of genetically nonencoded amino acids.

[0230] In some embodiments, once released from the activatable antibody and in a free state, the MM may have a biological activity or a therapeutic effect, such as binding capability. For example, the free peptide may bind with the same or a different binding partner.

[0231] In certain embodiments, the free MM may exert a therapeutic effect, providing a secondary function to the compositions disclosed herein. In some embodiments, once uncoupled from the activatable antibody and in a free state, the MM may advantageously not exhibit biological activity. For example, in some embodiments the MM in a free state does not elicit an immune response in the subject.

[0232] MMs that are suitable for use in the antibodies of the present invention include any that are known in the art, including those described in, for example, PCT Publication Nos. WO 2013 / 163631, WO 2013 / 192550, WO 2014 / 052462, WO 2015 / 066279, WO 2016 / 014974, WO 2016 / 149201, WO 2016 / 179285, WO 2016 / 179257, WO 2016 / 179335, WO 2017 / 011580, WO 2016 / 014974, WO 2019 / 075405, and WO 2019 / 213444, each of which are incorporated herein by reference in their entireties. Anti-CD3 masking moieties that are suitable for use in the practice of the present disclosure include any of those that are known in the art, including those described in, for example, PCT Publication Nos. WO 2016 / 014974, WO 2019 / 075405, WO 2019 / 213444, and WO 2023 / 064929, each of which is incorporated herein by reference in its entirety.

[0233] In some embodiments, the MM for masking the CD3-binding domain (MM1) comprises a sequence of SEQ ID NO: 30.

[0234] In some embodiments, the MM for masking the MUC1 -binding domain (MM2) comprises a sequence selected from the group consisting of SEQ ID NO: 31; SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.

[0235] In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 31. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 32. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 33. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 34. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 35. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 36. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 37. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 38. In some embodiments, the MM2 comprises a sequence of SEQ ID NO: 44.

[0236] Suitable MMs may also be identified and / or further optimized through a screening procedure from a library of candidate activatable antibodies having variable MMs. For example, an antigen-binding domain and a CM may be selected to provide for a desired enzyme / target combination, and the amino acid sequence of the MM can be identified by the screening procedure described below to identify a MM that provides for a switchable phenotype. For example, a random peptide library (e.g., of peptides comprising 2 to 40 amino acids or more) may be used in the screening methods disclosed herein to identify a suitable MM.

[0237] In some embodiments, MMs with specific binding affinity for an antigen-binding domain may be identified through a screening procedure that includes providing a library of peptide scaffolds comprising candidate MMs wherein each scaffold is made up of a transmembrane protein and the candidate MM. The library may then be contacted with an entire or portion of a protein such as a full length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing a protein (also capable of binding the binding partner of interest), and identifying one or more candidate MMs having detectably bound protein. The screening may be performed by one more rounds of magnetic- activated sorting (MACS) or fluorescence-activated sorting (FACS), as well as determination of the binding affinity of MM towards the antigen-binding domain and subsequentdetermination of the masking efficiency, e.g., as described in W02009025846 and US20200308243A1, which are incorporated herein by reference in their entireties.Cleavable Moieties (CMs)

[0238] The activatable antibody may comprise one or more cleavable moieties (CMs). The terms “cleavable moiety” and “CM” are used interchangeably herein to refer to a peptide, the amino acid sequence of which comprises a substrate for a sequence- specific protease. In some embodiments, the CM may be positioned between an antigen-binding domain and a MM.

[0239] The CM and the antigen-binding domain of the activatable antibodies may be selected so that the antigen-binding domain represents a binding moiety for a given target, and the CM represents a substrate for one or more proteases, where the protease is co-localized with the target in a tissue (e.g., at a treatment site or diagnostic site in a subject). The protease may cleave the CM in the activatable antibody when the activatable antibody is exposed to the protease. In some embodiments, the activatable antibodies may find particular use where, for example, one or more proteases capable of cleaving a site in the CM, is present at relatively higher levels in target-containing tissue of a treatment site or diagnostic site, e.g., a tumor environment, than in tissue of non-treatment sites, e.g., in healthy tissue.

[0240] In some embodiments, the CMs herein may comprise substrates for proteases that have known substrates have been reported in a number of cancers. See, e.g., La Roca et al., British J. Cancer 90(7): 1414- 1421 , 2004. Substrates suitable for use in the CM components employed herein include those which are more prevalently found in cancerous cells and tissue. Thus, in some embodiments, the CM may comprise a substrate for a protease that is more prevalently found in diseased tissue associated with a cancer. In some embodiments, the CM components comprise substrates for protease(s) that is / are more prevalent in tumor tissue. For example, the protease(s) may be produced by a tumor in a subject.

[0241] Suitable CMs for use in the activatable antibody herein include any of the protease substrates that are known the art. In some embodiments, the CM may comprise a substrate of a serine protease (e.g., u-type plasminogen activator, uPA, also referred to as urokinase, matriptase, also referred to herein as MT-SP1 or MTSP1). In some embodiments, the CM may comprise a substrate of a matrix metalloprotease (MMP). In some embodiments, the CM may comprise a substrate of cysteine protease (e.g., legumain).

[0242] In some embodiments, the bispecific activatable antibodies of the present invention comprise a first cleavable moiety (CM1) coupled to a CD3-binding domain and MM1, and asecond cleavable moiety (CM2) coupled to a MUC1 -binding domain and MM2. The antibodies are activated when the CM1 and the CM2 are cleaved, thereby releasing the MM1 and the MM2 from the antibodies. The resulting activated antibodies is thus free to bind to CD3 and MUC1.

[0243] In some embodiments, the CM1 and the CM2 may comprise the substrates of the same protease. In some embodiments, the CM1 and the CM2 may comprise the substrates of different proteases. In some embodiments, the CM1 and the CM2 may comprise or consist of the same sequence. In some embodiments, the CM1 and the CM2 may comprise or consist of different sequences.

[0244] In some embodiments, the CM1 and / or the CM2 is a polypeptide that functions as a substrate of a matrix metalloproteinase (MMP), a cysteine protease, and a serine protease. In some embodiments, the CM1 and / or the CM2 is a polypeptide that functions as a substrate of a matrix metalloproteinase (MMP), or a cysteine protease, or a serine protease.

[0245] In some embodiments, the CM may comprise a substrate for a disintegrin and metalloproteinase (ADAM) or disintegrin and metalloproteinase with thrombospondin motifs (AD AMTS) (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5), aspartate protease (e.g., BACE, Renin), aspartic cathepsin (e.g., Cathepsin D, Cathepsin E), Caspase (e.g., Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14), cysteine cathepsin (e.g., Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P), cysteine proteinase (e.g., Cruzipain, Legumain, Otubain-2), Chymase, DESCI, DPP-4, FAP, Elastase, FVIIa, FIXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoatase, Hepsin, HtrAl, Human Neutrophil Elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), metallo proteinase (e.g., Meprin, Neprilysin, PSMA, BMP-1), Lactoferrin, Marapsin, Matriptase-2, MT-SPl / Matriptase, NS3 / 4A, PACE4, Plasmin, PSA, a MMP (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27), TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11, tPA, Thrombin, Tryptase, and uPA.

[0246] In some embodiments, the protease substrate in the CM may comprise a peptide sequence

[0247] that is not substantially identical (e.g., no more than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence that is naturally cleaved by the same protease.

[0248] In some embodiments, the CM may comprise a total of 3 amino acids to 25 amino acids. In some embodiments, the CM may comprise a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids.

[0249] CMs that are suitable for use in the antibodies of the present invention include any that are known in the art, including those described in, for example, PCT Publication Nos. WO 2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020236679, WO2020176672, W02020118109, WO2019213444, WO2019183218, WO2019173771, WO2019046652, WO2019018828, WO2019014586, WO2018085555, W02017011580, WO2016179335, WO2020252349, WO2020252358, W02020092881, W02020086665, WO2019165143, W02019075405, WO2018222949, WO2018165619, WO2016179285, WO2016179257, W02016149201, WO2016014974, each of which is incorporated herein by reference in its entirety.

[0250] In some embodiments, the CM for the CD3-binding domain (CM1) comprises a sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54, and SEQ ID NO: 55. In some embodiments, the CM1 comprises a sequence of SEQ ID NO: 51. In some embodiments, the CM1 comprises a sequence of SEQ ID NO: 52. In some embodiments, the CM1 comprises a sequence of SEQ ID NO: 53. In some embodiments, the CM1 comprises a sequence of SEQ ID NO: 54. In some embodiments, the CM1 comprises a sequence of SEQ ID NO: 55.

[0251] In some embodiments, the CM for the MUC1 -binding domain (CM2) comprises a sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO:55. In some embodiments, the CM2 comprises a sequence of SEQ ID NO: 51. In some embodiments, the CM2 comprises a sequence of SEQ ID NO: 52. In some embodiments, the CM2 comprise a sequence of SEQ ID NO: 53. In some embodiments, the CM2 comprise a sequence of SEQ ID NO: 54. In some embodiments, the CM2 comprise a sequence of SEQ ID NO: 55.

[0252] In some embodiments, the MM1 is linked in an amino- (N-) terminal to carboxyl- (C- ) terminal direction to the CM1, either directly or indirectly, e.g., via one or more linkers, to form an MM1-CM1 construct, and the C-terminus of the MM1-CM1 construct is linked to the N- terminus of the CD3-binding domain (AB1). In some embodiments, the MM1-CM1 is linked either directly or indirectly to the VH of the AB 1.

[0253] In some embodiments, the MM2 is linked in an N- to C-terminal direction to the CM2, either directly or indirectly, e.g., via one or more linkers, to form an MM2-CM2construct, and the C-terminus of the MM2-CM2 construct is linked to the N- terminus of the MUC1 -binding domain (AB2). In some embodiments, the MM2-CM2 is linked either directly or indirectly to the VH of the AB2.Linkers

[0254] The antibodies may comprise one or more linkers. The linkers may comprise a stretch of amino acid sequence that link two components (e.g., between the heavy chain variable domain and the light chain variable domains, between the antigen -binding domain and a cleavable moiety, between the antigen-binding domain and a masking moiety, between a masking moiety and a cleavable moiety, or between the antigen-binding domain and another component in the antibodies). In some embodiments, the linker is disposed between a pair of components selected from the group consisting of MM1 and CM1, CM1 and AB1 VH, AB1 VH and AB1 VL, AB1 VL and a hinge region, MM2 and CM2, and CM2 and AB2 VH.

[0255] The linkers may be non-cleavable by any protease, or non-cleavable by any protease naturally occurring in humans. In some embodiments, the linker(s) may be flexible linkers, which may be introduced into the antibodies to provide flexibility at one or more of the junctions between domains, between moieties, between moieties and domains, or at any other junctions where a linker would be beneficial. In some embodiments, where the antibody is provided as a conformationally constrained construct, a linker may be inserted to facilitate the formation and maintenance of a structure. Any of the linkers described herein may provide the desired flexibility to facilitate the inhibition of the binding of a target, or to facilitate cleavage of a cleavable moiety by a protease. In some embodiments, the linkers may be all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired antibody. Some linkers may include cysteine residues, which may form disulfide bonds and reduce flexibility of the construct.

[0256] In some embodiments, a linker coupled to a masking moiety may have a length that allows the masking moiety to be in a position in the tertiary or quaternary structure of the antibody to effectively mask the antigen-binding domain. For example, in the tertiary or quaternary structure, the masking moiety may be proximal to the antigen-binding domain to be masked.

[0257] In most instances, the length of a linker may be determined by counting, in a N- to C- direction, the number of amino acids from the N-terminus of the linker adjacent to the C- terminal amino acid of the preceding component, to the C-terminus of the linker adjacent tothe N-terminal amino acid of the following component (z.e., where the linker length does not include either the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component).. In some embodiments, the linker may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0258] In some embodiments, a linker may be rich in glycine (Gly or G) residues. In some embodiments, the linker may be rich in serine (Ser or S) residues. In some embodiments, the linker may be rich in glycine and serine residues. In some embodiments, the linker may have one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs).

[0259] In some embodiments, the linker may have one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 59) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS (SEQ ID NO: 59) sequences). In some embodiments, the linker may have one or more Gly-Gly-Gly-Gly- Ser (GGGGS) (SEQ ID NO: 71) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS (SEQ ID NO: 71) sequences). In some embodiments, the linker may have one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 62) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG (SEQ ID NO: 62) sequences). Examples of the linkers may include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 133) and (GGGS)n (SEQ ID NO: 59), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be relatively unstructured, and therefore may be able to serve as a neutral link between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). In one embodiment, the linker is a GS linker. In one embodiment, the linker is a GGS linker. In one embodiment, the linker is a GGGS (SEQ ID NO: 59) linker. In one embodiment, the linker is a GSSGGSGGSGG (SEQ ID NO: 58) linker. In one embodiment, the linker is a GGGSSGGS (SEQ ID NO: 61) linker. In one embodiment, the linker is a GS linker of (Gly-Gly-Gly-Gly- Ser)n (SEQ ID NO: 134), and n is either 3 or 4.

[0260] Exemplary flexible linkers include one of or a combination of one or more of: GS, GGS, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, or SEQ ID NO: 82.

[0261] Examples of linkers may further include a sequence that is at least 90% identical (e.g., , at least 90%, , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the example linkers described herein. An ordinarily skilled artisan will recognize that design can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired antibody structure.

[0262] In some embodiments, an antibody may include one, two, three, four, five, six, seven, eight, nine, or ten linker(s) (e.g., the same or different linker sequences of any of the exemplary linker sequences described herein or known in the art). In some embodiments, a linker may comprise non-amino acid-based linkers including but not limited to sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl)aminobenzoate), SMPB (succinimidyl 4-(N- maleimidophenyl)butyrate), and sulfo-SMPB (sulfosuccinimidyl 4-(N- maleimidophenyl)butyrate), wherein the linkers react with primary amines sulfhydryls.Spacer

[0263] In some embodiments, the activatable antibodies may comprise components in addition to those described above. Such components can include a spacer. The term “spacer” refers herein to an amino acid residue or a peptide incorporated at a free terminus of a polypeptide of the activatable antibody. Spacers that are suitable for use in the practice of the present disclosure include any single amino acid residue or any peptide. Suitable spacers include any of those described in, for example, International Publication Nos.: WO 2016 / 014974, WO 2019 / 075405, and WO 2019 / 213444, each of which is incorporated herein by reference in their entireties.

[0264] In some embodiments, a spacer can comprise from about 1 amino acid to about 10 amino acids (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) or any number there between. In some embodiments, the spacer is N-terminally positioned relative to the MM1 and / or MM2. In some embodiments, the spacer has a sequence of SEQ ID NO:56. In some embodiments, the spacer has a sequence of SEQ ID NO: 57.

[0265] As provided herein, the format or structure of a bispecific activatable antibody of the present invention can include any number of optional additional components, including linkers and spacers. By way of example only, the structures set forth below are among thecontemplated embodiments. However, the embodiments shown below are not meant to limit the disclosure in any way.

[0266] In some embodiments, the bispecific activatable antibody comprises (a) a first antibody component (FAC) that specifically binds CD3 comprising a first optional spacer, a masking moiety (MM1), a first linker, a cleavable moiety (CM1), a second linker, a heavy chain variable domain (AB1 VH), a third linker, a light chain variable domain (AB1 VL), a fourth linker, a first hinge region, and a Fcl region; (b) a second antibody component (SAC) that specifically binds MUC1 comprising (i) a first polypeptide comprising a second optional spacer, a masking moiety (MM2), a fifth linker, a cleavable moiety (CM2), a sixth linker, a heavy chain variable domain (AB2 VH), a CHI constant domain, a second hinge region, and a Fc2 region; and (ii) a second polypeptide comprising a light chain variable domain of the MUCl-binding domain (AB2 VL), and a light chain constant domain.

[0267] In some embodiments, the FAC comprises an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 17, (b) SEQ ID NO:20, (c) SEQ ID NO:22, and (d) an amino acid sequence at least 90% identical thereto.

[0268] In some embodiments, the FAC comprises an amino acid sequence of SEQ ID NO: 17, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0269] In some embodiments, the FAC comprises an amino acid sequence of SEQ ID NO:20, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0270] In some embodiments, the FAC comprises an amino acid sequence of SEQ ID NO:22, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0271] In some embodiments, the SAC comprises an amino acid sequence of selected from the group of (a) SEQ ID NO: 18 and SEQ ID NO: 19, (b) SEQ ID NO:21 and SEQ ID NO: 19, (c) SEQ ID NO:23 and SEQ ID NO: 19, and (d) an amino acid sequence at least 90% identical thereto.

[0272] In some embodiments, the SAC comprises (i) an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto); and (ii) an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0273] In some embodiments, the SAC comprises (i) an amino acid sequence of SEQ ID NO:21, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto); and (ii) an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0274] In some embodiments, the SAC comprises (i) an amino acid sequence of SEQ ID NO:23, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto); and (ii) an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 90% identical thereto (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto).

[0275] In some embodiments, the bispecific activatable antibody comprises a set of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; (b) SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO: 19; (c) SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO: 19; and (d) an amino acid sequence at least 90% identical thereto.

[0276] In some embodiments, the bispecific activatable antibody comprises the amino acid sequences of: SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0277] In some embodiments, the bispecific activatable antibody comprises the amino acid sequences of: SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO: 19.

[0278] In some embodiments, the bispecific activatable antibody comprises the amino acid sequences of SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO: 19.

[0279] In some embodiments, the bispecific activatable antibody comprises the amino acid sequences of residues 7-522 of SEQ ID NO: 17, residues 8-488 of SEQ ID NO: 18, and SEQ ID NO: 19.

[0280] In some embodiments, the bispecific activatable antibody comprises the amino acid sequences of residues 7-522 of SEQ ID NO:20, residues 8-493 of SEQ ID NO:21, and SEQ ID NO: 19.

[0281] In some embodiments, the bispecific activatable antibody comprises the amino acid sequences of residues 7-527 of SEQ ID NO:22, residues 8-492 of SEQ ID NO:23, and SEQ ID NO: 19.

[0282] In some embodiments, the antibody of the present invention may be post- translationally modified. In one embodiment, under stress conditions, the post-translational modifications comprise deamidation of asparagine ( Asn— >Asp / iso-Asp), isomerization of aspartic acid ( Asp— riso-Asp) and oxidation of Met / Trp residues. Deamidation of Asn and isomerization of Asp residues are among the most commonly observed spontaneous post- translational modifications (PTMs) in proteins in accelerated and forced stability testing. In some embodiments, the methionine residues (e.g., at position 4 of the light chain, or at positions 34, 48, 70, 81, 83, 85, 106, 251, 284, 390, 427, and / or 460 of the heavy chain) of the antibody undergo oxidation under forced oxidation conditions. In some embodiments, the asparagine residues (e.g., at positions 324, 357, 383 and / or 416 of the heavy chain) of the antibody undergo deamination under forced deamination conditions.Conjugation Agents

[0283] In some aspects, the antibodies may further comprise one or more additional agents, e.g., a targeting moiety to facilitate delivery to a cell or tissue of interest, a therapeutic agent (e.g., an antineoplastic agent such as chemotherapeutic or anti-neoplastic agent), a toxin, a radioisotope, a small molecule, a diagnostic agent, a targeting moiety, or a detectable moiety, or a fragment thereof. The additional agents may be conjugated to the antibodies. The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0284] Those of ordinary skill in the art will recognize that a large variety of possible agents may be conjugated to any of the antibodies described herein. The agents may be conjugated to another component of the antibody by a conjugating moiety, which may be a linker, a CM, or other molecule or fragment thereof capable linking two molecules. In some examples, the conjugating moiety may be cleavable by an enzyme (e.g., a protease). In some examples, the conjugating moiety may be uncleavable by an enzyme (e.g., a protease).

[0285] Conjugation may include any chemical reaction that binds the two molecules so long as the antibody and the other moiety retain their respective activities. Conjugation mayinclude many chemical mechanisms, e.g., covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In some embodiments, the binding may be covalent binding. Covalent binding may be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents may be useful in conjugating any of the antibodies described herein. For example, conjugation may include organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzenes, and hexamethylene diamines. In some embodiments, the antibodies may include, or otherwise introduce, one or more non-natural amino acid residues to provide suitable sites for conjugation.III. Nucleic acids, Vectors, and Cells

[0286] In some aspects, the present disclosure further provides nucleic acids comprising sequences that encode the antibodies, or components or fragment thereof. The nucleic acids may comprise coding sequences for the heavy chain variable domains, light chain variable domains, the antigen-binding domains, the CMs, the MMs, and the linker(s) in the antibody.

[0287] The present disclosure includes a polynucleotide encoding a protein as described herein or a portion thereof, and use of such polynucleotides to produce the proteins and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.) that encode a protein as defined herein. The present disclosure includes compositions comprising such polynucleotides. In some embodiments, such compositions may be used therapeutically or prophy tactically.

[0288] Unless otherwise specified, a “nucleic acid sequence encoding a protein” includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence. The term “nucleic acid” refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid comprise a mixture or hybrid of DNA and RNA.

[0289] Modifications may be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)- mediated mutagenesis. Conservative amino acid substitutions are ones in which the aminoacid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with acidic side chains (e.g., aspartate and glutamate), amino acids with basic side chains (e.g., lysine, arginine, and histidine), non-polar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include: aliphatic -hydroxy amino acids (e.g., serine and threonine), amide family (e.g., asparagine and glutamine), aliphatic family (e.g., alanine, valine, leucine and isoleucine), and aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0290] In some embodiments, the present invention provides an isolated nucleic acid molecule encoding the bispecific activatable antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;.(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO: 18;(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO: 19;(h) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20;(i) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO:21;(j) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22; and(k) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO:23.

[0291] In some embodiments, the present invention provides an isolated nucleic acid molecule encoding the bispecific activatable antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:83;(b) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:84;(c) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:85;(d) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:86;(e) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:87;(f) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:88;(g) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:89;(h) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:90;(i) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:91;(j) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:92; and(k) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:93.

[0292] In some embodiments, the present invention provides an isolated nucleic acid molecule encoding the bispecific antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding anAB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;.(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 Fc fusion consisting of an amino acid sequence of SEQ ID NO:24;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 HC consisting of an amino acid sequence of SEQ ID NO:25; and(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 LC consisting of an amino acid sequence of SEQ ID NO: 19.

[0293] In some embodiments, the present invention provides an isolated nucleic acid molecule encoding the bispecific antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:83;(b) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:84;(c) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:85;(d) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:86;(e) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:26;(f) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:27; and(g) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:89.

[0294] In some embodiments, the present invention provides an isolated nucleic acid molecule encoding the monoclonal antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 15;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO: 16;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 28; and(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO:29.

[0295] The present disclosure further provides vectors and sets of vectors comprising any of the nucleic acids described herein. One skilled in the art will be capable of selecting suitable vectors or sets of vectors (e.g., expression vectors) for making any of the antibodies described herein, and using the vectors or sets of vectors to express any of the antibodies described herein. For example, in selecting a vector or a set of vectors, the type of cell may be selected such that the vector(s) may need to be able to integrate into a chromosome of the cell and / or replicate in it. Example vectors that can be used to produce an antibody are also described herein. As used herein, the term “vector” refers to a polynucleotide capable of inducing the expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any of the cells described herein). A “vector” is able to deliver nucleic acids and fragments thereof into a host cell, and includes regulatory sequences (e.g., promoter, enhancer, poly(A) signal). Exogenous polynucleotides may be inserted into the expression vector in order to be expressed. The term “vector” also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0296] Methods for constructing suitable vectors that comprise any of the nucleic acids described herein, and suitable for transforming cells (e.g., mammalian cells) are well-known in the art. See, e.g., Sambrook et al., Eds. “Molecular Cloning: A Laboratory Manual,” 2nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et al., Eds. “Current Protocols in Molecular Biology,” Current Protocols, 1993.

[0297] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector may, for example, include sufficient cis-acting elements for expression; other elements for expression may be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any antibodies described herein.

[0298] In some embodiments, the antibodies may be made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0299] In some embodiments, the present invention provides a vector comprising the isolated nucleic acid molecule encoding the bispecific activatable antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO: 18;(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO: 19;(h) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20;(i) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO:21;(j) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22; and(k) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO:23(m.

[0300] In some embodiments, the present invention provides a vector comprising the isolated nucleic acid molecule encoding the bispecific activatable antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:83;(b) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:84;(c) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:85;(d) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO:86;(e) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO:87;(f) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:88;(g) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:89;(h) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:90;(i) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:91;(j) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:92; and(k) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:93.

[0301] In some embodiments, the present invention provides a vector comprising the isolated nucleic acid molecule encoding the bispecific antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;.(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 Fc fusion consisting of an amino acid sequence of SEQ ID NO:24;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 HC consisting of an amino acid sequence of SEQ ID NO:25; and(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 LC consisting of an amino acid sequence of SEQ ID NO: 19.

[0302] In some embodiments, the present invention provides a vector comprising the isolated nucleic acid molecule encoding the bispecific antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:83;(b) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO:84;(c) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:85;(d) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:86;(e) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:26;(f) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:27; and(g) an isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:89.

[0303] In some embodiments, the present invention provides a vector comprising the isolated nucleic acid molecule encoding the monoclonal antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 15;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO: 16;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 28; and(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO:29.

[0304] In some embodiments, the present disclosure provides host cells comprising any of the vectors or nucleic acids described herein. The cells may be used to produce the antibodies described herein. In some embodiments, the cell may be an animal cell, a mammalian cell (e.g., a human cell), a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, or a guinea pig cell), a non-human primate cell, an insect cell, a bacterial cell, a fungal cell, or a plant cell. In some embodiments, the cell may be a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells includeChinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some embodiments, the cell may be a prokaryotic cell.

[0305] Methods of introducing nucleic acids and vectors (e.g., any of the vectors or any of the sets of vectors described herein) into a cell are known in the art. Examples of methods that can be used to introducing a nucleic acid into a cell include: lipofection (e.g., lipid transfection or liposome-based transfection), transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0306] In some embodiments, the introducing step includes introducing into a cell a vector (e.g., any of the vectors or sets of vectors described herein) including a nucleic acid encoding the monomers that make up any antibodies described herein.

[0307] In some embodiments, the present invention provides cells comprising a nucleic acid molecule encoding a the bispecific activatable antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO: 18;(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO: 19;(h) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20;(i) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO:21;(j) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22; and(k) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO:23.

[0308] In some embodiments, the present invention provides cells comprising a nucleic acid molecule encoding a BAA selected from the group consisting of:(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 comprising an amino acid sequence of SEQ ID NO: 13 and an amino acid sequence of SEQ ID NO: 14, and a nucleotide sequence encoding an AB2 comprising an amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO: 16;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO: 18 and an amino acid sequence of SEQ ID NO: 19;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO:21 and an amino acid sequence of SEQ ID NO: 19; and(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO:23 and an amino acid sequence of SEQ ID NO: 19.

[0309] In some embodiments, the present invention provides cells comprising a nucleic acid molecule encoding a bispecific antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 comprising an amino acid sequence of SEQ ID NO: 13 and an amino acid sequence of SEQ ID NO: 14, and a nucleotide sequence encoding an AB2 comprising an amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO: 16; and(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 Fc fusion consisting of an amino acid sequence of SEQ ID NO:24, and a nucleotide sequence encoding an AB2 HC consisting of an amino acid sequence of SEQ ID NO:25 and an AB2 LC consisting of an amino acid sequence of SEQ ID NO: 19.

[0310] In some embodiments, the present invention provides cells comprising a nucleic acid molecule encoding a monoclonal antibody selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 15 and a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO: 16; and(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 28 and a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO:29.IV. Compositions and kits

[0311] The present disclosure also provides compositions and kits comprising the antibodies described herein. The compositions and kits may further comprise one or more excipients, carriers, reagents, instructions needed for the use of the antibodies. In some embodiments, the compositions may be pharmaceutical compositions, which comprise the antibodies, derivatives, fragments, analogs and homologs thereof. The pharmaceutical compositions may comprise the antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0312] A pharmaceutical composition may be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); bufferssuch as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some, any of the antibodies described herein are prepared with carriers that protect against rapid elimination from the body, e.g., sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collage, poly orthoesters, and poly lactic acid. Methods for preparation of such pharmaceutical compositions and formulations are apparent to those skilled in the art. For example, the antibodies may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatinmicrocapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions.

[0313] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0314] In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor or phosphate buffered saline (PBS). The composition may be sterile and should be fluid to the extent that easy syringe ability exists. It may be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride may be included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0315] In some embodiments, the pharmaceutical composition may comprise a sterile injectable solution. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0316] In some embodiments, the pharmaceutical composition may be formulized for systemic administration. For example, systemic administration may be by intravenous, intradermal, intraperitoneal or intramuscular as well by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds may be formulated into ointments, salves, gels, or creams as generally known in the art.

[0317] In some embodiments, the pharmaceutical composition may be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0318] In some embodiments, the pharmaceutical composition may be prepared with carriers that protect the compound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[0319] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure may be dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0320] In some embodiments, the compositions (e.g., pharmaceutical compositions) may be included in a container, vial, syringe, injector pen, pack, or dispenser, optionally together with instructions for administration.

[0321] Also provided herein are kits that include any of the antibodies described herein, any of the compositions that include any of the antibodies described herein, or any of the pharmaceutical compositions that include any of the antibodies described herein. Also provided are kits that include one or more second therapeutic agent(s) in addition to an antibody described herein. The second therapeutic agent(s) may be provided in a dosage administration form that is separate from the antibodies. Alternatively, the second therapeutic agent(s) may be formulated together with the antibodies.

[0322] Any of the kits described herein can include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the antibodies described herein. In some embodiments, the kits can include instructions for performing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein.

[0323] Also provided herein are antibodies produced by any of the methods described herein. Also provided are compositions (e.g., pharmaceutical compositions) that comprise any of the antibodies produced by any of the methods described herein. Also provided herein are kitsthat include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.V. Methods of Producing Antibodies

[0324] In some aspects, the present disclosure provides methods of producing the antibodies of the present disclosure comprising: (a) culturing a host cell comprising one or more polynucleotides encoding the antibodies of the present disclosure, as well as vector(s) comprising the polynucleotides, in a liquid culture medium under conditions sufficient to produce the antibodies; and (b) recovering the antibodies.

[0325] In a particular aspect, provided herein are methods for producing an antibody of the present disclosure, comprising expressing the antibody thereof in a host cell. More specifically, provided herein is a method of producing an antibody comprising: (a) culturing a host cell comprising one or more polynucleotides encoding the antibody of the present disclosure in a liquid culture medium under conditions sufficient to produce the antibody; and (b) recovering the antibody.

[0326] Methods of culturing cells are well known in the art. In some embodiments, cells may be maintained in vitro under conditions that favor cell proliferation, cell differentiation and cell growth. For example, cells may be cultured by contacting a cell (e.g., any of the cells described herein) with a cell culture medium that includes the necessary growth factors and supplements sufficient to support cell viability and growth.

[0327] In some embodiments, the method may further includes isolating the recovered antibodies. The isolation of the antibodies may be performed using any protein separation or purification techniques. Examples of methods of isolation include: isolation using a protein purification tag (e.g., His tag), ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand- affinity chromatography, ion-exchange chromatography (e.g., anion or cation), and hydrophobic interaction chromatography.VI. Methods of Treatment

[0328] In some aspects, the present disclosure further provides methods of using the antibodies herein. In some embodiments, the present disclosure provides methods of the treating a disease or a disorder (e.g., a cancer, e.g., a low-MUCl expressing cancer) in a subject including administering a therapeutically effective amount of any of the antibodies described herein to the subject. In some embodiments, the disclosure provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwiseameliorating disease in a subject by administering a therapeutically effective amount of antibodies described herein to a subject in need thereof.

[0329] In some embodiments, the methods include selecting a subject having a low-MUCl expressing cancer. A subject is determined to have a low-MUCl expressing cancer based on the expression level of MUC1 on the cancer cells. The MUC1 expression level can be determined by any methods known in the art, for example, by flow cytometry. In some embodiments, a low-MUCl expressing cancer may express MUC1 at a level of less than about 0.1 x 106, e.g., less than about 0.09 x 106, about 0.08 x 106, about 0.07 x 106, or about 0.065 x 106, as measured by AMFI (mean fluorescence intensity). In some embodiments, a low-MUCl expressing cancer expresses MUC1 at a level of about 0.001 x 103to about 0.1 x 106as measured by AMFI (mean fluorescence intensity).

[0330] A therapeutically effective amount of an antibody of the disclosure relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigens that, in certain cases, interferes with the functioning of the targets. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific target, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody of the disclosure may be, by way of nonlimiting example, from about 0.001, 0.01, 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / kg body weight or higher. The structure of the antibody of the present disclosure makes it possible to reduce the dosage of the antibody that is administered to a subject compared to conventional antibodies. For example, the administered dose on a unit dosage basis or total dosage over a dosage regimen period may be reduced by 10, 20, 30, 40, or 50% compared to the corresponding dose of a corresponding conventional antibody.

[0331] Common dosing frequencies may range, for example, from once or twice daily, weekly, biweekly, or monthly.

[0332] Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0333] The antibody used in any of the embodiments of these methods and uses may be administered at any stage of the disease. For example, such an antibody may be administered to a patient suffering cancer of any stage, from early to metastatic. In some embodiments, theantibody and formulations thereof may be administered to a subject suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity.

[0334] A subject suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity may be identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic condition may be identified using any of a variety of clinical and / or laboratory tests such as, physical examination and blood, urine and / or stool analysis to evaluate health status. For example, subjects suffering from inflammation and / or an inflammatory disorder may be identified using any of a variety of clinical and / or laboratory tests such as physical examination and / or bodily fluid analysis, e.g., blood, urine and / or stool analysis, to evaluate health status.

[0335] In some embodiments, administration of an antibody to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration of an antibody to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if one or more of the symptoms associated with the disease or disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of an antibody to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state.

[0336] As used herein, the term “treat” includes reducing the severity, frequency or the number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., a cancer) in the subject. In some embodiments where the disease is cancer, treating results in reducing cancer growth, inhibiting cancer progression, inhibiting cancer metastasis, or reducing the risk of cancer recurrence in a subject having cancer.

[0337] In some embodiments, the disease may be a cancer. In some embodiments, the subject may have been identified or diagnosed as having a cancer. Non-limiting examples of cancer that may be treated by the compositions and methods include: blood cancers such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell lymphoma, multiple myeloma and T cell lymphoma, solid cancers such as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, lung cancer, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, skin T cell lymphoma, brain cancer, breast cancer, prostate cancer, bladder cancer, head and neck cancer, uterine cancer, liver cancer,gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, rectal cancer, small intestine cancer, gastric cancer, esophageal cancer, testicular cancer, ovarian cancer, vaginal cancer, cervix cancer, myelodysplastic syndromes, cancers of bone tissues, cartilage tissues, adipose tissues, muscle tissues, vascular tissues and blood-forming tissues, sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcoma, and blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma and retinoblastoma. Metastases of the aforementioned cancers may also be treated or prevented by the compositions and methods of the present invention. In some embodiments, the methods herein may result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of cancer in the subject prior to treatment).

[0338] The methods may further comprise administering to a subject one or more additional agents. In some embodiments, the additional agent(s) may be a chemotherapeutic agent. A “chemotherapeutic agent”, as used herein, refers to a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy. Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi- Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N .J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9- triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(l,2-diphenylbut-l-enyl)phenoxy]-N,N-dimethyl-ethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYC1NO), Akti-1 / 2, HPPD, and rapamycin.

[0339] More examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL- 147 (PI3K inhibitor, Exelixis), PTK787 / ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, calicheamicin gammall, calicheamicin omegall (Angew Chem. Inti. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L- norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0340] Also included in the definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007 / 044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0341] Also included in the definition of “chemotherapeutic agent” are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / B iogen Idee), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

[0342] In some embodiments, the additional agent comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor may be an inhibitor of a protein such as CTLA-4, LAG-3, PD-1, PD-1, TIGIT, TIM-3, B7H4, BTLA, or Vista. In some embodiments, the kinase inhibitor may be B-RAFi, MEKi, Btk inhibitors, ibrutinib, or crizotinib. In some embodiments, the tumor microenvironment inhibitor may be an IDO inhibitor, an a-CSFIR inhibitor, an a-CCR4 inhibitor, a TGF-beta, a myeloid-derived suppressor cell, or a T- regulatory cell. In some embodiments, the agonist may be 0x40, GITR, CD 137, ICOS, CD27, or HVEM. In some embodiments, the antibody may be administered during and / or after treatment in combination with one or more additional agents. In some embodiments, the antibody may be formulated into a single therapeutic composition, and the antibody and additional agent(s) may be administered simultaneously. Alternatively, the antibody and additional agent(s) may be separate from each other, e.g., each is formulated into a separate therapeutic composition, and the antibody and the additional agent are administered simultaneously, or the antibody and the additional agent are administered at different times during a treatment regimen. For example, the antibody may be administered prior to the administration of the additional agent, subsequent to the administration of the additional agent, or in an alternating fashion. The antibody and additional agent(s) may be administered in single doses or in multiple doses.

[0343] One of more of the antibodies herein may be co-formulated with, and / or coadministered with, one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzymatic inhibitors. Examples of the drugs or inhibitors that may be used include one or more of: IL-6 antagonists, e.g., tocilizumab (TOCI), sarilumab, and satralizumab; nonsteroidal anti-inflammatory drug(s) (NSAIDs), e.g., ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such as prednisolone; cytokine suppressive anti-inflammatory drug(s) (CSAIDs); inhibitors of nucleotide biosynthesis, e.g., inhibitors of purine biosynthesis, folate antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl] methylamino] benzoyl] -L-glutamic acid); and inhibitors of pyrimidine biosynthesis, e.g., dihydroorotate dehydrogenase (DHODH) inhibitors.

[0344] The antibodies herein may also be used in diagnostic and / or imaging methods. In some embodiments, such methods may be in vitro methods. In some embodiments, such methods may be in vivo methods. In some embodiments, such methods may be in situ methods. In some embodiments, such methods may be ex vivo methods. For example, antibodies having a CM may be used to detect the presence or absence of an enzyme capable of cleaving the CM. Such antibody may be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies in a given cell or tissue of a given host organism. Such accumulation of activated antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated antibody binds.

[0345] For example, the CM may be selected to be a protease substrate for a protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like. Using methods familiar to one skilled in the art, a detectable label (e.g., a fluorescent label or radioactive label or radio tracer) may be conjugated to an antigen-binding domain or other region of an antibody. Suitable detectable labels may be discussed in the context of the above screening methods and additional specific examples are provided below. Using an antigen-binding domain specific to a protein or peptide of the disease state, along with a protease whose activity is elevated in the disease tissue of interest, antibodies may exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at adetectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated antibodies in the disease tissue may be enhanced relative to non-disease tissues.

[0346] In some embodiments, the antibodies may be useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the antibodies. The in vivo imaging may be used to identify or otherwise refine a patient population suitable for treatment with antibodies of the disclosure.

[0347] For example, patients that test positive for both the target and a protease that cleaves the substrate in the CM of the antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an antibody comprising such a CM. Likewise, patients that test negative may be identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the antibody being tested). In some embodiments, such patients that test negative with respect to a first antibody can be tested with other antibodies comprising different CMs until a suitable antibody for treatment is identified (e.g., an antibody comprising a CM that is cleaved by the patient at the site of disease).

[0348] In some embodiments, in situ imaging may be useful in methods to identify which patients to treat. For example, in in situ imaging, the antibodies may be used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site. In some embodiments, in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with antibodies of the disclosure.

[0349] It is to be understood that this invention is not limited to particular assay methods, or test agents and experimental conditions described, as such methods and agents may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0350] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents andpublished patent applications cited throughout this application, as well as the Figures, are hereby incorporated herein by reference.EXAMPLES

[0351] Example 1: Generation of Mouse Monoclonal Antibodies against Human MUC1 Antigen

[0352] To generate monoclonal antibodies against human and cynomolgus (cyno) MUC1, four ABV hyperimmune mice were immunized with a human MUC1 peptide following a rapid immunization schedule. Two of the four mice received extended immunization schedule. Titer analyses were performed by flow cytometry against ZR-75 and HP AC cells and ELISA against MUC1 antigen to determine target- specific response. Hybridoma generation was performed on all four titer-positive mice.Hybridoma Screening and Subcloning

[0353] Hybridoma screening was performed using a flow cytometry binding assay with ZR- 75, HP AC, HCT116-cynoMUCl, and wildtype HCT116 cells. In brief, cells were incubated with the hybridoma supernatant for 1 hour at 4°C. Cells were then washed, incubated with FITC-labeled anti mouse IgG, washed, and analyzed using FACS array. The hybridomas with specific reactivity to human and cyno MUC1 antigen were expanded, and the supernatants were rescreened by flow cytometric binding assay using four independent cell lines: HP AC, HCT116-huMUCl, HCT116-cynoMUCl and wild type HCT116 cells. The hybridomas with positive binding to human and cyno MUC1 antigens but negative on wild type HCT116 were further subcloned by limiting dilution. One subclone from each hybridoma, which showed specific binding to human and cyno MUC1 antigens, was selected for subsequent analysis. A total of 4 fusions were conducted over the course of this investigation. 70 hybridomas specific for both human and cynomolgus MUC1 antigens were generated and 18 hybridomas were subcloned.Example 2: Bispecific chimericMUCl x huCD3s AntibodiesCloning of VL and VH regions

[0354] A set of bispecific chimeric MUC1 x huCD3s (CD3-epsilon) antibodies in the scFv- Fab-Fc format (see, e.g., FIG. 1A) was generated, each identical with the exception that each containing a different anti-MUCl antibody component, with each anti-MUCl antibody component obtained from one of the 18 subcloned hybridomas. The anti-MUCl antibodycomponents of the bispecific antibodies were selected from 4 different epitope bins (A, B, C, and D). A benchmark clone, 3D1, was used as a control. 3D1 is described in WO 2015 / 116753, the contents of which are hereby incorporated by reference in their entirety. The huCD3s scFv used in each bispecific MUC1 x huCD3s is described in WO 2023 / 192606, the contents of which are hereby incorporated by reference in their entirety. Each bispecific MUC1 x CD3E antibody had the structure depicted in the cartoon of FIG. 1A. The MUC1 and huCD3s binding properties of each bispecific antibody were assessed by flow cytometry on HCT116-huMUCl cells and Jurkat cells (clone E6-1 (TIB- 152), ATCC).

[0355] HCT116-huMUCl cells were cultured in McCoy's 5A (Modified) Medium (Gibco, Cat# 16600082) with 10% Heat Inactivated-Fetal Bovine Serum (HI-FBS, Life Technologies, Cat# 10438-026). Jurkat cells were cultured in RPMI-1640+glutamax (Life Technologies, Cat# 72400-047), 10% Heat Inactivated-Fetal Bovine Serum (HI-FBS, Life Technologies, Cat# 10438-026). The HCT116-huMUCl cells were detached with Versene™ (Life Technologies, Cat# 15040-066), washed, and plated in 96 well plates.

[0356] In the binding assay, IxlO5cells per sample were incubated with varying concentrations of MUC1 TCB in 100 pL FACS Stain Buffer + 2% FBS (BD Pharmingen, Cat# 554656). For both cell lines, cells were incubated at 4°C with shaking for about 1 hour, harvested, and washed twice with 200 pL of FACS Stain Buffer. Cells were resuspended in 50 pL of AF488-conjugated anti-human IgG Fc specific antibody (1.5 pg / ml, Jackson ImmunoResearch, Cat# 109-546-098) and incubated at 4°C with shaking for about 1 hour. Cells were harvested, washed, and resuspended in a final volume of 150 pL of FACS Stain Buffer containing 0.25 pg 7-AAD (BD Biosciences, Cat# 559925). Data was acquired on an Attune NxT Flow Cytometer and the median fluorescence intensity (MFI) of viable cells was calculated using FlowJo® V10.10.0. Raw MFI data was graphed in GraphPad Prism using curve fit analysis. Binding EC50 values are summarized in Table 1.Table 1. Chimeric MUC1 x huCD3 TCB on-cell binding EC50ND = not determined due to low on-cell binding.NA = not available due to failed protein expression.

[0357] Biological activity of chimeric anti-MUCl x anti-CD3 bispecific antibodies was assayed using cytotoxicity assays. Human PBMCs (Charles River Laboratories, Wilmington, MA) were co-cultured with MUC1 -expressing cancer cell line HCT116-huMUCl at a ratio of 10:1 in RPMI-1640+glutamax supplemented with 5% heat inactivated human serum (Life Technologies, Cat# 10438-026). Dose response was evaluated at starting concentrations as 10'2PM followed by 3.5-fold serial dilutions in the co-culture media. After 48 hours, cytotoxicity was evaluated using the CytoTox-GloTM Cytotoxicity Assay (Promega,Madison, WI Cat# G9291). Cytotoxicity for the luminescence was measured on SpectraMax M5 (Molecular Devices, San Jose, CA). Percent cytotoxicity was calculated and plotted in GraphPad PRISM with curve fit analysis. Cytotoxicity EC50 values are summarized in Table 2.Table 2. Chimeric MUC1 x huCD3 TCB cytotoxic EC50N / A = EC50 value could not be determined due to lack of cytotoxic activityExample 3: Anti-MUCl Antibody HumanizationCDR grafting

[0358] CDR regions were defined according to the AbM definition (Martin, A.C.R., Cheetham, J.C. and Rees, A.R. (1989) Proc. Natl Acad. Sci. USA, 86, 9268-9272; Martin, A.C.R., Cheetham, J.C. and Rees, A.R. (1991) Methods Enzymol., 203, 121-153; Pedersen, J.T et al. (1992) Immunomethods, 1, 126; and Rees, A.R. et al. (1996) In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172). The two cyno-binding clones 21D3 and 74G5 were chosen for humanization based on observed on- cell binding, cytotoxicity and cyno cross -reactivity. The VH and VL sequences of 74G5 and 21D3 were compared to a library of known human germline sequences from human VH genes and human Vlkappa genes (IMGT). The closest in sequence human germlines were chosen as the acceptor sequences for CDR grafting. For 74G5, IGKVl-39*01 and IGHV1- 46*01 were chosen as acceptors and the CDR sequences from 74G5E9_VL and 74G5E9_VH were grafted, respectively. For 21D3, IGKVl-39*01 and IGHV1-46*O1 were chosen as acceptors and the CDR sequences from 21D3E10_VL and 21D3E10_VH were grafted, respectively. Substitution amino acids were highlighted in humanized sequencealignment. Alignments of the humanized sequences with 74G5 and 21D3, with substituted mutations highlighted, are provided in FIGS. 2A and 2B.Binding affinity of humanized anti-MUCl antibodies

[0359] To screen the humanized 21D3 and 74G5 anti-MUCl antibodies, different pairs of VH and VL were cloned into an antibody format and culture supernatants were screened for binding to human and cyno MUC1. By Octet BLI, humanized antibodies were captured from culture supernatants using AHC biosensors (Sartorius 18-5060). The tips were associated with either human MUC1 protein (AcroBiosy stems MU1-H52H5) or cyno MUC1 protein (AcroBiosystems MU1-C52H5) for 250 seconds and dissociated in buffer for 300 seconds. Of the 15 21D3 humanized antibodies screened, ProC1892, ProC1894, and ProC1896 were selected for reformatting into the bispecific scFv-Fab-Fc format as they showed similar binding to human and cyno MUC1 as compared to the chimeric 21D3 antibody (ProC1883). Of the nine 74G5 humanized antibodies screened, all showed reduced binding to both human and cyno MUC1 compared to that of 74G5 chimeric antibody (ProC1884). Two antibodies, ProC1906 and ProC1909, were selected for reformatting into the bispecific scFv-Fab-Fc format.In vitro characterization of humanized MUC1 TCBs

[0360] On-cell binding and cytotoxic assays were carried out to compare chimeric 74G5 bispecific scFv-Fab-Fc (ProC1465) and humanized MUC1 bispecific scFv-Fab-Fc (ProC2231). FIG. 3A depicts binding curves with human HP AC and cyno small intestine epithelial cells. Cynomolgus monkey primary small intestinal epithelial cells (CellB iologics, catalog MK-6051) were culture in Human Epithelial Cell Medium with growth supplement (CellBiologics, catalog H6621). FIG. 3B depicts cytotoxic activity of the bispecifics to HP AC and cyno small intestine epithelial cells. The data indicate that the humanized MUC1 x CD3s bispecific antibodies retain strong binding and cytotoxic activity towards human and cyno MUC1 -expressing cells.Example 4: Glycosylation Modification of Humanized Anti-MUCl AntibodyN- glycosylation and de-glycosylation variants

[0361] Accelerated and forced stability studies and peptide mapping identified N- glycosylation on hu21D3 and hu74G5 TCB heavy chains. More specifically, the heavy chain CDR2 of both h21D3 and h74G5 contained N-glycosylation motif variably glycosylated onresidue N56. Heavy chain CDR2 of both h21D3 and h74G were engineered to remove the N- glycosylation motif by substitution mutations at N56, T58, or both N56 and T58. In each instance when substituted, the asparagine residue at position 56 was substituted with one of glutamine, serine, alanine, or glycine. In each instance when substituted, the threonine residue at position 58 was substituted with one of alanine, glycine, or lysine.

[0362] MUC1 x CD3s bispecific antibodies comprising humanized 74G5 de-glycosylation variants were expressed. Culture supernatants were screened by Octet BLI for binding to human and cyno MUC1. Substitutions at N56 and / or T58 didn’t have an effect on binding as compared to WT. Full binding characterization of MUC1 de-glycosylation variants was then performed.On-cell binding of affinity variants

[0363] The binding affinity hu74G5 de-glycosylated variants was compared to that of the parent hu74G5 TCBs. Binding affinities are summarized in Table 3. Flow cytometry binding assays and cytotoxic assays were carried out and analyzed as described above. FIG. 4A depicts binding curves of the variants with HCT116-huMUCl, HP AC, and cyno small intestine epithelial cells. FIG. 4B depicts the cytotoxic activity of the variants on HCT116- huMUCl and HP AC cells. The data demonstrate that de-glycosylation variants retain similar binding affinity and cytotoxicity relative to the parental antibody. Variant hu74G5-N56A was selected for further evaluation.Table 3. On-cell binding of huMUCl x huCD3 bispecific de-glycosylation variantsExample 5: Bispecific Humanized, De-Glycosylated anti-MUCl x anti-CD3 AntibodiesBinding affinity to recombinant human (WT and single nucleotide polymorphism SNP)) and cyno MUC1_N / C_ED proteins

[0364] ProC2689 (humanized 74G5 with substitution N56A in the variable heavy domain x humanized CD3s bispecific scFv-Fab-Fc antibody) was used for assaying binding kinetics torecombinant WT human (AcroBiosystems MU1-H52H5) and cyno (AcroBiosystems MU1- C52H5) MUC1 and human (AcroBiosystems CDE-H5223) and cyno (AcroBiosystems CDE- C5226) CD3s proteins. Using immobilized anti-histidine antibody to capture the his-tagged MUC1 ligand, serial dilutions of ProC2689 were associated for 250 seconds at a flow rate of 30 mL / min, followed by a dissociation phase of 500 seconds. At the end of each cycle, the immobilized sensor surfaces were regenerated using 10 mM Glycine-HCl pH 1.5 for 60 seconds. Binding kinetics for ProC2689 for the four ligands are summarized in Table 4.Table 4. ProC2689 Binding Kinetics | | | | |*KD values averaged for two biological replicates

[0365] Binding affinity of the clone 74G5 hybridoma supernatant for human, cyno and MUC1 SNP variants was determined to be strong in Koff screening by Octet BLI. As determined by ELISA, ProC2689 demonstrated comparable high binding to that of WT and SNP MUC1 variants.Binding to human HCT116-huMUCl, HPAC, and cyno primary small intestine epithelial cells

[0366] To assess huMUCl and cynoMUCl -binding by humanized MUC1 x CD3s bispecific antibodies, a flow cytometry-based binding assay was performed as described above. Table 5 provides averaged binding EC50 values based on binding assay results from multiple replicates.Table 5. Humanized MUC1 x CD3 bispecific antibody binding*Estimate EC50; binding curve does not plateauBiological Activity ( Cytotoxicity)

[0367] Biological activity of ProC2689 was assayed using cytotoxicity assays. Human PBMCs (Charles River Laboratories, Wilmington, MA) were co-cultured with MUC1 expressing cancer cell lines HCT116-huMUCl or HP AC, or cyno primary small intestine epithelial cells. Table 6 provides averaged EC50 values based on the cytotoxicity assay results from multiple replicates. ProC2689 exhibited potent cytotoxic activity in both human and cyno MUC1 target cells.Table 6. Humanized MUC1 x CD3 bispecific antibody cytotoxic EC50*Estimate EC50; binding curve does not plateauExample 6: Characterization of Dually Masked Bispecific anti-MUCl x CD3F Antibodies

[0368] The studies provided herein were designed to identify and characterize masking peptides for use in activatable humanized bispecific anti-MUCl x CD3s antibodies. A number of different candidate anti-MUCl masking peptides were evaluated. The masking peptides are listed in Table 7.

[0369] Table 7. Candidate anti-MUCl masking peptidesMasking efficiency of anti-MUCl masking peptides

[0370] The ability of candidate anti-MUCl masks to inhibit h74G5 binding was evaluated in an activatable antibody format, wherein each antibody arm comprises a h74G5 Fab linked to the masking peptide via a protease cleavable linker. For the studies described herein, a cleavable moiety comprising the amino acid sequence ISSGLLSGRSDNP (1490DNP, SEQ ID NO:54) was used.

[0371] Masking efficiencies (ME) were determined by ELISA. High binding plates were coated with either diluted MUC1 antigen (Aero Biosystems MU1-H52H5) in PBS to 2ug / mL or diluted CD3 antigen (Aero Biosystems CDE-H5223) to 2ug / mL in carbonate-bicarbonate buffer.

[0372] Plates were incubated at 4°C overnight. After overnight incubation, the antigens were washed from the plates 3X with plate washer. The plates were then blocked with Blocking Buffer (1% BSA, 0.05% Tween 20 in PBS) for one hour at room temperature. Plates were dumped and tapped vigorously to remove. Samples were diluted in a 4-fold dilution series in Blocking Buffer manually and applied to the blocked plates. The last column or row was keptas blank. The plates were allowed to incubate for one hour. The plates were then washed 6X with plate washer before adding HRP-conjugated anti-human-IgG-Fc (Sigma A0170) dilution in blocking buffer. The plates incubated for one hour at room temperate. After the hour incubation, the plates were washed 6X with plate washer. To develop the ELISA, TMB substrate was added and incubated for 2-14 minutes without shaking. To stop the ELISA, IN HC1 was added and the plates were immediately read in a plate reader at 450 nm. To analyze the data, the blank signal was subtracted from experimental signals. The data was pasted into GraphPad Prism. From the Prism software, masking efficiency (ME) was calculated as the ratio of masked molecule Kd signal over the unmasked molecule Kd signal. Masking efficiencies for the candidate anti-MUCl masking peptides in the activatable antibody format are summarized in Table 8.Table 8. Masking efficiencies for candidate anti-MUCl masking peptides in an activatable antibody (actvAb) formatMasking efficiency of select anti-MUCl masking peptides in a masked, activatable bispecific antibody format

[0373] A set of masked, activatable bispecific antibodies targeting human MUC1 and human CD3s was generated, in which each antibody comprised an anti-MUCl masking peptide, a linker comprising a first cleavable moiety linking the anti-MUCl masking peptide to a deglycosylated h74G5 Fab via the heavy chain, an anti-CD3s masking peptide, a linker comprising a second cleavable moiety linking the anti-CD3s masking peptide to an anti- CD3s scFv, a hinge region, and an Fc domain. The masked, activatable bispecific antibodies were identical with the exception the anti-MUCl masking peptide was varied between the antibodies. A description of the masked, activatable bispecific antibodies is provided in Table 9.Table 9. Activatable bispecific antibodies targeting human MUC1 and human CD3s with various anti-MUCl masking peptides

[0374] The ability of candidate anti-MUCl masks to inhibit h74G5 binding was evaluated in the masked, activatable bispecific antibody format. For the studies described herein, a linker comprising a first cleavable moiety comprising the amino acid sequence ISSGLLSGRSDNP (1490DNP, SEQ ID NO:54) was used to link the anti-MUCl masking peptide to the heavy chain of the anti-MUCl Fab and a linker comprising a second cleavable moiety comprising the amino acid sequence GLSGRSDNP (GL1204DNP, SEQ ID NO:53) was used to link the anti-CD3s masking peptide (20GG) to the anti- CD3s scFv.

[0375] Masking efficiencies for the candidate anti-MUCl masking peptides in the masked, activatable bispecific antibody format are summarized in Table 10.Table 10. Masking efficiencies of candidate anti-MUCl and anti-CD3s masking peptides in a masked, activatable bispecific antibody formatMUC1 ME = EC50 of masked molecules / EC50 of unmasked 74G5 (ProC2689)Results shown as mean ± SEM, N>3

[0376] Masking peptide M84 was selected for advancement into dually masked activatable bispecific antibodies targeting human MUC1 and human CD3 a .Example 7: Bispecific anti-MUCl x anti-CD3 Activatable Antibodies

[0377] A set of masked, activatable bispecific antibodies targeting human MUC1 and human CD3a was generated, in which each antibody comprised an anti-MUCl masking peptide, a linker comprising a first cleavable moiety linking the anti-MUCl masking peptide to a deglycosylated h74G5 Fab via the heavy chain, an anti-CD3s masking peptide, a linker comprising a second cleavable moiety linking the anti-CD3s masking peptide to an anti-CD3s scFv, a hinge region, and an Fc domain. The masked, activatable bispecific antibodies were identical with the exception that the first and second cleavable moieties were varied between the antibodies. The masked, activatable bispecific antibodies are set forth in Table 11, while Table 12 provides a description of the components of the masked, activatable bispecific antibodies utilized in the subsequent studies.Table 11. Amino acid sequences for masked, activatable bispecific antibodies targeting human MUC1 and human CD3sTable 12. Components of the activatable bispecific antibodiesBinding kinetics ofProC3405, ProC3687 and ProC3699 to human MUC1 / CD3 by SPR

[0378] SPR binding kinetics were run in duplicates for each of ProC3405, ProC3687 and ProC3699 to human MUC1 / CD3. 50 pg / mL of anti-histidine antibody (Cytiva Cat# 28995056) prepared in 10 mM sodium acetate buffer (Cytiva Cat# BR100349) was immobilized using a standard amine coupling kit (Cytiva Cat# BR100050) to a series Ssensor chip CM5 (Cytiva Cat# BR100530) in HBS-EP buffer (Cytiva cat# BR100669) at 25°C. 5 nM of His-tagged human (AcroBiosystems MU1-H52H5) or His-tagged human CD3s (AcroBiosystems CDE-H5223) was captured over the immobilized surfaces. Serial dilutions of ProC3405, ProC3687 or ProC3699 were associated for 250 seconds at a flow rate of 30 pL / min followed by a dissociation phase of 500 seconds. At the end of each cycle, the immobilized sensor surfaces were regenerated using 10 mM Glycine-HCL pH 1.5 for 60s. Data obtained from the sensorgrams were analyzed using the Biacore T200 evaluation software, 1:1 binding model. Attenuated binding to MUC1 and CD3 recombinant proteins was observed, with results summarized in Table 13.Table 13. Binding kinetics of ProC3405, ProC3687 and ProC3699 to human MUC1 / CD3 by SPR*Note: KD measurement is average of two technical replicates t Note: for these measurements, kinetic constant ka at limit of measurement by instrument and high analyte concentrations result in high bulk contributionsAttenuated on-cell binding to MUC1 and CD3 on target cells

[0379] The dually masked, activatable bispecific antibodies ProC3405, ProC3699, and ProC3687 were evaluated for on-cell binding to MUC1 and CD3 on target cells using a flow cytometry-based binding assay. Results for binding to HCT116-huMUCl, HP AC, and cyno primary small intestine epithelial cells (MUC1 binding) are depicted in FIGS. 5-7, respectively. Results for binding to Jurkat cells (CD3 binding) are depicted in FIG. 8.

[0380] FIGS. 5-7 show that the masking peptides employed in the dually masked bispecific antibodies were effective at reducing the ability of the bispecific antibody to bind to MUC1 relative to the unmasked bispecific antibody, ProC2689. Likewise, FIG. 8 shows that the masking peptide 20GG, employed to mask the scFv CD3-binding domain, was effective at reducing the ability of the bispecific antibody to bind to CD3s, relative to the unmasked bispecific antibody, ProC2689. The on-cell binding results are summarized in Table 14.Table 14. Attenuated on-cell binding to MUC1 and CD3 by dually masked, activatable bispecific antibodiesND = not determined; EC50 cannot be defined, no activity observed in this cell lineAttenuated cytotoxic activity against human and cyno MUC1 target cells

[0381] The biological activity of dually masked, activatable bispecific and unmasked bispecific antibodies was assayed using cytotoxicity assays. The results are depicted in FIG.9 (HHCT116-huMUCl cell line), FIG. 10 (HP AC cell line), and FIG. 11 (cyno small intestine epithelial cell line). EC50 values were determined, and masking efficiencies were computed. The results are provided in Table 15.Table 15. Cytotoxic EC50 and Masking EfficiencyND = not determined; EC50 cannot be defined, no activity observed in this cell lineMasking Efficiency = EC50 of activatable / masked TCB / EC50 of unmasked TCB ProC2689Protease activation of dually masked activatable bispecifics restores binding and cytotox activity

[0382] To activate dually masked activatable bispecific antibodies in vitro, a desired amount of activatable bispecific antibody was prepared in PBS, and recombinant protease was addedto the solution to a final concentration of 1:5 or 1:10 protease to antibody molar ratio. The solution was then incubated at 37 °C in a humidified 5% CO2 incubator overnight.

[0383] The binding affinity of MUC1 x CD3s activatable bispecific antibodies to HCT116- huMUCl, HP AC, cyno small intestine epithelial cells, and Jurkat cells was compared to that of the unmasked antibody ProC2689 and between activatable antibodies in the activated and masked forms. Flow cytometry binding assays were carried out and analyzed as described in Example 2.

[0384] As shown in FIGS. 12 and 13, activatable antibodies without activation had very poor binding to MUC1 on target cells, while the activated activatable antibodies retained binding affinity at a level similar to that of the parent antibody. Similarly, the masked activatable antibodies demonstrated very poor binding to CD3 on Jurkat cells, and although the activated antibodies showed reduced binding Bmax for CD3, they retained a level of binding affinity similar to that of the parent antibody (FIG. 14). On-cell binding results for the protease activated activatable bispecifics are summarized in Table 16.Table 16. On-cell binding of protease activated dually masked activatable anti-MUCl x anti-CD3s bispecific antibodies

[0385] The biological activity of the dually masked activatable bispecific antibodies was assayed using cytotoxicity assays. Cytotoxic potency of the dually masked activatable bispecific antibodies was compared to that of the unmasked antibody ProC2689 and between activatable antibodies in the activated and masked forms. The results are depicted in FIG. 15(HCT116-huMUCl cell line) and FIG. 16 (HP AC cell line). EC50 values are summarized in Table 17.Table 17. Cytotoxicity of protease activated dually masked activatable anti-MUCl x anti-CD3s bispecific antibodiesExample 8: In vivo Efficacy of Dually Masked Activatable anti-MUCl x anti-CD3s Bispecific AntibodiesAnti-tumor efficacy in HCT116-huMUCl xenograft model

[0386] Dually masked activatable bispecific antibodies ProC3405, ProC3699, and ProC3687, and unmasked ProC2689 were analyzed for their ability to induce regression or reduce growth of established human xenograft tumors in human PBMC engrafted NSG mice. The activatable bispecific antibodies differed from each other with respect to the cleavable moiety on each arm (see Table 12).

[0387] Human colorectal cancer cell line HCT116-huMUCl was cultured in McCoy’s media + 10% FBS according to established procedures. Purified, frozen human PBMCs were obtained from Charles River Laboratories, Wellington, MA (Donor ID # D340193.1; Lot# 21070559). NSG™ (NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ) mice were obtained from The Jackson Laboratories (Bar Harbor, ME). On day 0, each mouse was inoculated subcutaneously at the right flank with 2xl06HCT116-huMUCl cells in 100 pL RPMI + Glutamax, serum-free medium. Previously frozen PBMCs from a single donor were thawedand administered (i.p.) on day 3 at a CD3+ T cell to tumor cell ratio of 1:1. When tumor volumes reached -125 mm3, mice were randomized, assigned to treatment groups, and dosed i.v. according to Table 18. The results are shown in FIG. 17, which depicts a plot of tumor volume versus days post initial treatment dose. The results indicate that three of the dually masked activatable bispecific antibodies, ProC3405, ProC3699, ProC3687, and the unmasked bispecific antibody ProC2689, induced regression of HCT116-huMUCl xenograft tumors.Anti-tumor efficacy in HPAC xenograft model

[0388] Human pancreatic adenocarcinoma cell line HPAC was cultured in DMEM:F12 Medium (ATCC, cat#30-2006) supplemented with 0.002 mg / ml insulin, 0.005 mg / ml transferrin, 40 ng / ml hydrocortisone, 10 ng / ml hEGF (Lonza, Cat# 00192152), and 5% FBS. Purified, frozen human PBMCs were obtained from Charles River Laboratories, Wellington, MA (Donor ID # D163477, Lot# 23082047 and 23083322 ). NSG™ (NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ) mice were obtained from The Jackson Laboratories (Bar Harbor, ME). On day 0, each mouse was inoculated subcutaneously at the right flank with 5xl06HPAC cells in 100 pL RPMI + Glutamax, serum-free medium. Previously frozen PBMCs from a single donor were thawed and administered (i.p.) on day 3 at a CD3+ T cell to tumor cell ratio of 1:1. When tumor volumes reached -125 mm3, mice were randomized, assigned to treatment groups and dosed i.v. according to Table 18. The results are shown in FIG. 18, which depicts a plot of tumor volume versus days post initial treatment dose. The results indicate that three of the dually masked activatable bispecific antibodies, ProC3405, ProC3699, ProC3687, and the unmasked bispecific antibody ProC2689, induced regression of HPAC xenograft tumors.Table 18. Xenograft cytotoxicity study designAnti-tumor efficacy in NCI-H1650 xenograft model

[0389] Human non-small cell lung cancerl cell line NCI-H1650 was cultured in RPMI-1640 medium. On day 0, each NOG (NOD.Cg-Prkdcscid I12rgtmlSug / ShiJic) mouse was inoculated subcutaneously at the right flank with 5xl06NCI-H1650 cells in 100 |aL PBS. Previously frozen PBMCs from a single donor (Lonza, cat#CC-2702, Lot#21TL076010) were thawed and administered (i.v.) on day 5 with 2.5xl06cells in 200 |aL PBS. When tumor volumes reached -100 mm3, mice were randomized, assigned to treatment groups and dosed i.p. according to Table 19. The results are shown in FIG. 19, which depicts a plot of tumor volume versus days post initial treatment dose. The results indicate that three of the dually masked activatable bispecific antibodies, ProC3405, ProC3699, ProC3687, and the unmasked bispecific antibody ProC2689, induced tumor growth inhibition (TGI) of NCLH1650 xenograft tumors.Table 19. Xenograft cytotoxicity study designExample 9: MUC1 Expression Analysis on Human Cancer Cell LinesFlowcytometry analysis

[0390] Although MUC1 expression is significantly increased when the cells became malignant, its expression level varies among cancer patients (Jing Tu et al., J Oncol. 2022; Vol. 2022:4181658; B. V. Sinn et al., Annals of Oncology. 2013, 24(9): 2316-2324). Cytotoxicity of bivalent T cell bispecific antibodies have been reported to be less potent on tumor cells with a low target density (Lennart K et al., MABS. 2023, 15( 1):2183540; T.T. Junttila et al., Cancer Res 2014, 174( 19):5561-71 ). Taken together, MUC1 -targeting T cell bispecific antibodies with anti-tumor efficacy against low MUC1 -expressing tumor cells have a higher potential to treat a wider range of patients.

[0391] To investigate the anti-tumor efficacy of the bispecific activatable antibodies in cancer cell lines with different MUC1 expression levels, three cell lines were selected based on different MUC1 expression levels determined by flow cytometry.

[0392] Human colorectal cancer cell line HCT116-huMUCl was cultured in McCoy's 5A medium including 10% of FBS (Hyclone, SH30070.03). Human pancreatic adenocarcinoma cell line HP AC was cultured in DMEM / F12 medium including 10% of FBS. Human nonsmall cell lung cancer cell line NCLH1650 was cultured in RPML1640 medium including 10% of FBS.

[0393] Each cell line was collected and suspended in autoMACS Running Buffer (Miltenyi Biotec, Cat# 130-091-221). Subsequently the buffer was replaced with liquid solution containing the anti-MUCl antibody, ProC2689, and incubated for 30 min at a final concentration of 5 pg / ml. After washing twice with the autoMACS Runnning Buffer, antihuman IgG-PE (Jackson Immuno Research, 109-116-098) was added and incubated for 30 minutes at a final concentration of 2.5 pg / ml. After the wash step, MUC1 expression on the cells was analyzed by CytoFLEXS (Beckman Coulter).

[0394] Mean fluorescence intensity (MFI) of each sample was calculated by FlowJo (Becton Dickinson). Relative fluorescence intensity (RFI) was calculated by the following formula: (MFI of a sample incubated with anti-MUCl antibody) / (MFI of a sample incubated with negative control). RFI acquired on each human cancer cell was shown in the Table 20.

[0395] HCT116-huMUCl cells were shown to have the highest MUC1 expression. In contrast, expression level of MUC1 in HP AC was moderate, and MUC1 expression level was relatively low in NCLH1650 cells.Table 20. RFI acquired on each human cancer cell

[0396] The maximum % tumor growth inhibition of the dually masked activatable bispecific antibodies, ProC3405, ProC3699, ProC3687, and the unmasked bispecific antibody ProC2689, in each of the three xenograft models was shown in Table 21. As demonstrated, these antibodies were able to inhibit tumor growth in all three mouse models including mice with a low MUC1 expression level (NCI-H1650). The significant anti-tumor efficacy against low MUC1 -expressing cells suggested that these antibodies have potential to treat a wider range of patients including those with a low MUC1 expression level on cancer cells.Table 21. %Tumor Growth Inhibition in each anti-tumor efficacy studySequence Table

Claims

Claims1. A bispecific activatable antibody (BAA), wherein the BAA comprises a first binding component (FBC) and a second binding component (SBC), wherein the FBC comprises(a) an antigen binding domain (AB1) that specifically binds to a CD3, wherein the AB 1 comprises a heavy chain variable domain (AB 1 VH) and a light chain variable domain (AB1 VL); and(b) a first masking moiety (MM1) linked to a first cleavable moiety (CM1), wherein the MM1 inhibits the binding of the AB1 to CD3, wherein the CM1 is a polypeptide that functions as a substrate for a first protease, wherein the MM1 is linked in an amino- (N-) to carboxyl- (C-) terminal direction to the CM1 to form an MM1-CM1 construct, and wherein the C-terminus of the MM1-CM1 construct is linked to the N- terminus of AB1; and wherein the SBC comprises(a) an antigen binding domain (AB2) that specifically binds to a MUC1, wherein the AB2 comprises a heavy chain variable region (AB2 VH) and a light chain variable region (AB2 VL), and(b) a second masking moiety (MM2) linked to a second cleavable moiety (CM2), wherein the MM2 inhibits the binding of the AB2 to MUC1, wherein the CM2 is a polypeptide that functions as a substrate for a second protease, wherein the MM2 is linked to an N- to C-terminal direction to the CM2 to form an MM2-CM2 construct, and wherein the C-terminus of the MM2-CM2 construct is linked to the N-terminus of AB2.

2. The BAA of claim 1, wherein the AB 1 VH comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO:3, and wherein the AB 1 VL comprises a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light chain complementarity determining region 2 (LCDR2) ofSEQ ID NO:5, and a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6.

3. The BAA of claim 1 or 2, wherein the AB 1 VH comprises a sequence of SEQ ID NO: 13 and the AB1 VL comprises a sequence of SEQ ID NO: 14.

4. The BAA of any one of claims 1-3, wherein the AB1 is a single chain fragment variable (scFv) comprising the AB 1 VH and the AB 1 VL.

5. The BAA of any one of claims 1-4, wherein the MM1 comprises an amino acid sequence of SEQ ID NO:30.

6. The BAA of any one of claims 1-5, wherein the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.

7. The BAA of claim 6, wherein the CM1 comprises an amino acid sequence of SEQ ID NO:51.

8. The BAA of claim 6, wherein the CM1 comprises an amino acid sequence of SEQ ID NO:53.

9. The BAA of claim 6, wherein the CM1 comprises an amino acid sequence of SEQ ID NO:54.

10. The BAA of any one of claims 1-9, wherein the AB2 VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and wherein the AB2 VL comprises a LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

11. The BAA of any one of claims 1-10, wherein the AB2 VH comprises a sequence of SEQ ID NO: 15 and the AB2 VL comprises a sequence of SEQ ID NO: 16.

12. The BAA of any one of claims 1-11, wherein the AB2 is a Fab fragment comprising the AB2 VH and the AB2 VL.

13. The BAA of any one of claims 1-12, wherein the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.

14. The BAA of claim 13, wherein the MM2 comprises SEQ ID NO:36.

15. The BAA of any one of claims 1-14, wherein the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55.

16. The BAA of claim 15, wherein the CM2 comprises an amino acid sequence of SEQ ID NO: 52.

17. The BAA of claim 15, wherein the CM2 comprises an amino acid sequence of SEQ ID NO: 54.

18. The BAA of claim 15, wherein the CM2 comprises an amino acid sequence of SEQ ID NO: 55.

19. The BAA of any one of claims 1-18, wherein the BAA comprises a first antibody component (FAC) and a second antibody component (SAC), wherein the FAC comprises the FBC, a first hinge region and a first Fc region (Fcl), and wherein the Fcl is linked in an N- to C-terminal direction to the FBC via the first hinge region, and wherein the SAC comprises the SBC, a second hinge region and a second Fc region (Fc2), and wherein the Fc2 is linked in an N- to C-terminal direction to the SBC via the second hinge region.

20. The BAA of any one of claims 1-19, wherein the SAC comprises a heavy chain (HC) and a light chain (LC), wherein the AB2 VH is present within the HC that further comprises a CHI constant domain, the second hinge region and / or the Fc2, and wherein the AB2 VL is present within the LC that further comprises a light chain constant domain.

21. The BAA of claim 19 or 20, wherein the first hinge region and / or the second hinge region comprise an amino acid sequence encoding a human IgG hinge region or a variant thereof comprising from one to ten substitutions.

22. The BAA of claim 21, wherein the first hinge region and / or the second hinge region comprise an amino acid substitution in at least one of amino acid positions at S228, L234, and L235, as numbered by the EU index as set forth in Kabat.

23. The BAA of any one of claims 19-22, wherein the Fcl and / or the Fc2 comprise an amino acid sequence encoding a human IgG Fc domain or a variant thereof comprising from one to ten substitutions.

24. The BAA of claim 23, wherein the Fcl and / or the Fc2 comprise an amino acid substitution in at least one of amino acid positions at S354, T366, E368, Y349 and Y407, as numbered by the EU index as set forth in Kabat.

25. The BAA of any one of claims 1-24, wherein the BAA comprises at least one linker present between a pair of components selected from the group consisting of MM1 and CM1, CM1 and AB1 VH, AB1 VH and AB1 VE, AB1 VE and a first hinge region, MM2 and CM2, and CM2 and AB2 VH.

26. The BAA of any one of claims 1-25, wherein the FAC comprises an amino acid sequence selected from the group consisting of(a) SEQ ID NO:17,(b) SEQ ID NO:20,Ill(c) SEQ ID NO:22, and(d) an amino acid sequence at least 90% identical thereto.

27. The BAA of any one of claims 1-26, wherein the SAC comprises an amino acid sequence selected from the group consisting of(a) SEQ ID NO: 18 and SEQ ID NO: 19,(b) SEQ ID NO:21 and SEQ ID NO: 19,(c) SEQ ID NO:23 and SEQ ID NO: 19, and(d) an amino acid sequence at least 90% identical thereto.

28. The BAA of any one of claims 1-27, wherein the BAA comprises a set of amino acid sequences selected from the group consisting of(a) SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19,(b) SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO: 19,(c) SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO: 19, and(d) an amino acid sequence at least 90% identical thereto.

29. The BAA of any one of claims 1-28, wherein the BAA comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

30. The BAA of any one of claims 1-29, wherein the first protease and / or the second protease is a matrix metalloproteinase (MMP), a cysteine protease, and / or a serine protease.

31. An isolated nucleic acid molecule encoding the BAA of any one of claims 1-30 selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;.(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO: 18;(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a LC of a SAC consisting of an amino acid sequence of SEQ ID NO: 19;(h) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20;(i) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO:21;(j) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22; and(k) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a HC of a SAC consisting of an amino acid sequence of SEQ ID NO:23.

32. A vector comprising the isolated nucleic acid molecule of claim 31.

33. A cell comprising the nucleic acid molecule of claim 31, or the vector of claim 32.

34. A cell comprising the nucleic acid molecule encoding the BAA of any one of claims 26-28 selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 comprising an amino acid sequence of SEQ ID NO: 13 and an amino acid sequence of SEQ ID NO: 14; and a nucleotide sequence encoding an AB2 comprising an amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO: 16;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO: 17, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO: 18 and an amino acid sequence of SEQ ID NO: 19;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:20, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO:21 and an amino acid sequence of SEQ ID NO: 19; and(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a FAC consisting of an amino acid sequence of SEQ ID NO:22, and a nucleotide sequence encoding a SAC consisting of an amino acid sequence of SEQ ID NO:23 and an amino acid sequence of SEQ ID NO: 19.

35. A pharmaceutical composition comprising the BAA of any one of claims 1-30 and a pharmaceutically acceptable excipient.

36. The pharmaceutical composition of claim 35 for use in combination with another therapeutic agent for treating cancer.

37. A kit comprising the BAA of any one of claims 1-30, or the pharmaceutical composition of claim 35 or 36 and instructions for use.

38. A method of producing the BAA of any one of claims 1-30 by culturing a cell under conditions that lead to expression of the BAA, wherein the cell comprises the nucleic acid molecule of claim 31 or the vector of claim 32.

39. A method of treating cancer comprising administering a therapeutically effective amount of the BAA of any one of claims 1-30, or the pharmaceutical composition of claim 35 or 36, to a subject in need thereof.

40. A method of treating cancer comprising administering a therapeutically effective amount of the BAA of any one of claims 1-30, or the pharmaceutical composition of claim 35 or 36, in combination with another therapeutic agent for treating cancer to a subject in need thereof.

41. Use of the BAA of any one of claims 1-30 in the production of a pharmaceutical composition for treating cancer.

42. A bispecific antibody comprising(a) a first antigen binding domain (AB1) that specifically binds to a CD3, wherein the AB 1 comprises a AB 1 VH and a AB 1 VL,wherein the AB 1 VH comprises a HCDR1 of SEQ ID NO: 1, a HCDR2 of SEQ ID NO:2, and a HCDR3 of SEQ ID NO:3, and wherein the AB1 VL comprises a LCDR1 of SEQ ID NO:4, a LCDR2 of SEQ ID NO:5, and a LCDR3 of SEQ ID NO:6, and(b) a second antigen binding domain (AB2) that specifically binds to a MUC1, wherein the AB2 comprises an AB2 VH and an AB2 VL.

43. The bispecific antibody of claim 42, wherein the AB1 VH comprises a sequence of SEQ ID NO: 13 and the AB1 VL comprises a sequence of SEQ ID NO: 14.

44. The bispecific antibody of claim 42 or 43, wherein the AB1 is a scFv comprising the AB1 VH and the AB1 VL.

45. The bispecific antibody of any one of claims 42-44, wherein the AB2 VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and wherein the AB2 VL comprises a LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

46. The bispecific antibody of any one of claims 42-45, wherein the AB2 VH comprises a sequence of SEQ ID NO: 15 and the AB2 VL comprises a sequence of SEQ ID NO: 16.

47. The bispecific antibody of any one of claims 42-46, wherein the AB2 is a Fab fragment comprising the AB2 VH and the AB2 VL.

48. The bispecific antibody of any one of claims 42-47, wherein the bispecific antibody further comprises a first hinge region and a Fcl, and a second hinge region and a Fc2.

49. The bispecific antibody of claim 48, wherein the first hinge region and the second hinge region comprise an amino acid sequence encoding a human IgG hinge region or a variant thereof comprising from one to ten substitutions.

50. The bispecific antibody of claim 49, wherein the first hinge region and the second hinge region comprise an amino acid substitution in at least one of amino acid positions atS228, L234, and L235, as numbered by the EU index as set forth in Kabat.

51. The bispecific antibody of claim 50, wherein the Fcl and the Fc2 comprise an amino acid sequence encoding a human IgG Fc domain or a variant thereof comprising from one to ten substitutions.

52. The bispecific antibody of claim 51, wherein the Fcl and the Fc2 comprise an amino acid substitution in at least one of amino acid positions at S354, T366, E368, Y349 and Y407, as numbered by the EU index as set forth in Kabat.

53. The bispecific antibody of any one of claims 42-52, wherein the bispecific antibody comprises an AB 1 Fc fusion, an AB2 HC and an AB2 EC, wherein the AB1 Fc fusion comprises the AB1, the first hinge region and the Fcl, wherein the AB2 HC comprises the AB2 VH, a CHI constant domain, the second hinge region and the Fc2, and wherein the AB2 EC comprises the AB2 VE and a light chain constant domain.

54. The bispecific antibody of claim 53, wherein the AB1 Fc fusion comprises an amino acid sequence of SEQ ID NO:24, or an amino acid sequence at least 90% identical thereto; wherein the AB2 HC comprises an amino acid sequence of SEQ ID NO:25, an amino acid sequence at least 90% identical thereto; and wherein the AB2 EC comprises an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 90% identical thereto.

55. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 42-54 selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VH consisting of an amino acid sequence of SEQ ID NO: 13;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 VL consisting of an amino acid sequence of SEQ ID NO: 14;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding anAB2 VH consisting of an amino acid sequence of SEQ ID NO: 15;(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 VL consisting of an amino acid sequence of SEQ ID NO: 16;.(e) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 Fc fusion consisting of an amino acid sequence of SEQ ID NO:24;(f) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 HC consisting of an amino acid sequence of SEQ ID NO:25; and(g) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB2 LC consisting of an amino acid sequence of SEQ ID NO: 19.

56. A vector comprising the isolated nucleic acid molecule of claim 55.

57. A cell comprising the nucleic acid molecule of claim 55, or the vector of claim 56.

58. A cell comprising the nucleic acid molecule encoding the bispecific antibody of claim 55 selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 comprising an amino acid sequence of SEQ ID NO: 13 and an amino acid sequence of SEQ ID NO: 14, and a nucleotide sequence encoding an AB2 comprising an amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO: 16; and(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding an AB1 Fc fusion consisting of an amino acid sequence of SEQ ID NO:24, and a nucleotide sequence encoding an AB2 HC consisting of an amino acid sequence of SEQ ID NO:25 and an AB2 LC consisting of an amino acid sequence of SEQ ID NO: 19.

59. A monoclonal antibody comprising an antigen binding domain that specifically binds to MUCl, wherein the antigen binding domain comprises a VH and a VL; wherein the VH comprises a HCDR1 of SEQ ID NO:7, a HCDR2 of SEQ ID NO:8, and a HCDR3 of SEQ ID NO:9, and wherein the VL comprises a LCDR1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12.

60. The monoclonal antibody of claim 59, wherein the VH comprises a sequence of SEQ ID NO: 15 and the VL comprises a sequence of SEQ ID NO: 16.

61. A monoclonal antibody comprising an antigen binding domain that specifically binds to MUCl, wherein the antigen binding domain comprises a VH and a VL; wherein the VH comprises a HCDR1 of SEQ ID NO:45, a HCDR2 of SEQ IDNO:46, and a HCDR3 of SEQ ID NO:47, and wherein the VL comprises a LCDR1 of SEQ ID NO:48, a LCDR2 of SEQ ID NO:49, and a LCDR3 of SEQ ID NO:50.

62. The monoclonal antibody of claim 61, wherein the VH comprises a sequence of SEQ ID NO:28 and the VL comprises a sequence of SEQ ID NO:29.

63. An isolated nucleic acid molecule encoding the monoclonal antibody of any one of claims 59-62 selected from the group consisting of(a) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 15;(b) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO: 16;(c) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VH consisting of an amino acid sequence of SEQ ID NO: 28; and(d) an isolated nucleic acid molecule comprising a nucleotide sequence encoding a VL consisting of an amino acid sequence of SEQ ID NO:29.

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

65. A cell comprising the nucleic acid molecule of claim 63, or the vector of claim 64.

66. A pharmaceutical composition comprising the bispecific antibody of any one of claims 42-54, or the monoclonal antibody of any one of claims 59-62, and a pharmaceutically acceptable excipient.

67. The pharmaceutical composition of claim 66 for use in combination with another therapeutic agent for treating cancer.

68. A kit comprising the bispecific antibody of any one of claims 42-54, or the monoclonal antibody of any one of claims 59-62, or the pharmaceutical composition of claim 66 or 67, and instructions for use.

69. A method of producing the bispecific antibody of any one of claims 42-54, or the monoclonal antibody of any one of claims 59-62, by culturing a cell under conditions that lead to expression of the bispecific antibody or the monoclonal antibody, wherein the cell comprises the nucleic acid molecule of claim 55 or 63, or the vector of claim 56 or 64.

70. A method of treating cancer comprising administering a therapeutically effective amount of the bispecific antibody of any one of claims 42-54, or the monoclonal antibody of any one of claims 59-62, or the pharmaceutical composition of claim 66 or 67, to a subject in need thereof.

71. A method of treating cancer comprising administering a therapeutically effective amount of the bispecific antibody of any one of claims 42-54, or the monoclonal antibody of any one of claims 59-62, or the pharmaceutical composition of claim 66 or 67, in combination with another therapeutic agent for treating cancer to a subject in need thereof.

72. Use of the bispecific antibody of any one of claims 42-54, or the monoclonal antibody of any one of claims 59-62, in the production of a pharmaceutical composition for treating cancer.

73. A method of treating a low-MUCl expressing cancer, the method comprising administering a therapeutically effective amount of the bispecific antibody of any one of claims 42-54, or the pharmaceutical composition of claim 66 or 67, to a subject in need thereof, thereby treating the low-MUCl expressing cancer in the subject.

74. A method of treating a low-MUCl expressing cancer, the method comprising selecting a subject having a low-MUCl expressing cancer, and administering a therapeutically effective amount of the bispecific antibody of any one of claims 42-54, or themonoclonal antibody of any one of claims 59-62, or the pharmaceutical composition of claim 66 or 67, to the subject, thereby treating the low-MUCl expressing cancer in the subject.

75. The method of claim 73 or 74, wherein the low-MUCl expressing cancer expresses MUC1 at a level of less than about 170 as measured by RFI (relative fluorescence intensity).

76. The method of claim 73 or 74, wherein the low-MUCl expressing cancer expresses MUC1 at a level of about 1 to 170, or about 1 to 150, or about 1 to 130, or about 1 to 100, or about 1 to 80, or about 1 to 60, or about 1 to 55, as measured by RFI (relative fluorescence intensity).

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